Scientific syllabus 2012 umdf

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Mitochondrial Medicine 2012: Capitol Hill From Genomics and Systems Biology to Translation

Bethesda North Marriott Hotel & Conference Center 5701 Marinelli Road, Bethesda, MD 20851

Scienti!c Meetings: June 13-16, 2012 Capitol Hill Advocacy Day: June 14, 2012

2012 Course Chair: Vamsi Mootha, M.D. 2012 CME Chair: Bruce H. Cohen, M.D.

A special thanks to those organizations that made this event possible:the Northeast Ohio Medical University, the Mitochondrial Medicine Society,

the Mitochondrion/Mitochondria Research Society, and the Mitochondrial Physiology Society

Scienti!c Program June 13-16, 2012

Mitochondrial Medicine 2012: Capitol Hill

2012 Course DescriptionThe United Mitochondrial Disease Foundation, the Northeast Ohio Medical University, the Mitochondrial Medicine Society, the Mitochondria Research Society, and the Mitochondrial Physiology Society have joined e!orts to sponsor and organize another multidisciplinary symposium. Mitochondrial diseases are more common than previously recognized, and mitochondrial pathophysiology is now a recognized part of many disease processes, including heart disease, cancer, AIDS, and diabetes. There have been signi"-cant advances in the molecular genetics, proteomics, epidemiology and clinical aspects of mitochondrial pathophysiology. This conference is directed toward the scientist and clinician interested in all aspects of mitochondrial science.

The content of this educational program was determined by rigorous assessment of educational needs and includes surveys, program feedback, expert faculty assessment, literature review, medical practice, chart review and new medical knowledge. The format will include didactic lectures from invited experts in-termixed with peer-reviewed platform presentations. There will be ample time for professional discussion both in and out of the meeting room, and peer-reviewed poster presentations will be given throughout the meeting. This will be a four-day scienti"c meeting aimed at those with scienti"c and clinical interests.

Accreditation StatementThis activity has been planned and implemented in accordance with the Essential Areas and Policies of the Accreditation Council for Continuing Medical Education (ACCME) and the Accreditation Council for Phar-macy Education (ACPE); through the co/joint sponsorship of Northeast Ohio Medical University and the United Mitochondrial Disease Foundation. Northeast Ohio Medical University is accredited by the ACCME and ACPE to provide continuing education for physicians and pharmacists.

CreditsNortheast Ohio Medical University designates this live activity for a maximum of 23.25 AMA PRA Category 1 CreditsTM. Physicians should claim only the credits commensurate with the extent of their participation in the activity.

Target AudienceScientists, physicians (neurologists, geneticists, pediatrics/generalists, nephrologists, cardiologists, endo-crinologists), nurses, physician assistants, advanced practice nurses, genetic counselors and allied health professionals.

AwardsCash awards of $125, $250, $500 and $1,000 will be given to the top four posters, courtesy of the Mito-chondria Research Society (MRS) and the Mitochondrial Medicine Society (MMS).

Grantor AcknowledgementThe Northeastern Ohio Universities Colleges of Medicine and Pharmacy and the United Mitochondrial Disease Foundation acknowledge educational grants for partial support of this activity from: The Burroughs Wellcome Fund.

Mitochondrial Medicine 2012: Capitol HillFrom Genomics and Systems Biology to Translation

Mitochondrial Medicine 2012: Capitol Hill Special Announcements

Name Badges All attendees must wear a name badge to all course functions. At the end of the course, please return your name badge at the registration desk for recycling.

Scienti!c Sessions All scienti"c sessions will be held in the Grand Ballroom D.

Refreshment Breaks Exhibits will be open in the Ballroom Foyer during all breaks and lunches. Posters will be in Salons A & B. Abstract presenters are asked to station themselves at their posters during breaks to "eld questions. Times are as follows: Wednesday (even numbers) at 4:15 p.m. and Thursday (odd numbers) at 4:30 p.m.

Lunch Lunch will be held in the White Oak Conference Room (lower level) on Wednesday and Thursday and in Salons E & F (upper level) on Friday and Saturday.

Friday Night Banquet and Awards Ceremony Scienti"c and family program attendees are invited to attend the Friday Night Banquet featuring Keynote Speaker, William A. Gahl, M.D., Ph.D., Clinical Director, National Human Genome Research Institute. The reception begins at 6:00 p.m. in the Grand Ballroom Foyer with dinner at 7:00 p.m. in the Grand Ballroom.

Evaluation FormsPlease complete the Participant Course Evaluation Form and return them to the registration desk at the conclusion of the symposium. We appreciate your comments and "nd your feedback very useful for future planning.

CME Certi!catesPhysicians: All physicians were given a 3-part CME Certi"cate at Registration. Fully complete, sign, and turn in the carbon copies of the form at the end of the meeting (or upon your departure if you will not be staying for the duration of the meeting). Keep the WHITE copy for your records. This serves as your Credit/Attendance Record.

Non-Physicians: All non-physicians were given a 3-part Attendance Certi"cate at registration. Fully complete, sign, and turn in the carbon copies of the form at the end of the meeting (or upon your departure if you will not be staying for the duration of the meeting). Keep the WHITE copy for your records. This serves as your attendance certi"cate.

Mitochondrial Medicine 2012: Capitol Hill2012 Scienti!c Planning Committee

Mikhail Alexeyev, Ph.D., University of South Alabama, Mobile, AL Leonardo Alves, University of California, Irvine/ Children’s Hospital of Philadelphia, Philadelphia, PA Renkui Bai, M.D., Ph.D., GeneDx, Gaithersburg, MD Penelope Bonnen, Ph.D., Baylor College of Medicine, Houston, TX Nicola Brunetti-Pierri, M.D., Telethon Institute of Genetics and Medicine, Naples, IT Carolyn Buzin, Ph.D ., MEDomics, Azusa, CA Anne Chiaramello, Ph.D., George Washington University Medical Center, Washington, DC Jeana DaRe, Ph.D., Transgenomic, New Haven, CT Antonio Davila, JR, University of California - Irvine, Irvine, CA Francisca Diaz, Ph.D., University of Miami, Miami, FL Lisa Emerick, M.D., Baylor College of Medicine, Houston, TX Amy Goldstein, M.D., University of Pittsburgh School of Medicine, Pittsburgh, PA Steve Hershman, M.S., Broad Institute of MIT/Harvard, Cambridge, MA Veronica Hinton, Ph.D., Columbia University, New York City, NY Tim Koves, Ph.D., Duke University, Durham, NC Ran Lin, Research Institute of Children’s Hospital of Philadelphia, Philadelphia, PA Chun Shi Lin, University of California - Irvine, Costa Mesa, CA Igal Madar, Ph.D., Johns Hopkins Medical Institutions, Baltimore, MD Kasturi Mitra, Ph.D., National Institute of Child Health and Human Development, NIH, Bethesda, MD Phil Morgan, M.D., University of Washington, Seattle, WA Neal Sondheimer, M.D., Ph.D., The University of Pennsylvania, Philadelphia, PA Peter Stacpoole, Ph.D., M.D., University of Florida, Gainesville, FL James Stewart, Ph.D., Max Planck Institute for the Biology of Ageing, Cologne, Germany Ashley Wolf, Mootha Lab, Massachusetts General Hospital, Boston, MA Fang Ye, Ph.D., Case Western Reserve University, Cleveland, OH Xiaoshan Zhou, Ph.D,. M.D., Karolinska Institutet, Stockholom, Sweden

2012 Abstract Presenters

Vamsi Mootha, M.D., Course Chair, Harvard Medical School, Boston, MA Bruce H. Cohen, M.D., 2012 CME Chair, Akron Children’s Hospital, Akron, OH William C. Copeland, Ph.D., NIEHS, Research Triangle Park, NC Erich Gnaiger, Ph.D., Innsbruck Medical University, Innsbruck, Austria Carla Koehler, Ph.D., University of California Los Angeles, Los Angeles, CA Giovanni Manfredi, M.D., Ph.D., Weill Medical College of Cornell University, New York, NY Carlos Moraes, Ph.D., University of Miami, Miami, FL Robert K. Naviaux, M.D., Ph.D., University of California San Diego, San Diego, CA Thomas O’Brien, Ph.D., University of Florida, Gainesville, FL Russell Saneto, D.O., Ph.D., Seattle Children’s Hospital/University of Washington, Seattle, WA Peter Stacpoole, Ph.D., M.D., University of Florida, Gainesville, FL Keshav K. Singh, Ph.D., University of Alabama, Birmingham, AL

Mitochondrial Medicine 2012: Capitol Hill2012 Scienti!c Meeting Faculty

Robert S. Balaban, Ph.D., National Heart Lung and Blood Institute, NIH, Bethesda, MD Sarah Calvo, Ph.D., Broad Institute of MIT/Harvard, Cambridge, MA Patrick Chinnery, Ph.D., FRCP, FMedSci, Newcastle University, Newcastle upon Tyne, UK

Ralph J. DeBerardinis, M.D., Ph.D., University of Texas-Southwestern Medical Center, Dallas, TX Gregory M. Enns, MB, ChB, Stanford University Medical Center, Palo Alto, CA Marni J. Falk, M.D., Children’s Hospital of Philadelphia, Philadelphia, PA William Gahl, M.D., Ph.D., National Human Genome Research Institute, NIH, Bethesda, MD Michael W. Gray, Ph.D., Dalhousie University, Halifax, Nova Scotia, Canada Richard H. Haas, MB, BChir, MRCP, University of California San Diego, San Diego, CA Ron Haller, M.D., University of Texas-Southwestern Medical Center, Dallas, TX

Michio Hirano, M.D., Columbia University Medical Center, New York, NY Charles Hoppel, M.D., Case Western Reserve University, Cleveland, OH Vamsi Mootha, M.D., Massachusetts General Hospital, Harvard Medical School, and The Broad Institute,

Boston, MA Dave Pagliarini, Ph.D., University of Wisconsin-Madison, WI Joshua Rabinowitz, M.D., Ph.D., Princeton University, Princeton, NJ Curt Scharfe, M.D., Ph.D., Stanford Genome Technology Center, Palo Alto, CA Eric A. Shoubridge, Ph.D., Montreal Neurological Institute, Montreal, Quebec, Canada Jan Smeitink, M.D., Ph.D., Nijmegen Center for Mitochondrial Disorders, Nijmegen, The Netherlands David Thorburn, Ph.D., Murdoch Children’s Research Institute, Victoria, Australia Akhil B. Vaidya, Ph.D., Drexel University College of Medicine, Philadelphia, PA Charles P. Venditti, M.D., Ph.D., National Human Genome Research Institute, NIH, Bethesda, MD Jennifer van Eyk, Ph.D., John Hopkins University, Baltimore, MD Anu Suomalainen Wartiovaara, M.D., Ph.D., University of Helsinki, Helsinki, Finland Lee-Jun C. Wong, Ph.D., Baylor College of Medicine, Houston, TX

Save the Date!Mitochondrial Medicine 2013:

Newport Beach

Wednesday, June 12, 2013 thru Saturday, June 15, 2013

Newport Beach Marriott Hotel & Spa900 Newport Center DriveNewport Beach, CA 92660

Mitochondrial Medicine 2012: Capitol HillDisclosure of Relevant Financial Relationships

Everyone involved in the planning, reviewing and teaching of this activity is required to complete a disclosure form indicating all relevant "nancial relationships with any ‘commercial interest’. A ‘commer-cial interest’ is any entity producing, marketing, reselling, or distributing health care goods or services consumed by, or used on, patients. This is done so that the audience can determine whether an indi-vidual’s relationships may in#uence the presentations.

Renkui Bai, M.D., Ph.D.Employee – GeneDX

Carolyn H. Buzin, PhDEmployee – Medomics, LLC

Bruce Cohen, M.D.Speaker, Consultant – Transgenomic, IncMedical Author and Reviewer – New Mentor

Jeana Dare, Ph.D.Ownership Interest – Transgenomic, Inc

Ralph Deberardinis, M.D.Speaker – Genentech, Agius, P"zer, Glaxo-Smith ClineConsultant – Calithera, Johnson and Johnson

Gregory M. Enns, MB, ChBInvestigator – Edison Pharmaceuticals

Erich Gnaiger, Ph.D. Owner/Director – Oroboros Instruments Corporation

Michio Hirano, M.D.Speaker – Athena DiagnosticsInvestigator – Santhera Pharmaceutical, Edison Pharmaceuticals

Robert K Naviaux, M.D., Ph.D.Scienti"c Founder – Clinical Metabolomics

Joshua Rabinowitz, M.D., Ph.D.Consultant – Kadmon Pharmaceuticals

Jan Smeitink, M.D., Ph.D.Founder and CEO – Khondrion BV

Mitochondrial Medicine 2012: Capitol HillBethesda North Marriott Hotel & Conference Center

Mitochondrial Medicine 2012: Capitol HillScienti!c Program Schedule

Day 1: Wednesday, June 13, 2012

SCHEDULE

NOTE

Morning Platform Session (1)Platform Title: Genomics of Mitochondrial Biology8:00 a.m. Speaker: Vamsi K. Mootha, M.D., Course Chair Topic: Identi!cation of the Calcium Uniporter via Comparative Genomics8:45 a.m. Speaker: Michael W. Gray, Ph.D. Topic: Genomics vs. Proteomics: Two Views of Mitochondrial Evolution9:15 a.m. Speaker: Akhil B. Vaidya, Ph.D. Topic: Remaining Relevant: The Minimalist Mitochondrion of Malaria Parasite9:45 a.m. Break10:15 a.m. Abstract Presentations (7)12:00 p.m. Lunch

Afternoon Platform Session (2) Platform Title: Mitochondrial Proteomics2:00 p.m. Speaker: Anu Suomalainen Wartiovaara, M.D., Ph.D. Topic: Omics Approaches Reveal Novel Tools for Mitochondrial Disease Diagnosis2:30 p.m. Speaker: Dave Pagliarini, Ph.D. Topic: Proteomic Insights into Mitochondrial Form and Function3:00 p.m. Speaker: Jennifer van Eyk, Ph.D. Topic: Protein Modi!cations in the Mitochondria - Driving Heart Disease3:30 p.m. Abstract Presentations (3)4:15 p.m. Break & Poster Sessions (Non CME) Even Numbers Stay at Posters6:30 p.m. Adjourn

-

SCHEDULE

NOTE

Day 2: Thursday, June 14, 2012

Day 2: Thursday, June 17, 2010Morning Platform Session (3)Chemical Biology and Metabolomics

8:00 a.m. Speaker: Gregory M. Enns, MB, ChB Topic: Redox Biomarkers in Mitochondrial Disease

8:30 a.m. Speaker: Marni J. Falk, M.D. Topic: Treating Regulatory Signaling Network Changes that Cause the Metabolic Sequelae of Mitochondrial Disease

9:00 a.m. Speaker: Joshua Rabinowitz, M.D., Ph.D. Topic: Probing Cytosol-Mitochondrial Interplay via Metabolomics

9:30 a.m. Speaker: Ralph J. DeBerardinis, M.D., Ph.D. Topic: The Versatility of Mitochondrial Metabolism in Tumor Cell Growth

10:00 a.m. Break

10:30 a.m. Abstract Presentations (6)

12:00 p.m. Lunch

Afternoon Platform Session (4)Platform Title: Systems Biology and Dynamics

2:00 p.m. Speaker: Robert S. Balaban, Ph.D. Topic: The Systems Biology of the Cardiac Mitochondrion

2:30 p.m. Speaker: Curt Scharfe, M.D., Ph.D. Topic: Genome Technology for Mitochondrial Disease

3:00 p.m. Abstract Presentations (5)

3:30 p.m. Special Clinical Directors’ Workshop - This session is a new6:30 p.m. opportunity for clinicians, in an open discussion format, to work on day-to-day challenges faced by clinicians in their practices (patient care and systems-related). (Non CME)

4:30 p.m. Break and Posters (Non CME) - Odd Numbers Stay at Posters Reception and Cash Bar

6:30 p.m. Adjourn

SCHEDULE

NOTE

Day 3: Friday, June 15, 2012

Morning Platform Session (5)Next-Generation Sequencing of Mitochondrial Disease8:00 a.m. Speaker: David Thorburn, Ph.D. Topic: MitoExome Sequencing of Children with Mitochondrial Disease8:30 a.m. Speaker: Lee-Jun C. Wong, Ph.D. Topic: Challenges of Bringing Next Generation Sequencing Technologies to CLIA/CAP Certi!ed Clinical Laboratories9:00 a.m. Speaker: Eric A. Shoubridge, Ph.D. Topic: Exome Sequencing of Mitochondrial Disorders9:30 a.m. Abstract Presentations (2)10:00 a.m. Break10:30 a.m. Speaker: Sarah Calvo, Ph.D. Topic: Diagnostic E"cacy of Targeted Sequencing in Infantile Versus Adult Mitochondrial Disease11:00 a.m. Abstract Presentations (5)12:15 p.m. Lunch

Afternoon Platform Session (6) Translational Mitochondrial Medicine2:00 p.m. Speaker: Patrick Chinnery, Ph.D., FRCP Topic: Monitoring Disease: the Newcastle Approach2:30 p.m. Speaker: Charles P. Venditti, M.D., Ph.D. Topic: The Mitochondropathy of Methylmalonic Acidemia: De!nition and Therapeutic Approaches3:00 p.m. Speaker: Jan Smeitink, M.D., Ph.D. Topic: Disease Severity Scores and Outcome Measures in Mitochondrial Disease3:30 p.m. Break3:45 p.m. Speaker: Michio Hirano, M.D. Topic: North American Mitochondrial Disease Consortium (NAMDC)4:15 p.m. UMDF 2012 Funded Grant Projects (Abstracts)5:30 p.m. Adjourn6:00 p.m. Reception7:00 p.m. Friday Night Banquet and Awards Ceremony Keynote Address: William Gahl, M.D., Ph.D.

-

SCHEDULE

NOTE

Day 4: Saturday, June 16, 2012

Platform Session (7)Clinical Algorithms, Biochemical Testing, Exercise Dx

8:00 a.m. Speaker: Charles Hoppel, M.D. Topic: Respiratory Chain Testing

8:30 a.m. Speaker: Richard Haas, MB, BChir Topic: Biochemical Pro!ling in Mitochondrial Disease

9:00 a.m. Speaker: Ron Haller, M.D. Topic: Exercise Diagnosis

9:30 a.m. Speaker: Jan Smeitink, M.D., Ph.D. Topic: Oxidative Phosphorylation Disorders: from Bench to Bedside

10:00 a.m. Break

10:15 a.m. Speaker: David Thorburn, Ph.D. Topic: Criteria for Diagnosis of Mitochondrial Disease in the Era of Next Generation Sequencing

10:45 a.m. Speaker: Patrick Chinnery, Ph.D., FRCP Topic: New Treatments for Mitochondrial Diseases

11:15 a.m. Break - pick up boxed lunches and return to seats for panel discussion

11:30 a.m. Moderator: Vamsi Mootha, M.D. Panel: Chuck Hoppel, M.D.; Richard Haas, MB, BChir; Ron Haller, M.D.; Jan Smeitink, M.D., Ph.D.; Eric Shoubridge, Ph.D.; David Thorburn, Ph.D.; Patrick Chinnery, Ph.D., FRCP; Anu Wartiovaara, M.D., Ph.D. and Lee-Jun Wong, Ph.D. Panel Discussion: How to Incorporate Next Generation Pro!les into Routine Clinical Diagnosis

1:30 p.m. Adjournment

Platform Session 1: Abstracts

1 10:15 a.m. Mikhail Alexeyev Mutagenesis of mouse mitochondrial DNA

12 10:30 a.m. Neal Sondheimer Spontaneous Elimination of Mitochondrial Mutations During The Induction of Pluripotency

48 10:45 a.m. Ashley Wolf A systematic search for mitochondrial RNA processing components

78 11:00 a.m. James Stewart The maternal mtDNA mutation load heavily in#uences phenotypes in mtDNA mutator mice

88 11:15 a.m. Antonio Davila Epigenetic Memory in the Mitochondria of Human Embryonic Stem Cells

93 11:30 a.m. Steve Hershman Mutations in MTFMT Underlie a Human Disorder of Formylation Causing Impaired Mitochondrial Translation

95 11:45 a.m. Leonardo Alves Leber Hereditary Optic Neuropathy (LHON) associated mutation 3394 is also a high- altitude adaptive polymorphism


16 3:30 p.m. Anne Chiaramello The Neurogenic Basic Helix-Loop-Helix Transcription Factor NeuroD6 Induces Mitochondrial Biogenesis and Bioenergetics in Neuronal Cells

11 3:45 p.m. Peter Stacpoole Rapid Breath Test for In Vivo Determination of Human Pyruvate Dehydrogenase Complex Activity

70 4:00 p.m. Fang Ye Diagnostic application of Measuring Oxidative Phosphorylation in Permeabilized Skin Fibroblasts

Mitochondrial Medicine 2012: Capitol HillAbstract: Presentation Schedule

ABSTRACTS

Wednesday, June 13, 2012

*All Abstracts are listed in the back of this syllabus.


# Time Presenter Title

13 10:30 a.m. Lisa Emerick Glucose kinetics in subjects with MELAS syndrome: interim results

45 10:45 a.m. Phil Morgan Speci"c Hypersensitivity to Volatile Anesthetics in a Mouse Lacking Ndufs4, a Subunit of Mitochondrial Complex I

63 11:00 a.m. Nicola Brunetti-Pierri Phenylbutyrate therapy for pyruvate dehydrogenase de"ciency

103 11:15 a.m. Tim Koves Absence of malonyl-CoA decarboxylase (MCD) impacts endurance exercise capacity and reprograms skeletal muscle mitochondrial metabolism

20 11:30 a.m. Peter Stacpoole Long-term Safety of Dichloroacetate in Congenital Lactic Acidosis

51 11:45 a.m. Igal Madar In Vivo Localization and Quanti"cation of Mitochondrial Dysfunction Using PET Imaging of the Novel Voltage Sensor 18F-FBnTP

Platform Session 4: Abstracts# Time Presenter Title15 3:00 p.m. Xiaoshan Zhou Thymidine phosphorylation by transgene expression of the Drosophila melanogaster nucleoside kinase rescues the pathology of mitochondrial TK2 de"ciency

80 3:15 p.m. Kasturi Mitra Mitochondrial "ssion-fusion activities regulate cell fate determination between proliferation and di!erentiation: a possible link to tumorigenesis

31 3:30 p.m. Lisa Emerick PDHA1 Mutations and Continued Clinical and Genetic Heterogeneity: Are there gender Di!erences?

41 3:45 p.m. Veronica Hinton No Evidence of Cognitive Decline among Carrier Relatives of MELAS Patients

82 4:00 p.m. Ran Lin Inactivation of the Drosophila TSPO Inhibits the mPTP, Increases Longevity, Alters Heme Metabolism and Modulates Mitochondrial Bioenergetics

ABSTRACTS

Thursday, June 14, 2012


$250 Cash Award Winner


ABSTRACTS


Friday, June 15, 2012

Kelsey Wright Cash Award Winner - $1,000


# Time Presenter Title

24 9:30 a.m. Chun Shi Lin A Mouse Model with a Missense Mutation in ND6 for Pre-Leber’s Hereditary Optic Neuropathy

43 9:45 a.m. Amy Goldstein Triheptanoin Therapy for Inherited Disorders of Fatty Acid Oxidation

47 11:00 a.m. Jeana DaRe Clinical re-sequencing of over 410 genes to diagnose mitochondrial disorders: Results from the "rst 78 patients

101 11:15 a.m. Francisca Diaz Metabolic Adaptations in Neurons with Complex IV De"ciency

102 11:30 a.m. Carolyn Buzin NextGen Sequencing of the Complete mtDNA Genome: 20% Estimated Positive Cases among a Recent 117 Patients

105 11:45 a.m. Penelope Bonnen Exome sequencing and functional biology reveal novel Mitochondrial Disease genes

108 12:00 p.m. Renkui Bai Comprehensive Analysis of Entire Mitochondrial Genome by Long-Range PCR and Next Generation Sequencing for the Diagnosis of Mitochondrial Disorders: Yield of 216 Cases



Abstract#: Abstract Title:




Abstract#: 7 Abstract Title:


3


Abstract#: 14Abstract Title:




Mitochondrial Medicine 2012: Capitol HillAbstract#: Posters* (non-CME)













Abstract#:Abstract Title:




Abstract#:Abstract Title:









Abstract#: 86 Abstract Title: SURF1



Abstract#: 111 WangAbstract Title:


Abstract:Abstract Title:

Abstract:# Iain Hargreaves Abstract Title: MULTIPLE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN ENZYME DEFICIENCIES ASSOCIATED WITH A DECREASE IN SKELETAL MUSCLE COENZYME Q10 STATUS

Wednesday, June 13, 2012


Platform Session 1

Identi!cation of the Calcium Uniporter via Comparative Genomics

Author:Institution:

Title:

Platform Session 1

Genomics vs. Proteomics: Two Views of Mitochondrial Evolution

Author:Institution:

Title:

Platform Session 1

Remaining Relevant: The Minimalist Mitochondrion of Malaria Parasite

Akhil B. Vaidya, Ph.D.

Platform Session 2

Omics Approaches Reveal Novel Tools for Mitochondrial Disease Diagnosis

Anu Suomalainen Wartiovaara, M.D., Ph.D.

Platform Session 2

Proteomic Insights into Mitochondrial Form and Function

Dave Pagliarini, Ph.D.

Authors: 1 1

1 3 1

1

Institution: 1 3

Title:

Platform Session 2

Protein Modi!cations in the Mitochondria - Driving Heart Disease

Jennifer van Eyk, Ph.D.

Authors:

Institution:Title:

Thursday, June 14, 2012


Platform Session 3

Redox Biomarkers in Mitochondrial Disease

Gregory M. Enns, MB, ChB

in vivo

Platform Session 3

Treating Regulatory Signaling Network Changes that Cause the Metabolic Sequelae

of Mitochondrial Disease

Marni J. Falk, M.D.

Authors: 1 1 1 1

1 1 1 1 1

3 3 3

1

Institutions: 13

Title:

kd/kd

Platform Session 3

Probing Cytosol-Mitochondrial Interplay via Metabolomics

Joshua Rabinowitz, M.D., Ph.D.

Authors: 1 1

Institution: 1

Title:

13 14

Platform Session 3

The Versatility of Mitochondrial Metabolism in Tumor Cell Growth

Ralph J. DeBerardinis, M.D., Ph.D.

Authors:

Institution:

Title:

Platform Session 4

The Systems Biology of the Cardiac Mitochondrion

Robert S. Balaban, Ph.D.

Platform Session 4

Genome Technology for Mitochondrial Disease

Curt Scharfe, M.D., Ph.D.

Authors: 1 1 3 1

Institutions: 1

3

Title:

Abstract:

Friday, June 15, 2012


Platform Session 5

MitoExome Sequencing of Children with Mitochondrial Disease

David Thorburn, Ph.D.

Authors: 1

1 4 6

13 7

Institutions:1

3

4

5

6

7

8

9

10

11

13

Title:

Abstract:

de novo

Platform Session 5

Challenges of Bringing Next Generation Sequencing Technologies to CLIA/CAP

Certi"ed Clinical Laboratories

Lee-Jun C. Wong, Ph.D.

Authors:

Institution:

Title:

Platform Session 5

Exome Sequencing of Mitochondrial Disorders

Eric A. Shoubridge, Ph.D.

Author:Institution:

Title:

Platform Session 5

Diagnostic E$cacy of Targeted Sequencing in Infantile Versus Adult Mitochondrial Disease

Sarah Calvo, Ph.D.

Authors:4 1

1 1 6 6

1

Institutions: 1

3

4

5

6

Title:

Platform Session 6

Monitoring Disease: the Newcastle Approach

Patrick Chinnery, Ph.D., FRCP

Institution:

Title:

Platform Session 6

The Mitochondropathy of Methylmalonic Acidemia: De"nition and Therapeutic Approaches

Charles P. Venditti, M.D., Ph.D.

Author:

Institution:

Title:

Description:

Platform Session 6

Disease Severity Scores and Outcome Measures in Mitochondrial Disease

Jan Smeitink, M.D., Ph.D.

Authors:

Institution:

Title:

North American Mitochondrial Disease Consortium (NAMDC)

Michio Hirano, M.D.

1

Authors: 1 1

3 4

5 6 7 7

8 9 9 10

10 10 11

13 13 14 14

15 16 1

Institutions: 13

4 5

6 7

8 9

10

11

13 14

15 16

Title:

2012 UMDF Grant Recipients

M


Department of Animal Biology,

University of Pennsylvania

Telethon Institute of Genetics and

Medicine, Fondazione Telethon, Rome, Italy

Department of Pharmacological

Sciences, Stony Book University, New York

Department of Neurology,

Beth Israel Deaconess Medical Center, Boston

mut0

2010 UMDF Grant RecipientsUniversity of British Columbia,

Vancouver

Johns Hopkins School of Medicine, Baltimore, MD

Hospital for Sick Children, Toronto, Canada

2009 UMDF Grant RecipientsInstitute of Physiology, University of Zurich

Medical Research Council Dunn Human Nutrition

Unit, Cambridge, UK

Harvard Medical School

Restriction

Biochemistry & Biophysics, University of

California, San Francisco

2008

2007

2006

2005

UMDF Grant Recipients 1998-2008

2004

2003

2002

2001

2000

1999

1998

UMDF Grant Recipients 1998-2008

Saturday, June 16, 2012


Platform Session 7

Institution:

Title:

Results:

Conclusions:

Platform Session 7

Author:

Institution:

Title:

Platform Session 7

Authors:

Title:

Platform Session 7

Authors: Institutions:1

3 4

Title:

Abstract:

Platform Session 7

Institution:

Title:

Mitochondrial Medicine 2012: Capitol HillAbstracts

Abstract #: 1Presenter: Mikhail AlexeyevAuthors: Mikhail Alexeyev and Ra"k Fayzulin Institution: University of South Alabama, 5851 USA Dr North, Mobile, AL

Title: Mutagenesis of mouse mitochondrial DNA

Mitochondrial diseases can be caused by mutations in both nuclear and mitochondrial DNA (nDNA and mtDNA, respectively) and have estimated prevalence of at least 1:5000. While many anecdotal reports describe positive prognoses for an occasional patient or patients treated with various vitamins, cofactors or reagents, there is no cure or reliable treatment for these often fatal disorders. Animal (mouse) models of human disease are instru-mental in developing and testing new therapeutic modalities. Although the development of mouse models of human mitochondrial diseases caused by mutations in nuclear DNA is relatively straightforward, the routine development of animal models for the diseases caused by mtDNA mutations is not yet feasible. Recently, several groups demonstrated their ability to transfer mutant mtDNA from cultured cells into mice thus creating transmi-tochondrial mice. However, routine modeling of mitochondrial disorders caused by mtDNA mutations remains limited by the unavailability of a reliable supply of mouse cell lines bearing mutations in mtDNA. To date, some mutations in mouse mtDNA were isolated employing selection for resistance to various inhibitors, or by chemi-cal mutagenesis. These strategies are inherently biased (e.g., by the spectrum of mtDNA mutations that can be induced by a given mutagen), and do not allow for the isolation of the full spectrum of mtDNA mutations. We developed a mtDNA mutagenesis strategy that utilizes proofreading-de"cient mutant of the mitochondrial DNA-polymerase gamma (mPolGexo-). Mouse cells are engineered for inducible (Tet-On) expression of the #oxed mPolGexo-. These cells are induced with doxycycline, and mutagenesis is terminated at about one mutation per mtDNA by excising mPolGexo- with Cre recombinase. Following excision, cells are subjected to depletion to a level of about one mtDNA molecule per cell followed by repletion to normal copy number and cloning. Resulting clones are expanded and their mtDNA is sequenced to determine the nature of the mutation. The distribution of mutations obtained using this technique is fairly unbiased, which enables systematic mutagenesis and genera-tion of banks of mtDNA mutations, which may be useful for both modeling of the human mitochondrial disease in mice and for the functional analysis of the Electron Transfer Chain. Using this approach, we isolated more than 40 homoplasmic mouse cell lines with single homoplasmic nonsynonymous substitutions in mtDNA. This ex-ceeds the combined number of similar cell lines generated to date using all other approaches.

Abstract #: 3Presenter: Saskia KoeneAuthors: Saskia Koene1, Saskia Wortmann1, Eva Morava1, Maaike de Vries1, Jan Smeitink1

Institution: 1Nijmegen Centre for Mitochondrial Disorders, Radboud University Nijmegen Medical Centre, Geert Grooteplein 10, 6500 HB, PO BOX 9101, Nijmegen, The Netherlands

Title: Optimizing patient care and outcome measures: which complaints are most burdensome to patients and their parents?

Body of Abstract:To optimize patient care and to target supportive care e$ciently, it is important to know what disabilities pa-tients experience in daily life. The obtained information will also be useful in identifying outcome measure domains for future clinical trials. We investigated which complaints and symptoms in paediatric mitochondrial disease patients and their families are present and which of these complaints are most disabling.A questionnaire, designed to assess which symptoms are most burdensome to patients and their parents, was sent to all known Dutch-speaking patients with a mitochondrial disorder. Inclusion criteria: one or more enzyme complex de"ciencies, a decreased ATP and PCr production in fresh muscle tissue, and/or a con"rmed pathogenic mtDNA mutation. The questionnaire contained three main questions and numerous answer possibilities. The main questions were: i) Which complaints or symptoms are present?; ii) Which symptoms are most burdensome and wanted to improve mostly, indicated by the child or expected by the parent(s); iii) Which three symptoms would the parents like to change?

The questionnaire response rate was 56% (83 out of 149). Eighty-nine percent (74 out of 83) of the parents and their children "lled main questions ii) and iii). Thirty-two percent would (think that the child would) like to change the lack of energy, followed by tiredness (31%), and reduced muscle power (22%). Symptoms the par-ents would like to change include tiredness (25%), lack of energy (23%), and behavioural problems (23%).

Of the 54 children with tiredness, 43% of the children and 39% of their parents rate this symptom as one of the three problems they would like to change most. In the 55 patients with lack of energy, these percentages are 42 and 35%, respectively. In the 53 patients with developmental delay, 32% of the parent rate this as one of the most important problems. Of the 39 patients with speech di$culties, 36% of the children and 30% of the par-ents rates this symptom in the top three. For the 27 children with epilepsy, these percentages are 37 and 41% respectively. For the 26 children with behavioural problems, 66% of the parents rate this symptom as a major point they would like to change.

In conclusion, behavioural, speech and language problems are more cumbersome to parents and children then we would have expected. We advise to take these aspects into account more frequently in supportive care management.

Abstract#: 4Presenter: Yasutoshi KogaAuthors: Yasutoshi Koga1, Toshi Abe2, Takayuki Taniwaki3, Povalko Nataliya1

Institution: 1Department of Pediatrics and Child Health, 2Department of Radiology, 3Department of Neurology, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka 830-0011, Japan

Title: MELAS and L-arginine therapy –therapeutic timing and long term e"ects -.

Body of Abstract: Investigator-initiated clinical trial of L-arginine to cure the symptoms of the acute phase of stroke-like episodes (SLE) and to prevent the SLE in the interictal phase of MELAS has been "nished by June 30, 2010. We are now cleaning the date and are processing the date for approval. However we still do not know what is the best therapeutic timing of IV on SLE, and whether it can prevent the disease progression of MELAS during long term administration. We used L-arginine infusion at hyper-acute phase of SLE in 2 MELAS patients and evaluate the therapeutic e!ects by clinical and the serial neuroimaging analysis. Patient 1 is a 6-year-old girl who was diagnosed with MELAS/Leigh overlap syndrome based on a "nding of clinical symptoms and 78% mutation in an A3243G in the mitochondrial tRNALeu(UUR) gene. Patient 2 is a 32-year-old girl who ful"lled the diagnostic criteria of MELAS and has a 55% mutation of A3243G in muscle. She has a bilateral sensorineu-ral hearing loss and diabetes mellitus at the age of 31 years-old. She has two histories of stroke-like episodes and was then followed as MELAS. Within 2 hours after the onset of SLE, we infused 0.5g/kg/dose of L-arginine within 1 hour after the onset and took the brain MRI seriously. All of clinical symptoms disappeared within 30 min after L-arginine infusion without using anti-convulsants. The series of MRI were performed 1st, 7th days and one month from onset of stroke-like episodes. MRI obtained at 24 hours after the onset showed high intensity signal in T2WI, and DWI and low in apparent di!usion coe$cient (ADC), however those abnormal "ndings were completely normalized on MRIs taken at 7 days and one month later. We also administered L-arginine on MELAS for more than 5 years and evaluated the long term e!ects of it by duration of hospitalization, by Japanese Mitochondrial Disease Rating Scale (JMDRS) compared with MELAS cohort study in Japan without using L-arginine. L-arginine therapy signi"cantly decreases the JMDRS and prevents the disease progression as compared with cohort study. Our data indicated that L-arginine infusion at hyper-acute phase shows promise in cure the stroke-like episodes seen in MELAS, and long term of L-arginine therapy prevent the disease pro-gression of MELAS.


Abstract #: 5Presenter: Nataliya PovalkoAuthors: Nataliya Povalko, MD, PhD1, Yoshihisa Nagatoshi, MD, PhD2, Hiroko Inada, MD, PhD1, Shuichi Yatsuga, MD, PhD1, Yasutoshi Koga, MD, PhD1

Institution: 1Department of Pediatrics and Child Health, Kurume University School of Medicine, 67 Asahi-Machi, Kurume, Fukuoka 830-0011, Japan, 2 Section of Pediatrics, National Kyushu Cancer Center, 3-1-1 Notame, Minamiku, Fukuoka 811-1395, Japan Title: A novel sequence variant of tRNAThr in mtDNA, transferred by bone marrow transplantation in patient with ALL

Body of Abstract: Bone marrow transplantation (BMT) is one of the possible choices for the therapy of mi-tochondrial disorders such as MINGIE. However current donor matching system is only focused on the HLA typing based on the major histocompatibility antigens to avoid the graft-versus-host disease (GVHD). We had a 5-year old patient with ALL (acute lymphoblastic leukemia) who showed recurrent stroke-like episodes before and after the BMT. We found the novel mtDNA sequence variant which has not been found in a patient mtDNA before BMT. Analysis of entire sequence of mtDNA was performed twice (before and after BMT). First analysis was done after the "rst stroke-like episode. Patient under the chemotherapy suddenly showed severe headache, and a left tonic seizure with right hemiplegia. MRI of the brain showed high intensity areas on T2 and FLAIR in occipital, frontal, parietal lobes. The parameters of endothelial function, including plasma level of L-arginine, and FMD (#ow mediated dilatation), were abnormal. No pathogenic mtDNA mutations were found at his original mitochondrial DNA. The second entire sequencing of mtDNA was performed when he got the second episode of stroke in 5 month after BMT. He showed cortical blindness, vomiting and headache. Since the parameters of endothelial function were abnormal, infusion of L-Arginine was immediately started. No abnormalities were found on MRI taken after the stroke-like episode. We found a novel A15929G in the mito-chondrial tRNAThr gene. The heteroplasmy of this mutation was 90% in white blood cell, 8% in nail, however this mutation was not detected in urine, and recipient’s bone marrow before BMT, WBCs from mother, brother and sister of the proband, which suggesting that this mutation was transfer by BMT. According to the haplogroup analysis of mtDNA, original mtDNA belonged to haplogroup D4a (superhaplogroup M), on the other hand, donor’s mtDNA belonged to haplogroup F1 (superhaplogroup N). Since mtDNA has a very high mutation rate and has at least 30 polymorphic sites, minor antigen mismatching caused by the amino acid sequence poly-morphism may occur to lead the GVHD. On the other hand, many oligo-symptomatic or asymptomatic carriers having a pathogenic mtDNA mutation may become a potential donor for BMT and thus transmit the mutation to the recipient. Current donor matching system may have a risk of transmission of pathogenic mtDNA muta-tion and may contribute the GVHD by the proteins which are generated by the transmitted mtDNA.

Abstract #: 6 Presenter: Meghan Buckley Authors: Caitlin A. Baker, Meghan F. Buckley, Taylor L. DeRosa, Jenna A. Hernandez, Erin L. Hillis, Caroline R. Luciani, Rita Matta, Alexandra R. Novak, Christine C. Smith, Qingyu Xu, Sr. Mary Jane Paolella.Institution: Sacred Heart Academy, 265 Benham St. Hamden, CT 06514.

Title: The Comparative Study of Actin and Myosin Genes in Molgula manhattensis, Styela clava, and Limu-lus polyphemus: Implication on Mitochondrial DNA Maintenance.

Body of Abstract: One of the bewildering observations about mitochondrial diseases to scientists and clini-cians alike is the absence of a direct link between mtDNA mutations and a speci"c abnormality in a cell or tissue. Di!erent diseases such as CCD (Central Core Disease) and cardiomyopathy are caused by mutations of the myosin (lt. chain) gene which directly results in the absence of mitochondria in the muscle "bers and is indicative of mitochondrial disease. In addition, %-actin gene silencing has also shown to cause a decrease in


mtDNA copy number (Reyes, 2011). The study also concluded that collectively, these results strongly implicate the actomyosin cytoskeleton in mammalian mitochondrial DNA maintenance. Myosin and actin are genes of interest in tunicates and shell"sh due to their conservative nature and their structural and functional similari-ties. Myosin’s dependence on actin, as cited above, caused the focus of research to be redirected to actin, a protein that functions in the cell as a contractile system for the muscles. Classi"ed under the phylum Chordata, tunicates are small, sea-dwelling organisms that are often de"ned by the presence of siphons. Molgula man-hattensis, the sea grape, and Styela clava, the sea squirt, are invasive to Long Island Sound and pose a threat as fouling organisms by depleting the resources of local shell"sh such as Limulus polyphemus, the horseshoe crab. The blue blood of the endangered Lp is highly coveted in the "eld of medicine as a detector for bacterial endotoxins. The tunicates and crabs are the targets of this actin research. DNA was extracted from the tunicate body wall and gonads using spin columns, and from the shell"sh blood clots using FTA cards. Then student-de-signed primers were created using the actin sequence similarities in M.citrina, M.occulata, and S.clava. After am-pli"cation through PCR, the products were puri"ed and quantitated to obtain the DNA concentration in prepa-ration for downstream analysis. Templates from M.manhattensis, S.clava and L.polyphemus were sequenced in the school-owned ABI Prism 310 Genetic Analyzer, a single capillary automated sequencer. Subsequent results were evaluated using NCBI and EMBL to determine the evolutionary relationships between the tunicates and the horseshoe crab.

Abstract #: 7Presenter: Marisa FerraroAuthors: Katie E. Arnone, Elizabeth H. Bailey, Clare J. Donohue, Marisa K. Ferraro, Andrea L. Grammatico, Anna V. Marren, Angela M. Onofrio, Emily R. Roth, Xenia D. Zueva, Sr. Mary Jane PaolellaInstitution: Sacred Heart Academy, 265 Benham St. Hamden, CT 06514

Title: Evolution of the Actin gene: Comparing DNA Sequences Of Limulus polyphemus and Argopecten irradians and the Implication in Mitochondrial Function.

Body of Abstract: Actin is a highly conserved protein found in cytoskeletons, thin "laments, and part of the contractile apparatus of the muscle. This gene interplays with mitochondrial function in the cytoskeletons of many types of cells. A recent study found that these interactions are essential for normal mitochondrial mor-phology, motility, and distribution. In neurons, actin is required for mitochondrial transport and the actin in the cytoskeleton is a prerequisite for mitochondrial movement and immobilization. Mutations in actin and the loss of ATP production result in defects of these functions and/or high rates of mitochondrial DNA loss (Bolbogh, Pon 2006). In certain myopathies, cells will contain aggregates of actin "laments with a decrease in mitochon-dria. The study organisms, Argopecten irradians and Limulus polyphemus, (the Bay Scallop the Atlantic Horse-shoe Crab respectively), are highly sought-after organisms indigenous to Long Island Sound. The Scallop serves as an important food source for the "shing industry; the Horseshoe Crab’s blood is of medicinal importance containing LAL, Limulus Amoebocyte Lysate, as a detector of bacterial endotoxins. The organisms were provid-ed to Sacred Heart Academy by NOAA and the University of New Haven for experimentation and analysis. DNA was extracted from the blue blood of L. polyphemus with FTA cards and from the adductor muscle and gonads of A. irradians using spin columns. These DNA samples were quantitated using a biophotometer to obtain a tar-geted concentration of 50-100ng and an A260:A280 of 1.7-1.9 as an indication of the purity of the sample, prior to PCR. To amplify the gene, students designed two sets of primers unique to the organisms utilizing GenBank and ClustalW as resources. The puri"ed products were re-quantitated and diluted in order to be sequenced using the school’s ABI Prism 310 Genetic Analyzer. The results, analyzed using the bioinformatics sources, NCBI and EMBL, will be used this spring to study how divergence and duplication play a role in the evolution of the actin gene. Sequences will be submitted to GenBank by June 2012.


Abstract #: 8Presenter: Danielle DiPerna Authors: Danielle DiPerna and Lori Buhlman Institution: Midwestern University- Glendale, AZTitle: Nicotine a!ects mitochondrial dynamics and function by modulating nicotinic acetylcholine receptor (nAChR) 3 4 and via nAChR independent mechanisms.

The neuroprotective role of nicotine in Parkinson’s disease (PD) has been documented in epidemiological studies and subsequent in vivo studies. Though the current cause of PD pathology remains unknown for both familial and idiopathic forms of the disease, there is reason to suspect mitochondrial pathology may contrib-ute to the development and/or progression of this neurodegenerative disorder. Research also has shown that protective e!ects of nicotine can occur via modulation of nicotinic acetylcholine receptors (nAChR), though the exact nAChR subtypes, ligands and potential downstream mechanisms involved remain largely unknown. Thus, identi"cation of speci"c nAChR subtypes involved and understanding of the mechanisms by which their modulation is protective would create an attractive avenue for pharmaceutical developments. This study inves-tigates the a!ect of acute and chronic nicotine exposure on the human 3 4 nAChR using an in vitro model system with SH-SY5Y cells. Additionally, receptor independent e!ects will be studied by blocking SH-SY5Y nAChR and though the use of SHEP-1 cells which are nAChR null. Research has demonstrated several protective e!ects of nicotine in regard to re-establishment of the mitochondrial network as well as inhibition of the gen-eration of reactive oxygen species and limiting mitochondrial damage. This investigation focuses on nicotine e!ects on mitochondrial dynamics electron transport chain enzyme function. To this end, we have determined the e!ects of nicotine on mitochondrial dynamics when only 3 4 nAChR binding sites are available and when all nAChR binding sites are blocked, demonstrating receptor independent e!ects of nicotine exposure. We have also analyzed the e!ects of alpha3beta4 nAChR modulation on Complex V activity by measuring ATP gen-eration using the same nicotine exposure parameters. Subsequently, our study investigates whether nicotine can preserve mitochondrial dynamics, and preserve ATP production following exposure to carbonyl cyanide m-chlorphenol-hydrozone (CCCP; a uncoupler of mitochondrial membrane potential and ATP production).

Abstract #: 10Presenter: Sarika SrivastavaAuthors: Sarika Srivastava1, Karina N. Gonzalez Herrera1, Vincent Proacccio2, Douglas C. Wallace3 and Marcia C. Haigis1*

Institutions: 1Department of Cell Biology, Paul F. Glenn Laboratories for the Biological Mechanisms of Aging, Harvard Medical School, Boston MA 02115 USA; 2Department of Biochemistry and Genetics, Angers University Hospital, School of Medicine, and UMR INSERM, U771-CNRS6214, Angers, France; 3Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA 19104, USA

Title: Low Level of MtDNA Mutation Promotes Mitochondrial Bioenergetics and Oxidative Metabolism via Retrograde Signaling

Body of Abstract: Point mutations in mitochondrial tRNA genes have been associated with mitochondrial en-cephalomyopathies. The mitochondrial 3243A>G tRNA Leu(UUR) gene mutation exceeding a critical threshold (> 85%) leads to a clinical onset of MELAS disease. Below the critical threshold, the mitochondrial DNA (mtDNA) mutation load is clinically asymptomatic with no deleterious impact on cellular bioenergetics. In this study, we report that the low level of m3243A>G mutation activates mitochondrial bioenergetics, biogenesis and cellular metabolism in the MELAS 28% heteroplasmic cybrid (M28) cells compared to the 143B wild-type control cells. Strikingly, we found that the rate of mitochondrial respiration, oxidative capacity, enzyme complex activities and mtDNA levels were signi"cantly higher in the M28 cybrid cells relative to the 143B control cells. The mi-croarray data analysis revealed increased expression of several genes involved in oxidative phosphorylation, TCA cycle and fatty-acid metabolism pathways in the M28 cybrid cells relative to the 143B control. Mechanisti-cally, we found that multiple transcription factors and coactivators involved in regulating mitochondrial gene


expression and lipid metabolism were markedly stimulated in M28 cybrid cells relative to the 143B control cells. Furthermore, the energy and nutrient sensing AMPK signaling pathway was signi"cantly activated in the M28 cybrid cells relative to the 143B control cells. Our "ndings suggest that the low level of mtDNA mutation is a potential signal for mitochondrial dysfunction and that the nuclear genome apparatus senses and responds via retrograde signaling which consequently leads to the activation of the transcriptional regulatory network to further enhance mitochondrial bioenergetics, biogenesis and metabolism. This overall positive impact of the low level of mtDNA mutation on mitochondrial function implicates that the pharmacological modulation of the underlying signaling pathways may boost mitochondrial bioenergetics and opens new avenues for mitochon-drial disease therapeutics.

Abstract #: 11Presenter: Peter W. Stacpoole, PhD, MDAuthors: Peter W. Stacpoole, PhD, MD1; David A. Wagner, PhD2

Institution: 1University of Florida College of Medicine, Division of Endocrinology, Diabetes and Metabolism, Gainesville, FL; 2Metabolic Solutions, Inc., Nashua, NH, USA

Title: Rapid Breath Test for In Vivo Determination of Human Pyruvate Dehydrogenase Complex Activity

Abstract: The pyruvate dehydrogenase complex (PDC) is a key regulatory enzyme in cellular energy metabo-lism. Under aerobic conditions, PDC catalyzes the rate-determining step in glucose oxidation by irreversibly decarboxylating pyruvate to acetyl CoA and CO2. In this way, the PDC links glycolysis with the citric acid cycle and gluconeogenesis, as well as both lipid and amino acid metabolism. Regulation of the PDC is achieved by reversible phosphorylation by families of pyruvate dehydrogenase kinases (PDKs) and phosphatases (PDPs), in which the phosphorylated form of the PDC is inactive. Prior human research on PDC regulation has mainly em-ployed in vitro assays of isolated human cells or invasive skeletal muscle biopsies. However, these approaches fail to provide a safe and facile means of serially assessing whole body PDC activity. We have addressed these shortcomings by developing the Pyruvate Breath Test (PBT) that uses a small, oral dose of sodium 1-13C-pyruvate to determine PDC activity based on the conversion of 13C-pyruvate to 13CO2.

A pilot study in two subjects was conducted to provide proof-of-concept for the use of an oral PBT as a tool for assessing mitochondrial activity of the PDC and in response to therapeutic intervention using dichloroacetate (DCA), a prototypic PDK inhibitor. 13CO2 production in exhaled air was measured from an oral 100 mg dose of 1-13C-pyruvate in the basal, overnight fasted state and, after one-week washout, again in the fasted state one hour after oral administration of 25 mg/kg of DCA.

Oral 13C-pyruvate administration resulted in a typical concentration vs. time-dependent curve, with 13CO2 detect-able within 5 minutes of dosing, reaching maximum levels in 20-40 minutes and decreasing gradually over 2+ hours. In both subjects, DCA exposure rapidly stimulated PDC activity during the initial 30 minutes of breath collections, as evidenced by the upward and leftward shift in the concentration-time curve. We determined in one subject whether a lower oral 1-13C-pyruvate dose could be utilized. Cumulative % recovery of the dose as 13CO2 over 30 minutes was the same following doses of 25, 50 and 100 mg of 1-13C-pyruvate, indicating that an oral dose of 25 mg could be employed in future studies, using fewer samples and a shorter duration. In sum-mary, these data suggest that the PBT may provide a safe and rapid in vivo measure of a dynamic and critically important mitochondrial bioenergetic reaction within 30 minutes of oral substrate administration and can be performed serially under physiological, pharmacological and pathological conditions.

Abstract#: 12Presenter: Neal SondheimerAuthors: Neal Sondheimer1, Ornella Zollo1, Jason A. Mills2, Catherine E. Glatz1, Paul Gadue2, Deborah L. French2

Institutions: 1Division of Child Rehabilitation and Biochemical Genetics and 2Center For Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104

Title: Spontaneous Elimination of Mitochondrial Mutations During The Induction of Pluripotency


Body of Abstract:

Introduction: The correction of genetic lesions in pluripotent stem cells represents an important "rst step in the development of cell-based therapies. Disorders due to mitochondrial DNA (mtDNA) mutations present an intriguing target for cell-based therapy because of unique features of mitochondrial genetics. mtDNA mutations are often heteroplasmic, where both wild-type and mutant sequences are present at the level of the organism, tissue or cell. The e!ect of these mutations is often determined by the level or load of heteroplasmy, and the reduction of pathological heteroplasmy is an important goal of mitochondrial therapies. The objective of this study was to determine the dynamics of mitochondrial heteroplasmy during the conversion of "broblasts to induced pluripotent stem cells (iPSCs).

Methods: We generated iPSCs from four patients with heteroplasmic disease-causing mutations, one with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes and three with Leigh syndrome and one una!ected infant. Mitochondrial function in iPSCs was assessed by #uorescence-associated cell sorting analysis of Mito Tracker CMXRos straining and oxygen consumption.

Results: The generated cell lines ful"lled criteria for pluripotency, including the expression of pluripotency markers and the ability to form teratomas in mice. An evaluation of mitochondrial genotype demonstrated the spontaneous elimination of heteroplasmy. The iPSC lines generated were homoplasmic, with mutations either entirely eliminated or with no remaining wild-type DNA. Generation of iPSCs from a cell line with non-patho-genic heteroplasmy gave a similar result, suggesting that this result was not due to any selection for or against pathogenic mutations. Studies of the cells from which iPSCs were derived suggested that the bottleneck could be due to the presence of homoplasmic "broblast clones within an otherwise mixed population. Functional studies of mitochondrial activity demonstrated that mitochondrial membrane potential was impaired in iPSCs bearing pathogenic mutations.

Conclusions: Our ability to generate homoplasmic, pluripotent cells from patients with disease may be a mechanism for eliminating disease-causing mutations in cells with future therapeutic potential

Abstract #: 13Presenter: Lisa EmrickAuthors: Ayman W. El-Hattab1, Lisa Emrick2, Jean W.C. Hsu3, Farook Jahoor3, Fernando Scaglia2, William Craigen2 Institutions: 1Division of Medical Genetics, Department of Child Health, University of Missouri Health Care, Columbia, MO, 2Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 3US Department of Agriculture/Agricultural Research Service–Children’s Nutrition Research Center, Baylor College of Medicine, Houston, Texas

Title: Glucose kinetics in subjects with MELAS syndrome: interim results

Background: The mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syn-drome is one of the most frequent maternally inherited mitochondrial disorders in which diabetes mellitus (DM) occurs in one third of a!ected individuals. The pathogenesis of DM in MELAS syndrome remains unclear. We hypothesize that DM develops in individuals with MELAS syndrome due to multiple defects in glucose me-tabolism, including decreased glucose utilization, increased glucose production, decreased insulin secretion, and increased insulin resistance. Individuals with MELAS syndromes who do not yet have DM may have altered glucose metabolism.

Methods: In this study we aim to measure the rates of endogenous glucose production, gluconeogenesis, glu-cose oxidation, and glucose clearance via stable isotope infusion technique in subjects with MELAS syndrome who have DM, subjects with MELAS syndrome who do not have DM, and in healthy control subjects. In addition, we measure the concentrations of fasting blood glucose, insulin, and glycosylated hemoglobin (HbA1c); and as-


sess insulin resistance using Homeostatic Model Assessment (HOMA). The research subjects are admitted to the General Clinical Research Center at Texas Children’s Hospital. After a 12 hour-fast, the isotope infusion is started with a priming dose of NaH13CO3 and U-13C6 glucose followed by continuous infusion of U-13C6 glucose for 6 hours. Blood and breath samples are collected and analyzed for isotopic enrichments.

Results: To date, 6 control subjects, 4 subjects with MELAS and DM, and 4 non-diabetic subjects with MELAS have completed the study. Both groups of subjects with MELAS (with and without diabetes) show increased glucose production and gluconeogenesis rates when compared to the control subjects. Diabetic subjects with MELAS exhibit higher insulin resistance as calculated by HOMA, whereas non-diabetic subjects with MELAS show a higher rate of glucose clearance.

Conclusions: This interim analysis reveals that subjects with MELAS syndrome have abnormalities in glucose metabolism. Subjects with MELAS who do not have DM have higher rates of glucose production and gluco-neogenesis that can predispose them to develop diabetes. Subjects with MELAS and diabetes showed both increased glucose production and higher insulin resistance, suggesting that DM develops due to multiple defects in glucose metabolism in MELAS. The completion of this study will result in a better understanding of the pathophysiological mechanisms of DM in subjects with MELAS syndrome, which can in#uence the manage-ment and prognosis of the disorder and may provide further insights into the pathogenesis of DM in mitochon-drial diseases in general.

Abstract #: 14Presenter: Chian Ju JongAuthors: Chian Ju Jong1, Ramila K.C.1, Takashi Ito2, Junichi Azuma2, Stephen Scha!er1

Institutions: 1University of South Alabama, Department of Pharmacology, College of Medicine, Mobile, AL 36688, 2Hyogo University of Health Sciences, School of Pharmacy, Kobe, Japan

Title: The Role of a Taurine-containing Wobble Modi!cation De!ciency in MELAS

Body of Abstract: MELAS (mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes) is a mitochondrial disease that usually coexists with a cardiomyopathy. The most common pathogenic mutation in MELAS is an A to G transition at position 3243 in the aminoacyl stem of mitochondrial tRNALeu(UUR). The A3243G mutation a!ects tRNALeu(UUR) structure and stability, aminoacylation and posttranscriptional modi"cation of a wobble base, all of which, presumably, lead to a decrease in the synthesis of mitochondrial proteins. However, it is unclear if wobble modi"cation de"ciency causes MELAS-like changes in the respiratory chain that lead to car-diac contractile dysfunction. To examine the e!ect of wobble modi"cation de"ciency in mitochondrial function, we developed a mouse model lacking taurine transporter (TauTKO), which diminishes the levels of a substrate (taurine) required for the conversion of uridine to 5-taurinomethyluridine at the wobble position of tRNALeu(UUR). We showed that TauTKO hearts demonstrated decreased levels of several complex I subunits, reduced complex I activity and suppressed oxygen consumption. There was also evidence of oxidative stress, as exempli"ed by a decrease in both aconitase activity and glutathione redox ratio, and an increase in MitoSox #uorescence. We also showed activation of protein kinase C (PKC)-& and PKC-' and an increase in the phosphorylation state of troponin I, which regulates contractile function. Taurine depletion was also associated with development of cardiac dysfunction, as characterized by a decrease in both fractional shortening and ejection fraction, a decline in Ca2+-dependent myo"brillar ATPase activity and an increase in mRNA levels of heart failure markers such as ANP, BNP and %-MHC genes. Our "ndings suggest that a wobble defect leads to impaired respiratory chain ac-tivity, resulting in oxidative stress. This is consistent with the idea that a wobble defect decreases the e$ciency of UUG decoding for synthesis of mitochondrial proteins. This leads to a defect in respiratory chain #ux and a diversion of electrons from the respiratory chain to the acceptor, oxygen, forming in the process superoxide. Oxidative stress is a potential trigger of contractile defects, in part through PKC activation, which results in the phosphorylation of troponin I, reducing the binding a$nity of calcium for troponin C. Therefore, we showed that a wobble defect is responsible for MELAS-like respiratory chain abnormalities. In addition, it triggers oxida-tive stress-mediated contractile dysfunction. These results support an important role of a wobble modi"cation de"ciency in the pathogenesis of MELAS.


Abstract #: 15 Presenter: Xiaoshan Zhou#* Authors: Shuba Krishnan#, Xiaoshan Zhou#*, Joao Paredes and Anna KarlssonInstitution: Department of Laboratory Medicine, Division of Clinical Microbiology, Karolinska Institutet, F68, SE-14186 Huddinge, Sweden

# These authors contributed equally to this work* Presenting author

Title: Thymidine phosphorylation by transgene expression of the Drosophila melanogaster nucleoside kinase rescues the pathology of mitochondrial TK2 de!ciency

AbstractA strategy to reverse the symptoms of thymidine kinase 2 (Tk2) de"ciency in a mouse model was investi-gated. The nucleoside kinase from Drosophila melanogaster (Dm-dNK) was expressed in Tk2 de"cient mice that previously were characterized to present a severe phenotype caused by mitochondrial DNA depletion. The mtDNA depletion in the Tk2 knockout mice was fully rescued by intercrossing with the Dm-dNK+/- trans-genic mice. The Dm-dNK+/- /Tk2-/- mice had a normal content of mtDNA, and survived for 6-month without any abnormal behavior observed so far. Mice expressing Dm-dNK showed a substantial increase in thymidine phosphorylating activity in investigated tissues. The Dm-dNK expression also resulted in highly elevated dTTP pools. The dTTP pool alterations did not cause speci"c mitochondrial DNA mutations or deletions when 6 months old mice were analyzed. The mitochondrial DNA was also detected at normal levels in the Dm-dNK+/- mice. In conclusion, the Dm-dNK+/- /TK2-/- mouse model illustrates how dTMP can be synthesized in the cytosol and that transport of thymidine nucleotides to the mitochondrial compartment can compensate for loss of intra mitochondrial synthesis. The data presented open new possibilities to treat the severe symptoms of TK2 de"ciency.

Abstract #: 16Presenter: Anne ChiaramelloAuthors: Martine Uittenbogaard, Kristin Baxter, Anne ChiaramelloInstitution: George Washington University, School of Medicine and Health Sciences, Department of Anatomy and Regenerative Biology, Washington, DC 20037

Title: The Neurogenic Basic Helix-Loop-Helix Transcription Factor NeuroD6 Induces Mitochondrial Biogen-esis and Bioenergetics in Neuronal Cells

Body of Abstract: Developing neurons are vitally dependent on mitochondria for energy due to their need of high ATP levels to fuel dynamic changes in cytoskeletal assembly and plasmalemmal biogenesis associated with axonal and dendritic development. Although great progress has been made toward elucidating the transcription regulation of mitochondrial biogenesis and bioenergetics, little is known about the identity of neuronal-speci"c transcriptional factors adapting mitochondrial homeostasis with the onset of neuronal dif-ferentiation. Gene set enrichment of our genome-wide microarray analysis has revealed a link between Neu-roD6 expression and a cluster of mitochondrial bioenergetic-related genes, while our confocal #uorescence microscopy analysis has shown NeuroD6-mediated increase of the mitochondrial biomass during the early stages of neuronal di!erentiation. Moreover, NeuroD6 concomitantly sustains the mitochondrial biomass and keeps low levels of ROS during oxidative stress. Thus, the goal of our present study was to determine whether NeuroD6 could coordinate mitochondrial biogenesis and bioenergetics during the early stages of neuronal di!erentiation using our in vitro cellular paradigm, the PC12-NeuroD6 (PC12-ND6) cell line. PC12-ND6 cells behave as neuronal progenitor cells poised to undergo cell cycle withdrawal and execute terminal di!erentiation upon neurotrophic cues. Our collective studies have shown that NeuroD6 induces cytoskeletal remodeling and neuritogenesis characteristic of the "rst four stages of neuronal di!erentiation independently of nerve growth factor, events that were accompanied by increased mitochondrial mass, as demonstrated


by confocal microscopy, #ow cytometry and mitochondrial fractionation. In this study, we report that NeuroD6 triggers mitochondrial biogenesis in the early stages of neuronal di!erentiation by inducing mtDNA replication and expression of the key regulator Tfam. Furthermore, we observed that NeuroD6 increases the expression levels of key subunits of the respiratory complexes, COXI, COXIV and COXV, which resulted in increased ATP levels produced by oxidative phosphorylation. Finally, we measured the mitochondrial membrane potential (()m) by live-cell imaging using two distinct cationic #uorescent lipophilic dyes, TMRM and JC-10, both of which accu-mulate in the mitochondrial matrix in a ()m-dependent manner. We found that NeuroD6 stimulates the mito-chondrial bioenergetic functions by increasing ()m, thereby generating a bioenergetic reserve. In conclusion, our results indicate that NeuroD6 plays an integrative role in regulating and coordinating the onset of neuronal di!erentiation with acquisition of adequate energetic capacity to sustain cytoskeletal remodeling, plasmalem-mal expansion and growth cone development, events that are highly ATP demanding.

Abstract #: 17Presenter: Victor Wei ZhangAuthors: Victor Wei Zhang, Hong Cui, Lee-Jun WongInstitution: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030

Title: Implementation of Next Generation Sequencing for Clinical Molecular Diagnosis of Mitochondrial Disor-ders

Body of Abstract:

The next generation sequencing (NGS) technology is being gradually adapted as the primary sequenc-ing platform for the molecular diagnosis in clinical laboratories. The ability to sequence a group of target genes by NGS is exceptionally useful for molecular diagnosis of genetically and clinically heterogeneous syndromes, such as complex mitochondrial disorders. Comprehensive molecular diagnosis of mitochondrial disorders requires the evaluation of both nuclear and mitochondrial genomes at high accuracy for clinical diagnosis. The next generation sequencing based panel testing becomes the major driving force for such application. However, the complexity of NGS based sequencing present unprecedented challenge for quality assurance and quality control to warrant the correct result for our patients.

We develop a one-step comprehensive NGS for molecular diagnosis of mitochondrial disorders in a clinical set-ting with the implementation of proper quantitative and qualitative controls. A target gene selection followed by high throughput “deep” coverage NGS approach was validated with the indexed qualitative and quantita-tive controls analyzed along with each sample for quality assurance. We demonstrated an average coverage of >500X for targeted nuclear genes and >20,000x for each of the 16,569 bases of the mitochondrial genome. Nucleotide changes are correctly called with quantitative information. The limit of detection of a heteroplasmic change is calculated to be about 1.5%. Small and large insertion/deletions were correctly detected with clear breakpoints and percentage of heteroplasmy.

We have demonstrated the feasibility and e!ectiveness of the QA/QC system for routine NGS based testing for up to tens to hundreds sample per sequencing run. In combination of newly developed capture based analysis of a group of target nuclear genes and the one-step comprehensive analysis of mitochondrial genome, we are able to deliver molecular testing results to our patients in a timely, accurate, and cost-e!ective manner using the state-of-the-art technology. The inclusion of quality control system assures the highest quality performance required in a certi"ed clinical laboratory.

Abstract #: 18 Presenter: Peter W. StacpooleAuthors: Peter W. Stacpoole, PhD, MD, for the CoQ10 Study Group.Institution: University of Florida, College of Medicine, Gainesville, FL, 32611

Title: Phase 3 trial of coenzyme Q10 in children with mitochondrial diseases.


Abstract: Treatment of mitochondrial diseases has been generally disappointing and has usually been ap-proached in an uncontrolled manner. Although there is no proven therapy for any congenital mitochondrial disease, coenzyme Q10 (CoQ10) is a potential treatment for speci"c de"ciencies of the respiratory chain, because of its apparent safety, its integral role in the processes of electron transport and cellular energetics and its antioxidant properties. We postulate that CoQ10 is a safe and e!ective treatment for children with inborn er-rors of mitochondrial energetics due to defects in speci"c respiratory chain (RC) complexes or mitochondrial DNA (mtDNA) mutations, and that this bene"cial action will be re#ected in improved motor, neurobehavioral and sleep function and in quality of life. This postulate is currently being tested by accomplishing the following speci"c aims:

Speci!c Aim 1. Complete a multicenter, prospective, randomized, double-blind, placebo controlled cross-over trial of oral CoQ10 in children with biochemically proven de!ciencies of complex I, II, III or IV of the RC or with mutations of a gene coding for an RC component (mtDNA and nDNA). This aim tests the hypothesis that supplementation with CoQ10 (10 mg/kg/d) is safe and more e!ective in improving outcome than placebo. Clini-cal Research Centers (CRCs) or similar facilities will be the venues for this phase 3 clinical trial.

Speci!c Aim 2. Determine the e"ectiveness of CoQ10 in improving the morbidity of a"ected patients. This aim addresses the postulate that high dose CoQ10 improves motor function and quality of life, as determined by a validated questionnaire for this patient population, and by objective, standardized measures of motor func-tion.

Speci!c Aim 3. Determine the safety of CoQ10 in the target population. This aim tests the postulate that the formulation and dose of CoQ10 employed is well tolerated and the administration of this product is not associ-ated with signi"cantly more numerous or more severe adverse events than is administration of placebo.

Abstract #: 19Presenter: Peter W. StacpooleAuthors: Peter W. Stacpoole for the DCA/PDC Collaborative GroupInstitution: Departments of Medicine (Division of Endocrinology and Metabolism) and Biochemistry and Molecular Biology, College of Medicine, University of Florida, Gainesville, FL, 32611

Title: Phase 3 Trial of Dichloroacetate for Pyruvate Dehydrogenase Complex De!ciency.

Abstract: We have organized a phase 3 trial of dichloroacetate (DCA) in young children with de"ciency of the pyruvate dehydrogenase complex (PDC). PDC de"ciency is one of the most common causes of congenital lactic acidosis; it is a frequently fatal disease of childhood that causes progressive neurological and neuromuscular degenera-tion for which no proven treatment exists. We predict that DCA represents targeted potential therapy for PDC de"ciency because of its ability to increase both the catalytic activity and stability of the enzyme complex. The conclusions of numerous laboratory investigations and open label clinical trials are consistent with this postu-late and have led to the designation of DCA as an Orphan Product for congenital lactic acidosis by the Food and Drug Administration.

We plan to conduct a double-blind trial of 67 evaluable children, aged 3 months through 17 years, with proven de"ciency of PDC. Subjects will be randomized to receive 9 months of DCA at a dose of either 12.5 mg/kg/12 hr (20 patients) or 25 mg/kg/12 hr (20 patients) or placebo (27 patients) by mouth. After 9 months, patients in the DCA treatment arms will be crossed over to receive the alternate dose and all patients will be studied for an additional 9 months, for a total of 18 months of exposure to DCA or placebo. At randomization, patients will be strati"ed according to age <2 years or *2 years and according to prior exposure to a ketogenic diet (KD). Our primary hypothesis, stated as the null, is that the three groups are equivalent vs. the alternate conclusion that there is a di!erence between the active treatments and placebo. As a secondary comparison we shall compare the two DCA doses in the crossover study. Our primary hypothesis will be tested by accomplishing the following speci"c aims:


Speci"c Aim 1 is to determine the e$cacy of DCA based on outcome from two primary e$cacy aims: 1) gross motor function and 1) dietary carbohydrate tolerance. Primary e$cacy aim 1 will be quantitated using the Gross Motor Function Measure (GMFM), version 88, which is a validated, age-appropriate tool applicable to children with severe neurological and neuromuscular impairment. Primary e$cacy aim 2 will determine carbohydrate tolerance by measuring blood lactate response to a standard carbohydrate-rich meal.

Speci"c Aim 2 is to evaluate the safety and tolerability of DCA by comparing 1) motor and sensory nerve con-duction of the lower extremities in patients to age-appropriate normative values; and 2) di!erences in stan-dard serum biomarkers of hepatic function, namely aspartate aminotransferase and alanine aminotransferase, between treatment and placebo groups.

Speci"c Aim 3 is to address the following secondary questions:

For e$cacy, does DCA di!er from placebo in a!ecting: 3a. quality of life? 3b. endogenous PDC activity? 3c. the frequency and severity of lactic acidosis or other clinical/metabolic events requiring hospitalization? 3d. survival?

For safety, does DCA di!er from placebo in: 3e. maintaining standard clinical and biochemical measures of general health? 3f. exhibiting toxicity that correlates with plasma drug concentrations?

Abstract #: 20Presenter: Peter W. StacpooleAuthors: Monica Abdelmalak, Alicia Lew, Ryan Ramezani, Albert L. Shroads, Bonnie S. Coats, Meena N. Shankar and Peter W. StacpooleInstitution: University of Florida, College of Medicine, Gainesville, FL, 32611

Title: Long-term Safety of Dichloroacetate in Congenital Lactic Acidosis.

We have followed 8 patients (4 males) with biochemically and/or molecular genetically proven de"ciencies of the E1+ subunit of the pyruvate dehydrogenase complex (PDC; 3 patients) or complexes I (1 patient), IV (3 pa-tients) or I+IV (1 patient) for 10.9 to 16.5 years. All subjects originally participated in randomized controlled trials for dichloroacetate (DCA) and were continued on an open-label chronic safety study. Patients (1 adult) ranged in age from 3.5 to 40.2 years at the start of DCA administration and are currently aged 16.9 to 49.9 years (mean ± SD: 23.5 ± 10.9 years). Subjects were either normal or below normal body weight for age and gender. They have been evaluated at least twice annually for routine tests of hematopoietic, renal, hepatic, lipid and lipoprotein metabolism, peripheral nerve conduction and plasma trough DCA concentrations. The 3 PDC de"cient patients consumed a modestly increased fat diet (~2 fat:1 carbohydrate+protein). Oral DCA was administered at a dose of 12.5 mg/kg every 12 hours. Potentially relevant concomitant medications included carbamazepine and clon-azepam (1 patient) and progesterone (1 patient). DCA was well-tolerated and maintained normal blood lactate concentrations, even in PDC de"cient children on essentially unrestricted diets. Hematological, renal and electrolyte status remained stable. Mean serum ala-nine transaminases (ALT) levels were modestly higher on DCA (1.609-fold above baseline; p=0.008), but serum aspartate transaminases and lipid and lipoprotein levels were not signi"cantly altered while on the drug. Nerve conduction either did not change or decreased modestly and led to reduction or temporary discontinuation of


DCA in 2 patients. We conclude that chronic DCA administration is e!ective in maintaining normal blood lactate levels and is generally well-tolerated, particularly in pediatric patients with PDC de"ciency and other congenital causes of lactic acidosis.

Abstract #: 21Presenter: Anna-Kaisa NiemiAuthors: Anna-Kaisa Niemi1, Tereza Moore2, Tina Cowan2, Viktoria Kheifets3, Gregory M Enns1

Institutions: 1Dept of Pediatrics, Division of Medical Genetics, Stanford University, Stanford, CA 94305, 2Dept of Pathology, Stanford University, Stanford, CA 94305, 3Edison Pharmaceuticals

Title: Improved redox status after liver transplantation in a patient with MMA mut0 subtype; functional evi-dence for EPI-743 therapy.

Abstract: Methylmalonic aciduria (MMA) mut0 subtype is caused by methylmalonyl-CoA mutase de"ciency and is characterized by severe metabolic acidosis and hyperammonemia. Long-term complications include basal ganglia damage,optic atrophy, and end-stage renal disease. The pathophysiology of long-term complications is not completely understood, although there is increasing evidence that abnormalities in mitochondrial metabo-lism may play a role. Mitochondrial dysfunction is evident in patients and experimental models of MMA (Atkuri et al 2009, Chandler et al. 2009, de Keyzer et al. 2009, Murphy et al. 2010, Treacy et al. 1996). In order to increase our understanding of the pathophysiology of MMA, we report further observations related to a patient with evidence of signi"cant redox imbalance including lactic acidosis, low blood GSH levels, elevated urine TCA cycle intermediates and 5-oxoprolinuria. Although ascorbate had no e!ect, supplementation with citrulline and N-ace-tylcysteine resulted in resolution of 5-oxoprolinuria and improved GSH levels (Cusmano-Ozog et al. 2010 SIMD abstract). She received an orthotopic liver transplantation at age 11 months at which point antioxidant therapy was discontinued. Post-transplantation her clinical status stabilized; she has not had further metabolic crises, has made signi"cant developmental gains and has normal renal function. Blood GSH levels dropped immediately after transplantation, but thereafter have shown improvement associated with absence of urine 5-oxoproline and TCA cycle intermediates. Because mitochondrial dysfunction and redox imbalance have been described in MMA, we investigated the e!ect of EPI-743 ( -tocotrienol quinone) on skin "broblasts derived from our patient. EPI-743, a novel drug candidate in clinical trials for mitochondrial disorders, protects cells subjected to oxidative stress in "broblast assays of mitochondrial patients (Shrader et al. 2011, Enns et al. 2012). Unlike wild-type cells, "broblasts from our patient were sensitive to oxidative stress induced by glutathione synthesis inhibitor L-buthi-onine-(S,R)-sulfoximine (BSO). Furthermore, EPI-743 rescued the MMA "broblasts from death caused by oxida-tive stress in a pattern similar to that observed in cells from patients with primary mitochondrial disease. This study provides further evidence of mitochondrial dysfunction in MMA mut0 and the "rst functional evidence of improved MMA mut0 cell survival following EPI-743 supplementation. Redox-modulating agents, such as EPI-743, should be further investigated as a treatment modality in MMA.

Abstract #: 22Presenter: Yongmin LiuAuthors: Yongmin Liu1, Phuonggiang Nguyen1, Alexander Gibbons1, James B. Mitchell2 and Miriam C. Poirier1 Institutions: Carcinogen-DNA Interactions Section1, LCBG, and Tumor Biology Section, RBB2, National Cancer Institute, NIH, Bethesda, MD, 20892

Title: Tempol as a potential protective agent for nucleoside reverse transcriptase inhibitor (NRTI)-induced mitochondrial toxicity

NRTIs are essential components of the successful antiretroviral combination therapies used for treatment of HIV-1. However, during long-term therapy NRTIs may damage heart mitochondria, thus limiting clinical use of these drugs. Consequently, reducing NRTI-induced mitochondrial toxicity may bene"t HIV-1-infected patients. Using H9c2 rat cardiomyocyte cultures exposed long-term to Zidovudine (AZT) or AZT plus Didanosine (ddI) we


demonstrated NRTI-induced mitochondrial compromise. In an attempt to protect mitochondria from this dam-age we have used the stable free radical Tempol and its metabolite Tempol-H, cyclic nitroxides with antioxidant properties. H9c2 cells were exposed to 50 µM AZT plus 50 µM ddI for 15 passages (P15) in the presence and ab-sence of 150 µM Tempol or 150 µM Tempol-H. The AZT/ddI combination caused moderate growth inhibition (< 30 %), and co-exposure with Tempol or Tempol–H did not restore cell survival. Mitochondrial oxygen consumption rate (OCR) and extracellular acidi"cation rate (ECAR) were measured using the Seahorse XF24 analyzer. As seen previously, AZT/ddI reduced maximal FCCP-uncoupled OCR. However co-exposure with Tempol and Tempol-H stimulated OCR, restoring the AZT/ddI-reduced uncoupled OCR by 22-91 %. Similarly, the uncoupled ECAR levels were increased by 15-46 % with Tempol or Tempol-H. Preliminary Western blot "ndings showed that Tempol and Tempol-H enhanced the expression of uncoupling protein-2 (UCP-2). Therefore Tempol and Tempol-H may protect cardiomyocytes from mitochondrial compromise induced by the NRTI combination AZT/ddI, and UCP-2 may play a role through mild uncoupling. We are currently evaluating potential mechanisms by which these com-pounds may act as mitochondrial protective agents.

Abstract #: 23Presenter: Richard E FryeAuthors: Richard E. Frye, Shannon Rose, S. Jill James Institution: Arkansas Children’s Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR, USA 72202

Title: Children with Mitochondrial Disease and Autism have Alterations in Pathways Involved in Response to Endogenous and Exogenous Stressors

Body of Abstract (300-400 words):

Background: Mitochondrial disease (MD) is an important medical co-morbidity associated with autism spectrum disorder (ASD). Children with ASD and MD (ASD/MD) make up an important subgroup of children with ASD but little is known about their physiological, developmental and genetics characteristics as compared to children with ASD but no MD (ASD/NoMD). In addition, the majority of ASD/MD cases cannot be explained by a known mitochondrial DNA abnormality leaving open the possibility that their MD could be secondary to other physi-ological abnormalities, such as oxidative stress, or associated with nuclear genetic lesions.

Methods: 18 ASD/MD children were compared to 18 ASD/NoMD children matched on age and gender. Measures of language development, adaptive behaviour, core autism symptoms, oxidative stress [plasma glutathione, methylation precursors and 3-nitrotyrosine (3-NT)], in#ammation [3-chlorotyrosine (3-CT)], genome-wide gene expression and candidate nucleotide polymorphisms were obtained. Expression of nuclear electron transport chain (ETC) complex genes was also examined. Enrichment analysis was conducted to match signi"cant genetic di!erences in expression and polymorphisms between the groups to functional ontologies derived from canoni-cal pathways.

Results: The ASD/MD group demonstrated higher 3-CT as compared to ASD/NoMD suggesting greater immune activation and in#ammation in the ASD/MD group. The relationship between 3-NT, a measure of chronic oxida-tive stress, and adaptive behavior, language development and core autism symptoms was di!erent for the two groups. Better adaptive behavior, language development and fewer core autism symptoms were associated with higher 3-NT levels in the ASD/MD group but lower 3-NT levels in the ASD/NoMD group. Similar, but fewer, relationships were also found for 3-CT. The relationship between the expression of ETC nuclear genes was also dif-ferentially related to 3-NT and 3-CT between the two groups. Enrichment analysis found that both expression and polymorphism di!erences strongly converged on the pathway responsible for maturation of pro-opiomelanocor-tin, a pathway that is essential for the production of adrenocorticotropic hormone.

Conclusions: Clear physiological di!erences were found between the ASD children with and without MD. Both chronic oxidative stress and in#ammation appear to be di!erentially related to measures of cognitive develop-ment and gene expression between the two groups. Lastly, children with ASD/MD appear to have genetic poly-


morphisms that e!ect gene expression related to hormonal pathways responsible for adaptation to physiological stressors. Together, these data suggest that children with ASD/MD have a unique pro"le of pathways involved in adapting to endogenous and exogenous stressors. These "ndings have signi"cant implications for treatment of this subgroup of ASD children.

Abstract #: 24Presenter: Chun Shi LinAuthors: Chun Shi Lin1,2, Mark Sharpley1, 2, Megan McManus2, Eric Sung3, Billy Phan4, Fred Ross-Cisneros4, Peter Baciu3, Valerio Carelli5, Alfredo Sadun4, Douglas Wallace1, 2 Institutions: 1Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, 2Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, 3Allergan Inc., Irvine CA 92623, 4Doheny Eye Institute, Department of Ophthalmology, USC-Keck School of Medicine, Los Angeles, CA 90089, 5Department of Neurological Sciences, University of Bologna, Via Ugo Foscolo 7, 40123 Bologna, Italy

Title: A Mouse Model with a Missense Mutation in ND6 for Pre-Leber’s Hereditary Optic Neuropathy

Body of Abstract: Mitochondrial DNA (mtDNA) mutations have long been implicated in ophthalmological and neuronal disorders. More speci"cally, mtDNA mutations in the subunits of NADH: ubiquinone oxidoreductase (complex I) cause Leber’s hereditary optic neuropathy (LHON). Roughly 95% patients harbor one of three point mutations in mtDNA encoding complex I subunit: G3460A of ND1, G11778A of ND4, and T14484C of ND6. LHON patients su!er acute vision losses due to massive retinal ganglion cell losses. Visual recovery is rare, and treat-ment is scarce. Clinical and pathological studies for LHON are limited to biochemical measurements of in vitro cybrids and pathology on post-mortem samples. Cybrids are immortalized cell lines that do not share physiologi-cal relevance and complexity of LHON patients. Histopathology does not address the progression, rather the end point, of the disease. As a result, attempts to understand the consequences of LHON mutations have yielded limited and con#icting interpretations. To adequately remedy this problem, a homoplasmic missense, G13997A, mtDNA mutation, has been introduced into the mouse germline. This mutation results in an amino acid change of P25L in the ND6 subunit of complex I. The ND6 mutant mice displayed many of the same characteristics of asymptomatic LHON mutation carriers and patients. While the vision was not impaired, the retinal response of the mutant mice was signi"cantly decreased under light and dark conditions. Pathology on the optic nerve of ND6 mutant mice revealed retinal cell death and swollen axonal population. In addition, ND6 axons possessed proliferative, yet abnormal mitochondria. Biochemical measurements determined that the ND6 mutant su!ered from reduced bioenergetic capacity driven by complex I and increased oxidative stress and injuries. This trans-genic strain is the "rst mouse model that genetically, phenotypically, and biochemically recapitulates all aspects of LHON. It provides an opportunity to both test the e$cacy of therapies and determine which factors modulate the penetrance of LHON.

Abstract #: 25Presenter: Markéta Tesa,ováAuthors: Markéta Tesa,ová1, Viktor Stráneck-2, Hana Kratochvílová1, Zuzana Hájková, Jana Sládková1, Jana Spá.ilová1, Hana Hansíková1, Tomá/ Honzík1, Hana Hartmannová2, Lenka Nosková2, Lenka Piherová2, Emilie Lalonde3, Jacek Majewski3, Stanislav Kmoch2, Ji,í Zeman1

Institutions: 1Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic. 2Institute of Inherited Metabolic Disorders, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Prague, Czech Republic. 3Department of Human Genetics, McGill University Health Centre, Montreal, Quebec, Canada.

Title: Novel phenotype associated with OPA1 mutations?



Mutations in OPA1 gene, encoding a dynamin-like mitochondrial GTPase indispensable for mitochondrial net-work structure and morphology, were found to be responsible for autosomal dominant optic atrophy (ADOA). Moreover OPA1 mutations may be responsible for a continuum of phenotypes ranging from mild disorders af-fecting only the optic nerve to severe and multi-systemic diseases.

In Caucasian non-consanguineous family, 3 boys were born with lactic acidosis, a severe muscle hypotonia, apa-thy, breathing and breastfeeding di$culties. Patient 1 died on day 9 during the early metabolic lactate acidosis. In his younger brothers, central hypotonic syndrome was present from the 2nd and 3rd month of life. Severe vision and hearing impairment developed at 4 months followed by hypertrophic cardiomyopathy. Brain atrophy was found by MRI in patient 2 at 4 month of age. In patient 3, brain atrophy and Leigh syndrome were observed at autopsy. Patient 2 died at the age of 10 months, patient 3 died at the age of 12,5 months. Metabolic examina-tion revealed lactic acidosis with increased excretion of Krebs cycle intermediates and 3-methylglutaconic acid (only patient 3). In isolated muscle mitochondria of patients 2 and 3, mild decrease in activity of complex II+III, complex IV and PDHc were observed. Mutation in mtDNA and TMEM70, TAZ, SCO2, SCO1 were excluded by direct sequencing in patient 3. Genome-wide copy number analysis was unremarkable. Subsequently, whole-exome sequencing in patient 2 and 3 revealed heterozygous mutations: c.1062_1064delAGA (p.E354del, novel mutation) inherited form father and c.1768C>T (p.R590W) inherited from mother in OPA1 as the most probable candidates. Steady-state levels of OPA1 isoforms were signi"cantly decreased in muscle, heart and frontal core of patient 3 with profound reduction of short isoforms. In cultured skin "broblasts of patients 2, 3, and their parents, OPA1 isoform levels were decreased in both patients´ and father´s sample. More pronounced reduction of short OPA1 isoforms (S3, S4, S5) was observed in patients compared to parents. Moreover, mitochondrial network was more fragmented in patients´ "broblasts compare to parental samples. Nevertheless in all "broblasts cell-lines, RCC activities were in reference range. To conclude, whole-exome sequencing revealed possibly pathogenic mutations in OPA1 gene. Despite ongoing analyses to con"rm OPA1 mutation as cause of the disease, symptoms described in our patients may enlarge phenotypic variability associated with OPA1. Supported by research project PRVOUK-P24/LF1/11 and grant IGA NT 13114-4/2012.

Abstract #: 26Presenter: Hana Kratochvilova Authors: Hana Kratochvilova1, Marketa Tesarova1, Tomas Mracek2, Katerina Hejzlarova2, Hana Hansikova1, Josef Houstek2, Jiri Zeman1

Institutions: 1Department of Pediatrics and Adolescent Medicine, First Faculty of Medicine, Charles University in Prague and General University Hospital in Prague, Czech Republic. 2Institute of Physiology, Academy of Sciences of the Czech Republic, Czech Republic.

Title: Topology of TMEM70 in the inner mitochondrial membrane

Body of Abstract (300-400 words): TMEM70 represents an ancillary factor involved in the biogenesis of mam-malian ATP synthase and is uniquely speci"c for higher eukaryotes. Its absence results in an isolated decrease of the content of fully assembled ATP synthase and reduction of enzyme activity. TMEM70 is a 21 kDa mitochondrial protein of the inner mitochondrial membrane synthesized as a 29 kDa precursor (Hejzlarova et. al, 2011). It con-tains two putative transmembrane regions in the central part of the structure.

The aim of the present study was to determine topology of TMEM70 in the inner mitochondrial membrane by two independent methods: trypsin digestion and quenching of #uorescence signal. HEK293 cell lines stabile expressing TMEM70_FLAG (FLAG: C_terminus) or TMEM70_GFP (GFP: C_terminus) fusion proteins were used to characterize TMEM70 orientation. Trypsin digestion: Freshly isolated mitochondria from HEK293 cells expressing TMEM70_FLAG were swollen using hypotonic medium to disrupt outer mitochondrial membrane but keeping the inner mitochondrial membrane intact. The swollen mitochondria were incubated with/without trypsin and with/without TRITON X-100. Samples were separated by 12% SDS-PAGE and immunoblotted with antibodies. OPA1 (located in the inner mitochon-drial membrane facing the intermembrane space) was completely digested when trypsin was added, whereas


TMEM70_FLAG and FH (located in matrix) was completely protected from trypsin digestion. NDUFB6 was used as loading control (protein protected before trypsin digestion by structure of complex I).

Quenching of #uorescence signal: In HEK293 cells expressing TMEM70_GFP the #uorescence signal was moni-tored during the whole experiment. In the "rst step the cytoplasmic membranes of the cells were disrupted by digitonin treatment. Treated HEK293 cells were incubated with Proteinase K (digests cytosolar proteins) and Trypan blue dye (quenches #uorescence signal of proteins which is not shielded by continuous membranes). Till this phase of the experiment the intensity of #uorescence signal was unchanged. Afterwards Triton X-100 was added to disrupt all membranes and #uorescence signal of TMEM70_GFP was quenched. This suggests that the GFP epitope was protected by the inner mitochondrial membrane untill the last step of assay.

Results from two independent methods indicated that C-terminus of TMEM70 is oriented to the mitochodrial matrix as predicted by Jonckheere et. al, 2011. Supported by research projects GA UK 37710, GA UK 370411, SVV 2012-502 and IGA MZ !R NT12370-5.

Abstract #: 28Presenter: Shilpa Iyer1

Authors: Raj R. Rao2, Erich Gnaiger3, and James P.Bennett, Jr.4Institutions: 1Center for the Study of Biological Complexity, Virginia Commonwealth University, 2Chemical and Life Science Engineering, Virginia Commonwealth University. 3Medical University of Innsbruck, Department of Visceral, Transplant and Thoracic Surgery, D. Swarovski Research Laboratory, 4Parkinson’s Disease Center, Virginia Commonwealth University.

Title: Mitochondrial gene therapy improves respiration and biogenesis in mitochondrial diseases of children and adults.

Body of Abstract

Mitochondrial genome manipulation is a potential therapy for many incurable mitochondrial diseases of children and adults resulting from mutant mitochondrial DNA (mtDNA) and impaired respiration. Leigh’s syndrome (LS) is a fatal neurodegenerative disorder of infants and Leber’s hereditary optic neuropathy (LHON) causes blindness in young adults. We treated LHON and LS cells respectively harboring G11778A and T8993G mutant mtDNA by >90%, with a single dose of healthy donor mtDNA complexed with recombinant human mi-tochondrial transcription factor A (rhTFAM). Our results showed improvement in mitochondrial respiration by ~1.2 fold in LHON cells and restoration of ~>50% ATP synthase function in LS cells. We also observed increases in mitochondrial replication, transcription and translation of key respiratory genes and proteins in the short term. Up regulation of NRF1, TFAMB1 and TFAMA expression demonstrated that mitochondrial biogenesis likely accounted for the mechanism for improving mitochondrial respiration. While much work needs to be done in determining the factors that might in#uence stability, transmission and maintenance of the introduced mtDNA; these results represent opportunities for therapeutic interventions for LHON and LS patients in the near future.

Abstract#: 29Presenter: Sha TangAuthors: Sha Tang1, Jing Wang1, Fangyuan Li1, Victor Wei Zhang1, Megan Landsverk1, Hong Cui1, Cavatina K. Truong1, Guoli Wang1, Eric S. Schmitt1, William J. Craigen1, Lee-Jun C. Wong1

Institution: 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030

Title: First Tier Molecular Diagnosis of Mitochondrial Disorders – the Experience of a Mitochondrial Diagnostic Laboratory pre NextGen Era


Mitochondrial disorders are a group of genetically and clinically heterogeneous diseases. For patients with suspected mitochondrial dysfunction, DNA-based molecular diagnosis generally starts with screening for mitochondrial DNA (mtDNA) common point mutations and large deletions (screening), followed by analysis of the entire mitochondrial genome (mtWGS) and/or POLG gene (POLG) by Sanger sequencing. These three tests are considered the “"rst tier” analyses in the routine molecular evaluation of mitochondrial diseases. From May 2005 to December 2011, the screening, mtWGS, and POLG tests were performed on 9,260, 2,851, and 4,240 independent patients at the Mitochondrial Diagnostic Laboratory, Baylor College of Medicine, respectively. A total of 343 patients (3.7%) were positive for the screening test, including 225 patients harboring a common mtDNA point mutation, 86 patients containing a single mtDNA large deletion, and 32 patients with mtDNA multiple deletions. Among common mtDNA point mutations, m.3243A>G remains the most frequent one (56.9%), followed by m.11778G>A (9.8%) and m.8993T>G (7.6%). 109 mtDNA mutations (3.8%), including 93 re-ported and 16 novel pathogenic changes, have been identi"ed in the patient cohort with mtWGS. We imposed stringent criteria, including results from clinical, biochemical, and molecular genetics studies of matrilineal family members, in the assessment of pathogenicity for the novel mutations. De"nitive molecular diagnosis of POLG-related disorder was made for 137 patients (3.2%) and a heterozygous POLG mutation, either reported (69) or novel (29), was detected in another 98 (2.3%) subjects. Conclusive molecular "ndings were detected in 23 out of 343 patients tested simultaneously for all the three tests. This detection rate (6.7%) almost doubled that of any single test alone (3.2 – 3.8%). In the remaining 320 patients negative for all three tests, no further molecular/biochemical tests were pursued in 40 (12.5%). Additional test(s) were performed for the other 280 negatives. The most common sequential tests are analyses of other mtDNA depletion genes (54.6%), ETC enzyme activity (49.3%), and mtDNA copy number (41.1%). These additional tests con"rmed the molecular defects in only two (MPV17 and PDHA1) patients (0.7%), demonstrating the low detection rate of pre-NextGen sequential approach. In summary, each of the screening, mtWGS, and POLG tests has a positive detection rate of ~3.5% and the combination of the three (6.7%) signi"cantly enhances the capability of making a de"nitive molecular diagnosis. We expect the #ourishing application of the NextGen technology investigating multiple loci in parallel to really usher in a more e$cient epoch of molecular diagnosis of mitochondrial disorders.

Abstract #: 30Presenter: Sha TangAuthors: Sha Tang1, Yu-Wen Huang1, Margherita Milone2, Xia Tian1, Hong Cui1, Victor Wei Zhang1, Jing Wang1, Lee-Jun C. Wong1

Institutions: 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030; 2Department of Neurology, Mayo Clinic, Rochester, MN, United States

Title: The Molecular Etiology of Progressive External Ophthalmoplegia (PEO) Associated with Mitochondrial Myopa-thy

Objective: To elucidate the molecular etiology of Progressive External Ophathalmoplegia (PEO) with associat-ed mitochondrial myopathy syndrome and to determine the prevalence of di!erent gene defects causing PEO.

Background: Progressive External Ophthalmoplegia (PEO) with associated mitochondrial myopathy can be caused by autosomal dominant mutations in POLG1 (adPEOA1), ANT1 (adPEOA2), and TWINKLE (adPEOA3). Two autosomal recessive POLG1 mutations can also result in autosomal recessive PEO (arPEO). Recently, muta-tions inPOLG2 (adPEOA4) and RRM2B (adPEOA5) have been reported to be associated with PEO. Autosomal dominant optic atrophy caused by mutations in the OPA1 gene can also present as PEO. The encoded products of these genes are involved in mitochondrial DNA (mtDNA) replication (POLG1, POLG2, and TWINKLE), distribution (OPA1), and the maintenance of deoxynucleotide pools (ANT1 and RRM2B).

Methods: The coding regions and exon-intron boundaries of POLG1, ANT1, and TWINKLE were sequenced in 187 patients (> 10 yr) suspected of PEO. RRM2B gene was sequence analyzed in 25 patients negative for mutations in the above three genes. Southern blot and/or PCR were used to detect mtDNA deletions in muscle specimens of 7 patients, 6 of which with two POLG1 mutant alleles and the 7th one harboring a dominant TWINKLE mutation.


Results: In this cohort, 21 patients (11.2%) had a de"nite molecular diagnosis of PEO and mitochondrial myo-pathy. These include 12 patients with arPEO caused by 2 mutant alleles in POLG1 and 9 patient with adPEO caused by a heterozygous mutation in POLG1 (1 patient), ANT1 (1 patient), or TWINKLE (7 patients). In addition, 5 patients were heterozygous for a POLG1 mutation usually found in compound heterozygosity with another mutation. No mutation was detected in the RRM2B gene in 25 patients negative for POLG1, ANT1, and TWINKLE mutations. In 6 out of the 7 patients with muscle specimen available for analysis, multiple mtDNA deletions were detected (85.7%).

Conclusions: Our data suggest that POLG1 (ar) and TWINKLE (ad) represent the most frequent molecular defects in PEO patients associated with mitochondrial myopathy. Multiple mtDNA deletion in the muscle is a cardinal feature for these patients, indicating that compromised mtDNA integrity plays an important role in the etiology of PEO.

Perspective: We are currently using the NextGen platform to simultaneously investigating all the 6 loci in pa-tients with clinical symptoms consistent with PEO and multiple mtDNA deletions. We expect such approach to be more e!ective for molecular diagnosis.

Abstract #: 31Presenter: Lisa EmrickAuthors: Sha Tang1, Lisa Emrick1, Inn-Chi Lee1,2, Guoli Wang1, Fangyuan Li1, Shao-Wen Weng3, William J. Craigen1, Lee-Jun C. Wong1

Institutions: 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030; 2Department of Pediatrics, Division of Pediatric Neurology, College of Medicine, Chung-Shan Medical University Hospital, Institute of Medicine of Chung-Shan Medical University, Taichung, Taiwan ; 3Department of Internal Medicine, Chang Gung Memorial Hospital, Kaohsiung Medical Center, Chang Gung University College of Medicine, Kaohsiung, Taiwan

Title: PDHA1 Mutations and Continued Clinical and Genetic Heterogeneity: Are there gender Di"erences?

Pyruvate dehydrogenase complex (PDHC) de"ciency is a genetically and clinically heterogeneous disorder. Mutations in the PDHA1 gene are responsible for the majority of cases of PDHC de"ciency. The PDHA1 gene is on the X chromosome, however, PDHC de"ciency appears to a!ect males and females equally due to skewed X chromosome inactivation in the a!ected females. Sequence analyses of the PDHA1 gene were performed for 624 patients, including 323 females and 301 males, with suspected PDHC de"ciency. Targeted sequence analyses for selected variants in relevant family members were also performed to aid in the assessment of pathogenicity. From this cohort, causative mutations were detected in 13.6% of females (44/323) and 8.3% of males (25/301) and 24 novel mutations have been identi"ed. In addition, we have used aCGH to investigate the possibility of large deletions in 7 female patients with biochemically proven PDHC de"ciency without identi"-able mutations by sequencing. However, copy number changes were not found.

The genetics and clinical features of the 69 positive cases were reviewed. The average age of onset was 3.5 years for females and 5.9 years for males. We have clinical information on 44/69 (32 female and 12 male) patients with mutations. Most cases presented with neurological and other system involvement. Seven of 44 patients, all females, had only neurological symptoms. Nine of 44 patients had abnormal PDHC activity (7/32 females and 2/12 males). The most common reason for testing was lactic acidosis (31/44). Developmental de-lay was the most common neurological "nding (19/32 females and 6/12 males). Microcephaly was recorded in 14/44 positive cases (12/32 female and 2/12 males). Among 16 patients with brain MRIs results, 4 had cortical atrophy (3 females and 1 male), 3 had brain malformations (all females), 2 had abnormalities of the basal gan-glia (1 female and 1 male), and 3 had undisclosed MRI abnormalities (all females). Female patients tend to have more severe mutations (34% with null mutations) while males have milder mutations (8% with null mutations).In summary, a higher mutation detection rate was observed in females compared to males tested. Females


tend to have more severe mutations than males, probably because null mutations are better tolerated due to X inactivation. In those patients where information is available, female patients tended to have more problems related to brain developments, in particular there was a higher rate of microcephaly and brain malformations in females in comparison to males.

Abstract #: 32Presenter: Matthew J BirdAuthors: Matthew Bird1,2, Ann Frazier1, Adrienne Laskowski1, David Thorburn1,2

Institutions: 1Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Melbourne, Australia 2University of Melbourne Department of Paediatrics, Melbourne, Australia

Title: Functional analysis of astrocytes in a complex I de!cient mouse model.Body of Abstract: Mitochondrial dysfunction is strongly associated with a range of neurological conditions. However the mechanisms underlying mitochondrial neuropathology are poorly understood. Accessing biologi-cally relevant neurological tissue has traditionally been di$cult, until the recent development of animal models. Our lab has characterised a mouse model of complex I de"ciency, the most common mitochondrial respiratory chain defect. Ndufs4fky/fky mice have a retroviral insertion resulting in a complete knockout of the Ndufs4 subunit of complex I.

The mice exhibit a progressive neurological disease and we have isolated and characterised a number of relevant primary cell lines including astrocytes, a glial cell type which supports neuronal cells. Likewise, making mouse embryonic "broblasts is a useful reference population as it is typical to receive human "broblasts in a diagnos-tic lab for pathological analysis and they are accordingly well characterised. To this end, astrocytes and MEFs have been isolated from Ndufs4fky/fky pups and embryos respectively. Primary cell cultures were established, then grown in media containing either glucose or galactose as primary carbon source for 24 hours prior to analysis. Galactose catabolism is rate limiting as compared to glucose, and it is expected that its utilisation could force the cells to rely more heavily on oxidative phosphorylation. Enzymatically, complex I activity is equally impaired in all cell types tested. Examination of the rates of ATP synthesis and reactive oxygen species generation in the primary cell lines suggest that the Ndufs4 knockout astrocytes have an underlying defect regardless of the car-bon source. This is in contrast to the MEFs, which appear phenotypically normal when cultured on glucose, only presenting a compromised phenotype when cultured on galactose as the primary carbon source. Membrane potential is apparently conserved in both cell types tested, as is gross mitochondrial morphology.

Abstract #: 33Presenter: Sophie Curbo Authors: Sophie CurboII,*, Xiaoshan ZhouII , Kristina KannistoII, Ulrika von DöbelnIII, Kjell HultenbyIV, Sindra IsetunIII, Mats GåfvelsII and Anna KarlssonI

Institution: IDivision of Clinical Microbiology F-68, IIDivision of Clinical Chemistry, C1-72, IIIDivision of Metabolic Diseases, IVDivision of Clinical Research Center, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden

Title: Thymidine kinase 2 de"ciency-induced mtDNA depletion in liver leads to defect %-oxidation and insu$cient supply of ketone bodies and glucose for brain function

Thymidine kinase 2 (TK2) de"ciency in humans causes mitochondrial DNA depletion syndrome for which there currently is no cure. To study the molecular mechanisms underlying the disease and search for treatment options we previously generated and described a TK2 de"cient mouse strain (TK2-/-). The TK2-/- mice were progressively hypothermic, showed no hypodermal fat layer at postnatal day 14, displayed heterogeneous adipocytes, and accumulated intracellular lipid vesicles in the liver. The attained data suggested alterations in lipid metabolism as a part of disease progression. In this study we show that the accumulation of fat vesicles in the liver cells of TK2-/- mice was accompanied by increasingly hypertrophic mitochondria and depletion of mtDNA. The levels of cholesterol and nonesteri"ed fatty acids were elevated and there was accumulation of long chain acylcarnitines in plasma of the TK2-/- mice. The hepatic cells of 14 days old TK2-/- mice exhibited less than 20 % mtDNA compared


to the wild type mice and they had acquired signi"cantly reduced mitochondrial beta-oxidation and ATP produc-tion rates. Furthermore, although the TK2-/- mice of postnatal day 14 were eating, the blood sugar and the ketone levels dropped signi"cantly compared to the levels in the wild type mice. Our hypothesis is that the gradually increasing failure of the liver to produce ketone bodies and glucose for the brain diminishes the remaining brain function and leads to loss of consciousness and ultimately death of the TK2-/- mice.

*Contact information: Sophie Curbo, Karolinska Institutet, Division of Clinical Microbiology F-68, Karolinska Uni-versity Hospital Huddinge, S-141 86 Stockholm, Sweden, Telephone +46-8-52483616 and fax +46-8-58587933, E-mail: [email protected]

Abstract #: 34Presenter: Marjan Amiri Authors: Marjan Amiri#, Nicola Solaroli and Anna KarlssonInstitution: Division of Clinical Microbiology, Department of Laboratory Medicine, Karolinska Institutet, Karolinska University Hospital, Huddinge, S-141 86 Stockholm, Sweden

Title: The residual deoxycytidine phosphorylating activity of thymidine kinase 2 may determine di"erences in clinical manifestations of thymidine kinase 2 de!ciency

Thymidine kinase 2 (TK2) is a nuclear encoded mitochondrial enzyme that catalyze the synthesis of thymidine and deoxycytidine nucleoside monophosphates for mitochondrial DNA (mtDNA). TK2 de"ciency is known to cause a myopatic form of mitochondrial DNA depletion syndrom (MDS), but some patients also present with progressive encephalomyopathy with mitochondrial DNA (mtDNA) depletion in many tissues. Although TK2 de"ciency mainly manifests in infancy and early childhood, it also can have later onset and cause disease in adult life. R130W and R183W are two TK2 mutated enzymes that previously have been described to give di!erent clini-cal symptoms. In patients, the R130W TK2 mutant results in severe muscle, brain and liver symptoms, with onset during the "rst month of life. The R183W TK2 mutant results mainly in muscle symptoms with onset at an aver-age 2 years of age. In this study we have investigated di!erences in dThd and dCyd phosphorylation e$ciency in these mutant enzymes. Our results demonstrate that the residual dThd phosporylation capacity of the two TK2 mutants was similar whereas the dCyd phosphorylation of the TK2 R130W mutant enzyme was signi"cantly lower compared to the TK2 R183W mutant enzyme. The di!erence in dCyd phosphorylating capacity correlate to the di!erences in onset and symptoms of a!ected patients. Our data demonstrate the importance to consider residual dCyd kinase activity of TK2 for the development of treatment strategies for TK2 de"ciency.

Contact information: Marjan Amiri, Telephone +46-8-52483616 and fax +46-8-58587933, E-mail: [email protected]

Abstract #: 35 Presenter: Shannon RoseAuthors: Shannon Rose, Stepan Melnyk, Richard E. Frye, and S. Jill James Institution: Arkansas Children’s Hospital Research Institute, University of Arkansas for Medical Sciences, Little Rock, AR 72202

Title: Increased Susceptibility to Reactive Oxygen Species in Autism Lymphoblastoid Cells is Mediated by Mitochondrial Dysfunction


Background: Mitochondrial disease (MD) is an important medical co-morbidity associated with autism, a behav-iorally de"ned neurodevelopmental disorder in which the etiology is unknown for the majority of cases. De-creased glutathione-mediated redox capacity has been shown in plasma, primary immune cells, lymphoblastoid cell lines (LCLs) and mitochondria isolated from LCLs derived from children with autism. Autism LCLs produce


signi"cantly higher levels of reactive oxygen species (ROS) and exhibit an increased susceptibility to mito-chondrial membrane depolarization following acute nitric oxide exposure relative to controls. This evidence suggests that glutathione redox capacity is insu$cient to counter endogenous ROS production in autism LCLs. This could increase susceptibility to oxidative damage and mitochondrial dysfunction following pro-oxidant exposures.

Hypothesis: We hypothesized that decreased glutathione redox capacity in autism LCLs renders them more susceptible to mitochondrial dysfunction following acute oxidative stress than control LCLs. Further, we hy-pothesized that pretreatment to increase the glutathione redox capacity would confer protection from mito-chondrial dysfunction. Methods: We used extracellular #ux analysis to measure mitochondrial oxygen consumption rate (OCR) at baseline (basal OCR) and upon the sequential addition of mitochondrial inhibitors to compare bioenergetic pro"les in autism and control LCLs before and after a 1 hr exposure to 5-15 0M DMNQ (2,3-dimethoxy-1,4-napthoquinone), an ROS generator. The autism LCLs were subsequently tested after 48 hr pretreatment with 1 mM N-acetyl-cysteine (NAC) to determine whether changes in mitochondrial bioenergetics after DMNQ expo-sure could be prevented by NAC-induced increase in intracellular glutathione redox capacity. Results: Prior to DMNQ treatment, autism LCLs exhibited signi"cantly increased mitochondrial reserve capacity (maximal OCR- basal OCR) relative to controls (p=0.04). Treatment with DMNQ resulted in signi"cantly higher basal OCR (10-15 0M; p10.05), OCR linked to ATP synthesis (5-15 0M; p10.02), and a greater depletion of reserve capacity (10-15 0M; p10.02) in autism LCLs relative to control LCLs. Pretreatment of the autism LCLs with NAC successfully increased the intracellular glutathione redox capacity to that of controls and normalized the mito-chondrial bioenergetic response to DMNQ to that of control LCLs.

Conclusions: Supporting our hypothesis, we demonstrate that acute exposure to an ROS producing agent results in a detrimental e!ect on mitochondrial bioenergetics that is greater for autism LCLs as compared to control LCLs. This suggests that the autism LCLs are more vulnerable to oxidative insults, most likely through in-duced mitochondrial dysfunction. Further, targeted treatment to restore intracellular glutathione redox capac-ity increases the ability of the autism LCLs to withstand excessive ROS exposure.

Abstract #: 36Presenter: Natalie S. HauserAuthors: Natalie S. Hauser1, Renkui Bai2

Institutions: 1Children’s Hospital Central California, Medical Genetics Department, Madera, CA 93704; 2GeneDx Inc., Gaithersburg, MD 20877

Title: Two novel RRM2B gene mutations in a patient with Autosomal Recessive Progressive External Ophthalmople-gia, Encephalopathy and Cytochrome C Oxidase De!ciencyAbstract: In this report we describe a male child born to healthy parents at 31-32 weeks gestation, the preg-nancy was a twin gestation, the twin experienced a fetal demise; consequently our patient was delivered. The baby was 5lbs 9oz. He remained in the NICU for several weeks after birth then was discharged home. He had di$culty with GERD in the "rst few months of life, but no other medical problems. The patient was the third child in a sibship of 3, the 2 older siblings are healthy. Mother described him as a placid baby, never demand-ing, with myopathic facial features. He was introduced to solid foods at six months, but seemed unable to master the oral coordination to eat solids from a spoon. Parents described him as “weaker” as compared to his other siblings. At 7 mos of age he was still working on head control, and had not yet achieved the ability to sit up. Five days prior to admission at 7.5mos of age, he lost head control and the ability to eat. Parents brought him to the local ER, where he was admitted. During his admission, he became progressively weaker. He eventu-ally required a tracheostomy and g-tube. Currently, he has minimal spontaneous movement, ophthalmople-gia, ptosis and seizures. The diagnosis of cytochrome C oxidase de"ciency was made from muscle biopsy. The muscle biopsy showed abnormal mitochondria with structural abnormalities, reduced cytochrome C oxidase by enzyme histochemistry and increased neutral lipid. Mitochondrial respiratory chain testing showed a repro-ducible de"ciency in cytochrome C oxidase activity to 13% of the normal reference mean. Complexes II-III were also reduced at 16% of the normal mean. Citrate synthase activity was elevated at 234% of control, a marker for


mitochondrial content. Molecular Genetic Testing: Sequencing results of the following individual genes-SCO1, SCO2, SURF1, COX10, COX15, COX6B1, and FASTKD2 were normal. Sequence analysis of a panel of 24 nuclear genes (BCS1L, COQ2, COX10, COX15, COX6B1, DGUOK, DLD, MPV17, OPA1, PDHA1, PEO1, POLG, POLG2, RRM2B, SCO1, SCO2, SLC25A4, SUCLA2, SUCLG1, SURF1, TACO1, TIMM8A, TK2, and TYMP) identi"ed two heterozygous novel missense mutations in the RRM2B gene- G200E (c.599 G>A, GGA>GAA) and E105K (c.313 G>A, GAA> AAA), both are non-conservative amino acid changes, at a highly conserved positions, not seen in the general population or any available online mutation/variant databases, and are predicted to be disease-causing. We conclude the cytochrome C oxidase de"ciency noted on this patients muscle biopsy is caused by these two novel mutations in the RRM2B gene.

Abstract #: 37Presenter: Jody K. TakemotoAuthors: Jody K. Takemoto1, Julia Choi1, Cecilia M. Shikuma1,2, Jintanat Ananworanich1,2,3,4, Kara Bennett5, Pairoa Praihirunkit2, Piranun Hongchookiat2, Pornpen Mathajittiphun3, Daniel E. LiButti1, Victor DeGruttola5, Nittaya Phanuphak2,3, Nitiya Chomchey2, Patcharawee Rungrojrat2, Nipat Teeratakulpisarn3, Praphan Phanuphak3, Tanate Jadwattanakul6, Mariana Gerschenson1

Institutions: 1Department of Cell and Molecular Biology, University of Hawaii, 651 Ilalo Street, Honolulu, Hawaii 96813, 2South East Asia Research Collaboration with Hawaii, Thai Red Cross AIDS Research Center 104, Tower 2, 2nd Floor Rajdumri Road, Pathumwan Bangkok 10330 Thailand, 3Thai Red Cross AIDS Research Center, 104 Ratchadumri Road, Bangkok, Thailand 10330, 4The HIV Netherlands Australia Thailand Research Collaboration, 104 Ratchadamri Road, Pathumwan, Bangkok, 10330, Thailand, 5Harvard School of Public Health, 677 Huntington Avenue, Boston, Massachusetts 02115, 6Queen Savang Vadhana Memorial Hospital, Si Racha Chon Buri 20110, Thailand

Title: BASELINE PBMC OXPHOS ENZYMES CAN PREDICT FAT CHANGES AT 24 OR 72 WEEKS IN THAI SUB-JECTS INITIATED ON HAART

Background: We have previously reported that peripheral blood mononuclear cells (PBMCs) mitochondrial (mt) oxidative phosphorylation (OXPHOS) protein levels correlate to the amount of adipose tissue OXPHOS in HIV patients on ART, and that lipoatrophic patients have lower levels of OXPHOS compared to seronega-tive controls. In this study, we examined whether baseline PBMC OXPHOS predict fat changes in limb or trunk from baseline to 24 week and from 24-72 week in naïve patients initiating di!erent HAART regimens.

Methods: PBMC OXPHOS NADH dehydrogenase (CI) and cytochrome c oxidase (CIV) activities were measured by immunoassays (Abcam, San Francisco, CA) and body composition parameters assessed by DEXA in ART-naïve subjects entering SEARCH 003, a 150 in an ARV clinical trial in Bangkok and Chonburi, Thailand. This study was designed to compare rates of mt toxicity-mediated syndromes among 3 di!erent ART regimens (stavudine vs. zidovudine vs. tenofovir). Associations between CI or CIV and DEXA parameters were assessed by linear regression; these analyses included adjustment for potential confounders, sex and age.

Results: 67 men and 81 women were evaluated at baseline and median (Q1,Q3) CD4 count was 177 (72,233) cells/mm3 and HIV RNA by PCR was 75460 (29873,201565) copies/ml. In separate univariate models, for each 1 unit (nmol/min/mg) increase in baseline PBMC CIV activity, DEXA limb fat changed by -6 gm (95% CI (-12,-1)) and trunk fat changed by -7 gm (-13,-1) from week 0 to 24. These associations remained after adjusting for sex and age. In univariate models for week 24-72 change in DEXA, for each 1 unit increase in baseline CI activity limb fat increased by 7 gm (-1,15) and trunk fat by 8 gm (1,15). Similarly, for each 1 unit increase in CIV activity limb fat increased by 8 gm (0,17) and trunk fat by 7 gm (-1,15). Signi"cant interactions were observed between treatment arm and CI or CIV (p<0.02), e.g. for each 1 unit increase in CIV among subjects from the d4T arm and from the TDF arm, limb fat increased by 22 gm (7,38) and 23 gm (9,38), respectively. By contrast, among subjects from the ZDV arm, for each 1 unit increase of CIV, limb fat changed by -6 gm (-20,7). DEXA/


BMI analyses showed similar results.

Conclusions: Baseline PBMC CIV activity predicts week 0-24 change in limb or trunk fat even after adjusting for confounders. Furthermore, CI or CIV predict changes in 24-72 week fat changes dependent on treatment arm.

Abstract #: 38Presenter: Dmitriy M. Niyazov, M.D.Authors: Dmitriy M. Niyazov, Diane Africk Institution: 1Ochsner Clinic Foundation, Children’s Ambulatory Care Center, 1315 Je!erson Hwy, New Orleans, LA 70121

Title: Diagnosis and Treatment of Secondary Mitochondrial Disease

Abstract: It’s been well-documented that mitochondrial disease (MtD) has incomplete penetrance and variable expressivity. Sometimes it’s easy to diagnose primary MtD if a patient "ts the phenotype and it’s con"rmed by a known or indisputably pathogenic mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) mutation. However, it’s extremely challenging to establish a de"nitive diagnosis in the vast majority of MD patients who don’t have an identi"able mtDNA or nDNA mutation or they have a variant of unclear clinical signi"cance. Even muscle biopsy can be markedly abnormal but still inconclusive because "ndings suggestive of mitochondrial dysfunction can be primary or secondary to some other disorder. In our clinical practice, we’ve seen many patients with signs of Mt dysfunction whether based on clinical phenotype, blood and urine markers, muscle biopsy or equivocal mtDNA or nDNA gene testing. In these cases, it’s tempting to assign a patient’s phenotype to MD but sometimes a primary disorder other than MD is present and Mt dysfunction is a part of that disorder. We’ll share our experi-ence with diseases which can be confused with primary MD but instead they cause secondary Mt dysfunction. It’s imperative to recognize that there’s another condition which may require speci"c treatment. One of the ex-amples would be our patient with the muscle biopsy consistent with complex III de"ciency who we subsequently found to have Lesch-Nyhan syndrome and additional workup revealed renal stones which required treatment with allopurinol to avert bouts of extreme pain. Another example would be a patient with complex I de"ciency who was subsequently found to have Cohen syndrome and the patient did well on growth hormone therapy. We’ll also show that treatment of Mt dysfunction in these disorders, consisting of a combination of proper nutri-tion, exercise and a vitamin cocktail, is safe and e!ective in ameliorating patient’s primary condition. We’ll utilize descriptions, pictures and videos of patients with diseases causing secondary Mt dysfunction who directly ben-e"t from the mitochondrial treatment. We argue that the knowledge learned from our experience is important to disseminate among doctors of various specialties as well as families, to improve care of patients with secondary Mt dysfunction.

Abstract #: 39Presenter: Barbara Ko#erAuthors: Edith E. Mueller1, Susanne M. Brunner1, Johannes A. Mayr2, Olaf Stanger3, Wolfgang Sperl2, Barbara Ko#er1 Institution: 1Research Program for Receptor Biochemistry and Tumor Metabolism, 2Department of Pediatrics, 3Department of Cardiac Surgery, Paracelsus Medical University, Salzburg, Austria.

Title: Functional comparison of Mitochondrial Haplogroup T and Haplogroup H in HEK293 Cybrid Cells.

Body of Abstract: Epidemiological case-control studies revealed that mitochondrial haplogroups are associated with onset and/or progression of multifactorial diseases. For instance, mitochondrial haplogroup T was previ-ously shown to be associated with vascular diseases including coronary artery disease and diabetic retinopathy. Whereas haplogroup H is the most frequent haplogroup in Europe and often found to be more prevalent in


healthy controls compared to patient study groups. However, justi"cations for the assumption that haplo-groups are functionally di!erent are rare. Therefore, we attempted to compare di!erences in mitochondrial function between haplogroup H and T cybrids.

Mitochondrial haplogroup H and T cybrids were generated by fusion of HEK293 cells devoid of mitochondrial DNA with isolated thrombocytes of individuals with the respective haplogroups (HEK H cybrids and HEK T cy-brids). These cybrid cells were analyzed for oxidative phosphorylation (OXPHOS) enzyme activities, mitochon-drial DNA (mtDNA) copy number, growth rates and susceptibility to reactive oxygen species (ROS). We observed that haplogroup T cybrids have lower complex V activity despite higher mtDNA copy number. The elevated mtDNA copy number of HEK T cybrids could be due to a compensatory mechanism of these cells in response to a lower OXPHOS capacity. The probably higher basal OXPHOS capacity of HEK H cybrids renders these cells capable of equal or faster growth in galactose medium in comparison to haplogroup T, which might produce adenosine triphosphate to a larger degree by glycolysis. HEK T cybrids showed a signi"cantly higher survival rate after treatment with hydrogen peroxide (H2O2) compared to HEK H cybrids. This "nding implies a higher production of ROS by HEK H cybrids, as a higher baseline production of ROS together with external addition of H2O2 might render HEK H cybrids to reach the threshold faster where cells undergo cell death. In conclusion, we were able to show that mitochondrial haplogroups H and T are functionally di!erent in our model system.

Abstract #: 40Presenter: Barbara Ko#erAuthors: René G. Feichtinger1, Serge Weis2, Johannes A. Mayr1, Franz A. Zimmermann1, Reinhard Geilberger1, Wolfgang Sperl1 and Barbara Ko#er1

Institutions: 1Department of Pediatrics, Research Program for Receptor Biochemistry and Tumor Metabolism, Paracelsus Medical University, Salzburg, Austria, 2Laboratory of Neuropathology, Department of Pathology and Neuropathology, State Neuropsychiatric Hospital Wagner-Jauregg, Linz, Austria.

Title: Alterations of the aerobic mitochondrial energy metabolism in brain tumors

Body of Abstract: Recently, for a subgroup of neuroepithelial brain tumors (NETs) therapeutic intervention by ketogenic diet was reported to be e!ective. To elucidate the functional explanation for the e$cacy of this high-fat low-carbohydrate diet we analyzed the mitochondrial energy metabolism in brain tumors. Mitochon-drial biogenesis and the status of the aerobic energy metabolism was evaluated by immunohistochemical staining (IHC) of porin, a mitochondrial outer membrane protein, and complexes of the oxidative phosphory-lation (OXPHOS) were analyzed in NETs (n=55) and meningiomas (n=76). Mitochondrial DNA (mtDNA) copy number was determined and the samples were screened for mtDNA deletions and mutations. All NET entities studied showed a striking ubiquitous combined reduction of the OXPHOS complexes I and IV compared to adjacent normal brain tissue. The mitochondrial mass as well as complexes II, III and V of the OXPHOS were compensatorily up-regulated in the tumor tissue compared to adjacent brain tissue. No patho-genic mutations and deletions were present in mtDNA encoded subunits of complex I, complex IV and mito-chondrial tRNA genes, which could explain the reduction of complex I and IV. We presume that genetic altera-tions in nuclear genes cause the de"ciency in aerobic mitochondrial energy metabolism found in NETs.

The meningioma entities (meningothelial, "broblastic, transitional, psammomateous, secretoric, atypic, anaplastic) di!ered with regard to their mitochondrial energy metabolism. Down-regulation of at least two complexes of the OXPHOS especially complex I and IV was found in tumors compared to brain controls. In ad-dition, reduced complex II expression was found in all entities with exception of secretory and anaplastic men-ingiomas. Complex V was only reduced in psammomatous and secretory meningiomas. Expression of complex III was heterogeneous among the tumor entities. In all tumors of the meninges up-regulation of mitochondrial mass was observed, whereas mtDNA copy number was reduced compared to normal brain tissue.

The loss of OXPHOS has potential therapeutic implications, since the OXPHOS de"cient tumor cells have to rely solely on glycolysis which might be selectively targeted by inhibitors of this pathway or a ketogenic diet.

This work was supported by the Cancer Foundation Salzburg and the Children’s Cancer Foundation Salzburg.


Abstract #: 41Presenter: Veronica J. Hinton Authors: Veronica J. Hinton1, Engelstad, K1, DiMauro S1, De Vivo, D1.Institution: Columbia University, Department of Neurology, New York City, NY 10032

Title: No Evidence of Cognitive Decline among Carrier Relatives of MELAS Patients.

Body of Abstract:The mitochondrial DNA m.3243A>G genotype can present with multiple symptoms including mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (or MELAS), as well as migraine headaches, hearing loss, exercise intolerance, diabetes and cognitive impairment. MELAS patients are fully symptomatic, having su!ered either a stroke-like episode or seizures. Maternally related relatives (carrier relatives) may have some or no symptoms, but have no history of stroke-like episodes or seizures. Whether the carrier relatives have cogni-tive de"cits associated with the genotype is unknown.

To examine whether cognitive function di!ers among MELAS patients, carrier relatives and controls (relatives by marriage), participants were administered a battery of neuropsychological tests and comparative statistics were run across the three groups. 130 matrilineal relatives, ages 18 years and older, from 54 families participated in a Natural History Study at Columbia University. 31 patients with MELAS (48% male, mean age 37 + 2 years), 76 carrier relatives (23% male, mean age 42 + 2 years) and 28 controls (77% male, mean age 50 + 2 years) received standardized tests examining a range of cognitive skills. The groups did not di!er in ethnic background (>90% Caucasian), but the controls were signi"cantly older than the MELAS patient and carrier relative groups, and the ratio of males to females varied across the groups. Consequently, both age and gender were entered as covari-ates in subsequent analyses. Univariate analysis of variance comparisons were run across the three groups on cognitive test scores, followed by post-hoc between-group t-tests. Results found consistent signi"cant di!er-ences in performance among the groups on measures of mental status, verbal memory, visual memory, lan-guage skills, visual spatial skills, attention and executive function. Moreover, across all measures, di!erences were found between the patients with MELAS and both carrier relatives and controls (the performance of MELAS patients was lower), but there was no di!erence in performance on any measure between carrier relatives and controls. As such, these data document that patients with MELAS have global cognitive impairments. Impor-tantly, these data also show no signi"cant cognitive decline in carriers who are not symptomatic with MELAS across cognitive domains. These "ndings suggest that therapeutic interventions in carrier relatives before they convert to MELAS phenotype may preserve cognitive function.

To examine whether cognitive function di!ers among fully symptomatic individuals (de"ned as having evi-dence of focal brain involvement in addition to lactic acidosis), carriers of the mutation, and controls (de"ned as xxxxx), were administered a battery of neuropsychological tests. 130 matrilineal relatives from 54 families who were over 18 years of age participated in an observational study at Columbia University Medical Center in the City of New York. 31 symptomatic individuals (48% male, mean age 37 + 2 years), 76 carriers (23% male, mean age 42 + 2 years) and 28 controls (77% male, mean age 50 + 2 years) received standardized tests examining a range of cognitive skills. The groups did not di!er in ethnic make-up ( >90% identi"ed themselves as Cauca-sian), but the controls were found to be signi"cantly older than the other two groups, and the ratio of males to females varied across the groups, so both age and gender were entered as a covariates in subsequent analyses. Univariate comparisons were run on cognitive test scores, followed by post-hoc between group t-tests. Result found consistent signi"cant di!erences in performance among the groups on measures of mental status, verbal memory, visual memory, language skills, visual spatial skills, attention and executive functions. Moreover, across all measures, di!erences were found between the symptomatic individuals and both carriers and controls (with symptomatic individuals’ performance being lower), but no di!erence in performance was observed on any measure between carriers and controls. As such, these data indicate that symptomatic individuals with MELAS have global cognitive impairments. Additionally, these data show no signi"cant cognitive declines associated with carrier status across cognitive domains.


Abstract #: 42Presenter: Xiao WangAuthors: Alicia M. Pickrell1#, Xiao Wang2#, Milena Pinto3, Sandra R. Bacman3, Aixin Yu5, Aline Hida3, Lloye M. Dillon4, Paul D. Morton6, Thomas R. Malek5, Siôn L. Williams3, and Carlos T. Moraes1,2,3,4*

Institutions: 1Neuroscience Graduate Program, 2Graduate Program in Cancer Biology, 3Department of Neurology, 4Department of Cell Biology and Anatomy, 5Department of Microbiology and Immunology, 6Department of Neurosurgery. University of Miami: Miller School of Medicine, Miami, FL 33136, USA.

* To whom correspondence should be addressed:Carlos T. Moraes, 1420 NW 9th Avenue, Rm.229, Miami, FL 33136; Tel: 305-243-5858; Fax: 305-243-6955; E-mail: [email protected]

# These authors contributed equally to this work.

Title: Mitochondrial DNA Damage Contributes to Premature Aging Through p53-Dependent Response MechanismsAbstract: Defects in the repair of single- and double-strand breaks of nuclear DNA have been associated with progeroid phenotypes. Such nuclear DNA damage activates the transcriptional regulator p53, leading to a signaling cascade resulting in DNA repair, cell cycle arrest, or apoptosis. In contrast to the well-studied role of nuclear DNA damage, the role of mitochondrial DNA (mtDNA) damage in senescence is largely unknown.We developed a mouse model that expresses an inducible restriction endonuclease, PstI, targeted to the mito-chondria, (mito-PstI). Ubiquitous expression mito-PstI was transiently induced for 5 days when the mice reached adulthood. Three months after the insult, these mice developed a phenotype resembling premature aging, most evidently in highly mitotic tissues, including thymic progenitor cells. We found that the mtDNA double strand breaks caused by mito-PstI expression in the thymus led to a rapid p53 response, causing transcriptional signal-ing through activation of downstream targets such as Mdm2, and cell cycle arrest protein p21. These cellular stressors eventually resulted in the stalling of progenitors maturation, leading to apoptosis and di!erentiation of premature thymocytes. We also found similar results in the Mutator mouse model expressing an error-prone proofreading mtDNA polymerase y.

We further con"rmed our "ndings ex vivo in ScaI cell culture, and determined that the p53-dependent pathway was activated through ROS signaling. Based on these pieces of evidence, we conclude that damages to mtDNA recruit the same machinery as employed by the nuclear DNA; we also speculate that stem cells and progenitor cells are preferentially a!ected by this mechanism in our mouse models.In summary, we determined that mtDNA damage activated a previously thought to be exclusively nuclear DNA damage pathway, the p53 response, contributing to aging. This study opens avenues for research in the inter-play between nuclear and mitochondrial damage signaling in aging, cancer, and stem cell "elds.

Abstract #: 43Presenter: Amy GoldsteinAuthors: Amy Goldstein1,2, Amanda R Barone1, Stephanie J DeWard1, Nicole Payne2 and Jerry Vockley1,2

Institutions: University of Pittsburgh School of Medicine1 and Children’s Hospital of Pittsburgh2, Pittsburgh, PA 15213

Title: Triheptanoin Therapy for Inherited Disorders of Fatty Acid Oxidation

Background: Fatty acid oxidation (FAO) disorders result in inadequate energy production from fatty acids dur-ing fasting and stress. Symptoms of long chain FAO defects include hypoglycemia, rhabdomyolysis, myopathy, and cardiomyopathy. Treatment consists of medium chain triglycerides (MCT), but is only partially e!ective. MCT oil contains 8 and 10 carbon fatty acids that enter mitochondria to allow generation of the end product acetyl-CoA, but may result in the secondary depletion of 3 carbon tricarboxylic acid (TCA) cycle intermediates. In contrast, the seven-carbon MCT heptanoate results in production of the three carbon TCA cycle precursor propionyl-CoA (C3) in addition to acetyl-CoA that can replenish odd carbon intermediates, thus improving pro-


duction of reducing equivalents for ATP synthesis by the respiratory chain. Triheptanoin has been proposed as superior to MCT oil for treatment of FAO disorders.

Methods: 22 patients are currently enrolled in a compassionate use trial of triheptanoin at Children’s Hospital of Pittsburgh. Diagnoses include de"ciencies of VLCAD (very long chain acyl dehydrogenase, 11 patients), LCHAD (long chain 3-hydroxyl CoA-dehydrogenase, 5 patients), TFP (trifunctional protein, 2 patients), CPT II (carnitine palmitoyltransfarase II, 3 patients), and CACT (carnitine-acylcarnitine translocase, 1 patient). Patients ranged in age from 1.5-58 years and received 1-2 g/kg/day of triheptanoin. Metabolic and nutritional parameters were collected upon study entry and at 2 months, 6 months, and every 6 months after that. Echocardiograms were monitored annually.

Results: Triheptanoin has been well tolerated overall with similar side e!ects to MCT oil. Metabolic parameters compared to MCT treated patients showed only elevated C3 carnitine levels in blood (mean 3.70, reference<0.77) and elevated 6-hydroxy heptanoic and 4-hydroxy-N-valeric acids in urine (not quantitated). Of the 20 patients who have participated at least 12 months in the study, there were on average 0.6 hospitalizations per year per person, decreased by report compared to prior to starting triheptanoin therapy.

Conclusions: Triheptanoin oil is a well-tolerated alternative to MCT oil in patients with long chain FAO disorders, and has led to marked clinical improvement in some patients. A double blind FDA Phase II is currently underway to directly compare the safety and e$cacy of these therapies in patients with FAO disorders.

Abstract#: 44Presenter: Siôn WilliamsAuthors: Siôn Williams1, Alicia Pickrell1, Lloye Dillon1, Sandra Bacman1, Deborah Mash1, Stephan Z2chner2, Carlos T. Moraes1

Institution: 1Department of Neurology, University of Miami; 2John P. Hussman Institute for Human Genomics, University of Miami, Miami, Florida 33136.

Title: Towards a clearer picture of somatic mitochondrial DNA mutation spectrums using next generation sequencing.

Body of Abstract: We have developed Mito-Seq a next generation sequencing (NGS) methodology for sequenc-ing mtDNA-enriched, native DNA samples. Analysis of assemblies and extracted sequences allows for detailed examination of single nucleotide variants (SNVs) and recombination. We have applied Mito-Seq to the study of mitochondrial genome variation in putamen of young and aged individuals. Transgenic mice were used to develop approaches to the study of mtDNA variation. NZB/BALBc heteroplasmic mice provided models of abundant heteroplasmy; A mitochondria-targeted PstI restriction enzyme model, the Mito-PstI mouse, was used as a model of mtDNA deletions and recombination; And the PolgD257A/D2557A mtDNA mutator mouse severed as a model of increased SNV and control region multimer (CRM) loads.

The mitochondrial genome of putamen was sequenced using Mito-Seq from cohorts of young (19-34 years, n=7) and aged (67-89 years, n=8) individuals. Bioinformatic analysis demonstrated that the mtDNA of aged individuals carries a complex spectrum of somatic mutations, some features of which are also present in a minority of young individuals. Clonally expanded SNVs were apparent in aged and young specimens resembling the patterns of heteroplasmy seen in NZB/BALBc heteroplasmic mice. However we did not "nd evidence of an age-related increase in somatic SNV load in aged putamen as seen in PolgD257A/D2557A mtDNA mutator mice. The common deletion, similar to clonal Pst1 deletions in mito-Pst1 mice, and abundant major arc deletions, were present in all aged specimens and one young specimen. Novel recombination hotspots were identi"ed in the control region at higher prevalence in aged specimens implying that they represent age-associated damage to mtDNA.

These studies reveal that despite many years of investigation, questions still remain in our understanding somat-ic mtDNA mutation spectrums. It is hoped that the mtDNA features identi"ed in these studies will provide useful biomarkers of mitochondrial dysfunction for research into mitochondrial diseases and aging.


Abstract #: 45Presenter: Phil G. MorganAuthors: Phil G. Morgan1, Albert Quintana2, Richard D. Palmiter2, Margaret M. Sedensky1

Institution: 1Dept Anesthesiology, Univ. Wash., Seattle, WA; 2HHMI, Dept Biochem, Univ. Wash., Seattle, WA

Title: Speci!c Hypersensitivity to Volatile Anesthetics in a Mouse Lacking Ndufs4, a Subunit of Mitochondrial Complex I.

Body of Abstract: In earlier studies we found that defects speci"cally in complex I of the mitochondrial electron transport chain altered sensitivity to VAs in the nematode C. elegans. We previously reported hypersensitivity to iso#urane and halothane in a mouse carrying a knockout (KO) mutation in a subunit of mitochondrial complex I (Ndufs4). A role for mitochondrial function underlying VA action was corroborated by studies showing that children with mitochondrial dysfunction in complex I are also sensitive to sevo#urane. We have now extended our studies of the Ndufs4 KO mouse to include sensitivities to propofol and ketamine using the righting re#ex as an endpoint. We "nd strikingly speci"c responses by the Ndufs4 KO mouse to these sedatives. Methods. Young (P23-27) Ndufs4 KO mice were compared to age-matched, wild-type littermates at an age be-fore any severe CNS phenotype is apparent. Genotypes were con"rmed by PCR for all mice. Mice were anesthe-tized with incremental doses of iso#urane or halothane in a closed chamber. VA concentrations were determined by gas chromatography. Lack of response to a non-crushing tail clamp stimulus was the anesthetic endpoint. Studies of propofol and ketamine were done by peritoneal injection and tested for loss of righting re#ex (LORR). All mice were either studied under a warming lamp such that ambient temperature was maintained at 36oC or on a warming blanket to maintain body temperature. Results. Ndufs4 KO animals showed a roughly 300% hypersensitivity to both iso#urane and halothane (EC50 iso = .44% in KO, 1.23% in wt; EC50 hal = .52% in KO, 1.28% in wt). Sensitivity to propofol increased by 100 % (ED50 prop = 35mg/kg in KO, 70mg/kg in wt). In contrast, the KO animals were 50% resistant to the e!ects of ketamine, with an ED50 of 100mg/kg compared to 70mg/kg for wt. Discussion. The mice with mitochondrial dysfunction have a signi"cant change in VA sensitivity that corrobo-rates data in both the nematodes and humans and reinforces the concept that mitochondrial complex I con-tributes to VA sensitivity. The results with propofol and ketamine indicate that the changes are not the result of nonspeci"c depression of the CNS. The homology between mitochondrial proteins in worms and in mice pre-dicts that fundamental processes within mitochondria are similar between these species. These mice a!ord the opportunity to identify speci"c regions within the brain that are responsible for this VA sensitivity by using cell speci"c knockouts of the Ndufs4 gene.

Abstract #: 46Presenter: Hong CuiAuthors: Hong Cui, Victor Wei Zhang, Lee-Jun WongInstitution: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030

Title: Diagnosis of diseases associated with mitochondrial DNA biosynthesis and maintenance of integrity by next generation sequencing

Body of Abstract (300-400 words): 385

Mitochondrial dysfunction can be caused by mutations in mitochondrial DNA (mtDNA) or nuclear genes that encode proteins function in mitochondria. Currently, about 80-95% of patients with clinically suspected primary mitochondrial disorders do not have an identi"ed pathogenic mutation in the mtDNA. These cases are usually further tested for mutations in nuclear-encoded genes associated with distinct clinical phenotypes of mitochon-drial disorders. Screening tests, such as analyses of mtDNA copy number, mtDNA multiple deletions, and respira-tory chain complex activities, as well as clinical evaluation, are helpful in the selection of a group of genes to be


analyzed. Panel testing can maximize the successful rate of obtaining a molecular diagnosis. This can be achieved by the application of next generation massively parallel sequencing (NGS) technique. A large number of genomic targets can be analyzed simultaneously. We have developed and validated an in-solution capture based method for analyzing a group of nuclear genes involved in mtDNA biosynthesis and maintenance of integrity, including POLG, DGUOK, MPV17, TK2, TYMP, SUCLA2,SUCLG1, SUCLG2, RRM2B, POLG2, OPA1, SLC25A4 (ANT1), C10orf2 (TWIN-KLE), and OPA3.

The NGS test of this panel of genes has been validated in 3 stages. In phase I, three unknown samples were sequenced by both NGS and Sanger method for the comparison of speci"city and sensitivity. Our results dem-onstrate that all target sequences are fully covered at > 100X with an average of 700X. The variants called by MPS are in 100% concordance with the list generated by Sanger sequencing. In phase II validation, samples with vari-ous mutation types, including single nucleotide change, small indels, and large deletions were selected for test-ing. Results demonstrated that the NGS approach is capable of detecting all types of mutations in these genes. Phase III validation was aimed to discover causative mutations in undiagnosed cases. Samples screened positive for mtDNA depletion or mtDNA multiple deletions are analyzed for mutations in genes responsible for mtDNA biosynthesis and maintenance. Results demonstrated high positive mutation detection rate in samples pre-screened for mtDNA depletion or multiple deletions. For quality assurance, we also instituted proper qualitative and quantitative controls to be analyzed along with each sample. In conclusion, massively parallel sequencing of a group of genes can e$ciently detect the underlying molecular defects of the mitochondrial disorders due to mtDNA depletion or multiple deletions, in a cost e!ective and timely manner.

Abstract #: 47Presenter: Jeana DaRe, Ph.D., FACMGAuthors: Jeana DaRe1, Valeria Vasta2, John Penn2, Thao Tran2, Lisa Susswein1, Jessica Booker1, Sihoun Hahn2,3 Institutions: 1Transgenomic, New Haven, CT, USA, 2Seattle Children’s Hospital Research Institute, Seattle, WA, USA, 3University of Washington, School of Medicine, Seattle, WA, USA

Title: Clinical re-sequencing of over 410 genes to diagnose mitochondrial disorders: Results from the !rst 78 patients

Body: The diagnosis of mitochondrial disorders (MD) is very challenging as they are clinically and genetically heteroge-neous diseases. We recently developed a clinical next-generation sequencing (NGS) test to diagnose mitochon-drial disorders that targets >410 nuclear genes encoding entire subunits of the mitochondrial respiratory chain complex (RCC), assembly factors, transcription/translation and biogenesis factors, enzymes, carrier proteins and genes causing secondary mitochondrial defects or presenting with similar phenotypes. Patient samples were submitted by physicians experienced with mitochondrial disorders. On average, 590 variants were found in the targeted genes including exons and 20bp at each intronic end. After removing variants with a relatively high minor allele frequency in our cohort or public SNP databases, we found an average of 7 variants per patient that could be pathogenic. Most of these were unpublished, single heterozygous variants of unknown signi"cance in genes with autosomal recessive (AR) disease inheritance, resulting in possible carrier status for the correspond-ing disease. In a total of 44 (56%) cases, we identi"ed variants considered to be possibly disease-causing, with 8 cases involving genes with AR inheritance, 27 cases with autosomal dominant (AD) inheritance, 4 cases with X-linked inheritance, and 5 cases with positive "ndings in both AR and AD disease genes. Parental testing was used to further evaluate the signi"cance of variants, which decreased the number of cases with plausibly causative mutations cases to 35 (45%), though incomplete penetrance or di!erences in expression remain possible. Of note, we found only 3 cases with variants in RCC subunits or assembly factors. Most variants were found in genes that cause secondary mitochondrial defects, or cause diseases with phenotypes similar to MD. These genes in-cluded metabolic disease genes such as OTC (urea cycle disorder), sul"te oxidase de"ciency, and Fabry disease, spastic paraplegia, spinocerebellar ataxia, and other genes such as CLN6 (neuronal ceroid lipofuscinosis), and UBE3A (Angelman syndrome). The gene category that we found the second most variants in was mitochondrial


DNA transcription/translation and mitochondrial biogenesis factors, possibly indicating that mutations in these genes may be more prevalent in primary mitochondrial disease than mutations in RCC subunits or assembly factors. Many cases also had positive "ndings in multiple genes, indicating that complex disease may be caused by mutations in several genes. While NGS shows promise for diagnosing suspected mitochondrial disease pa-tients, the challenges remain high as the underlying genetic heterogeneity may be greater than suspected and de"ning the signi"cance of variants detected may require additional studies.

Abstract #: 48Presenter: Ashley R. WolfAuthors: Ashley R. Wolf1,2,3 and Vamsi K. Mootha1,2,3

Institutions: 1Center for Human Genetic Research and Department of Molecular Biology, Massachusetts General Hospital, 185 Cambridge Street, Boston, MA 02114, USA. 2Department of Systems Biology, Harvard Medical School, 200 Longwood Avenue, Boston, MA 02115, USA. 3Broad Institute of Harvard and Massachusetts Institute of Technology, 7 Cambridge Center, Cambridge, MA 02141, USA.

Title: A systematic search for mitochondrial RNA processing components

Abstract: Mammalian mitochondria house a dedicated transcriptional and translational machinery to produce vital mitochondrial-encoded components of the respiratory chain. Defects in mitochondrial DNA replication, mRNA maintenance, and protein synthesis have all been linked to heritable mitochondrial disorders. Since mitochondrial DNA only encodes thirteen subunits required for oxidative phosphorylation, nuclear-encoded proteins are required for all reactions within the organelle, including mRNA processing. Both strands of the human mitochondrial genome are transcribed in large multi-genic units. These precursor RNAs are cleaved into distinct mRNAs and tRNAs by the RNase P complex and the RNase Z activity of ELAC2. Additional unidenti"ed nuclear proteins are likely required for chaperoning the mRNAs to translation and eventual degradation. A recent inventory of mitochondrial-localized proteins serves as a launching point for characterizing this poorly understood process. We have used shRNAs to knock down 100 putative RNA-binding proteins localized to the mitochondria. By simultaneously measuring the levels of all mitochondrial-encoded transcripts and precur-sors in knockdown samples, we can identify proteins necessary for the processing and stability of speci"c transcripts. With this method, we clarify the in vivo role of recently classi"ed processing enzymes and identify new e!ectors of RNA processing. Further, we identify novel regulators of mRNA levels that may work alongside degradation machinery to speci"cally control mRNA half-lives.

Abstract #: 49Presenter: Rasika S. VartakAuthors: Rasika S. Vartak1, Janice Deng1, Yidong Bai1

Institution: 1University of Texas Health Science Center at San Antonio, Department of Cellular and Structural Biology, San Antonio , TX 78229

Title: Identifying factors promoting Complex I assembly in mammalian cells.

Body of Abstract: Respiratory Complex I de"ciency is often an underlying cause of mitochondrial disorders. Hence understanding the assembly pathway of Complex I has been of signi"cant interest. Previous studies have established that assembly of Complex I takes place in a modular manner: the peripheral hydrophilic arm and the membrane arm assemble in a coordinated fashion, with the help of chaperone proteins, to form the L shaped Complex I. Certain structural subunits of Complex I such as ND1, ND4 and ND6 are of critical importance to the assembly of Complex I. These have been found frequently mutated in patients su!ering from Complex I de"cient disorders. To understand how exactly these mutations a!ect Complex I assembly, we previously iso-lated a mouse A9 derivative, 4A, which contains a homoplasmic frameshift mutation in the ND6 gene leading to


the loss of ND6 protein. Complex I assembly is also completely abolished in these cells as shown by Blue Native PAGE. Time point [S]35 pulse chase analysis in 4A cells show presence of a high molecular band at an early time-point, implying that Complex I can form to a certain extent in these cells. Another spontaneous revertant was isolated, termed 4AR, which remained homoplasmic for the ND6 mutation and no ND6 protein, but regained the ability to assemble Complex I. The 4AR mitochondria when transferred to another nuclear background lost this ability, suggesting that a change in 4AR nuclear background plays a crucial role in the assembly of Com-plex I in these cells. Western blot and pulse chase analysis of Complex I in 4AR cells reveals additional Complex I speci"c bands as compared to control cells suggesting a change in assembly pattern. We conducted mass spectroscopy analysis to identify the proteins associated with the Complex I bands, exclusively in 4AR cells. The proteins enriched in the Complex I band in 4AR cells but not parental A9 and 4A cells were OXPHOS subunits, metabolic enzymes as well as chaperone proteins. Co-Immunoprecipitation con"rmed presence of some of these proteins in association with complex I in 4AR cells but not A9 cells. We will further conduct knockdown studies to determine the impact of the selected proteins on Complex I assembly/stability. We hypothesize that atleast one of these proteins could act as a novel assembly/stability factor that is helping Complex I assemble in 4AR cells despite lack of a critical subunit.

Abstract #: 50Presenter: Mary Elizabeth ParkerAuthors: Mary Elizabeth Parker1, Matthew F. Moran2, John T. Foley3, Michael J. Weiss4

Institutions: 1Texas State University (San Marcos, TX), 2Sacred Heart University (Fair"eld, CT), 3State University of New York at Cortland (Cortland, NY), 4Fair"eld University (Fair"eld, CT)

Title: Mitochondrial Disorders and Autism: A New Avenue of Research on Movement

Body of Abstract: Two areas of novel research have collided with the collaboration of the authors. With a growing body of research on mitochondrial disorders and autism, and the interest in identi"cation of autism markers, it seems prudent to start to compare research on movement dysfunction in both populations. This novel line is in a preliminary stage as the authors are disseminating their original research on movement dysfunction in autism.

Utilizing the Bruininks-Oseretsky Test of Motor Performance (BOT2) Short Form and the Timed Up and Go (TUG) as well as motion analysis, the authors have been able to demonstrate markers of movement dysfunc-tion in a sample population of individuals with autism. Areas of impairments include gait, hopping, jumping, throwing, as well as core strength.The primary author has an interest in di!erential diagnosis individuals with complex multisystem disorders, a hallmark example being mitochondrial disorders; in addition, this author has an interest in education health care professionals on the various presentations of mitochondrial disorders including those with autism as a clinical feature.

At this juncture in the collaboration the authors are interested in seeking feedback on the work performed to date and gather support on our future direction of research in movement dysfunction in autism, and the rela-tionship of autism, mitochondrial disorders, and movement dysfunction.

Abstract #: 51Presenter: Igal MadarAuthors: Igal Madar1, Yong Du1, Ting Liu2, Hayden Ravert1, Michael Crow2, Joshua Hare2, Brian O’Rourak2, Richard Wahl1, Robert Dannals1, Institution: Johns Hopkins Medical Institutions, Departments of 1Radiology and 2Medicine, Baltimore, MD 21205

Title: In Vivo Localization and Quanti!cation of Mitochondrial Dysfunction Using PET Imaging of the Novel Voltage Sensor 18F-FBnTP


The mitochondrial membrane potential ((3m) is the central parameter of the organelle’s bioenergetic func-tion, and the unifying measure of multiple key cellular pathways underlying mitochondrial dysfunction, including ATP synthesis, Ca homeostasis and ROS production and detoxi"cation. Therefore, (3m a!ords a unique sensitive marker for an early and comprehensive identi"cation of mitochondrial dysfunction. To ad-dress this hypothesis, we developed the PET imaging (3m-targeting voltage sensor 18F-#uorobenzyl triphenyl phosphonium (18F-FBnTP), and characterized its voltage performance, in vitro and in vivo.

18F-FBnTP exhibited linear relationship over a wide range of potassium di!usion potentials and dose-depen-dent relationship with uncoupling-induced (3m loss in cardiomyocyte mitochondria and single cells. 18F-FBnTP voltage performance was nearly identical to that of 3H-tetra phenyl phosphonium (TPP), a most accu-rate and standard tool for measuring (3m in vitro. PET imaging documented a massive washout of 18F-FBnTP during selective collapse of (3m using pharmacologic and physiological uncoupling protocols in Langendor! heart and intact animal models, respectively. Here, we demonstrate the excellent capacity of 18F-FBnTP PET to localize and quantify mitochondrial dam-age in vivo in animal models of heart failure and myocardial aging. Heart failure was induced in mongrel dogs using 4-wk rapid pacing. 18F-FBnTP PET unraveled a complex, heterogeneous topology of mitochondrial damage in the LV wall of the dilated heart. Both global and area-speci"c patterns of mitochondrial damage were identi"ed. 18F-FBnTP spatial pro"le suggests that mitochondrial damage is a propagating process initi-ated at discrete site/s. Mitochondria in the endocardial aspect of the lateral wall were the most vulnerable to workload induced by chronic rapid pacing. 18F-FBnTP’s regional uptake correlated in space and magnitude with segmental LV wall contractility and apoptosis. In the aging heart, 18F-FBnTP demonstrated a progressive global and focal decrease of uptake with increasing age (6, 12, 18 and 24 mos). A strong spatial and quantita-tive correlation was found between 18F-FBnTP myocardial distribution and extent of oxidative stress assayed by aconitase activation, and the frequency of apoptotic myocytes measured by TUNEL staining.

Together, these "ndings validate that 18F-FBnTP PET can accurately assess the localization and extent of mito-chondrial membrane dysfunction in the intact organism, and in so doing presents a novel diagnostic tool to assess mitochondrial disease progression and response to therapy.

Abstract #: 52Presenter: Rahul PandeyAuthors: Rahul Pandey1, Divya Mehrotra1, Vimal Choubey2, Rajiv Sarin3, Abaas Ali Mahdi4, Devendra Parmar5

Institutions: 1Department of Oral & Maxillofacial Surgery, Faculty of Dental Sciences, CSM Medical University, Lucknow, 2Department of Urology, CSM Medical University, Lucknow, 3Advanced Centre for Treatment Education & Research in Cancer, Mumbai, 4Department of Biochemistry, CSM Medical University, Lucknow, 5Developmental Toxicology, Indian Institute of Toxicological Research, Lucknow, India

Title: Mitochondrial DNA Displacement Loop (CA)n Dinucleotide Repeat Polymorphism in Oral Precancer: A marker for Risk and Progression

Background: Oral cancer is the 6th most common cancer. WHO 2004 report accounted 7.1 million deaths in 2003 and estimated 50% rise in new cases by next 20 years. Mitochondrial genome alternations may be in-volved in carcinogenesis. DNA damage persists longer in the mitochondrial genome. The noncoding region of the mitochondrial DNA (mtDNA) displacement loop (D-loop) has emerged as a mutational hotspot. MtDNA is highly polymorphic (Mitomap). One of the most informative mtDNA variations is the (CA)n dinucleotide repeat polymorphism between nucleotide position 514 and 523 in the third hypervariable region, which has been well-recognized as a marker of mitochondrial genome instability. Hence, this study was planned to assess the relationship of the germline (CA)n dinucleotide repeat polymorphism with risk for progression in oral precan-cer.


Materials/Methods: In a case control study at dental hospital, CSM Medical University, Lucknow, India 50 his-topathologically con"rmed cases of oral precancer cases were enrolled. Their blood and biopsy samples were collected after consent. 50 blood and tissue samples were taken from otherwise normal healthy individuals who came for third molar extractions. Genomic DNA was extracted from both tissue and blood. Genotyping for the (CA)n polymorphism was done and subsequently products were put to polyacrlyamide electrophoresis.

Results/Statistics: A total of 8 (CA)n repeat alleles were observed in our study population, ranging from 4 re-peats [(CA)4] to 11 repeats [(CA)11]; Alleles (CA)6 (56.0%) and (CA)7 (37.0%) were the two most common alleles in controls. Allele frequencies of 8 to 11 repeats were low (3% in cases and 0.8% in controls). Increasing number of multiple alleles of the mtDNA D-loop (CA)n polymorphism was signi"cantly associated with increasing histo-pathological grades (42 for each successive stages (mild, moderate, severe: 5, 4.25, 7).

Conclusions/Clinical Relevance: Given the high susceptibility of mitochondrial DNA to undergo damage it is probable that such mitochondrial changes are early events in the process of carcinogenesis. The presence of multiple alleles (heteroplasmy) of the (CA)n repeat may be an indicator of mitochondrial genome instability and mtDNA malfunction and, thus, may be associated with oral precancer progression. This study suggests that the mtDNA D-loop (CA)n dinucleotide repeat polymorphism may play a signi"cant role in oral cancer etiology.

Abstract #: 53Presenter: Sergey M. KorotkovAuthors: Sergey M. Korotkov, Vladimir P. Nesterov, Irina V. Brailovskaya, Svetlana A. Konovalova, Victor V. FuraevInstitution: Sechenov Institute of Evolutionary Physiology and Biochemistry, the Russian Academy of Sciences, Thorez ave. 44, 194223 St. Petersburg, Russia

Contact information: [email protected]

Title: Joint E"ects of Pinacidil and Ca2+ on Succinate-energizated Rat Heart Mitochondria

It is known that pinacidil (PIN), non-selective modulator of the mitochondrial KATP channels, showed e!ect of the pharmacologic preconditioning on myocardium at ischemia/reperfusion experiments in the use of Lan-gendorf-perfused rat heart. However, hitherto mechanism of the preconditioning is vague. Among attempts to clear the mechanism is that increase ion permeability of the inner mitochondrial membrane (IMM) and fol-lowed swelling of mitochondria produce some decrease of the electrochemical potential (()mito) which prevent calcium overload of heart mitochondria after the ischemia/reperfusion experiments. We studied simultaneous action of 100 0M PIN and 100 0M Ca2+ on swelling, respiration, and ()mito of isolated rat heart mitochondria (RHM) in the presence of succinate, respiratory complex II substrate. PIN substantially accelerated swelling of energized calcium loaded in media containing of NH4NO3 or K acetate. The swelling extremely decreased in the presence of cyclosporine A (CsA) or ADP, mitochondrial permeability transition pore (MPTP) inhibitors. Selec-tive inhibitor of the channels, 5-hydroxydecanoate (5-HD), slightly prevented the swelling of RHM in K acetate medium. Dissipation of ()mito after 5a2+ injection was retarded by PIN and completely inhibited by ADP and CsA. Carboxyatractyloside, a "xer of the adenine nucleotide translocase in c conformation, eliminated a PIN-induced stimulation of state 4 in succinate-energized RHM regardless of the presence of Ca2+. PIN in contrast to Ca2+ has not decreased state 3 or 2,4-dinitrophenol -uncoupled respiration of RHM. It was concluded that pinacidil facilitates K+ #ux in energized mitochondria and promotes MPTP opening in a low conduction state.

Abstract #: 54Presenter: Dina Mehaney1

Authors: Laila Selim1, Fayza Hassan2,Dina Mehaney2

Institution: 1Pediatrics Department and 2Clinical and Chemical Pathology Department, Faculty of Medicine, Cairo University, Egypt, 11562


Title: Mitochondrial encephalopathy with lactic acidosis and stroke-like episodes (MELAS) in a Japanese child : Clinical, Radiological and Molecular genetic analysis.

Body of Abstract: Mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS) is a mitochondrial multisystemic disorder. This disease has mainly been associated to the mitochondrial desoxyri-bonucleic acid (mtDNA) mutation A3243G located in the tRNA Leu(UUR) gene.

Here we report the clinical, radiological and molecular diagnosis of a 10 year old child, with the classical MELAS phenotype.

A 10 year old male, Japanese child presented to the Inherited Metabolic Disease Unit at Cairo University Chil-dren’s hospital with recurrent episodes of headache, nausea and vomiting of 5 years duration. These episodes were associated with motor weakness on the right side, with di$culties in language and memory and visual disturbance, mostly right sided homonymous hemianopia, Neurological examination revealed generalized muscle weakness, with mild right sided hemiparesis. These clinical manifestations were reliable to Magnetic Resonance Imaging (MRI) of infarction of left posterior parietal and left occipital regions and to the left medial temporal, without visible vascular abnormality on Magnetic Resonance Angiography (MRA).Laboratory Investigations revealed hyper lactic acidemia and a discrete increase in Hepatic transaminases. The diagnosis of MELAS was con"rmed with the genetic study of mitochondrial DNA. DNA was extracted from whole blood of the child and his mother and tested for the most common point mutations related to MELAS A3243G,T3271C, G13513A using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR- RFLP) analysis and direct sequencing for con"rmation. Genetic analysis of the mitochondrial DNA revealed a heteroplasmic A to G substitution at position 3243 in the tRNS Leu (UUR) gene. The A3243G mutation was also detected in his Chinese mother who is asymptomatic. The maternal uncle of the patient su!ered from the same clinical picture and died by the age of 19 years. L- Arginine is reported to be bene"cial for MELAS patients and a preventive treatment was given in the form of Arginine 500 mg Twice per day.

Abstract #: 55Presenter: Dina Mehaney1

Authors: Dina Mehaney1, Laila Selim2, Fayza Hassan1

Institution: 1Clinical and Chemical Pathology Department, 2Pediatrics Department, Faculty of Medicine, Cairo University, Egypt, 11562.Title: Screening of the most common mitochondrial DNA mutations among Egyption Pediatric patients with Mitochondrial disorders: A one year study.

Body of Abstract: Mitochondrial diseases (MCD) are an important cause of morbidity and mortality in both adults and children. These Disorders are clinically and genetically heterogeneous. Mitochondrial disorders can be caused by mutations in both nuclear or mitochondrial encoded genes. There are a set of common point mutations in the mitochondrial DNA (mtDNA) that are responsible for common mitochondrial diseases. As a result of the clinical overlap, it is usually necessary to analyze more than one mutation for a patient suspected of a mitochondrial disorder. Screening for these common point mutations could be used as a preliminary non-invasive testing for common syndromic forms of mitochondrial disorders. The spectrum of mitochondrial DNA mutations among Egyption pediatric patients with mitochondrial disorders was not previously studied.

This report is the "rst report to describe the screening service for mitochondrial diseases routinely started at the lab of Inherited Metabolic Disease Unit at the Cairo University Children’s hospital from January 2011 to January 2012.

DNA was extracted from whole blood samples of 54 Egyption Pediatric patients using standard protocol. Nine Patients were clinically and radiologically suspected to have Myoclonus epilepsy & ragged-red "bers (MERRF), Fifteen patients with mitochondrial encephalomyopathy , lactic acidosis & stroke-like episodes (MELAS), Twenty four patients with and Leigh’s syndrome/Neuropathy Ataxia Retinitis Pigmentosa syndromes (LS/NARP), three with Leber’s hereditary optic neuropathy (LHON) ,and one with chronic progressive external oph-thalmoplegia (CPEO).


Mitochondrial DNA mutations screened included point mutations associated with LHON (G3460A, G11778A and T14484C), MELAS (T3271C, G13513A, A3243G), MERRF (A8344G, T8356C, G8363A), LS/NARP (T9176 C, T8993C/G) and patient with CPEO was screened for the A3243G mutation. Methods of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR- RFLP) were performed to screen for the mutations.

The detection rate of mtDNA point mutations was low. Positive rate could be improved by better diagnos-tic algorithm including the histochemical and Biochemical analysis of the mitochondrial Respiratory Chain enzymes in appropriate tissues and sequencing of selected regions in the mtDNA in patients with a high likelihood of mtDNA disease. As Egypt is a country with high rate of consanguineous marriage, The molecular pathogenesis of mitochondrial respiratory chain disorders is suspected to be of nuclear genetic origin.

Abstract #: 56Presenter: Joungil ChoiAuthors: Joungil Choi1,2, Vera Venkatanaresh Kumar Batchu1,2, Manfred Schubert3, Rudolph J. Castellani4, and James W. Russell1,2

Institutions: 1Department of Neurology, University of Maryland, Baltimore, MD, 21201; 2Veterans A!airs Medical Center, Baltimore, MD, 21201; 3National Institute of Neurological Disorders and Stroke, Baltimore, MD, 21201; 4Department of Pathology, University of Maryland, Baltimore, MD, 21201. Title: Activation of mitochondrial bioenergetics by 35-kDa PGC-1#/PINK1 signaling pathway

Body of Abstract: Peroxisome proliferator-activated receptor-gamma co-activator 1 (PGC-1 ) and PTEN-induced putative kinase 1 (PINK1) have recently emerged as key molecules to the maintenance of mitochon-drial integrity and linking metabolic abnormality and neurodegeneration. PGC-1 is a powerful regulator of mitochondrial energy metabolism. Down-regulation of PGC-1 is associated with mitochondrial dysfunction and impaired lipid metabolism in obesity and diabetes, both of which increases the risk of neurodegenerative diseases. Polymorphisms in PINK1 are associated with altered plasma fatty acid concentration and oxidative energy metabolism in diabetes. Loss-of-function mutations in PINK1 have been linked to Parkinson’s disease. We have recently found that a previously unrecognized, novel 35-kDa PGC-1 localizes to brain mitochon-dria. Western blot analysis shows that the level of 35-kDa PGC-1 protein is signi"cantly decreased in human Alzheimer disease (AD) and diabetic animal brains. The truncated, 35-kDa PGC-1 protein is the only PGC-1 isoform found in mitochondria, and is generated by post-translational cleavage of full-length PGC-1 in the cytosol. Immunoelectron microscopy of mouse mitochondria and import experiments in vitro revealed that 35-kDa PGC-1 colocalizes and interacts with voltage-dependent anion channel (VDAC), and its import de-pends on VDAC. The 35-kDa PGC-1 binds and colocalizes with PINK1 in the brain mitochondria. Overexpres-sion of PGC-1 decreases lipid-droplet accumulation and increases mitochondrial fatty acid oxidation upon exposure to lipid overload; down-regulation of PINK1 abolishes these e!ects. Together, these results provide new insights into the role of 35-kDa PGC-1 in mitochondrial beta oxidation in a PINK1-dependent manner and implicate impaired mitochondrial signaling in the pathogenesis of AD and diabetes.

Abstract #: 57 Presenter: Nagendra YadavaAuthors: Chul Kim1, Prasanth Potluri2, Daria Gout2, Lisa M. Minter3, Douglas C. Wallace2, Nagendra Yadava1

Institutions: 1Pioneer Valley Life Sciences Institute, Spring"eld, MA 01107, USA; 2Center for Mitochondrial and Epigenomic Medicine at The Children’s Hospital of Philadelphia, Philadelphia, PA, 19104, USA; 3Department of Veterinary and Animal Sciences at University of Massachusetts-Amherst, MA 01003, USA

Title: A novel mouse model for nuclear-encoded partial respiratory chain Complex I de!ciency.


Abstract: Respiratory chain Complex I (NADH ubiquinone oxidoreductase) de"ciency is the most common cause of mitochondrial diseases. To model pathophysiological e!ects of partial Complex I de"ciency, we have created a mouse model by knocking in a point mutation within the X-chromosome-linked Ndufa1 gene, which encodes an essential subunit, the MWFE protein. We have recently shown that the conversion of serine-55 to alanine (S55A) reduces Complex I assembly by ~50%. The S55A mutation was introduced into mouse genome at the Ndufa1 locus by homologous recombination in 129/SvEv-derived embryonic stem (ES) cells. An ES cell clone with correct targeting of the Ndufa1S55A allele, identi"ed by PCR and Southern blot analyses, was injected into balstocysts of pseudopregnant C57BL/6 mice. Resulting chimera were mated with 129 FLP mice to si-multaneously generate progeny with germ-line transmission of the Ndufa1S55A allele and delete the Neomycin (Neo) selection cassette from the knocked-in locus. Tail DNA from pups with agouti coat color was analyzed for the presence of the Ndufa1S55A allele, and deletion of the Neo cassette. These progeny were crossed with wild type 129 mice. All possible genotypes were obtained and no infertility was observed. A genetic bias against female progeny is noticed in certain crosses (Chi2 test, <0.05). Biochemical characterization of the mutant male mice using blue-native polyacrylamide electrophoresis (BN-PAGE) and respirometry assays con"rm the pres-ence of Complex I de"ciency with variable penetrance in brain, heart, liver, muscle, and embryonic "broblasts. Mutant mice do not show any signi"cant di!erence in weight gain during 7-12 weeks after birth and survive up to 10 months without any sign of increased mortality. A mild insulin resistance is noticed at ~ 7 months of age in male mice, which we plan to expose by feeding high fat-diet. A detailed pathophysiologial characteriza-tion of these mice with reference to mitochondrial encephalomyopathies under normal and stressed condi-tions is in progress. In summary, we have successfully created mice with a nuclear-encoded partial Complex I de"ciency, which will serve as a valuable tool: (i) to model spontaneous vs. triggered mitochondrial disease onset; (ii) to assess deregulation of in#ammatory and autoimmune responses, and (iii) to study alterations of signaling pathways relevant to cell fate and proliferation due to Complex I de"ciency in the context of mito-chondrial encephalomyopathies and other diseases.

Research is supported by a CAER Translational grant and institutional startup funds to NY, and an NIH grant (NS21328) to DCW.

Abstract #: 58Presenter: Alessandra MarescaAuthors: Alessandra Maresca1,2, Claudia Zanna3, Sara Vidoni2, Michela Rugolo3, Patrizia Amati-Bonneau4, Valerio Carelli1, Guy Lenaers2, Cecile Delettre2.Institutions: 1IRCCS Istituto delle Scienze Neurologiche di Bologna, Dipartimento di Scienze Neurologiche, Università di Bologna, Italy; 2Institut de Neurosciences de Montpellier, INSERM U583, France; 3Departimento di Biologia Sperimentale e Evoluzione, Università di Bologna, Italy; 4INSERM U694, Angers, France.

Title: OPA3, a new regulator of mitochondrial !ssion?

Body of abstract: OPA3 (Optic Atrophy 3) is a protein encoded by the nuclear genome and targeted to mi-tochondria. The OPA3 gene maps on chromosome 19 and consists of three exons: two transcript variants (OPA3V1, OPA3V2) have been described, due to alternative splicing of exon 2 and exon 2b. Recessive muta-tions in exon 2 are associated with Coste! syndrome, whereas dominant mutations in exon 2 are associated with Autosomal Dominant Optic Atrophy plus Cataract (ADOAC). A clear function for this protein and the pathogenetic mechanisms leading to these diseases have not been clari"ed yet.

We investigated the e!ects of the overexpression and silencing of OPA3 isoforms in HeLa cells, evaluating the mitochondrial network morphology using Mitotracker Red, mitochondrial membrane potential using JC1, esti-mating susceptibility to apoptosis by counting apoptotic cells after treatment with staurosporine and quan-tifying the mtDNA content by Real Time-PCR. Moreover, we investigated the fusion-"ssion system in ADOAC "broblasts carrying two di!erent OPA3 dominant mutations by Western blot and Real Time-PCR analysis.


Overexpression of OPA3V1, OPA3V2 and OPA3V1 carrying an ADOAC mutation (G93S) produced an extensive mitochondrial fragmentation, a complete loss of membrane potential and a signi"cant increase of sensitivity to staurosporine, whereas it had no consequences on mtDNA content. On the contrary, the lack of both OPA3 isoforms or only OPA3V2 produced an unbalance of fusion-"ssion leading to increased "lamentous mitochon-dria, whereas silencing of OPA3V1 alone cause an opposite e!ect on mitochondrial morphology, raising the number of fragmented organelles. The membrane potential did not result compromised by the loss of OPA3 expression, but a signi"cant increase of apoptotic cells number was found silencing OPA3V1 and OPA3V2 independently. Interestingly, we observed in HeLa cells a compensatory mechanism based on the increase of OPA3V1 mRNA expression when OPA3V2 was suppressed, and vice versa. As for the overexpression, the absence of OPA3 variants did not in#uence mtDNA content. Preliminary data on ADOC "broblasts showed an up-regulation of the fusion proteins OPA1, MFN1 and MFN2, and a down-regulation of the "ssion proteins Fis1 and MTP18, although these tendencies did not reach any statistical signi"cance.Our data support the hypothesis that OPA3 may regulate, directly or indirectly, mitochondrial "ssion and moreover we highlight the existence of a compensatory mechanism regarding the OPA3 variants, that may be relevant for the pathogenetic mechanism leading to ADOAC, as well as Coste! syndrome.

Abstract#: 60Presenter: Sunil SahdeoAuthors: Sunil Sahdeo1, Mark Pook2, Marek Napierala3, Joe Sarsero4, Gino Cortopassi1

Institution: 1University of California, Davis, Molecular Biosciences, School of Veterinary Medicine, Davis, CA 95616, 2Division of Biosciences, School of Health Sciences and Social Care, Brunel University, Uxbridge, UB8 3PH, UK, 3University of Texas MD Anderson Cancer Center, Department of Molecular Carcinogenesis and Center for Cancer Epigenetics, 1515 Holcombe Boulevard, Houston, TX, 4Cell and Gene Therapy, Murdoch Childrens Research Institute, Royal Children’s Hospital, Parkville, Victoria, Australia

Title: Re-purposing small-molecule drugs to treat the mitochondrial disease Friedreich’s ataxia Body of Abstract: Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disease that results from GAA triplet repeat expansion, that likely as a result of chromatin condensation, results in de"cient expres-sion of the mitochondrial protein frataxin. Frataxin has been shown to support the biogenesis of iron-sulfur clusters in mitochondria. De"cient frataxin expression results in degeneration of dorsal root ganglion and cerebellar neurons, resulting in ataxia, and increased insulin-resistance and diabetes. Currently, there is no cure for this disease. Based on microarrays that identi"ed a decrease in antioxidants as a consequence of decreased frataxin expression, we hypothesized that FRDA patient cells would be more sensitive to the glutathione oxidizer diamide than cells from healthy patients. Using this di!erence, we screened clinically approved small molecule drugs to identify one that rescued cells from oxidative stress. Of the protective drugs identi"ed, the glucocorticoid dexamethasone (DEX) was particularly interesting because we previously showed it to rescue in#ammatory death of Schwann cells mediated by frataxin de"ciency. Since one possible mechanism of dex-amethasone’s protection could be an increase in FXN expression, we tested DEX’s e!ect on FXN protein levels using traditional and in-cell western blot techniques in FRDA patient cells, as well as using a FXN-GFP cell line, and observed increases. In order to elucidate the mechanism of FXN increase, we examined the e!ects of DEX on decondensation of chromatin in a GAA560-GFP cell line, and observed activity. Our last proof-of-concept was to try DEX in the YG8 mouse model of FRDA. Although dexamethasone did decrease splenocyte number after 6 days of intraperitoneal dosing, it did not increase FXN levels in cerebellum or spleen. Thus although the drug increases frataxin in patient cells and decreases frataxin-de"cient death in Schwann cells, it does not appear to increase frataxin in an animal model. Upon completion of these studies, we have validated a novel screen to identify potential therapeutics for this debilitating mitochondrial disease.


Abstract#: 62Presenter: Leonardo CaporaliAuthors: Leonardo Caporali1-2, Carla Giordano3, Luisa Iommarini1, Alessandra Maresca1-2, Pio D’adamo4, Solange R. Salomao5, Rubens Belfort Jr.5, Alfredo Sadun6, Valerio Carelli1-2

Institutions: 1Department of Neurological Sciences, University of Bologna, Italy, 2IRCCS Istituto delle Scienze Neurologiche di Bologna, Bologna, Italy, 3Department of Experimental Medicine and Pathology, Sapienza, University of Rome, Rome, Italy, 4Division of Medical Genetics, IRCCS Burlo Garofolo, University of Trieste, Trieste, Italy, 5Department of Ophthalmology, Federal University of Sao Paulo, UNIFESP, Sao Paulo, Brazil, 6Departments of Ophthalmology and Neurosurgery, Keck School of Medicine at USC, Los Angeles, CA, USA

Title: Searching for genetic modi!ers of Leber’s hereditary optic neuropathy penetrance

Body of Abstract

Leber’s hereditary optic neuropathy (LHON) is a maternally inherited blinding disease due to point mutations in complex I subunit genes of mtDNA. In most LHON families the mtDNA mutation is homoplasmic, but only a limited number of subjects becomes a!ected. Thus, the mtDNA mutation is necessary but not su$cient to cause optic neuropathy. Environmental triggers and genetic modifying factors have been considered to ex-plain the variable penetrance in LHON.

We have measured mtDNA copy number in blood cells from LHON a!ected individuals and una!ected muta-tion carriers from a large LHON pedigree, harboring the 11778/G>A mutation in MT-ND4 gene on a haplo-group J background. The subjects belonging to the mutant maternal lineages (25 a!ected and 38 carriers) presented a signi"cantly higher content in mtDNA, as compared to controls (n= 54). By sub-grouping the individuals in a!ected and carriers we showed that the mtDNA copy number was signi"cantly higher in carri-ers. These results strongly support a role for mitochondrial biogenesis in modulating LHON penetrance.

Thus, we investigated 9 candidate genetic variants (SNPs) in 5 of the genes regulating mtDNA replication and biogenesis (PGC-1+, PGC-1%, p53, TFAM, PARL). PGC-1+ and PGC-1% are co-transcriptional activators involved upstream to NRF1, NRF2 and TFAM, thus driving the master regulation mitochondrial biogenesis; p53 is a tumor suppressor gene with a large number of additional functions, including a role in regulating mtDNA replication; TFAM is the major transcription factor for mtDNA; PARL is a mitochondrial protease implicated in regulating mtDNA copy number in a control population and, interestingly, PARL gene have been recently as-sociated with LHON in a linkage study of LHON pedigrees of Asian ancestry. None of the nine genetic variants analyzed resulted signi"cantly associated with the status of a!ected or carrier.

Our study provides a valuable mechanism to explain variability of penetrance in LHON and clues for high throughput genetic screening to identify the nuclear modifying gene/s, opening an avenue to develop predic-tive genetic tests on disease risk and therapeutic strategies.

Abstract#: 63Presenter: Nicola Brunetti-PierriAuthors: Rosa Ferriero1, Eleonora Lamantea2, Edoardo Nusco1, Luisa Bonafè3, Brendan Lee4,5, Massimo Zeviani2, and Nicola Brunetti-Pierri16

Institutions: 1Telethon Institute of Genetics and Medicine, Naples, Italy; 2Unit of Molecular Neurogenetics, The Foundation “Carlo Besta” Institute of Neurology-IRCCS, Milan, Italy; 3Division of Molecular Pediatrics, Centre Hospitalier Universitaire Vaudois, Lausanne, Switzerland; 4Department of Molecular and Human Genetics, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030; USA; 5Howard Hughes Institute, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030; USA; 6Department of Pediatrics, Federico II University of Naples, Italy

Title: Phenylbutyrate therapy for pyruvate dehydrogenase de!ciency


Body of Abstract: De"ciency of pyruvate dehydrogenase complex (PDHC) is the most common disorder lead-ing to lactic acidemia. Phosphorylation of speci"c serine residues of the E1-alpha subunit of the PDHC by pyru-vate dehydrogenase kinase (PDK) inactivates the enzyme, whereas dephosphorylation restores PDHC activity. We recently found that phenylbutyrate prevents phosphorylation of the E1-alpha subunit of the branched-chain ketoacid dehydrogenase complex (BCKDC) and reduces plasma concentrations of neurotoxic branched chain amino acids in patients with maple syrup urine disease (MSUD), due to the de"ciency of BCKDC. We hypothesized that, similarly to BCKDC phenylbutyrate enhances PDHC enzymatic activity by increasing the portion of unphosphorylated enzyme. To test this hypothesis, we have "rst treated wild-type human "broblasts at di!erent concentrations of phenylbutyrate. Following drug treatment, cells were harvested for Western blot analysis using antibodies speci"c for the E1+ subunit of the PDHC phosphorylated at each of the three phos-phorylation sites of the E1 (Ser293, Ser232, and Ser300). Western blot showed a signi"cant reduction in the levels of phosphorylated E1+ subunit of PDHC in phenylbutyrate treated cells compared to untreated cells. Several reports show that phenylbutyrate cross the blood-brain-barrier and it has pharmacological e!ects in brain cells. Therefore, we next investigated the e!ect of phenylbutyrate in vivo on brain PDHC phosphorylation. To this end, we have given saline or phenylbutyrate (250 and 500 mg/kg/day, which are doses given to humans) orally by gavage to C57B6 wild-type mice and after three days of treatment the animals were sacri"ced for analyses. Western blot on brain mitochondrial extracts showed that phenylbutyrate resulted in a signi"cant reduction of the levels of phosphorylated E1+ subunit of Pdhc compared to saline treated mice, while it did not have e!ect on total amount of E1+. Pdhc enzyme activity in brain mitochondrial extracts was increased, thus showing that phenylbutyrate enhances cerebral Pdhc enzyme activity in vivo in a disease relevant tissue. These results are extremely promising because they show that phenylbutyrate increases the activity of the Pdhc in the brain which is the most a!ected tissue in PDHC de"ciency. Moreover, we found that phenylbutyrate increases PDHC residual enzyme activity in 3 out of 4 "broblast cell lines from PDHC de"cient patients.

Given that phenylbutyrate is a drug already approved for use in humans and its safety pro"le is well characterized, our "ndings have the potential to be rapidly translated into treatment for patients with PDHC de"ciency.

Abstract#: 64Presenter: Jarred W. RensvoldAuthors: Jarred W. Rensvold1, Shao-En Ong2, Athavi Jeevananthan1, Steven A. Carr3, Vamsi K. Mootha3,4 and David J. Pagliarini1

Institutions: 1Department of Biochemistry, University of Wisconsin-Madison, Madison, WI 53706, 2Department of Pharmacology, University of Washington, Seattle, WA 98195, 3Broad Institute of MIT and Harvard, Cambridge, MA 02142, 4Departments of Systems Biology and Medicine, Harvard Medical School, Boston, MA 02446

Title: Complementary RNA and protein pro!ling identi!es iron as a key regulator of mitochondrial biogenesis.

Abstract: The production of mitochondria, known as mitochondrial biogenesis, involves the orchestrated transcription, translation, import and assembly of more than one thousand proteins encoded by two genomes. The num-ber and composition of mitochondria varies across tissues and changes in response to environmental condi-tions and nutrient demands, indicating that mitochondrial biogenesis can be tailored to meet speci"c cellular requirements. Defects in this process are associated with a broad range of human diseases and age-related disorders. The transcriptional regulators that control mitochondrial biogenesis, including the peroxisome proliferator-activated receptor gamma, coactivator-1 (PGC-1) family of transcriptional coactivators and their associated transcription factors, are well described. However, other aspects of the cellular control of mitochon-drial biogenesis remain less clear. These include post-transcriptional processes that control mitochondrial gene expression, peripheral pathways that are important for supporting mitochondrial biogenesis and signals that communicate a need for mitochondrial restructuring. To better de"ne the mitochondrial biogenesis program, we performed matched, quantitative SILAC (stable


isotope labeling by amino acids in cell culture)-based proteomics and microarray analyses of mouse muscle cells following overexpression of PGC-1+. From these analyses we "nd that proteins involved in cellular iron homeostasis are highly regulated during mitochondrial biogenesis, and that depletion of cellular iron results in a coordinated down-regulation of mitochondrial protein levels. Immunoblot and quantitative PCR analy-ses con"rm that depletion of cellular iron through various mechanisms results in a rapid, dose-dependent decrease in nuclear- and mitochondrial-encoded gene expression and oxidative capacity in a variety of mam-malian cell lines that is fully recoverable within 3-4 days following the reintroduction of iron. Furthermore, we "nd that the e!ects of iron deprivation are distinct from previously reported HIF-1+ (hypoxia inducible factor)-driven reductions in mitochondrial biogenesis. Collectively, our work shows that cellular iron concentration is a key parameter in calibrating the mitochondrial biogenesis program and suggests that cellular iron deprivation initiates a reversible, adaptive transcriptional response to remodel the mitochondrial proteome and reduce mi-tochondrial respiratory function. Our data serve as a resource for investigating genes subject to post-transcrip-tional regulation, and for identifying additional auxiliary pathways that might be important for modulating the mitochondrial biogenesis program.

Abstract#: 65Presenter: Jason A. Mears Authors: Frances Joan D. Alvarez1, Louie Zhou1, Jason A. Mears1 Institution: 1Department of Pharmacology, Center for Mitochondrial Disease, Cleveland Center of Membrane and Structural Biology, Case Western Reserve University, Cleveland, OH 44106- 4965

Title: Structural studies of Dnm1/Drp1 provide mechanistic insight into mitochondrial !ssion.

Body of Abstract: Dynamin-related protein 1 (Drp1) belongs to a family of large GTPase proteins that regulate membrane dynamics and morphology. Drp1 localizes to mitochondrial constriction sites in vivo to facilitate outer membrane "ssion, and mutations that inhibit its activity lead to hyper-fused mitochondria in vivo. Direct inhibition of Drp1 protects against cell death by limiting increased mitochondrial "ssion associated with apop-tosis. Previously, we have studied the yeast homolog of Drp1, Dnm1, using electron microscopy (EM) to deter-mine its structural properties. We found that Dnm1 forms large (>100 nm in diameter) helical oligomers that constrict upon GTP hydrolysis to generate a contractile force on the underlying membrane (1). We are using similar methods to gain mechanistic insight into the mammalian mitochondrial "ssion complex, and several di!erences exist between the yeast and mammalian systems. We "nd that recombinant Drp1 forms stable te-tramers, which represent the pre-assembled state of Drp1. The size of this complex (~330 kDa) provides a suit-able target for 3D image reconstruction. Interactions with GTP analogs and/or synthetic liposomes promote additional Drp1 self-assembly into helical oligomers. The diameter of Drp1 helices in a GTP-bound state (~40 nm) places considerable strain on the underlying lipid bilayer. We are examining the e!ects of GTP hydrolysis on the Drp1 oligomer to determine how this structure promotes outer mitochondrial membrane "ssion. Future studies will examine interactions between Drp1 and partner proteins in the mitochondrial "ssion complex.

1. Mears, J. A., Lackner, L. L., Fang, S., Ingerman, E., Nunnari, J., and Hinshaw, J. E. (2011) Conformational changes in Dnm1 support a contractile mechanism for mitochondrial "ssion, Nat Struct Mol Biol 18, 20-26.

Abstract#: 66Presenter: Yutaka SuzukiAuthors: Yutaka Suzuki1, Jan Riemer1

Institution: 1Department of Biology, University of Kaiserslautern, 67663 Kaiserslautern, Germany

Title: Regulation of Superoxide Dismutase 1 in Intermembrane Space of MitochondriaReactive oxygen species (ROS) are implicated in human pathology and aging, as damaging agents and/or

6), is generated by the respiratory chain, mitochondrial DNA mutations and mitochondrial diseases can be causes and/or results of production of superoxide. Super-


oxide can be converted to molecular oxygen and hydrogen peroxide by superoxide dismutases. Superoxide dismutase 1 (SOD1) localizes to the cytosol with a minor portion targeted to the intermembrane space (IMS) of mitochondria. The activity of SOD1 is regulated by the copper chaperone for SOD1 (CCS1), which introduces copper ions and disul"de bonds to SOD1. CCS1 also localizes to cytosol and IMS. Knock out of either of those proteins in yeast and in mouse results in hypersensitivity to oxidative stress.

We are interested in the mechanism of SOD1 and CCS1 targeting to the IMS and how the distribution between cytosol and IMS is regulated. In yeast, we showed that oxidation of a pair of cysteine residues has a critical role in the IMS localization of CCS1 and thereby also in providing SOD1 activity for the IMS [1]. The oxidation of CCS1 depends on Mia40, a component of a redox-regulated import machinery. Although localization of CCS1 to the IMS in mammalian cells has been reported to depend on Mia40, the cysteines that are oxidized in yeast CCS1 are not conserved in mammalian proteins. This indicates a di!erent mechanism of CCS1 distribution in mam-malian cells compared to yeast cells which we currently investigate in mechanistic detail. Our observations will contribute to a better understanding of the physiological and pathological role of superoxide in the IMS.

Klöppel C*, Suzuki Y*, Kojer K, Petrungaro C, Longen S, Fiedler S, Keller S, Riemer J. (2011). Mol Biol Cell. 3749-57.

Abstract #: 68Presenter: Hisashi FujiokaAuthors: Hisashi Fujioka1,2, Bernard Tandler2,3, Laura Konczal4, Mark L. Cohen5, Mariana Rosca2,6, Charles L. Hoppel2,6,7

Institutions: 1Electron Microscopy Facility and 2Center for Mitochondrial Diseases, School of Medicine; 3Department of Biological Sciences, School of Dental Medicine; 4Center for Human Genetics, and 5Department of Pathology, University Hospitals of Case Medical Center; 6Departments of Pharmacology and of 7Medicine, School of Medicine, Case Western Reserve University, Cleveland, Ohio, 44016, USA

Title: A myopathy with retiform mitochondria

Body of Abstract

A surgical biopsy was obtained from the left quadriceps muscle of a 40-year-old male who exhibited pro-gressive fatigue and unexplained exercise intolerance. Light microscopic examination of the specimen was unremarkable. Electron microscopic (EM) examination of the biopsy revealed an extremely unusual mitochon-drial arrangement. In normal muscle, mitochondria are found in clusters beneath the sarcolemma or in rows between myo"brils. In the case in question, the muscle mitochondria were disposed in what at low magni"-cation appeared to be a network. Some of the component organelles measured more than 19 µm in length. The cristae in these elongated organelles were for the most part obliquely oriented, and ranged from lamellar to tubular in form. The consensus among biochemists is that mitochondria form a structural network in the cytoplasm, i.e., these networks consist of but a single continuous mitochondrion that rami"es throughout the cell. This notion is based on confocal microscopy (with its limited resolution) of living cultured cells. At "rst blush, our case seems to "t this idea. When, however we examined, the muscle mitochondria in these putative networks using EM (with its high degree of resolution), it became apparent that long, ramifying mitochondria were joined together end-to-end and that there was no direct continuity of membranes from one organelle to another. Such “networks” were apparent only in traverse or slightly oblique sections of the myo"brils. In some cases, an element of sarcoplasmic reticulum was interposed between the ends of two closely apposed mito-chondria. It is obvious that mere propinquity does not necessarily equal continuity. However this does not rule out the possibility these organelles form a functional syncytium. Mitochondria isolated from the biopsy lost their elongated morphology to become spheroidal and were metabolically normal. It can be concluded that the muscle symptoms exhibited by our patient did not originate in defective mitochondria per se, but rather in the peculiarity of their arrangement within myo"brils.


Abstract#: 69Presenter: Hisashi FujiokaAuthors: Hisashi Fujioka1,2, Bernard Tandler2,3, Laura Konczal4, Mark L. Cohen5, Mariana Rosca2,6, Charles L. Hoppel2,6,7

Institution: 1Electron Microscopy Facility and 2Center for Mitochondrial Diseases, School of Medicine; 3Department of Biological Sciences, School of Dental Medicine; 4Center for Human Genetics, and 5Department of Pathology, University Hospitals of Case Medical Center; 6Departments of Pharmacology and of 7Medicine, School of Medi-cine, Case Western Reserve University, Cleveland, Ohio, 44016, USA Title: Misplaced mitochondria in a human myopathy Body of Abstract A specimen of the right quadriceps muscle was obtained by surgery from a 23-year-old female who displayed muscle weakness among other symptoms. Light microscopy revealed abnormal mitochondrial distribution, consistent with primary mitochondrial disease. Electron microscopic examination of the biopsy revealed areas of typical structure, as well as areas showing morphological alterations. In the normal portions, small mitochondria participated in the formation of triads, which were situated at the Z disk; these triads consisted of paired mitochondria in close relation to elements of the sarcoplasmic reticulum (SR).

In pathologically a!ected areas, typical triads were absent. Instead, relatively long, cylindrical mitochondria were oriented at right angles to the long axis of the myo"brils at the level where Z-discs normally are pres-ent. These mitochondria measured up to 7 µm in length and usually less than 0.2 µm in width. Their cristae showed a normal range of variation. Many of these transverse mitochondria had extremely narrow segments, giving them a nodal appearance. In these narrow regions, the cristae were largely una!ected. Scattered ele-ments of SR occasionally were present in relation to the transverse mitochondria, but rather than forming conventional triads, single SR elements were distributed unilaterally alongside the transverse mitochondria. In those myo"brils with transverse mitochondria, there were no mitochondria in the conventional inter"brillar position.

Interestingly, biochemical examination of mitochondria isolated from the original biopsy revealed normal oxidative metabolism and completely normal metabolic parameters. In the face of normal mitochondrial func-tion, it seems reasonable to suppose that the patient’s muscle symptoms are based on malpositioning of the muscle mitochondria and on e!acement of Z disks. The latter are sites of interaction between +-actinin, titin, and actin "laments. Because sarcomeric contraction depends on linkage to Z disks and on interplay between SR calcium, ATP, and heavy meromyosin, the aberrant morphology of the myo"brils and their attendant organ-elles might a!ect di!usion of the requisite molecules, leading to impaired contractility, hence weakness.

Abstract#: 70 Presenter: Fang Ye Authors: Fang Ye and Charles L. Hoppel Institution: Center of Mitochondrial Disease, Department of Pharmacology and Medicine, Case Western Reserve University, Cleveland, Ohio, 44106

Title: DIAGNOSTIC APPLICATION OF MEASURING OXIDATIVE PHOSPHORYLATION IN PERMEABILIZED SKIN FIBROBLASTS


The laboratory investigation in patients suspected of having a mitochondrial disease includes clinical labora-tory metabolomics, genetic analyzing of mtDNA and nuclear DNA, and muscle biopsy. We asked whether cultured skin "broblasts, a less invasive procedure, could provide a diagnostic step, possibly circumventing an open muscle biopsy in some patients. To approach this goal, we used human skin "broblasts cultured in glu-cose containing medium to examine oxidative phosphorylation (OXPHOS), added to the measurement of elec-tron transport chain (ETC) complex activities. We developed a method composed of two protocols using a high resolution respirometer (O2K, Oroboros). Protocol 1 measures oxygen consumption rates of Complex I and II under the ADP-stimulated state and maximal respiration with an uncoupler, FCCP. Following this, Complex IV rate is measured with TMPD plus ascorbate. In protocol 2, we assess fatty acid oxidation with palmitoylcarni-tine and Complex III rate with duroquinol under state 3 condition, in addition to Complex III maximal respira-tion with FCCP. We have obtained reproducible OXPHOS data from twenty-three normal human skin "broblast lines with multiple repeats and built reference intervals for normal OXPHOS function. These data also provide validation for the analysis. To initially verify the utility of skin "broblasts, we analyses a patient with a previ-ously diagnosed skeletal muscle mitochondrial complex I defect. A Complex I defect was clearly identi"ed by measuring OXPHOS in the "broblasts. Moreover, four "broblast lines from four patients show normal OXPHOS function which correlates with normal OXPHOS in isolated skeletal muscle mitochondrial from these patients. Currently, this approach has been applied for clinical diagnosis. Fifty-two highly selected patient samples have been analyzed. We identi"ed 24 samples with normal OXPHOS function, 15 with Complex I related defects, 5 with ADP phosphorylation system defects, 3 with fatty acid oxidation defects, 2 with Complex III related de-fects, and one each for defect in CoQ, pyruvate related oxidation or Complex IV. Interestingly, there are three subtypes of Complex I defects detected: 1) Decreased state 3 and maximal respiration (pyruvate, malate and glutamate) with decreased fatty acid oxidation. 2) Decreased state 3 and maximal respiration (pyruvate, malate and glutamate), but with normal fatty acid oxidation. 3) Normal state 3 oxidation (pyruvate, malate, glutamate and palmitoylcarnitine), but with decreased maximal respiration. Of diagnostic importance, this new method uncovered ADP phosphorylation defects as well as defects in substrate transport (pyruvate oxidation) and fatty acid oxidation. It is our belief that by identifying defects with a skin biopsy, we can avert an open muscle biopsy, a worthwhile endeavor.

Abstract#: 71Presenter: Daniel S. LieberAuthors: Daniel S. Lieber1-4, Sarah E. Calvo1-4, Nancy G. Slate1,2, Shangtao Liu1,2, Mark L. Borowsky1, Steven G. Hershman1,-4, Nina B. Gold1,2, Gerard T. Berry5, David M. Mueller6, Jeremy D. Schmahmann7, Katherine B. Sims2,7, Vamsi K. Mootha1-4

Institutions: 1Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114 2Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114 3Department of Systems Biology, Harvard Medical School, Boston, MA 02115 4Broad Institute of Harvard and MIT, Cambridge, MA 02141 5The Manton Center for Orphan Disease Research, Children’s Hospital Boston, Boston, MA 02115 6Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago, Illinois 60064 7Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston MA 02114

Title: Targeted exome sequencing of suspected mitochondrial disorders in a hospital-based cohort

Body of Abstract:Advances in next-generation sequencing (NGS) have revolutionized genetic research and are poised to be-come routine tools for clinical diagnosis. However, given the di$culty of interpreting genetic variants, the util-ity of such tools for clinical diagnosis remains unclear, particularly for diseases that are characterized by clinical and genetic heterogeneity. We have developed a targeted “MitoExome” sequencing approach for the molecu-lar diagnosis of suspected mitochondrial disorders in which we sequence the mitochondrial DNA (mtDNA) and the exons of ~1600 nuclear genes encoding the mitochondrial proteome or implicated in Mendelian disor-ders with phenotypic overlap to mitochondrial disease. Previously, we have shown that targeted sequencing enabled con"dent molecular diagnoses in 24% of severe, infantile cases with biochemically-proven mitochon-drial disease whose molecular basis was not found through traditional genetic testing. We have now ap-


plied targeted exome sequencing to 102 phenotypically diverse patients referred to our hospital with clinical suspicion of mitochondrial disease. This cohort is far more diverse than our previous cohort, with a broader age distribution and many individuals lacking strong biochemical evidence of mitochondrial disease. Here, we report the results of this analysis as well as the experimental follow-up of candidate disease genes identi"ed in this study. These results shed light on the utility of replacing candidate gene sequencing with NGS-based approaches, and underscore the promises and challenges of clinical sequencing for the molecular diagnosis of suspected mitochondrial disorders.

This project was supported by American Recovery and Reinvestment Act (ARRA) funds through grant number RC2HG005556 from the NHGRI/NIH.

Abstract#: 72Presenter: Reetta HinttalaAuthors: Reetta Hinttala1,2, Johanna Uusimaa2,3, Hana Antonicka1, Hannaleena Kokkonen3, Jukka S. Moilanen2,3, Heikki Rantala2,3, Kari Majamaa2,3, Eric A. Shoubridge1

Institutions: 1Department of Human Genetics, Montreal Neurological Institute, McGill University, Montreal, QC, Canada, 2University of Oulu, Institute of Clinical Medicine, Oulu, Finland, 3Oulu University Hospital, Oulu, Finland

Title: Biogenesis of the mitochondrial respiratory chain in children with severe multiorgan disorders. Background: Mitochondrial disorders are the most frequent cause of inborn metabolic errors, a!ecting 1 individual in 5000. Symptoms begin at birth or in early childhood in most cases and generally present as multi-system disorders with a fatal outcome. Mutations have been reported in both nuclear and mitochondrial structural genes encoding subunits of the respiratory chain, but the genetic defects in the majority of the cases still remain unresolved. Thus the origin of the defect in these cases seems to lie in accessory factors rather than in structural genes. Very little is known about the mechanisms of mitochondrial translation and the assembly of respiratory chain at present.

Objectives: Our goal is to discover novel factors involved in the mitochondrial biogenesis and to better under-stand the mechanisms involved in the respiratory chain de"ciency in children.

Patients. The cohort of this study is composed of ten Finnish pediatric patients with encephalomyopathy, mus-cular hypotonia and/or epilepsy in combination with either isolated or combined mitochondrial respiratory chain enzyme de"ciency in muscle.

Results: Here we report the results of the exome sequencching carried out in our cohort of patients.

Abstract#: 73Presenter: Hema S AluriAuthors: Hema S Aluri1, Sai S Koka2, Edward J. Lesnefsky1,2,3 and Rakesh C Kukreja1,2

Institutions: 1Departments of Physiology and Biophysics and 2Internal Medicine, Virginia Commonwealth University, School of Medicine, Richmond, VA 23298; 3McGuire Department of Veterans A!airs Medical Center, Richmond, VA 23249.

Title: Chronic treatment with the long acting phosphodiesterase-5 Inhibitor, Tadala!l, decreases mito-chondrial damage in diabetic mice

The PDE-5 inhibitor tadala"l (Cialis) acts by increasing tissue levels of cGMP. PDE-5 inhibitors attenuate dys-function in pulmonary hypertension, cardiomyopathy, cardiac hypertrophy and heart failure. In diabetes, alterations in cardiac energy metabolism and mitochondrial dysfunction occur early and precede the develop-


ment of cardiac dysfunction. In addition, increased delivery of fatty acids to the diabetic myocardium increases lipotoxic and redox stress potentially contributing to contractile dysfunction. Thus, identi"cation of early changes in mitochondrial function and energy metabolism in the diabetic myocardium will facilitate interven-tion before the onset of cardiac dysfunction in diabetic patients.

AIM: To evaluate the e!ect of chronic treatment with tadala"l on mitochondrial function in leptin knock out diabetic mice.

METHODS: Ten-month old adult homozygous db/db mice (strain B6.Cg-m +/+Leprdb/J) with fasted blood glucose levels above 300 mg/dl were studied against their respective control strain mice using Control (un-treated); Control+tadala"l (1mg/kg in 10% DMSO, i.p.); Db/Db; Db/Db+tadala"l groups. Following eight weeks of treatment with tadala"l in the respective groups, hearts were excised and mitochondria isolated. Oxidative-phosphorylation (OXPHOS) was measured using substrates that donate to speci"c sites in the electron trans-port chain. The generation of reactive oxygen species (ROS) and susceptibility to calcium-induced membrane permeability transition (MPT) was studied.

RESULTS: OXPHOS with the complex I substrate glutamate was decreased in diabetic mice by 50% (Table). Treatment of diabetic mice with tadala"l protected OXPHOS with improved glutamate state 3 respiration rates. Treatment with tadala"l did not a!ect on the susceptibility to MPT. The increased ROS production from com-plex I in diabetic mice was decreased by tadala"l treatment.

±SEM; one-way ANOVA; *p<0.05 vs. Control; @ p<0.05 vs. Db/Db; maximal state 3 respiration (nAO/min/mg); succinate and TMPD-ascorbate with rotenone; ROS-glutamate+rotenone (pmol/mg/min)

CONCLUSIONS: Chronic treatment of diabetic Db/Db mice with the PDE-5 inhibitor tadala"l improved diabet-ic-related impairment of OXPHOS. The impaired respiration with increased ROS production at complex I was improved.

Abstract#: 74Presenter: John ChristodoulouAuthors: W.A Gold1, S.L. Williamson1, S. Kaur, S1, 3, J.H Gibson1, G.J Pelka2, I.P Hargreaves5, J.M Land5, P.P.L Tam2, J. Christodoulou1,3,4

Institutions: 1NSW Centre for Rett Syndrome Research, Western Sydney Genetics Program, Children’s Hospital at Westmead, Sydney, Australia, 2Embryology Unit, Children’s Medical Research Institute, Sydney, Australia, 3Discipline of Paediatrics & Child Health, University of Sydney, Australia, 4Discipline of Genetic Medicine, University of Sydney, Australia, 5Neurometabolic Unit, National Hospital and Department of Molecular Neuroscience, Institute of Neurology, London

Title: Mitochondrial Defects In Rett Syndrome

Group Glutamate Succinate TMPD-ascorbate ROS (complex I) (complex II) (complex IV) (complex I) Control (n=4) 441±43 832±63 2063±205 77±15Db/Db (n=5) 222±28* 670±35 1808±191 227±80*Db/Db+Tadala"l (n=6) 358±20@ 702±34 1903±52 123±15@Control+Tadala"l (n=6) 469±33 835±29 2210±110 72±3


Body of Abstract:Rett syndrome (RTT) is a severe neurodevelopmental disorder, predominantly caused by mutations in the X-linked Methyl-CpG-binding protein 2 (MECP2) gene. RTT patients share a number of common clinical features with primary mitochondrial respiratory chain (RC) disorder patients, in addition to the abnormal neuronal mitochondrial morphology and RC function, which have previously been identi"ed in RTT patients.

We have shown evidence of RC gene dysfunction in post mortem brains of RTT patients where the mito-chondrial cytochrome c oxidase I (MTCO1) gene was down-regulated. Mitochondrial abnormalities were also detected in the skeletal muscle from the RTT Mecp2tm1Tam mouse model. RC enzyme activity of COII+III and COIV complexes and reduced glutathione levels were signi"cantly reduced in symptomatic mice. Microarray analysis of symptomatic mouse samples revealed a down-regulation of the nuclear encoded gene, cardiolipin synthase (Crls1), which codes for an enzyme crucial for the formation of RC supercomplexes. Proteomic stud-ies have not shown any alterations in CRLS1 expression in whole cell lysates and so we are currently examining CRLS1 expression in mitochondrial enriched lysates.

Our "ndings suggest that mitochondrial abnormalities in skeletal muscle may well be contributing to the pathogenesis of RTT, possibly through the dysregulation of CRLS1. This could in turn a!ect the organization and stability of RC supercomplexes, and/or through the accumulation of free radicals, as evidenced by the decrease in reduced glutathione. Free radical accumulation has been implicated in a number of neurological disorders and potentially may be a novel target area of future RTT therapeutic approaches.

Abstract#: 75Presenter: Laura StrittmatterAuthors: Laura Strittmatter1,2, Valerio Leoni3, Giovanna Zorzi4, Federica Zibordi4, Sabrina Dusi5, Barbara Garavaglia5, Paola Venco5, Claudio Caccia3, Amanda L. Souza2, Amy Deik2, Clary B. Clish2, Marco Rimoldi3, Emilio Ciusani3, Enrico Bertini6, Nardo Nardocci4, Vamsi K. Mootha1,2, Valeria Tiranti5

Institutions: 1Departments of Systems Biology and Medicine, Harvard Medical School and Massachusetts General Hospital, Boston, MA 02114, USA, 2Broad Institute, Cambridge, MA 02142, USA, 3Laboratory of Clinical Pathology and Medical Genetics IRCCS Foundation Neurological Institute “C.Besta”, Milan, Italy, 4Unit of Child Neurology IRCCS Foundation Neurological Institute “C.Besta”, Milan, Italy, 5Unit of Molecular Neurogenetics, Pierfranco and Luisa Mariani Center for the study of Mitochondrial Disorders in Children, IRCCS Foundation Neurological Institute “C.Besta”, Milan, Italy, 6Unit of Molecular Medicine, Department of Neurosciences, Bambino Gesù Pediatric Research Hospital, Rome, Italy

Title: Global metabolic pro!ling reveals metabolic consequences of mitochondrial coenzyme A de!ciency in patients with PANK2 mutations

Body of Abstract: Biochemical studies of mitochondrial disease have traditionally been limited to a focused set of clinically measured metabolites. Global metabolic pro"ling – spanning hundreds of small molecules – holds the potential to reveal not only novel biomarkers but also provide insight into disease pathogenesis. Here, we apply our previously described LC-MS/MS platform to plasma from a cohort of 14 genetically de"ned patients with pantothenate kinase-associated neurodegeneration (PKAN) and 18 controls. PKAN is a rare, inborn error of metabolism caused by mutations in pantothenate kinase 2 (PANK2), the rate-limiting enzyme in mitochondrial coenzyme A biosynthesis. Patients typically present with iron accumulation in the basal ganglia, dystonia, dysarthria, and retinal degeneration, but the link between these symptoms and mutations in PANK2 is unknown. We found that lactate is elevated in PKAN patients, suggesting dysfunctional mitochon-drial metabolism. We also found that pantothenate, the substrate of PANK2, is elevated in patients with early stop codons in this gene, a predicted but never previously reported result. Global metabolic pro"ling and follow-up studies in patient-derived "broblasts also reveal defects in bile acid conjugation and lipid metabo-


lism, pathways that require coenzyme A. These "ndings raise the novel therapeutic hypothesis that a dietary intervention, with fat and bile acid supplementation, could ameliorate some of the symptoms of PKAN. More broadly, our study illustrates the value of metabolic pro"ling as a tool for systematically exploring the bio-chemical basis of mitochondrial disease and motivates future studies of larger, more varied patient cohorts.This work was supported by a grant from the NIH NIDDK (R01DK081457).

Abstract#: 76Presenter: Prasanth PotluriAuthors: Prasanth Potluri1, Immo E Sche7er2 and Douglas C Wallace1 A$liations: 1Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, 2Division of Biology, University of California-San Diego, La Jolla, CA 92093

Title: Characterization of a Chinese hamster mutant cell line with a complete defect in mitochondrial protein synthesis. Abstract: It is being estimated that there are about one thousand nuclear genes and their products directly a!ecting the mitochondrial biogenesis and function. E!orts are being made, by several research groups, to conclusively identify some of these genetic components a!ecting the mitochondrial functions by exploiting the de"cient phenotype either in human patient cell lines or various rodent and other model systems. We have earlier identi"ed a series of respiration de"cient Chinese hamster mutant cell lines and grouped them into seven complementation groups. All of their mutations are nuclear in origin and have a severe phenotype of one or several missing oxidative phosphorylation complexes. We identi"ed and reported the de"cient genes in two of complex I mutant cells after relevant suspected genes had been mapped and their cDNAs could be tested. In the more recent work we have for the "rst time used a Chinese Hamster cDNA retroviral library to successfully complement several of our mutant cell lines. Among them was the "rst mammalian mitochondrial protein synthesis mutant to be reported, with almost no detectable levels of proteins made in the mitochon-dria. The complementing cDNA from the library restored the mitochondrial protein synthesis in this mutant, and it encodes the mRPS6 subunit of the 28S small ribosome. Mammalian mitochondrial ribosomes are a product of two genomes. While their RNA is transcribed from the mitochondrial genome, all of their 77 pep-tides are nuclear encoded. mRPS6 is a small (14kDa), highly conserved, RNA interacting protein. The hamster mRPS6 null mutant could also be complemented by the mRPS6 orthologs from either mouse or human. The mRPS6 gene is located on the chromosome 16 of the mouse genome and on chromosome 21 (21q21.3-q22.1) of human genome. It appears that in the diploid hamster parental cells one of its alleles is silenced through CpG methylation; the remaining active allele is mutated in the mutant. Tagging the hamster protein at its C-terminus by HA, His or GFP leaves it fully functional. Fluorescent microscopy of the GFP tagged protein shows that the protein is exclusively localized in the mitochondria. Through site directed mutagenesis we identi"ed the probable RNA interacting domains of the mRPS6 protein, in agreement with structures determined for the corresponding prokaryotic small ribosomal subunit. The highly conserved RPS6 protein is found in prokaryotes as well as eukaryotes. Curiously, a truncated pro-tein in which the highly conserved mammalian-speci"c 29 C-terminal amino-acids are deleted could also fully restore ribosome function.

Abstract#: 77Presenter: Morten Scheibye-Knudsen Authors: Morten Scheibye-Knudsen1, Karsten Scheibye-Alsing2, Chandrika Canugovi1, Deborah L. Croteau1, Vilhelm A. Bohr1

Institutions: 1Laboratory of Molecular Gerontology, National Institute on Aging, National Institutes of Health, Baltimore, Maryland, USA 2Section of Genetics and Bioinformatics, IBHV, Faculty of Health and Medical sciences, University of Copenhagen, Denmark


Title: An evolving bioinformatics tool reveals mitochondrial pathology in diseases and aging

Bona "de mitochondrial diseases represent a heterogeneous group of genetic syndromes with a combined in-cidence of around 1:5000. The complex phenotype of these disorders poses a signi"cant diagnostic challenge for clinicians and an analytical barrier for scientists. Further, mitochondrial dysfunction has been proposed to be an underlying origin of aging through free radical production. These highly volatile species will readily react with any lipid, protein and nucleic acid. The mitochondrial theory of aging states that an accumulation of damage to these macromolecules throughout the lifetime of an organism leads to cellular decay, loss of tissue homeostasis, and "nally death. If mitochondrial dysfunction is causative in aging, we would expect diseases displaying accelerated aging to exhibit signs and symptoms of mitochondrial diseases.

To investigate this hypothesis, we compiled an evolving online database, www.mitodb.com, of the signs and symptoms observed in mitochondrial diseases. We then developed an array of bioinformatics tools allowing us to test if a disorder can be characterized as mitochondrial. These tools can be found on www.mitodb.com and consist of various hierarchical clustering algorithms, a support vector machine, a scoring system and others. Using these applications we detected mitochondrial dysfunction in Xeroderma Pigmentosum, group A (XPA); a disease with a DNA repair defect not previously associated with mitochondrial dysfunction. We then experi-mentally tested whether several primary XPA patient "broblasts displayed mitochondrial dysfunction relative to normal control primary "broblasts. Speci"cally, we found increased oxygen consumption (~2-fold, p<0.001), mitochondrial content (~20 %, p=0.007), mitochondrial membrane potential (~50 %, p<0.001) and free radical production (~50 %, p=0.02 / p=0.006) in XPA cells, relative to controls; demonstrating mitochondrial dysfunc-tion in XPA, thus validating the usefulness of the database.

Next, we tested the progerias, in addition to normal aging, using our database and found that several of the segmental progerias (Cockayne Syndrome, Ataxia-Telangiectasia and XPA) as well as aging display features of mitochondrial dysfunction. In closing, we have developed a unique tool that will signi"cantly aid clinicians and scientists in uncovering mitochondrial dysfunction in diseases and in correlating complex disease phenotypes with mitochondrial pathology.

Abstract#: 78Presenter: James B. StewartAuthors: James B. Stewart, Christoph Freyer, Arnaud Mourier, Erik Hagstrom, Marie Lagouge, Anna Wredenberg & Nils-Göran LarssonInstitution: Max Planck Institute for the Biology of Ageing. Gleueler Str. 50a D-50931 Cologne, Germany.

Title: The maternal mtDNA mutation load heavily in%uences phenotypes in mtDNA mutator mice

Body of Abstract: The mtDNA mutator mice express only a proof-reading-de"cient version of mitochondrial DNA polymerase, which creates high levels of mtDNA point mutations, leading to ageing-like phenotypes and premature death. Wildtype littermates have been used as controls in most mtDNA mutator studies. However, these wildtype mice are o!spring of females heterozygous for the PolgAmut allele. Since the heterozygous fe-males are also inducing mtDNA mutations, it is possible that their developing oocytes will carry mtDNA muta-tions to the resulting o!spring. If clonally expanded, these germline transmitted mutations may constitute an important source of mtDNA mutations in the mutator mice and apparent wild-type littermate controls.

We tested this hypothesis by breeding heterzoygous knockout females (PolgA+/KO), that do not have elevated levels of mtDNA mutations in the germline, to heterozygous mtDNA mutator males (PolgA+/mut), thus creating hemizygous mtDNA mutator mice of the genotype PolgAKO/mut. By comparing hemizygous (PolgAKO/mut) and standard (PolgAmut/mut) mtDNA mutators, as well as other genotypes from these crosses, we were able to investigate the amount of mtDNA mutations contributed by heterozygous mothers (PolgA+/mut) to their o!spring. We did observed clonal expansion of mtDNA molecules in o!spring to the heterozygous mothers. The physiological consequences of


these mutations were assessed by comparing the standard mtDNA mutator mice to the hemizygous mtDNA mutators. The absence of maternally transmitted mutations in the hemizygous mtDNA mutators led to a delayed onset of respiratory chain de"ciency, which, in turn, resulted in a delayed onset of weight loss and alleviation of male sterility. This suppression of characteristic symptoms in mtDNA mutator mice is reminiscent of recent results obtained by using PGC1-+ overexpression or exercise as an intervention in mtDNA mutator mice. In contrast, the anemia and blood abnormalities were consistent between these two types of mtDNA mutator mice, suggesting a direct role of somatic mutagenesis in creating stem cell phenotypes.

Our results demonstrate the importance of using control animals that are free of inherited mtDNA mutations in studies comparing e.g. life span or physiological phenotypes. Our results suggest that subset of the mutations derived from the female germline will be prone to clonal expansion, and therefore may drift to high levels in certain tissues, as observed in ageing humans. Thus, it will be critically important for our understanding of the role of mtDNA mutations in the ageing process, to disentangle the relative contribution of inherited mtDNA mutations versus the ongoing mutation load supplied by the PolgAmut polymerase.

Abstract#: 79Presenter: Lawrence GrossmanAuthors: Lawrence Grossman1, Siddhesh Aras1, Pak Oleg2, Natascha Sommer2, Maik Hüttemann1, Norbert Weissmann2. Institutions: 1Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI. 2University of Giessen Lung Center, Giessen, Germany.

Title: GENE REGULATION OF CYTOCHROME C OXIDASE SUBUNIT 4 ISOFORM 2: A TALE OF THREE FACTORS

Cytochrome c oxidase (COX) is the terminal enzyme of the electron transport chain, made up of 13 subunits encoded both in mitochondrial as well as in nuclear DNA. Subunit 4 (COX4), a key regulatory subunit, exists as two isoforms, the ubiquitous isoform 1 (COX4I1) and the predominantly lung speci"c isoform 2 (COX4I2). We have previously identi"ed a highly conserved 13-bp sequence in the proximal promoter region of COX4I2 that functions as an oxygen responsive element (ORE), maximally active at an oxygen concentration of 4%. We also have identi"ed three transcription factors that bind this conserved ORE, namely RBPJ , CHCHD2, and CXXC5. Interestingly, our data indicate that CHCHD2, an activator of the ORE, has a dual localization both in the mitochondria and the nucleus, possibly with distinct functions. To validate the results derived from cul-tured cells, we show using RNA interference the role of these transcription factors in the gene regulation of COX4I2 in primary pulmonary artery smooth muscle cells. We present a model proposing that nuclear CHCHD2 regulates COX4I2 gene expression and that the mitochondrial CHCHD2 regulates COX function. Finally, under hypoxic stress, a concerted action of the three transcription factors enhances the expression of COX4I2 that in turn could be responsible for the augmentation both of COX activity and of its properties in order to cope with the altered cellular energy requirements.

Abstract#: 80Presenter: Kasturi MitraAuthors: Kasturi Mitra 1,2, Richa Rikhy1, Mary Lilly1, Jennifer Lippincott-Schwartz1, David Chess2 and Bob Balaban2

Institution: 1NICHD, NIH 2NHLBI, NIH

Title: Mitochondrial !ssion-fusion activities regulate cell fate determination between proliferation and dif-ferentiation: a possible link to tumorigenesis


Body of Abstract:Mitochondrial architectural state rapidly inter-converts between "lamentous and fragmented forms in prolif-erating cells, which go through controlled cell cycle events. Certain mitochondrial "ssion-fusion proteins bring about such dynamism in mitochondrial architecture, the signi"cance of which is not well understood. We have shown that mitochondrial form changes dramatically during cell cycle with majority of the mitochondria fus-ing together to form a singular structure (hyperfused mitochondria) just prior to initiation of DNA synthesis in normal "broblastic cells. This structure is maintained by down-regulation of mitochondrial "ssion protein Dy-namin Related Protein 1 (DRP1). Down-regulation of DRP1 is critical for cyclin E build up that is required to ini-tiate DNA synthesis in proliferating cells. In Drosophila, we found that DRP1 down-regulation leads to uncon-trolled proliferation and blocks di!erentiation. On the other hand, enhanced DRP1 dependent mitochondrial "ssion lowers cyclin E levels and promotes precocious exit from cell cycle to allow for di!erentiation. Therefore, mitochondrial "ssion-fusion states control proliferation and entry into di!erentiation. This raises a possibility that mitochondrial "ssion-fusion rate is higher in actively proliferating cells than resting di!erentiated tissue. To be able to monitor the degree of mitochondrial "ssion-fusion in terminally di!erentiated tissue we have generated a transgenic mouse ubiquitously expressing a photo-convertible #uorescent protein, EosFP, tar-geted to the mitochondrial matrix (mitoEosFP). This allows us to highlight small pools of mitochondria and follow their fate by using #uorescence imaging. As mitochondrial "ssion-fusion events should equilibrate and dilute this highlighted mitochondrial pool, one can quantitatively assay for mitochondrial "ssion-fusion events by monitoring rate of dilution of the highlighted mitochondrial pool. Using intravital #uorescence microscopy on ventilated mitoEosFP mice we have been able to follow highlighted mitochondrial pools in resting skeletal muscle. The results suggest that inter"brillar mitochondria in skeletal muscle "bers are largely immobile and do not undergo appreciable dilution of the highlighted mitochondrial pool in a period of 30 minutes. This is in contrast with the proliferating primary embryonic "broblasts isolated from the mitoEosFP mouse, where the highlighted mitochondrial pool disappears within 30 minutes. To understand whether such increase in mito-chondrial "ssion-fusion is a property of actively proliferating cells, we are now performing similar experiments in quiescent primary mitoEosFP "broblasts. Also, e!orts to monitor mitochondrial "ssion-fusion in other tis-sues are underway. This study highlights the importance of mitochondrial "ssion-fusion dynamics in proliferat-ing cells and indicates that alteration of mitochondrial "ssion-fusion dynamics can potentially revert quiescent di!erentiated cells into proliferating tumorigenic cells.

Abstract#: 81Presenter: Raj R. Rao1

Authors: Raj R. Rao1 and Shilpa Iyer2 Institution: 1Chemical and Life Science Engineering, Virginia Commonwealth University 2Center for the Study of Biological Complexity, Virginia Commonwealth University.

Title: Development of stem cell-based models for neuro-mitochondrial disorders.Body of Abstract

Neuro-mitochondrial disorders (NMDs) are neurological conditions characterized by the presence of mito-chondrial dysfunction, often at the level of the mitochondrial genome (mtDNA), in neural cell lineages such as neurons and glia. Research on the mechanism and pathogenesis of these conditions and development of treatments is hampered by lack of adequate, valid in vitro models. Cytoplasmic hybrid cell models created from patient derived platelets containing pathogenic mtDNA have found use; however their tumorigenic host backgrounds limit their usefulness and pose potential problems for obtaining meaningful results. A way of avoiding the confounding e!ects of these models is to utilize human pluripotent stem cells (hPSCs) and use them as #exible sources of multiple cell lineages that closely parallel the genome and (dys)function of cells in the whole organism. However, generation of stem cells de novo is technically challenging and the analysis of speci"c mtDNA contribution to the pathomechanism is fraught with interpretative challenges. We have used a recombinant human mitochondrial transcription factor A (rhTFAM) to deliver pathogenic mtDNA from Leber’s hereditary optic neuropathy (LHON) into previously generated hPSC derived human neural progenitor (hNP) cells that have been depleted of their own mtDNA. Entry into mitochondria of G11778A mtDNA was observed when complexed with rhTFAM. Restriction digestion analysis con"rmed continued presence and expression


of the G11778A mtDNA mutation in the hNEPs. Based on expression of neuron-speci"c class III beta-tubulin, neuronal di!erentiation occurred. Uptake of the pathogenic mtDNA into hNP cells would generate cell lin-eages that would both reprise mtDNA dysfunction speci"c to the investigated conditions and be able to act as #exible sources of research material, which can later be di!erentiated into neurons and glia. Such disease-speci"c stem cells would be a valuable resource for the research community and for use in high-throughput drug screening and in molecular diagnosis platforms.

Abstract#: 82Presenter: Ran LinAuthors: Ran Lin, Alessia Angelin, Douglas C. WallaceInstitution: Center for Mitochondrial and Epigenomic Medicine (CMEM), Children’s Hospital of Philadelphia Research Insitute, Philadelphia, PA 19104

Title: Inactivation of the Drosophila TSPO Inhibits the mPTP, Increases Longevity, Alters Heme Metabolism and Modulates Mitochondrial Bioenergetics

Abstract:

The human mitochondrial translocator protein 18kDa (TSPO), formerly named peripheral benzodiazepine re-ceptor (PBR), has been proposed to be a component of the mitochondrial permeability transition pore (mPTP), implicated in heme biosynthesis, and found to be increased in neurodegenerative diseases and cancer. In wild type Drosophila TSPO RNA and protein levels increased with age. Inhibition of Drosophila TSPO with speci"c ligands PK11195 or Ro5-4864 or inactivation of the tspo gene by P-element insertion both extended lifespan. Drosophila was found to have a mPTP based on #y mitochondrial swelling when exposed to calcium, which was dependent on moderate concentration of phosphate. The mutation of previously recognized mPTP components, VDAC (voltage-dependent anion channel, or porin) partially blocked the swelling. But neither Drosophila CypD point mutation nor CypD ligand CyclosporinA (CsA) altered permeability transition. By con-trast, inhibition of TSPO by ligands, insertion mutant, or RNAi knock-down did inhibit swelling. Tspo mutant and knock-down #ies showed reduced activity of caspase 3/7, the most downstream caspase in apoptosis pathway. At 3-6 days post-eclosion, tspo de"cient (-/-) #ies exhibited increased mitochondrial and decreased cytosol heme content. At 2-5 days after eclosion, the tspo -/- #ies manifest increased state IV respiration and decreased mitochondrial inner membrane potential suggesting increased proton leak. By days 7-10 post eclo-sion tspo -/- #ies exhibited reduced OXPHOS complex activity and increased mitochondrial reactive oxygen species production. These data indicate that TSPO is a core component of the mPTP, it is important in heme transport, and its inactivation extends life span, perhaps through reduced cell death.

Abstract#: 83Presenter: Atsushi TanakaAuthors: Atsushi Tanaka1, Izumi Tanaka1, Motoi Nagayoshi1, Hiroshi Kusunoki2, Seiji Watanabe3 Institutions: 1Saint Mother Obstetrics and Gynecology Clinic and Institute for ART, Fukuoka, Japan, 2Faunal Diversity Sciences, Graduate School of Agriculture, Kobe University, Kobe, Japan, 3Department of Anatomy, Hirosaki University School of Medicine, Hirosaki, Japan

Title: The future is now:oocyte plasmic exchange for aged oocytes

Body of Abstract:The main causes of repeated failure in assisted reproduction such as IVF-embryo transfer are believed to be ooplasmic de"ciencies, abnormalities and ageing rather than nuclear de"ciencies. It is a common phenom-enon that pregnancy rates decrease, but miscarriages increase, as women grow older. Also, the percentage of fetal chromosomal abnormalities in miscarriages increases according to female age, reaching >90% when women are over 40 years old; surprisingly, about 90% of them are cases of autosomal trisomy. Such aneuploidy


is mainly induced by the chromosomal pre-division, in which homologous chromo-somes fail to pair during meiosis I and segregate before it is complete, resulting in disomic gametes. Nuclear transfer into the meta-phase-II(M-II) oocytes shows promise as a means of repairing female infertility due to ooplasmic de"ciency and abnormalities. We therefore conducted nuclear transfer of in vitro matured metaphase-II oocytes (recipi-ent oocytes) into enucleated freshly ovulated metaphase-II oocytes(donor oocyte).

In both in-vitro matured oocytes and freshly ovulated oocytes, the M-II chromosomes were easily recognized as a round transparent substance in which the chromosome body was centrally located, and they were usually beneath or adjacent to the 1PB with the aid of an inverted microscope equipped with a Normarski di!erential interference contrast system. The aspirated M-II karyoplast of recipient oocytes was transferred into the perivi-telline space of an enucleated donor oocyte. The grafted oocyte was transferred in Zimmerman cell fusion medium. Membrane fusion was facilitated by electrical stimulation (10V for 1 second AC + 10V for 45 micro-second DC) with an electro cell fusion generater (LF 201). After fusion, the constructed oocytes were cultured in HTF medium for 2 hours and ICSI was performed.

The percentage of identi"cation of M-II chromosome was 91.1 %(41/45) in freshly ovulated oocytes and 96.0%(48/50) in vitro-matured oocytes.

All of 35 karyoplasts of recipient oocytes were replaced in the periviteleine space of enucleated donor oocytes and 28 of these 80.0% were fused to form a reconstituted oocyte. The fertilization rate, cleavage rate and blas-tocyst formation rate following ICSI for constructed oocytes and recipients oocytes were[77.1%(27/35),65.7%(23/35),25.7%(9/35)],[59.0%(58/98),26.1%(25/98),3.4%(3/98)]respectively. Chromosomal analysis of 4 embryos following nuclear transfer indicated they were all diploid sets of 46 chromosome.

In conclusion, it has been demonstrated that oocytes constructed following the karyoplast transfer of in-vitro matured M-II oocytes into enucleated freshly ovulated M-II oocytes clearly had more e$cient and chromoso-mally normal embryonic development than did in-vitro matured oocytes after ICSI.

Abstract#: 84Presenter: Masashi Tanaka Authors: Masashi Tanaka1, Kumpei Tanisawa1, Eri Mikami1, Noriyuki Fuku1, Yutaka Nishigaki1, Yasutoshi Koga2, Akira Ohkake3, Yasushi Okazaki4, Kei Murayama5

Institutions: 1Department of Genomics for Longevity and Health, Tokyo Metropolitan Institute of Gerontology; 2Department of Pediatrics, Kurume University School of Medicine; 3Department of Pediatrics, Saitama Medical University; 4Research Center for Genomic Medicine, Saitama Medical University; 5Department of Metabolism, Chiba Children’s Center

Title: Rapid Genetic Diagnosis and Pyruvate Therapy for Mitochondrial Diseases

1. We have developed a rapid screening system for 61 mutations of mitochondrial DNA (Nishigaki et al. Mi-tochondrion 10: 300–308, 2010). We analyzed 2419 autopsied cases (average ages at death: 81 years) in our geriatric hospital. We detected LHON (Leber’s hereditary optic neuropathy)-related mutations in 8 cases (m.3460G>A in 1 case, m.7444G>A in 1 case, m.11778G>A in 3 cases, m.14484T>C in 3 cases). The m.1555A>G was detected in 2 cases. Pathogenic mutations in tRNA genes were detected in 23 cases (m.3243A>G in 10 cases, m.3271T>C in 6 cases, m.5703G>A in 1 case, m.5814T>C in 2 cases, m.8348A>G in 3 cases, m.12297T>C in 1 case). The overall detection rate of mtDNA mutations was unexpectedly high in autopsied geriatric pa-tients (1.57%. 38/2419).

2. We utilized a semiconductor sequencer Ion PGM for rapid diagnosis of mitochondrial diseases. In one patient with complex I de"ciency, we detected a heteroplasmic m.3677A>G mutation in the MT-ND1 gene


causing a N124S substitution. The mutation load was 85% (A:G = 107:612). The mutation was detected neither in the sister, mother, nor father of the patient. The Asn-124 was conserved among 61 mammalian species. The m.3677A>G mutation is a novel disease causing mutation at the coenzyme Q-binding site of complex I.

3. We previously proposed sodium pyruvate therapy for mitochondrial diseases (Tanaka et al. Mitochondrion 7: 399–403, 2007). We demonstrated e$cacy of sodium pyruvate in a myopathic patient with cytochrome c oxidase de"ciency (Kokaze et al. Biochim Biophys Acta 1800: 313–315, 2010), in a patient with Leigh syndrome due to pyruvate dehydrogenase E1+ de"ciency (Koga et al. Brain Develop 34: 87–91, 2012), and in 6 patients with MELAS due to m.3243A>G mutation and 1 patient with m.13513G>A mutation (Koga et al., in press). The Ministry of Health of Japan has decided to initiate a clinical trial of sodium pyruvate for mitochondrial diseases.

Abstract#: 85Presenter: Valerio CarelliAuthors: Valerio Carelli1, Piero Barboni2, Leonardo Caporali1, Alessandra Maresca1, Guy Miller3, Maria Lucia Valentino1

Institutions: 1IRCCS Institute of Neurological Sciences of Bologna and Department of Neurological Science, University of Bologna, Bologna, Italy, 2Studio Oculistico D’azeglio, Bologna, Italy, 3Department of Anesthesiology, Critical Care Medicine, Stanford University, Stanford, California

Title: EPI-743: preliminary report on Italian experience in open label study of three patients with acute Leber’s hereditary optic neuropathy

Body of Abstract

EPI-743 is a therapeutic under development for the treatment of mitochondrial disease, whose activity de-pends upon reversible two-electron redox-cycling. In a recently reported clinical series, EPI-743 arrested dis-ease progression and reversed vision loss in four out of "ve patients with Leber’s hereditary optic neuropathy (LHON) in the US. Three Italian LHON patients were enrolled in a prospective trial and treated with EPI-743 (900 mg/day). Visual acuity (VA), computerized visual "elds (VF) and serum lactate levels during rest and exercise were measured on each subject. One of the three patients (female, age 17) had a 3460/MD1 mutation and the other two patients (males, ages 17 and 27) had the 11778/ND4 mutation. Therapy was initiated in all three subjects at the stage of mono-ocular clinical involvement.

Lactic acid was pathologically elevated after exercise in all three patients before therapy (25.7, 33.1, and 51.4; normal range 5-22mg/dl). After three days of therapy, all patients showed decrease of these values and cur-rently, after 11 months of therapy for the 3460/ND1 patient and eight months for the other two patients, the lactic acid values are 20.5, 15.3, and 31.6 mg/dl. Two patients progressed to vision loss in the second eye (two months after start of therapy for the 3460/ND1 patient, and three months after start of therapy for one 11778/ND4 patient). In both cases, VF defects preceded the clinical onset of visual loss. Currently, VA is counting "ngers in the second eye. At last evaluation, a slight improvement of the "rst eye (as assessed by both VA and VF) was present in both patients. The third patient is unusual in having a homonymous hemianopsia, of which he was not aware, due to a cerebral lesion in the visual cortex, probably congenital in origin. Despite this VF defect, he noticed loss of central vision in one eye, with a scotoma clearly distinguishable from the hemianopic defect. This patient started therapy three months after onset of visual loss, and he maintained normal VA in the second eye after eight months of EPI-743 administration. He also improved his VA in the "rst eye (from .2 to .25). EPI-743 improved plasma lactate levels in all three subjects following three days of therapy. All three patients demonstrated improved visual function in the "rst eye and at this time there is absence of disease in the second eye in one of the patients. Upcoming prospective trials will evaluate the reported EPI-743 therapeutic response.


Abstract#: 86Presenter: Inn-Chi LeeAuthors: Inn-Chi Lee1,2, Ayman W. El-Hattab3, Jing Wang1, Fang-Yuan Li1, Shao-Wen Weng1,4, William J Craigen1, Lee-Jun C. Wong1

Institutions: 1Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA; 2Department of Pediatrics, Institute of Medicine, School of Medicine, Chung Shan Medical University and Hospital, Taichung, Taiwan; 3Division of Medical Genetics, Department of Child Health, University of Missouri Health Care, Columbia, Missouri, USA; 4Department of Internal Medicine, Chang Gung Memorial Hospital, Kaohsiung, Taiwan

Title: SURF1-associated Leigh syndrome: A case-series and novel mutations

ABSTRACT Leigh syndrome (LS) is a mitochondrial disease that typically presents in infancy with subacute neurodegen-erative encephalopathy. It is genetically heterogeneous, but mutations in the complex IV assembly genes, particularly SURF1, are an important cause.

In this study, SURF1 gene was sequenced in 590 subjects with a clinical suspicion of LS, complex IV de"ciency, or clinically features of mitochondrial disorders. We identi"ed 21 patients with clinical features of LS who are either homozygous or compound heterozygous for SURF1 mutations. Twenty two di!erent mutations were identi"ed, including 13 novel mutations. The 13 novel mutations include 5 frameshift mutations, 2 missense variants, and 6 splice site mutations. The novel framshift mutations are c.807_810del4ins9 (p.E270QfsX9), c.169delG (p.E57KfsX15), c.555_556delGA (p.K186SfsX4), c.183_186delTCTT (p.L62SfsX9), and c.472_473delAG (p.S158WfsX21). The novel missense mutations are c.614G>A (p.G205E) and c.769G>A (p.G257R). The novel splice site mutations are c.55-1G>A, c.833+1G>A, c.106+1G>C, c.107-2A>G, c.516-2A>G, c.324-11T>G. Of the 42 mutant alleles, 36 (86%) are null mutations (frameshift, splicing, or nonsense) and 6 (14%) are missense. The c.312_321del10insAT (p.L105X) allele is the most common mutation (26%, 11/42), followed by c.845_846delCT (p.S282CfsX9) (10%, 4/42) and c.574_575insCTGC (p.R192PfsX8) (7%, 3/42). We have also reviewed the previ-ously reported SURF1 mutations and observed a clustering of mutation in exon 8 of SURF1 suggesting a vital function for this region.

Although mutations in SURF1 have been mainly associated with typical LS, 5 of the patients in this report had an atypical course of LS. There is no de"nite genotype-phenotype correlation; however, frameshift mutations resulting in protein truncation closer to the C-terminus may carry a better prognosis.

Among the patients who did not have SURF1 mutations, heterozygous variants in SCO1, SCO2 and COX10 were identi"ed in 2, 2, and 6 patients, respectively. Only one patient had homozygous variant in SCO2. In addition, 67 patients had whole mitochondrial genome sequencing with 3 patients identi"ed to carry the m.13153G>A mutation described in patients with LS.

Key words: mitochondrial disorders, complex IV de"ciency, complex IV assembly, electron transport chain, atypical Leigh syndrome.

Abstract#: 87Presenter: Sonia CortassaAuthors: Niraj Bhatt1, Miguel A. Aon1, Xiaoxu Shen2, Brian O’Rourke1, Weidong Gao2 and Sonia Cortassa1

Institution: Johns Hopkins University, School of Medicine, 1Division of Cardiology and 2Department of Anesthesiology and Critical Care Medicine, Baltimore, Maryland

Title: Contractile and mitochondrial dysfunction in heart trabeculae from diabetic rats.


Cardiomyopathy is a common complication in non-insulin dependent diabetes mellitus (NIDDM), but the mechanisms involved in heart dysfunction are not well understood. This study is aimed at investigating the mechanisms underlying heart dysfunction in the Zucker diabetic fatty (ZDF) rat, an animal model of NIDDM. Herein, we present a comparative bioenergetic characterization of working heart trabeculae from ZDF and its lean control when subjected to changes in workload, under euglycemic (EG, 10mM glucose) and hypergly-cemic (HG, 30mM glucose) conditions. We monitored simultaneously force and mechanical work, respiratory rate (VO2), reduced glutathione (GSH), and oxidative stress in the absence and presence of increasing levels of insulin. ZDF trabeculae displayed lower developed force than the lean ones, under all conditions of insulin or glucose levels. The rate of respiration as a function of pacing frequency was lower in trabeculae from ZDF rats compared to lean controls under EG conditions. In addition, under HG, VO2 did not respond to increasing frequency in either ZDF or lean trabeculae. The lower VO2 displayed by ZDF with respect to control trabeculae is in agreement with measurements performed in isolated mitochondria from these hearts. In parallel, HG-chal-lenged trabeculae displayed a decrease in NADH #uorescence and mitochondrial membrane potential in the presence of insulin. Increasing insulin concentration led to higher oxidative stress as revealed by MitoSOX but not CMH2DCF #uorescence. The MitoSOX signal showed a larger dependence on the hormone dose under HG than EG conditions both in lean and ZDF. Concomitantly, glutathione (GSH) levels decreased in the presence of insulin in muscles from diabetic as compared with lean animals irrespective of the level of glucose. Taken together, these results suggest that impaired mitochondrial respiration in conjunction with elevated oxidative stress and diminished GSH are involved in the impairment of contractility exhibited by heart trabeculae from the diabetic ZDF rat.

Abstract#: 88Presenter: Antonio Davila, Jr.Authors: Antonio Davila, Jr.1,2, Douglas C. Wallace2

Instituitions: 1Center for Molecular and Mitochondrial Medicine and Genetics, Department of Biological Chemistry, University of California, Irvine, Irvine, CA 92697, 2Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, University of Pennsylvania, Philadelphia, PA 19104-4302

Title: Epigenetic Memory in the Mitochondria of Human Embryonic Stem Cells

To investigate the role of mitochondrial function in Human Embryonic Stem Cells (hESC), we generated a col-lection of transmitochondrial cybrid cell lines composed of mitochondria derived from hESC lines (H1 or H9) and the nuclear background of two unique somatic rho 0 (8°) cell types which had previously been voided of their endogenous mitochondrial DNA (143B TK- 8° osteosarcoma line or Wal2A 8° lymphoblast line). For controls, we established cybrid cell lines in parallel containing mitochondria derived from adult "broblasts. We characterized each of these lines through sequencing of the mtDNA, RFLP, FAM-labeled fragment analysis, and through microsatellite analysis of nuclear repeat regions.

Using this transmitochondrial cybrid system, we demonstrated that the suppressed OXPHOS function, char-acteristic of hESC, was maintained and transmitted into two distinct somatic 8° cell types. This e!ect was not observed in the somatic cell-derived cybrids. High-resolution respirometry in intact cells and mitochondrial membrane potential measurements in isolated mitochondria revealed that the coupled basal respiration and the uncoupled maximal respiration of our hESC and induced Pluripotent Stem Cells (iPSC) lines were signi"cantly decreased by half compared to all of the somatic parental lines and cybrids. This phenotype was mirrored by all cybrid lines containing hESC-derived mitochondria. Moreover, the ratio of the basal to the maximal respiration values of all of the cell types used- derived from either somatic cells, pluripotent hESC, or iPSC- shows that the mitochondria are functionally intact with equal OXPHOS capacity. This suggests that there is a suppression of function that is independent of mtDNA sequence that is unique to the mitochondria of pluripotent cells.


Supporting this functional study was gene expression data showing that the hESC-derived mitochondria conferred properties to the somatic parental cells that are consistent with those of pluripotent and iPSC. These include a similarly reduced expression of mtDNA-encoded OXPHOS subunits and a cell signaling pat-tern characterized by increased proliferation and self-renewal. Interestingly, the hESC cybrids showed similar gene expression pattern to that of somatic cells in regards to nDNA-encoded OXPHOS subunits, mitochondria biogenesis, and mtDNA synthesis.

Our study demonstrates for the "rst time a heritable phenotype that is maintained and transmitted inde"nitely through the mitochondria of pluripotent hESC. This phenotype was not achieved by using mitochondria from adult, somatic cells but is a property exclusive to the hESC-derived mitochondria. Further studies will seek to elucidate the mechanisms underlying the suppressed OXPHOS phenotype in hESC-derived mitochondria.

Abstract#: 90Presenter: Kothari Sonam R1

Authors: Bindu PS1, Gayathri N1,Taly AB1,Govindaraju C1, Arvinda HR1, Khan N2,Thangaraj K2.Institutions: 1National Institute of Mental Health and Neurosciences (NIMHANS), Bangalore, India 2Centre for cellular and molecular biology (CCMB), Hyderabad, India

Title: Mitochondrial Encephalomyopathies: Phenotypic spectrum in a cohort of sixty !ve patients from South India. Background: Muscle biopsy is an important tool for the diagnosis of mitochondrial respiratory chain disorders (RCD) disorders but, reports on the complete clinical spectrum of biopsy proven cases are rather limited. This study describes the clinical spectrum of the biopsy positive mitochondrial RCD from a tertiary care hospital in South India.

Patient and methods: 65 patients in the age range 18 months to 65 years (Male: Female -36:29) were evaluated over a period of 8 years (2005 -2012). Inclusion criteria was histopathologically proven mitochondrial respirato-ry chain disorders; ragged red "bers (RRF) and ragged blue "bers (RBF) and /or cox de"ciency/SDH de"ciency on biceps or quadriceps muscle biopsy. Patients were phenotypically classi"ed depending on the clinical and imaging features. Results:

Clinical data: Common presentations included ptosis-36, muscle weakness (proximal and distal muscle -32, bulbar-15),seizures -12( Myoclonus-5,focal-2,GTCS-4,febrile-1), ataxia-8, regression and/or developmen-tal delay-8 and involuntary movements-4.On evaluation, external opthalmoplegia -26,cardiomyopathy-7,sensorimotor axonal neuropathy-6,limb girdle syndrome-6 ,optic atrophy-3,retinitis pigmentosa-3 and right bundle branch block-2 were noted. Phenotypic markers like deafness, short stature, baldness and endocrine dysfunction were noted in eighteen patients. Family history of similar illness was reported in 7 patients. Neu-roimaging available in 26 patients (Brain: CT-2, MRI-25 and spine: MRI-1), was normal in 8. T2W and FLAIR signal hyperintensities were observed in 12 cases in one or more areas: basal ganglia -6, cerebellum-6, brain stem-5 and cortex-4. Atrophy of cerebellar-3, cortex-2 and basal ganglia-1 was noted. Other features were basal gan-glia calci"cation-2 and spinal cord signal changes-1.

Patients were broadly grouped into Chronic progressive external opthalmoplegia(CPEO)-15, CPEO plus syndrome-15, Mitochondrial myopathies-13,Leigh and Leigh like illness-7,Myoclonic encephalopa-thy with ragged red "bers(MERRF)-5,Kearn Sayre syndrome(KSS)-3, Mitochondrial neurogastrointestinal encephalopathy(MNGIE)-2,Mitochondrial encephalopathy, lactic acidosis, stroke like episodes(MELAS)-1,Hypertrophic cardiomyopathy with myopathy-1, Benign infantile cardiomyopathy with myopathy-1,herediatory spastic paraplegia with ragged red "ber myopathy-1.


Histological !ndings: Presence of ragged red "bers (62), with partial or complete COX de"ciency (53) and COX de"ciency with no RRF (2) was seen. One case with total SDH absence, on immunohistochemistry for complex 2 showed de"ciency. Features of both muscular dystrophy and mitochondrial disease were present in six.

Genetic analysis by complete mitochondrial DNA, SURF and POLG sequencing done in 20/65 showed novel and known mutations.

Conclusion: This study brings out signi"cant variability in clinical spectrum, imaging and histopathological features of patients with mitochondrial respiratory chain disorders.

Abstract#: 91Presenter: Qun ChenAuthors: Aijun Xu1, Karol Szczepanek1, Ying Hu1, Qun Chen1, Edward J. Lesnefsky1,2,3

Institutions: Departments of Medicine (Division of Cardiology)1 and Biochemistry2, Virginia Commonwealth University, Richmond, VA, 23298; McGuire Department of Veterans A!airs Medical Center3, Richmond, VA 23249

Title: Blockade of electron transport before ischemia provides an additive protection in bcl-2 overexpres-sor mice following prolonged ischemia-reperfusion

Body of Abstract (300-400 words): Cardiac ischemia (ISC) damages the mitochondrial electron transport chain that leads to increased myocardial injury during ISC and reperfusion (REP). Blockade of electron trans-port before ISC using amobarbital (AMO) decreases injury by preserving mitochondrial bcl-2 content and de-creasing the susceptibility to the mitochondrial permeability transition pore (MPTP) opening during REP. Bcl-2 overexpression decreases myocardial injury following ISC-REP. Depletion of bcl-2 favors MPTP opening during REP in ISC-damaged mitochondria. We asked (1) if bcl-2 overexpression decreased cardiac injury by preventing the MPTP opening during REP; (2) if AMO treatment provided an additional protection in bcl-2 overexpression mice during ISC-REP.

Langendor! perfused mouse hearts were subjected to 40 min global ISC (37°C) and 30 min REP. In AMO treated bcl-2 overexpressor hearts, AMO (2.0 mM) was given for 1 min before ISC. Subsarcolemmal (SSM) were isolated at end of REP. Lactate dehydrogenase (LDH) release from coronary e7uent was used to re#ect injury, and CRC (calcium retention capacity) was used to detect the MPTP opening. Overexpression of bcl-2 decreased LDH release during REP compared to wild type, supporting that bcl-2 overexpression decreases car-diac injury. However, overexpression of bcl-2 itself did not improve phosphorylation (OXPHOS) or CRC in SSM following REP (Table). AMO decreased the LDH release during REP vs. untreated bcl-2 overexpression hearts, suggesting that blockade of electron transport provides an additive protection in bcl-2 overexpressor hearts. AMO protected OXPHOS in SSM following REP when glutamate + malate was used as complex I substrate compared to untreated bcl-2 hearts. AMO did not improve OXPHOS in SSM oxidizing succinate compared to untreated hearts. AMO improved the CRC in SSM following REP vs. untreated bcl-2 hearts (Table). Thus, bcl-2 overexpression decreases cardiac injury during REP independent of the improved mitochondrial function. Blockade of electron transport can decrease injury in bcl-2 hearts following prolonged periods of ISC-REP by decreasing the MPTP opening by an additive mechanism in addition to bcl-2 content.


ISC-REP LDH OXPHOS OXPHOS CRC (mU/mg/min) Glutamate (20 mM)+ Succinate (20 mM)+ (nmol CaCl2/mg) malate (10 mM) Rotenone (7.5 uM)

Wild type 812 ± 50 199 ± 11 264 ± 17 240 ± 0 (n=4)

Bcl-2 (n=5) 615 ± 58* 158 ± 19 260 ± 17 272 ± 17

Bcl-2 + AMO 441 ± 21* † 218 ± 21 † 270 ± 24 312 ± 9 * (n=4)

Mean ± SEM: * p<0.05 vs. untreated wild type. † p<0.05 vs. bcl-2 hearts.

Abstract#: 92Presenter: Qun ChenAuthors: Thomas Ross1, Karol Szczepanek1, Ying Hu1, Qun Chen1, Edward J. Lesnefsky1,2,3

Institutions: Departments of Medicine (Division of Cardiology)1 and Biochemistry2, Virginia Commonwealth University, Richmond, VA, 23298; McGuire Department of Veterans A!airs Medical Center3, Richmond, VA 23249

Title: Partial inhibition of complex I does not prevent reverse %ow-induced ROS generation

Body of Abstract (300-400 words): The reverse electron #ow (REF) from complex II to complex I increases reactive oxygen species (ROS) generation in isolated mitochondria using succinate as sub-strate. In vivo, however, both complex I and complex II substrates simultaneously feed electrons into the respiratory chain. We asked if the REF-mediated ROS generation can occur when both complex I and complex II substrates are used together. Inhibition of complex I with rotenone decreases the REF-induced ROS generation. Overexpression of STAT 3 targeted to mitochondria (MLSTAT3E mouse) decreases complex I activity but maintains the inner mitochondrial membrane potential (93). We used mitochondria isolated from STAT3E mouse heart to test if only partial inhibition of complex I a!ects the REF-induced ROS generation.

The REF-induced ROS generation still occurred when glutamate and succinate were used together as substrates (Table), supporting the notion that the REF-induced ROS generation was independent of the NADH/NAD+ ratio. The REF-induced ROS generation was not increased in MLSTAT3E mouse compared to wild type [mean ± SEM, 1592 ± 147 (wild type, pmol/mg/30min) vs. 1846 ± 448 (ML-STAT3E), p=NS, n=4]. These results indicated that partial inhibition of complex I alone did not prevent REF-mediated ROS generation. Complete depolarization of the 93 by dinitrophenol (DNP) prevented REF-induced ROS generation (Table). Only a 16% decrease in 93 by DIDS (an ion channel inhibitor) also prevented REF-induced ROS generation (Table). DIDS did not a!ect the glutamate oxidation in the isolated mitochondria (data not shown), indicating that the decreased ROS generation by DIDS


was not due to decreased complex I activity. Taken together, the 93 contributed a key role in REF-mediated ROS generation.

The REF-induced ROS generation was less likely to occur in vivo in that 93 is depolarized to support constant ADP phosphorylation. The increased ROS generation from mitochondria contributes to myocardial injury during ischemia-reperfusion. Cardiac ischemia damages the electron transport chain that led to decreased complex I activity and partially depolarized 93. Therefore, the REF-in-duced ROS generation should not occur during ischemia-reperfusion. Thus, the REF-mediated ROS generation is an unlikely potential source of cardiac injury during reperfusion.

Rat heart Succinate Succinate (5 mM) Succinate (5 mM) + DNP Succinate (5 mM)+ DIDS mitochondria (5 mM) + glutamate (5 mM) (0.3 mM) (50 uM)

H2O2 958 ± 112 990 ± 107 37 ± 10 * 35 ± 11 *(pmol/mg/30min)

Mean ± SEM: * p<0.05 vs. Succinate or succinate + glutamate. N=5 in each group.

Abstract#: 93Presenter: Steven G. HershmanAuthors: Steven G. Hershman1,2,3, Elena J. Tucker4,6, Caroline Köhrer7, Casey A. Belcher-Timme1,2,3, Jinal Patel3, Olga A. Goldberger1,2,3, John Christodoulou8,9,10, Jonathon M. Silberstein11, Matthew McKenzie12, Michael T. Ryan13,14, Alison G. Compton4, Caterina Garone15,16, Beatriz Garcia-Diaz15, Salvatore DiMauro15, Jacob D. Ja!e3, Steven A. Carr3, Sarah E. Calvo1,2,3, Uttam L. RajBhandary7, David R. Thorburn4,5,6, Vamsi K. Mootha1,2,3

Institutions: 1Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, USA, 2Department of Systems Biology, Harvard Medical School, Boston, MA 02115, USA, 3Broad Institute, Cambridge, MA 02142, USA, 4Murdoch Childrens Research Institute, Royal Children’s Hospital, Melbourne, VIC 3052, Australia, 5Genetic Health Services Victoria, Royal Children’s Hospital, Melbourne, VIC 3052, Australia, 6Department of Paediatrics, University of Melbourne, Melbourne, VIC 3052, Australia, 7Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02142, USA, 8Genetic Metabolic Disorders Research Unit, Children’s Hospital at Westmead, Sydney, NSW 2006, Australia, 9Discipline of Paediatrics and Child Health, University of Sydney, Sydney, NSW 2006, Australia, 10Discipline of Genetic Medicine, University of Sydney, Sydney, NSW 2006, Australia, 11Department of Neurology, Princess Margaret Hospital for Children, Perth, WA 6008, Australia, 12Centre for Reproduction and Development, Monash Institute of Medical Research, Monash University, Melbourne, VIC 3168, Australia, 13Department of Biochemistry, La Trobe University, Melbourne, VIC 3086, Australia, 14ARC Centre of Excellence for Coherent X-Ray Science, La Trobe University, Melbourne, VIC 3086, Australia, 15Department of Neurology, Columbia University College of Physicians and Surgeons, New York, NY 10032, USA, 16Human Genetics Joint PhD Programme, University of Turin, 10125 Turin, Italy and University of Bologna, 40125 Bologna, Italy

Title: Mutations in MTFMT Underlie a Human Disorder of Formylation Causing Impaired Mitochondrial Translation

Body of Abstract: Of the ~ 90 protein components of the oxidative phosphorylation (OXPHOS) machinery, 13 are encoded by the mitochondrial DNA (mtDNA) and translated within the organelle. Defects in mitochondrial protein synthesis lead to combined OXPHOS de"ciency. Although the mtDNA encodes two of the ribosomal and 20 of the transfer RNAs, all remaining components of the mitochondrial translational machinery are en-


coded by nuclear genes and imported into the organelle. To date, mutations in more than ten di!erent nuclear genes have been shown to cause defective mitochondrial translation in humans. Using targeted sequencing of the mtDNA and nuclear exons encoding the mitochondrial proteome (MitoExome), we identi"ed mutations in the mitochondrial methionyl-tRNA formyltransferase (MTFMT) in unrelated children presenting with combined OXPHOS de"ciency and Leigh syndrome.

The metazoan mitochondrial translation machinery is unusual in having a single tRNA(Met) that ful"lls the dual role of the initiator and elongator tRNA(Met). A portion of the Met-tRNA(Met) pool is formylated by MTFMT to generate N-formylmethionine-tRNA(Met) (fMet-tRNA(met)); however, the requirement of formylation for initia-tion in human mitochondria is still under debate.

Patient "broblasts exhibit severe defects in mitochondrial translation that can be rescued by exogenous ex-pression of MTFMT. Furthermore, patient "broblasts have dramatically reduced fMet-tRNA(Met) levels and an abnormal formylation pro"le of mitochondrially translated COX1. Our "ndings demonstrate that MTFMT is criti-cal for e$cient human mitochondrial translation and reveal a human disorder of Met-tRNA(Met) formylation.

This work was funded by NIGMS (GM077465 and GM097136) and was published in the September 2011 issue of Cell Metabolism.

Abstract#: 94Presenter: Phil G. MorganAuthors: Margaret M. Sedensky1, Marni J. Falk2, Eugene Kolker1, Roger Higdon1, Chris Newgard3, Phil G. Morgan1

Institutions: 1Center for Developmental Therapeutics, University of Washington, Seattle, WA, 2Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, 3Departments of Pharmacology and Cancer Biology, Biochemistry and Internal Medicine, Duke University, Durham, NC

Title: A Systems Biology Approach to Mitochondrial Disease in C. elegans

Body of Abstract: Mitochondrial function is emerging as the keystone of an ever-growing and fairly extraordi-nary ensemble of clinical conditions. However, mitochondrial diseases remain di$cult to diagnose. Although many other valuable approaches are being used to understand how mitochondria function, the use of molecu-lar genetics in C. elegans possesses powerful and unique advantages. The excellent genetics, superb catalogue of mutations, well-de"ned behaviors, and invariant cell lineage led to our choice of C. elegans for study. Stud-ies in C. elegans have shown that mitochondrial function in the nematode is in extremely similar to that seen in mammals. This has led us to study mitochondrial function in disease by analyzing C. elegans mutants with defects in di!erent steps of the electron transport chain (ETC).

Methods: We combined the use of gene set expression analysis (GSEA), protein set enrichment analysis (PSEA; proteomics) and metabolomics to study the global changes in three mutants a!ecting the ETC in comparison to wildtype. The mutants studied were a complex I mutant (gas-1), a complex II mutant (mev-1) and a mutant in coenzyme Q synthesis (clk-1). For GSEA and metabolomics, we isolated RNA and metabolites from whole worms as "rst day adults. For proteomics, we isolated protein from mitochondria of "rst day adults. Pathways identi"ed as changed in GSEA and PSEA platforms were studied further by introducing mutations directly a!ecting those pathways. The expression platforms were then compared to the metabolomic results.

Results: Comparison of gene expression and proteomic arrays were revealed multiple concordant trends. For example pathways of 1) glycolosis/gluconeogenesis, 2) valine, isoleucine and leucine metabolism (both synthesis and degradation), 3) ubiquinone metabolism, 4) arginine/ proline metabolism, 5) TCA cycle, 6) fatty acid metabolism and 7) glyoxylate metabolism were found upregulated in gas-1 and mev-1. The expression results reported earlier by Falk et al corroborate the proteomic results reported here. Inhibition of ubiquinone


metabolism by the clk-1 mutation, which by itself does not cause a strong phenotype, caused a sterile pheno-type when combined with gas-1 or mev-1. Inhibition of the glyoxylate pathway (gei-7), upregulated in both the GSEA and PSEA in gas-1, was lethal when combined with gas-1. Studies combining gei-7 with mev-1 are in progress.

Discussion: A systems-based approach to characterizing the phenotypes of mitochondrial mutants may aid both in diagnosis of the diseases and understanding the interactions of multiple defects. Such understanding is likely to be more useful than any one platform used alone.

Abstract#: 95Presenter: Leonardo S. AlvesAuthors: Leonardo S. Alves1, 2, Fuyun Ji3, Mark S. Sharpley1,2, Olga Derbeneva1,2, Dimitra Chalkia1,2, Maria Lvova1,2, Guisheng Qian3, Lorna G. Moore3, Douglas C. Wallace1,2

Institutions: 1Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, University of Pennsylvania, 3501 Civic Center Boulevard Philadelphia, PA 19104-4302, 2Center for Molecular and Mitochondrial Medicine and Genetics, University of California, Irvine, Irvine, CA 92697-3940 USA; 3lnstitute of Human Respiratory Disease, Xinqiao Hospital, Third Military Medical University, Chongqing 400037, China; 4Graduate School of Arts & Sciences, Wake Forest University, Winston-Salem, North Carolina 27157-1001 USA

Title: Leber Hereditary Optic Neuropathy (LHON) associated mutation 3394 is also a high-altitude adaptive polymorphism.


The distinction between mild pathogenic mtDNA mutations and population polymorphisms can be ambigu-ous since both are homoplasmic, alter conserved functions, and correlate with disease. One possible expla-nation for this ambiguity is that the same variant may have di!erent consequences in di!erent contexts. Although not one of the original mtDNA disease causing mutations, 3394 has been found in signi"cantly increased frequencies in LHON pedigrees (1). A case in point is the mtDNA ND1 T3394C (Y30H) missense mutation. This mutation has been associated with LHON when occurring on macrohaplogroup N mtDNAs (2). However, in Asia the 3394C variant is most commonly associated with the M9 haplogroup which is rare at low elevations but increases in frequency with elevation, to an average of 25% in residents of the Tibetan Plateau (OR = 23.7). In high-altitude Tibetan and Indian populations, the 3394C variant occurs on "ve di!erent macro-haplogroup M haplogroup backgrounds and it is enriched on the M9 background in Tibet and the C4a4 back-ground on the Indian Deccan Plateau (OR = 21.9). When occurring on macrohaplogroup N mtDNAs including B4c and F1 this variant causes reduced complex I and reduced cellular respiration on macrohaplogroups N haplogroups B4c and F1 mtDNAs but not in the M9 haplogroup. On the M9 background the 3394C variant is associated with a complex I activity that is equal to or higher than that of the 3394T variant on the B4c and F1 backgrounds. Hence, the 3394C variant can either be deleterious or bene"cial depending on its haplogroup and environmental context. Thus this mtDNA variant ful"lls the criteria for a common variant that predisposes to a “complex” disease.

1 Brown MD, Torroni A, Reckord CL, & Wallace DC (1995) Phylogenetic analysis of Leber’s hereditary optic neuropathy mitochondrial DNA’s indicates multiple independent occurrences of the common mutations. Human Mutation 6(4):311- 325.

2 Liang M, et al. (2009) Leber’s hereditary optic neuropathy is associated with mitochondrial ND1 T3394C mutation. Biochemical and Biophysical Research Communications 383(3):286-292.


Abstract#: 96Presenter: Célia NogueiraAuthors: Célia Nogueira1, José Barros2, Maria José Sá3, Luísa Azevedo4, Filippo M. Santorelli5, Laura Vilarinho1

Institutions: 1National Institute of Health, Genetics Department, INSA, Oporto, Portugal, 2Oporto Hospital Center, Neurology Unit, Hospital S. António, Oporto, Portugal, 3Hospital S. João, Neurology Unit, Hospital S. João, Oporto, Portugal, 4IPATIMUP, Population Genetics, Oporto University, Oporto, Portugal, 5Fundazione Stella Maris, Molecular Medicine & Neurogenetics, IRCCS, Pisa, Italy.

Title: Identi!cation of a novel TTC19 mutation in a Portuguese family with complex III de!ciency

Abstract:Defects of mitochondrial complex III (CIII) are a relatively rare cause of mitochondrial dysfunction. CIII or ubiquinol-cytochrome c reductase is the third component of the mitochondrial respiratory chain and catalyzes the electrons transfer from reduced coenzyme Q to cytochrome c and is composed of 11 subunits; one encod-ed by mitochondrial DNA (MT-CYB) and the remaining by nuclear genes. BCS1L gene is a CIII assembly factor. Mutations in MT-CYB and BCS1L genes account for the vast majority of mutations leading to CIII de"ciency, and are associated with a wide range of neuromuscular disorders. The human tetratricopeptide 19 (TTC19), encodes a poorly understood member of tetratricopeptide repeat domain 19 located on chromosome 17 and appears to be involved in the correct assembly of CIII. Recently, mutations in TTC19 have been described in three unrelated Italian kindred in association with a severe neurodegenerative disease.

Here we present a consanguineous Portuguese family where a severe biochemical de"ciency of complex III en-zyme activity occurred in four siblings in association with neurological manifestations suggestive of cerebellar ataxia combined with relentless psychiatric manifestations. Variability in age at onset and disease course was associated with a novel homozygous mutation in TTC19.

We had "rst detected a biochemically de"cient enzyme activity in the family, we had analyzed all structural genes part of CIII as well as BCS1L. Only the recent description of mutations in TTC19 raised high the suspect of a similar condition in the present family. The novel TTC19 mutation identi"ed in this family, was homozygous in the four patients, heterozygous in their parents and in two healthy relatives, and it was absent in ethnically-matched controls. The mutation predicts a frameshift, resulting in a truncated protein by the insertion of a premature stop codon.

In summary, we are describing the 4th family identi"ed in the world carrying a novel TTC19 mutation. Our data corroborate the genotype and phenotype variability presented by the a!ected family members and hopefully will contribute to a deeper understanding of the CIII-related disorders.

Abstract#: 97Presenter: Ligia S AlmeidaAuthors: Lígia S Almeida1, Mariana Ferreira1, Célia Nogueira1, Fátima Furtado2, Teresinha Evangelista3, Filippo M Santorelli4 and Laura Vilarinho1

Institution: 1Research Unit, Dept. of Genetics, Medical Genetics Center, INSA, Porto, Portugal; 2Serviço de Pediatria, Hospital Distrital de Beja, Portugal, 3Hospital Sta. Maria, Neurologia, Lisboa, 4Molecular Medicine & Neurodegenerative Diseases Diseases, IRCCS Fondazione Stella Maris, Pisa, Italy

Title: “Double-trouble” or digenic disorder in complex I de!ciency


Complex I (CI) de"ciency is a defect of OXPHOS caused by mutations in the mitochondrial or nuclear genomes. To date disease-causing mutations have been reported in all mitochondrial-encoded subunits and 22 nuclear genes. In about 50% of the patients no mutations are found, suggesting that undiscovered factors are an im-portant cause of disease. In this study we report a consanguineous family from Southern Portugal with three a!ected children where CI de"ciency could not be clari"ed.

The a!ected children presented similar clinical "ndings with early onset of the disease having the three of them a fatal outcome. A reduced activity of CI was detected in two of the patients that led us to investigate, at the molecular level, some CI associated genes. After studying some CI associated genes no mutations were detected. Interestingly, all patients presented 3-methylglutaconic acid in the urinary organic acids and it is known that POLG gene is involved in the etiology 3-methylglutaconic acidurias. In only one of the patients, presenting also with mtDNA depletion, the p.G848S/p.Q1236H mutations were found in POLG.

Besides the e!orts done it remains unsolved whether this family due to the high consanguinity could have two di!erent disorders or if a yet unknown gene, leading to CI de"ciency, could be involved.

Abstract#: 101Presenter: Francisca DiazAuthors: Francisca Diaz1, So"a Garcia1 and Carlos T Moraes1, 2Institution: 1Department of Neurology, 2Department of Cell Biology and Anatomy, University of Miami, Miller School of Medicine, Miami, Florida, 33136, USA.

Title: Metabolic Adaptations in Neurons with Complex IV De"ciency


Abstract#: 102Presenter: Carolyn H. BuzinAuthors: 1Carolyn H. Buzin, 1Christian Neckelmann, 1Melissa Gasper, 1Juliana O. Barba, 1William A. Scaringe, 1Steve S. SommerInstitution: 1MEDomics, 426 N. San Gabriel Ave., Azusa, CA 91702

Title: NextGen Sequencing of the Complete mtDNA Genome: 20% Estimated Positive Cases among a Recent 117 Patients Body of Abstract: The complete mitochondrial genome (mtDNA), including all 37 genes and the control region, was sequenced 10,000+ times by Next Generation Sequencing for 117 patients recently submitted to a clinical laboratory (MEDomics). The sensitivity of NextGen Sequencing is at least 30-fold higher than that of Sanger sequencing and mtDNA mutant heteroplasmy levels can be detected down to a level of 1%. Unique algorithms and a proprietary bioinformatics pipeline developed at MEDomics, as well as expert interpreta-tion of the functional signi"cance of sequence alterations, were used to detect and interpret mutations in the patients. Based on literature review, mtDNA databases, and evolutionary conservation of the wild type amino acid or nucleotide, variants were grouped into four categories: Group 1- Positive (known mutations or very likely positive); Group 2 - May be positive (heteroplasmic variants at < 8% frequency that may drive the clinical phenotype if present at threshold levels in target tissues); Group 3 - Variants of uncertain signi"cance (un-likely to drive, the phenotype, but may cause some e!ects in some individuals, especially in conjunction with other mtDNA or nuclear gene mutations); Group 4 – Likely to be negative (known polymorphisms or variants unlikely to contribute to the clinical phenotype). Of the 117 patients, 21% had a Group1 mutation (positive), 10% had a Group 2 variant that was heteroplasmic at <8% in blood, 27% had a Group 3 variant (uncertain signi"cance), and 41% had only Group 4 variants (negative). Among the Group 1 mutations, some examples of typical subgroups are:

(1) A novel heteroplasmic missense change (not found in ~2700 mostly healthy controls) in the CYTB gene at 16% frequency in blood. The wild type human amino acid is highly conserved, in 99.2% of ~1400 vertebrate and invertebrate species.

(2) A heteroplasmic large rearrangement at ~50% frequency in blood composed of a 7.1 kb deletion and con-comitant 972 bp insertion.

(3) A heteroplasmic missense mutation in the ND4 gene at 18% frequency in blood; known to be a primary cause of LHON; not completely penetrant. The wild type human sequence is highly conserved, in 99.9% of spe-cies. This patient is a 14 year old male with severe photophobia, seizures, and other neurological symptoms who has not developed LHON (note that LHON generally presents in the late teens and twenties.

(4) A rare homoplasmic missense change in the ND5 gene. The wild type human sequence is highly conserved and is present in 99.9% of ~1000 species. A sibling with a similar phenotype also shares this homoplasmic vari-ant.

In summary, NextGen Sequencing has identi"ed a known causative mutation or one that is highly likely to drive the clinical phenotype in ~20% of a group of 117 patients clinically suspected to have a mitochondrial disorder.


Abstract#: 103Presenter: Tim KovesAuthors: Tim Koves1,3, Sarah Seiler3, Karen DeBalsi3, April Whitman3, Deborah Muoio1,2,3

Institution: Duke University, Depts. of 1Medicine, 2Pharmacology & Cancer Biology, 3Sarah W. Stedman Nutrition and Metabolism Center, Durham, NC 27708

Title: Absence of malonyl-CoA decarboxylase (MCD) impacts endurance exercise capacity and reprograms skeletal muscle mitochondrial metabolism

Malonyl-CoA decarboxylase (MCD) catalyzes the degradation of cytoplasmic malonyl-CoA, a multifaceted molecule that serves as a precursor for de novo fatty acid synthesis and a potent biological inhibitor of the outer mitochondrial membrane enzyme, carnitine palmitoyltransferase I (CPT1). Because CPT1 activity is absolutely essential for entry of long-chain fatty acids into the mitochondria, MCD plays a pivotal role in pro-moting beta-oxidation. Humans with mutations in the MCD gene (MLYCD) are unable to derive energy from fats and present with malonic aciduria, developmental delay, cardiomyopathy, vomiting and hypoglycemia. Our previous work established that young mice lacking MCD shift fuel use in favor of glucose oxidation and resist high fat diet-induced insulin resistance. In the current studies we sought to understand how lifelong inhibition of beta-oxidation a!ects whole-body energy metabolism, skeletal muscle and mitochondrial func-tion in older aged mice. Fifteen month old control and MCD-/- mice underwent VO2max testing in metabolic treadmills. MCD-/- mice displayed equivalent running capacities with lower sub-maximal VO2 and higher RER values versus littermate controls. By contrast, when challenged with endurance exercise, MCD-/- mice dis-played a markedly reduced running time to exhaustion, consistent with a relative inability to produce energy from fat catabolism. In vitro fatigue studies on electrically-stimulated soleus and extensor digitorum longus muscles revealed elevated lactate release and reduced force production by MCD de"cient muscles compared to controls. Interestingly however; when assessed in isolated skeletal muscle mitochondria, a system that lacks cytosolic malonyl-CoA, rates of 14C-palmitate oxidation and palmitoylcarnitine-supported state 3 respiration were actually higher in the MCD-/- group. This increase in mitochondrial fat oxidation capacity was accom-panied by elevated acyl-CoA dehydrogenase activity; whereas respiratory function of the electron transport system was largely una!ected by loss of MCD. These studies support the idea that MCD de"ciency results in adaptive reprogramming of skeletal muscle mitochondria toward enhanced capacity for beta-oxidation with no overt de"cits in mitochondrial function. Nonetheless, remodeling at the mitochondrial level appears insuf-"cient to overcome malonyl-CoA-mediated inhibition of CPT1 in intact working skeletal muscles. We conclude that MCD is essential for optimal endurance performance and skeletal muscle function. Future studies aim to understand potential interactions between MCD activity and age-related perturbations in oxidative stress and insulin resistance.

These studies were supported in part by grants from The Ellison Medical Foundation (TK) and R01HL101189 (DM).

Abstract#: 104Presenter: Steve S. SommerAuthors: 1Steve S. Sommer, 1Harris S. Soifer, 1Shogher Nalbandian, 1Krishna S. Morampudi, 1William A. Scaringe, 1Nicolas Nekelmann, 1Juliana O. BarbaInstitution: 1MEDomics, 426 N. San Gabriel Ave., Azusa, CA 91702

Title: MitoNuc2Dx – Analyzing the Mitochondrial Genes Encoded in the Nuclear Genome to Assess their Possible Association with Mitochondrial Disorders


Body of Abstract: MitoNuc2Dx is a diagnostic test designed to identify mutations in nuclear genes of patients suspected to be a!ected by a mitochondrial disease. The test, based on the power of Next Generation Se-quencing coupled with advanced bioinformatics methods and interpretation strategies, analyzes 509 nuclear genes associated with mitochondrial structure and/or function, about half of which are known to be mito-chondrial disease-associated. MitoNuc2Dx testing, in conjunction with the MitoDx test which detects genetic variants encoded in the mitochondrial genome, will provide a comprehensive and speci"c analysis that estab-lishes the foundation for a genetic diagnosis, and can elucidate a patient’s pathophysiology and guide tar-geted treatment. The results obtained may also assist with the enrollment of patients into research and drug treatment trials that bene"t from the improved understanding of de"ned disease mechanisms.

About 25% of our previous MitoNucleomeDx tests analyzing 312 genes are de"nitely/very likely to be positive. In addition, for the MitoDx test, about 20% of the samples analyzed are de"nitely positive or very likely to be positive in the mitochondrial genome. This is consistent with the mitochondrial genome having a mutation rate that is 10-20 fold higher than that of the nuclear genome.

The original 312 genes tested (of which 166 are known to cause mitochondrial diseases) include (1) those cod-ing for the proteins in Complexes I-V, (2) OXPHOS Complex assembly factors, (3) OXPHOS co-enzyme synthesis, (4) factors involved in mtDNA replication/transcription/translation (including the 86 ribosomal protein genes), (5) genes involved in maintaining mitochondrial copy number (mutations in which cause mitochondrial DNA depletion), (6) genes involved in the urea cycle, Krebs cycle, and fatty acid oxidation pathways, which prepare molecules for input into the OXPHOS pathway, and (7) genes coding for additional likely relevant mitochondri-al proteins that are not yet associated with speci"c diseases. An additional 197 genes have been added to the new MitoNuc2Dx test (of which 81 are known to be mitochondrial disease-associated. These genes include (1) mitochondrial tRNA synthesis genes, (2) solute carriers, (3) mitochondrial membrane transporters, (4) mito-chondrial maintenance genes, and (5) additional genes that include those discovered in the last two years.

In conclusion, analysis of 104 recent samples for the initial 312 genes indicates that an estimated 25% are posi-tive; a higher percentage is expected for the 509 genes in MitoNuc2Dx. Examples of some of the cases positive for a mutation in nuclear mitochondrial genes will be discussed.

Abstract#: 105Presenter: Penelope E. Bonnen Authors: Penelope E. Bonnen, Arnaud Besse, Tarak Donti, Adithya Raghavan, Seema Lalani, Fernando Scaglia, William J Craigen, Brett H Graham Institution: Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX 77030

Title: Exome sequencing and functional biology reveal novel Mitochondrial Disease genes

Mitochondrial disease is a diverse group of disorders that are estimated to occur with a combined incidence of 1/5,000. Approximately ninety percent of pediatric onset cases are caused by high penetrance recessive mutations in the nuclear genome, however after exhausting all available diagnostic tests most patients remain without a molecular diagnosis. Using a combination of sequencing, bioinformatics, and cellular and molecu-lar biology we have discovered high-con"dence pathogenic mutations. The Mitochondrial Respiratory Chain Disorders Collection at Baylor College of Medicine (MRCD-BCM) contains over 15,000 patients with suspected MRCD with accompanying DNA, diagnostic and clinical information. From this database we identi"ed pediatric patients who have been pre-screened and shown to be negative for mitochondrial and known nuclear gene candidates but have overwhelming evidence supporting a mitochondrial disease diagnosis. A battery of mito-chondrial functional assays have been performed on all samples with available tissues including electron trans-port chain analysis, FACS based analysis of relative mitochondrial membrane potential and mitochondrial DNA copy number. Exome sequencing has been completed on 20 patients. We have discovered and validated the pathogenic mutation in "ve patients. A causal relationship has been established using cDNA complementation


and mitochondrial functional assays. This work has resulted in the discovery of novel disease genes, improved molecular diagnosis, and new insights into the pathogenetic mechanisms underlying mitochondrial disease.

Abstract#: 106 Presenter: Brett A. KaufmanAuthors: Jill E. Kolesar, Catherine Y. Wang, Shih-Hsuan Chou and Brett A. KaufmanInstitution: Department of Animal Biology, University of Pennsylvania School of Veterinary Medicine, 3800 Spruce St. VET220E, Philadelphia PA 19104.

Title: Two-dimensional intact mitochondrial DNA agarose electrophoresis demonstrates the complexity of mtDNA structural forms and in vivo supercoiling by TFAM

The circular mitochondrial genome’s structural and topological conformations make the study of mitochondri-al DNA (mtDNA) compaction notoriously di$cult. The principal double-stranded DNA binding protein in the mitochondria is TFAM, which has been shown to supercoil and compact circular DNA in vitro, but not yet in vivo due to a lack of suitable methods to resolve myriad mtDNA structures that vary in both shape and compaction state.

Here we describe two-dimensional intact mtDNA agarose gel electrophoresis (2D-IMAGE) pro"ling, which detected approximately two-dozen whole genome structures, which we have characterized through multiple molecular approaches. The major topoisomer forms were well conserved across many cell and tissue types, but di!erences unique to certain cells and tissues were also present. Linear mtDNA, which is not adequately resolved from circular DNA by conventional 1D electrophoresis, is increased by oxidative stress. Increased steady-state expression levels of TFAM altered the relative distribution and absolute abundance of topoiso-mers, by increasing covalently closed circles and extent of their supercoiling. Additional studies are underway to examine mtDNA by 2D-IMAGE in mouse models of human disease.

Abstract#: 107Presenter: Brett A. KaufmanAuthors: Jill E. Kolesar1, Yumiko V. Taguchi1, Rebecca L. Cramer1, Catherine Wang1, Shih-Hsuan Chou1, Scott A. Soleimanpour2, Doris A. Sto!ers2 and Brett A. Kaufman1

Institution: 1Department of Animal Biology, School of Veterinary Medicine, and 2Department of Medicine and the Institute for Diabetes, Obesity, and Metabolism University of Pennsylvania, Philadelphia, PA 19104

Title: EVIDENCE FOR MITOCHONDRIAL ADAPTATION TO CHRONIC GLUCOSE EXPOSURE IN BETA CELLS

Pancreatic beta cells increase in number, size, and insulin synthesis/secretion to compensate for higher endo-crine demands driven by increased circulating glucose. We have found that mitochondrial DNA (mtDNA) in-creases in response to chronic high glucose exposure of pancreatic insulinoma cells, and developed TAQMAN expression arrays to examine changes in mitochondrial factors in these cells. We identi"ed PIF1 and FEN1 as the candidate factors regulating the observed increase in mtDNA content; the full transcript (cDNA) encoding PIF1, a DNA helicase, is glucose responsive and encodes two translation products, which are targeted to both the nucleus or mitochondria by an alternative translation start mechanism. In vitro, PIF1 has been shown to enhance the Okazaki fragment processing activity of FEN1, the #ap endonuclease, which is known to localize to both the nucleus and mitochondria. PIF1 also has G-quadruplex helicase activity, and has been identi"ed at telomeres. In the current study, we investigate the mechanisms by which PIF1 increases mtDNA and insulin content, and how PIF1 expression is glucose responsive.


Abstract#: 108Presenter: Renkui BaiAuthors: Renkui Bai1, Jaimie Higgs1, Sharon Suchy1, Mustafa Sai"1, Melanie Knight1, Sabrina Buchholz1, Sonia Benhamed1, Dolores Arjona1, Barbara Boggs1, Craig Chinault1, Yuriy Shevchenko1, Nizar Smaoui1, Gabriele Richard1, John Compton1, Sumit Parikh2, Mark Tarnopolsky3,Wendy Chung4, Sherri Bale1

Institutions: 1 GeneDx, Gaithersburg, MD 20877 2 Department of Neurology, Cleveland Clinic, Cleveland, OH 44195 3 McMaster University Medical Center, Hamilton, Ontario, L8N 3Z5 4 Columbia University Medical Center, CHONY 6N-601A New York, NY 10032

Title: Comprehensive Analysis of Entire Mitochondrial Genome by Long-Range PCR and Next Generation Sequencing for the Diagnosis of Mitochondrial Disorders: Yield of 216 Cases

Body of Abstract:

Background: Mutations in the mitochondrial genome account for about 10-20% pediatric patients and up to 40% of adult patients with a primary mitochondrial disorder (MTD). To date, more than 120 disease-causing large single deletions and more than 400 point mutations in the mitochondrial genome have been reported and recorded in the mtDNA mutation databases as disease-causing mutations (www.mitomap.org). Although dozens of known recurrent, de"nitive point mutations and large single deletions account for the majority of patients with a primary mtDNA disorder, many patients and families have private mutations. Traditional meth-ods of testing the mitochondrial genome were insensitive, labor intensive, time-consuming and costly and no single method could detect both point mutations and large deletions. By using whole mitochondrial genome ampli"cation and next-generation sequencing, over 99% of the primary pathogenic mutations in the mtDNA, including point mutations, small insertions/deletions, and large-scale single deletions, can be detected.

Samples and Methods: Samples (11 muscle, 4 liver, 201 blood or DNA) of 216 unrelated patients clinically suspected of a MTD (39 de"nite or "t into a discrete clinical syndrome, 61 probable, 78 possible, others with clinical information not provided) were tested for their mitochondrial genome sequence at GeneDx. All vari-ants identi"ed were compared with mtDNA mutation databases in Mitomap, other online resources, and GeneDx’s general & haplogroup-speci"c mtDNA variant databases. Each individual’s mtDNA haplogroup was also determined. All novel variants were thoroughly evaluated and classi"ed as novel mutations (nMut), vari-ants of unknown signi"cance: likely mutation (VLM), undetermined (VUS), or likely benign (VLB), or as a benign polymorphism (Poly). The “Reported” pathogenic mutations in Mitomap without solid supporting evidence were re-evaluated.

Results: Altogether 22 (10.2%) of the 216 cases were positive for a de"nitive mutation/nMut/VLM: 18 point mu-tations (17 in blood, 1 in muscle, heteroplasmy 6%-100%) and 4 single deletions (2.3 kb and 12.8kb in muscle; 4.5kb and 5.2 kb in blood with heteroplasmy of 50%-88%) In addition, 25 (11.6%) cases were positive for a VUS. The majority (~80%) of the de"nitive mutations and nMut/VLMs were identi"ed in patients with a de"nitive/probable MTD or fell into a discrete clinical syndrome.

Conclusion: Comprehensive Analysis of Entire Mitochondrial Genome by Long-Range PCR and Next Genera-tion Sequencing is an e$cient method to detect primary mtDNA mutations; the positive detection rate is ap-proximately 20-25% for patients with a de"nitive/probable MTD or with clinical features that fall into a discrete clinical syndrome.


Abstract#: 109Presenter: Renkui BaiAuthors: Renkui Bai1, Jaimie Higgs1, Sharon Suchy1, Mustafa Sai"1, Melanie Knight1, Federica Gibellini1, Sabrina Buchholz1, Sonia Benhamed1, Dolores Arjona1, Barbara Boggs1, Craig Chinault1, Yuriy Shevchenko1, Nizar Smaoui1, Gabriele Richard1, John Compton1, Sherri Bale1, Sumit Parikh2, William J. Rhead3,David Dimmock3, Susan Sparks4, Adeline Vanderver5, Natalie S. Hauser6, Jaya Ganesh7, Marni J. Falk7

Institutions: 1GeneDx, Gaithersburg, MD 20878, 2Department of Neurology, Cleveland Clinic, Cleveland, OH 44195, 3Medical College of Wisconsin Milwaukee, WI 53226, 4Carolinas Medical Center, Charlotte, NC 28203, 5Department of Neurology, Children’s National Medical Center Washington, DC 20010, 6Children’s Hospital Central California, Medical Genetics Department, Madera, CA 93704, 7The Children’s Hospital Of Philadelphia & Perelman School of Medicine at the University of Pennsylvania Philadelphia, PA 19104

Title: PCR-Based Target Sequence Enrichment and Next Generation Sequencing of 24 Nuclear Genes for the Diagnosis of Mitochondrial Disorders: Yield of 262 Cases

Body of Abstract:Background: Most primary mitochondrial disorders (MtD) are caused by mutation(s) in nuclear genes. Muta-tions in many di!erent nuclear genes cause similar clinical features. Mutations in about 100 nuclear genes have been associated with MtD. In practice, molecular diagnosis of MtD using single gene testing has been laborious and time-consuming. To increase the e$ciency and cost-e!ectiveness of mutation detection in MtD, we used a multiplex PCR-based targeted enrichment approach and next generation sequencing to simultane-ously resequence completely 24 nuclear genes which account for over 60% of known nuclear gene mutations associated with MtD. This test has a technical sensitivity comparable to that of Sanger sequencing.

Samples and Methods: Blood or DNA samples of 262 unrelated patients suspected of an MtD (52 de"nite or falling into a discrete clinical syndrome, 68 probable, 106 possible, others with clinical information not pro-vided) were tested at GeneDx. All variants identi"ed were compared with speci"c mutation databases includ-ing HGMD, LSDBs, GeneDx internal variant databases and many other online resources. All novel variants were thoroughly evaluated and classi"ed as novel mutations (nMut), variants of unknown signi"cance [likely muta-tion (VLM), undetermined (VUS), or likely benign (VLB)], or as a benign polymorphism. The published “patho-genic” mutations without solid supporting evidence were re-evaluated.

Results: Of the 262 samples tested, disease-causing mutations were identi"ed in one gene and con"rmed the diagnosis for 24 (9.2%) of the patients; Seven of them also harbor a heterozygous nMut/VLM/VUS in 1-2 other genes in the panel. In 15 (5.7%) patients, heterozygous nMut/VLM/VUS were identi"ed in 1-3 genes but were insu$cient to make a molecular diagnosis. The types of mutations identi"ed include point mutations and insertions/deletions of 1-24 bps. The majority (~80%) of the disease-causing mutations and VLMs were identi"ed in patients with a de"nitive/probable MtD or a discrete clinical syndrome. Some of the 24 cases with diagnostic mutations identi"ed by this panel had either previously undergone extensive biochemical and/or molecular (single gene sequencing) evaluations without a de"nitive diagnosis, or mutation(s) were identi"ed in a gene unexpected from the results of prior clinical, histo-pathological or electron transport chain (ETC) enzyme evaluations.

Conclusion: The sensitivity of a targeted next generation sequencing panel of selected nuclear genes is signi"cantly higher than single gene sequencing for the molecular diagnosis of MtD. The clinical sensitivity of this 24-nuclear gene panel is approximately 15% for patients with a de"nitive/probable MtD or with clinical features that fall into a discrete clinical syndrome.


Abstract#: 110Presenter: Renkui BaiAuthors: Renkui Bai1, Jaimie Higgs1, Sharon Suchy1, Federica Gibellini1, Melanie Knight1, Sabrina Buchholz1, Sonia Benhamed1, Dolores Arjona1, Craig Chinault1, Rhonda Brandon1, Nizar Smaoui1, Gabriele Richard1, Sherri Bale1

Institution: 1GeneDx, Gaithersburg, MD 20877

Title: PCR-Based Targeted Enrichment and Next-Generation Sequencing of 101 Nuclear Genes for the Diag-nosis of Mitochondrial Disorders

Body of Abstract:

Background: The majority of primary mitochondrial disorders (MtD) is caused by mutations in nuclear genes. To date, only about 100 nuclear genes have reported mutations associated with a primary MtD. About 25-30% of these genes have 1-35 pseudogenes or homologous sequences in other genes in the genome (www.pseudogene.org). Whole exome sequencing (WES), which uses hybridization-based capture as the targeted enrichment method making it unable to avoid pseudogenes or homologous sequences, creates many false positive and false negative results as well as many low/no coverage regions (in the exome data, usually about 7-10% of the exons of the genes of interest have insu$cient sequence reads to make a variant call). By design-ing unique primers that selectively amplify only the gene of interest and using droplet-based multiplex PCR and massive parallel sequencing, we are able to simultaneously re-sequence 101 nuclear genes which account for over 95% of known nuclear genes/mutations associated with a primary MtD. This panel includes 976 exons with adjacent intronic sequences, and 25-50 bps into the promoters and 3’ UTR regions, as well as common mutations in deep intronic regions.

Method: To validate this test, twelve DNA samples with mutations identi"ed by single gene Sanger sequenc-ing or by multiplex PCR-Based targeted enrichment and Next-generation sequencing of a Mito24 nuclear gene panel and con"rmed by Sanger sequencing, were blinded and evaluated. Inter-run and intra-run varia-tions were assessed for 3 of the samples. Results were compared to the original data.

Results: Eight of the 12 samples had no low coverage, with 40x-8000x read depth per nucleotide, resulting in average of approximately 2000x per nucleotide. The other four samples each had insu$cient coverage for only 1-3 exons. 100% of all pathogenic mutations and previously detected sequence variants in the regions of interest were identi"ed. Totally, each sample had 90-110 variants identi"ed with 0-3 mutations or variants of unknown signi"cance.

Conclusion: This panel allows complete re-sequencing of all the exons and adjacent intronic sequences of 101 nuclear genes simultaneously in a timely manner, with a technical sensitivity and speci"city comparable to Sanger sequencing. In combination with exon level deletion/duplication testing of all these genes, it is an ideal panel for ruling out pathogenic mutations in genes known to be associated with a primary mitochondrial disorder before considering whole exome sequencing (WES).


Abstract#: 111Presenter: Hsing-Wen WangAuthors: Han Wen Guo1, Jia Sin Yu1, Shu Han Hsu2, Yau Huei Wei2,3, Oscar K. Lee1,4, Hsing Wen Wang1 Institutions: 1Institute of Biophotonics, National Yang Ming University, Taipei 112, Taiwan 2Department of Biochemistry and Molecular Biology, National Yang-Ming University, Taipei 112, Taiwan; 3Department of Medicine, Mackay Medical College, San-Jhih, Taipei County 252, Taiwan; 4Institute of Clinical Medicine, National Yang Ming University, Taipei 112, Taiwan

Title: NADH %uorescence lifetime as an intrinsic biomarker of the metabolic change during osteogenic di"erentiation of human mesenchymal stem cells (hMSCs)

Fluorescence lifetime of NADH had been proposed to use as an intrinsic biomarker for monitoring cellular me-tabolism. In our pervious studies, we have demonstrated that NADH #uorescence lifetime of hMSCs increase gradually with time of osteogenic di!erentiation [1]. However, the cause of NADH #uorescence lifetime change is not clear. In this study, we performed NADH lifetime measurement of hMSCs from a di!erent donor to study its association with several metabolic indices such as ATP level, oxygen consumption, and lactate release. We also measured the quantity of Complex I, III, IV and V during hMSC di!erentiation. NADH #uorescence lifetime images were performed as our previous studies [1] that treated hMSC cells were imaged with a two-photon laser scanning microscope by a 60 : 1.45 NA PlanApochromat oil objective lens (Olympus Corp., Japan). NADH #uorescence was excited at 740 nm by a Verdi pumped modelocked femtosecond Ti:sapphire laser (Coherent, Inc., Santa Clara, California) at 76 MHz and the emitted #uorescent light was detected at 450±40 nm by a band-pass "lter (Edmund Optics, Inc., Barrington, New Jersey). Fluorescence photons were detected by a photon-counting photomultiplier H7422P-40 (Hamamatsu Photonics K.K., Hamamatsu, Japan). Time-resolved detec-tion was conducted by the single-photoncounting SPC-830 printed circuit board (Becker & Hickl GmbH, Berlin, Germany). Data were analyzed with the commercially available SPCImage v2.8 software (Becker & Hickl GmbH, Berlin, Germany) via a convolution of the two component exponential decay function and the instrument response function (IRF), and then the convolved result were "tted to the actual data to derive lifetime param-eters t1 (NADH short lifetime component),t2 (NADH long lifetime component), a1 (amplitude related to t1), a2 (amplitude related to t2), and tm. Mean lifetime tm is de"ned as (a1t1+a2t2)/(a1+a2). IRF was measured using a second-harmonic generated signal from a periodically poled lithium niobate crystal. The cell respiration rate was measured by a 782 Oxygen Meter as previously reported [2] and intercellular ATP level was measured by the bioluminescent somatic cell ATP assay kit (Sigma-Aldrich, St. Louis, Missouri).

The results show that more oxygen consumption, higher ATP level expressed and less lactate released during di!erentiation. Similar to our previous study, NADH #uorescence lifetime increased gradually up to 4 weeks after osteogenic di!erentiation. We observed a good correlation between the increase of NADH #uorescence lifetime and ATP level and oxygen consumption (R2= 0.88 and 0.95 respectively). Signi"cant higher expression of the total Complex protein than controls was observed at 3 and 4 weeks after di!erentiation. However, Com-plex I expression, which was directly related to NADH, did not show signi"cant correlation with the increase of NADH #uorescence lifetime. In summary, we demonstrated that the change of NADH lifetime has potential as an intrinsic biomarker to monitor the metabolic change during osteogenic di!erentiation of hMSCs. The increase of NADH lifetime was in part due to the increased Complex protein interaction in mitochondria during di!erentiation.

References1. Guo HW, Chen CT, Wei YH, Lee OK, Gukassyan V, Kao FJ, and Wang HW*, “Reduced Nicotinamide Adenine Dinucleotide (NADH) Fluorescence Lifetime Separates Human Mesenchymal Stem Cells from Di!erentiated Progenies,” J Biomedical Optics (Letters), 13(5), 050505, 2008.

2. Chen CT, Shih YR, Kuo TK, Lee OK, and Wei YH, “Coordinated changes of mitochondrial biogenesis and anti-oxidant enzymes during osteogenic di!erentiation of human mesenchymal stem cells,” Stem cells, 26(4), 960-8, 2008.


Abstract#: 112Presenter: Hyongi ChonAuthors: Hyongi Chon, Lina A. Gugliotti, Kiran Sakhuja, Mariya London, Susana M. Cerritelli and Robert J. CrouchInstitutions: Program in Genomics of Di!erentiation, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health. 6 Center Dr. Bethesda, Maryland 20892, USA

Title: Mitochondrial DNA depleted mouse B cells are defective in the response to antigen-stimulation

RNase H1 is one of the enzymes that degrade RNA of RNA/DNA hybrids. Two di!erent forms of the protein, localizing to nuclei and mitochondria, are translated from a single mRNA. RNase H1 knockout mice are em-bryonic lethal, resulting from loss of mitochondrial DNA (mtDNA) in the knockout mice, indicating that RNase H1 is essential for mtDNA replication. To examine the importance of this gene in later stages of development, speci"cally in B cells, we constructed a mouse whose Rnaseh1 gene is conditionally knocked-out (KO) by a Cre recombinase uniquely expressed in B cells (mb1-Cre), which has been used to e$ciently delete conditional alleles at early stage of B cell development (1). To respond to diverse antigens, large varieties of B cells are con-stantly produced from hematopoietic stem cells. Some of these B cells undergo developmentally determined apoptosis, for which mitochondria are essential. Puri"ed resting B cells from the conditional knockout mice showed more than 90% depletion of both RNase H1 protein and mtDNA levels. Surprisingly, B cell develop-ment in the bone marrow and spleen from the conditional Rnaseh1-KO mice were nearly normal, although an-tibodies in the blood serum were signi"cantly decreased. In agreement with the blood serum antibody level, Rnaseh1-KO B cells were defective in the response to LPS/IL4 stimulation in the in vitro cultures. Additionally, stimulation did not occur even when supplements (high glucose, pyruvate and uridine) that allow the growth of cells in the absence of mtDNA were included in the medium. Analysis of the membrane potential of the puri"ed B cells from WT and the conditional knockout did not show a di!erence prior to stimulation. However, a clear di!erence was observed following 1 day stimulation. This variation in membrane potential may corre-spond to the defect of Ca2+ signaling essential to B cell response to antigens.(1) Hobeika E. et al., PNAS (2006), 103, 13789-13794

Abstract #: 113Presenter: Susana PeraltaAuthors: Peralta S., Torraco A., Wenz T., Moraes C.T.Institution: Department of Neurology, University of Miami Miller School of Medicine

Title: Ablation of the gene coding for Complex I subunit NDUFA5 in CNS results in a mild phenotype

Mitochondrial complex I de"ciencies are associated with severe pathologies which are often lethal in early childhood. To design new therapies, animal models are needed for the study of the pathophysiology and adaptive responses in complex I defects. The NADH-ubiquinone oxidoreductase 1 subcomplex subunit 5 (NDUFA5) is a nuclear encoded structural subunit of complex I. This 13 kDa protein is located in the periph-eral arm of complex I. We have created a conditional NDUFA5 knock-out ablating the endogenous alleles and including a rescuing transgene #anked by loxP sites. The #oxed transgene was deleted selectively in neu-rons by expressing cre driven by the CamK-II promoter. NDUFA5-CMK-II conditional knock-out (cKO) mice presented lethargy and loss of motor skills at the age of 12 month old and death by the 16 month. These mice presented high levels of lactate levels in the blood at the age of 12 month. Blue Native gels electrophoresis showed that the amount of intact complex I was greatly reduced in cortex and no accumulation of complex I subassemblies was observed. Accordingly, complex I proteins levels were markedly reduced in the NDUFA5-CMK-II knock-out mice. Biochemical measurement showed that complex I activity was reduced to 60% of control levels in cortex at the same age. The activity levels of the other OXPHOS complexes and citrate syn-thase activity remained unchanged. Our data suggest that NDUFA5-CMK-II cKO is a useful mouse model to study the molecular mechanism of mild mitochondrial encephalophathies.


Abstract #: 114 Presenters: Iain HargreavesAuthors: Iain Hargreaves and John LandInstitution: Neurometabolic Unit, National Hospital for Neurology and Neurosurgery, London, UK

Title: MULTIPLE MITOCHONDRIAL ELECTRON TRANSPORT CHAIN ENZYME DEFICIENCIES ASSOCIATED WITH A DECREASE IN SKELETAL MUSCLE COENZYME Q10 STATUS

Coenzyme Q10 (CoQ10) functions as an electron carrier in the mitochondrial electron transport chain (ETC) as well as serving as a potent lipid soluble antioxidant. Diagnosis of a CoQ10 de"ciency normally follows a "nding of decreased activity of the linked ETC complex II+III and/or I-III which utilise endogenous CoQ10 and normal activities of complex I, II and complex III. Diagnosis is con"rmed by HPLC analysis of muscle CoQ10. However, since all ETC complexes are susceptible to free radical induced oxidative damage a de"cit in CoQ10 status may in theory result in multiple ETC de"ciencies. In order to investigate this hypothesis we assessed the muscle CoQ10 status of 12 patients with combined ETC I (0.075 ± 0.006; ref interval: 0.104-0268), II-III (0.023 ± 0.004 ; ref interval: 0.040-0.204) and IV (0.006 ± 0.001; ref interval: 0.014-0.034) de"ciencies of as yet unknown ge-netic cause. ETC activities expressed as a ratio to citrate synthase activity. On assessment, 8 of the 12 patients showed evidence of decreased muscle CoQ10 status (82.0 ± 16.6 pmol/mg; ref interval: 140-580 pmol/mg).

In conclusion, evidence of decreased muscle CoQ10 status has been detected in a subset of patients with mul-tiple ETC de"ciencies. These results may have therapeutic implications.


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