Marni Falk, Convegno Mitocon 2015

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New perspectives in research & therapies for mitochondrial disease in the US: Roles of networks, databases & biobanks Marni J. Falk, M.D., FACMG Assistant Professor of Pediatrics Division of Human Genetics The Children’s Hospital of Philadelphia University of Pennsylvania Perelman School of Medicine Philadelphia, Pennsylvania, USA & Chair, Scientific and Medical Advisory Board United Mitochondrial Disease Foundation (UMDF)

Transcript of Marni Falk, Convegno Mitocon 2015

Page 1: Marni Falk, Convegno Mitocon 2015

New perspectives in research & therapies for mitochondrial disease in the US:

Roles of networks, databases & biobanks

Marni J. Falk, M.D., FACMGAssistant Professor of Pediatrics

Division of Human GeneticsThe Children’s Hospital of Philadelphia

University of Pennsylvania Perelman School of MedicinePhiladelphia, Pennsylvania, USA

&Chair, Scientific and Medical Advisory Board

United Mitochondrial Disease Foundation (UMDF)

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DISCLOSURES

Marni J. Falk, M.D. is

•Chair, Scientific and Medical Advisory Board & Member, Board of Trustees, United Mitochondrial Disease Foundation

• Organizer, Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium

• SAB Member, The Genesis Project

• Consultant, Mitobridge

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OUTLINE• Mitochondrial Disease Clinical Care & Research in US

– CHOP Mito-Genetics Diagnostic Clinic experience

• Crossing the line from clinical care to human research– Establishing local and/or national biospecimen repositories– Enrolling subjects in national registries and biobanks

• North American Mitochondrial Disease Consortium (NAMDC)• Mitochondrial Disease Community Registry (MDCR)• Mitochondrial Disease Sequence Data Resource (MSeqDR)

• Translational research with mito disease biospecimens– Etiology-based studies of genetics and metabolism– Subgroup specific pathophysiology and therapies

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n = 152 patients referred from 2008-2011 to CHOP Mito-Genetics Diagnostic Clinic

Ages 6 weeks to 81 years old.

CHOP Mito-Genetics Clinic: Referral Indication

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Genetic diagnoses made in 28% of cases referred for suspected mito disease

Diagnostic Yield “PRE” Next Gen Sequencing:

DEFINITE MITO DISEASE

-Defined molecular etiology 14%

-Biochemical findings without evident molecular etiology* 4%

PROBABLE/POSSIBLE MITO DISEASE

-mtDNA variant of unknown significance 5%

-Normal tissue biochemistry + no clear molecular etiology 29%

-UNLIKELY PRIMARY MITO DISEASE 39%

-PROVEN OTHER GENETIC DISORDER 9%

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Improved diagnostic yield from whole mtDNA genome sequencing

• Pathogenic mtDNA mutations identified in 19 patients (10 kindreds):

– tRNALEU 3243A>G heteroplasmy (3 patients)– tRNALEU 3288A>G heteroplasmy (6 patients) Hadjigeorgiop GM et al, 1999

– tRNALYS 8344A>G heteroplasmy (1 patient)– tRNASER(AGY) 12264C>T heteroplasmy (1 patient) Schrier SA et al, 2012

– tRNATRP 5537_5538insT heteroplasmy (2 patients) Santorelli FM et al,1997

– ND4 and ND6 11778G>A /14484T>C het/homo (3 patients) Brown MD et al, 2001

– ND4 11778G>A homoplasmy (1 patient)– ND5 13513G>A heteroplasmy (2 patients)

* mtDNA common mutation panel

• Potentially pathogenic mtDNA mutations (VUS) in 7 patients (5 kindreds):

– tRNATYR 5836A>G homoplasmy (2 patients)– ND2 4936C>T heteroplasmy (1 patient)– ND2 and ATP8 4960C>T /8472C>T homo/homo (2 patients) – ATP6 155A>T homoplasmy (1 patient)– COXII 7962T>C homoplasmy (1 patient)

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15 other genetic diagnoses made• Primary mitochondrial disease (2 patients)

– mtDNA deletion in muscle of 1 isolated CPEO patient– POLG-related disease in 1 kindred

• Non-primary mitochondrial disease (8 patients)– Molybdenum cofactor deficiency (MOCS2)– CPT2 deficiency– WFS1-related hearing loss– Myotonia congenita– Congenital myasthenic syndrome (CHRNE)– SEPN1-related myopathy– Ullrich muscular dystrophy (COL6A1 deletion/mutation)– Gitelman syndrome

• Chromosomal copy number abnormalities (5 patients)– MEF2C deletion (SNP array)– IL1RAPL2 deletion (SNP array)– 7.91 Mb deletion on chromosome 7q31.32q32.2 (SNP array)– 3-way unbalanced translocation (Karyotype/SNP array)– Isochromosome Xp (Karyotype)

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Gene-by-gene diagnostic approach in mito disease has limited success

• A dedicated Mitochondrial-Genetics Diagnostic Clinic improves the diagnosis of primary mitochondrial diseases

• Recognize “classic” but complex phenotypes

– mtDNA disorders > nDNA disorders

• Guide optimal utilization/interpretation of metabolic screening labs, genetic testing, and tissue biopsy studies

• Identify wide variety of phenotypically overlapping conditions

– Time, labor, and testing intensive• Few metabolic specialists to meet growing clinical demand

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MITO DISEASE DIAGNOSTICS IN THE ERA OF NGS

• Mitochondrial disease is highly heterogeneous in causes and features

‒ Traditional single gene testing has had limited diagnostic success

‒ Newer genomics technologies enable comprehensive and efficient testing for all known genetic causes in dual genomes

‒ >200 nuclear genes

‒ All 37 mtDNA genesm

‒ Diagnose >50% of complex mitochondrial diseases in one test*

‒ Novel disease gene discovery

• We have entered a computationally sophisticated molecular diagnostic age for understanding subclasses of mitochondrial disease**:

**Calvo S, Mootha R, Ann Rev Genom Hum Genet, 2010; **McCormick E et al, 2012, Disc Med

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NGS diagnostic approach comparison

• Sequence ~100+ known mito disease and related disease genes• Prior unsolved cases of infantile Leigh syndrome• 23% (13/60) diagnostic rate (Calvo S et al, Nature Genetics, 2010)

• Sequence ‘MitoExome’ of mitochondria-localized genes• Targeted capture of 1,381 nuclear genes + mtDNA genome• 291 OXPHOS patients (Calvo S et al, Sci Transl Med, 2011)• 24% (10/42) diagnostic rate for unsolved cases

• 47% extrapolated diagnostic rate to all cases

• Sequence whole nuclear exome• 38% (13/34) cases analyzed have clear genetic etiology (Baylor)

– Only analyze exome of probands, not of family members– Not include analysis of mtDNA genome

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Emerging diagnostic approach for suspected mitochondrial disease

• Careful clinical evaluation/ phenotype description– History and Exam– Pedigree– Blood/urine metabolic screening laboratory studies– Tissue analyses in some

• mtDNA whole genome sequence analysis

+ mtDNA deletions, mtDNA copy number

• nDNA copy-number alterations (genome-wide SNP array)

• nDNA exome capture/Next Gen sequencing analysis

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OUTLINE• Mitochondrial Disease Clinical Care & Research in US

– CHOP Mito-Genetics Diagnostic Clinic experience

• Crossing the line from clinical care to human research– Establishing local and/or national biospecimen repositories– Enrolling subjects in national registries and biobanks

• North American Mitochondrial Disease Consortium (NAMDC)• Mitochondrial Disease Community Registry (MDCR)• Mitochondrial Disease Sequence Data Resource (MSeqDR)

• Translational research with mito disease biospecimens– Etiology-based studies of genetics and metabolism– Subgroup specific pathophysiology and therapies

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Challenges and benefits to PI of establishing a local biorepository

• Time-Intensive, Detail-Intensive, and Labor-Intensive– Genetic Counselor as common clinic & study coordinator– Local IRB establishment, approval, oversight/audits– Database establishment/maintenance/mining

• Excel REDCap– Local acquisition of tissue samples and establishment of cell lines and

derived materials

• High potential pay-off– High local knowledge of subject phenotypes & prior testing– Rich material for translational research at discretion of PI

• Genetic etiology, Pathophysiology, and Therapeutic modeling

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>600 mito disease + control subjects enrolled since 2008 in CHOP IRB-approved

tissue research study protocolMITO DISEASE

CATEGORYLCL FCL Muscle KNOWN DIAGNOSES

Definite 21 18 5

mtDNA mutations (MELAS, MERRF, LHON, other tRNAs, CI subunits)

nDNA mutations (POLG, MPV17, RRM2B, TIMM44 , SLC25A12 )

Probable/Possible 14 17 1mtDNA variant of ? pathogenicity,

RC deficiency of ? etiology, Lactic acidemia

Other Disease 3 8 5

PDH or PC deficiency, SEPN1 mutation, MEF2C deletion,

NMNAT1 mutation

Healthy Control 14 14 10 -

Blood DNA Blood RNA Muscle DNA Muscle RNA FCL DNA FCL RNAEXTRACTED

SAMPLE #33 19 47 28 26 29

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CHOP Bioinformatics efforts to improve patient-linked registry and tissue biorepository

• Sample/Data Tracking LIMS

• Manage PI Level data REDCap

• Clinical Data EPIC

• Clinical Trial Management Oncore

• Data Query + Exploration Harvest

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Incorporate Phenotype Capture & Display Tools

• REDCap– Research electronic data capture tool– Free, web-based, clickable data entry– Custom design tools to capture any desired data type

• Common data elements (CDE) optimized for mitochondrial disease• Integrate with NAMDC data capture tools and fields

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REDCap-based Mito Disease Data Capture

Claire Sheldon, MD, PhD, Elizabeth McCormick, MS, Jeff Miller, PhD

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Central Issues in Multi-Investigator Biorepository• Trust

– Data quality: accurate and updated information entered?– Sample quality: handling and shipment protocols?– Ongoing resource funding and sustainability?

• Incentive– Why and what should local PIs contribute from own tissue repositories?– Local PI support mechanism for samples acquisition and data entry?– Ongoing, widely available source of relevant human disease subjects + tissues

• Unequal burden:benefit ratio for established vs new investigators?

• Data and sample governance/access– How will data be stored and accessed? – What are hurdles and costs to obtain samples out of repository?– Should/how will contributing PI be acknowledged in resulting publications?

• Data transparency and systems ease-of-use– What samples are in biorepository, available for study and by whom?

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North American Mitochondrial Disease Consortium (NAMDC)

• Established in 2011, now 17 sites in US + Canada– NICHD/NINDS R01: PI – Dr. Michio Hirano (Columbia University, NY)

• Major goals:1.Mito Disease Patient Registry (790 enrolled by June 1)

A. Natural history studies (IND pre-requisite)B. Patient base for clinical trialsC. Detailed phenotypic information requested from physician

2.Mito Disease Specimen and DNA BiorepositoryA. Central IRB protocol biorepository at Mayo Clinic (MN)B. Any tissue type accepted

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Mitochondrial Disease Community Registry

Who? Patients, caregivers & family membersConfirmed diagnosis is NOT required

Why? Need patient data collected over time in order to improve diagnoses and develop treatments

Key Considerations:Registrants fully control privacy settings

• Allow, deny or “ask me”• Who can see anonymous data• Who can analyze anonymous data• Who can contact you about

research studies and clinical trials

www.umdf.org/registry

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Mitochondrial Disease Community Registry Key Points

• MDCR is sponsored by UMDF, but meant to be a community asset– UMDF pledges to steward the project and serve as guardian of collected

data

• MDCR seeks input from patients, caregivers and relatives of those affected by mitochondrial disease– Living or deceased / Any location– Confirmed diagnosis NOT required

• MDCR is meant to collect health information over time– Surveys will be presented on a regular basis– No demands or expectations- participate when you can and want

• MDCR is not meant to replace any other registry– Each registry has unique goals and capabilities

• Registrants are in full control of privacy settings– Adaptable settings about who may see and do what with your information

Philip Yeske, PhDUMDF Science and Alliance Officer

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Mitochondrial Disease Community RegistryStatus & Future Directions

Present:•~1000 accounts, ~1200 health profiles, ~120K data points (questions answered)•First survey: basic demographic info, diagnostic state, opinions on state of mitochondrial research and future MDCR directions•If already registered, please sign in again and confirm first survey complete

Near Future:•Creation of FAQ based on community feedback•Selection of “Navigators”- peers willing to help peers with registry•Additional surveys presented on topics of interest

In-Planning:•Uploading and sharing of genetic data (whole exome sequencing)•Importation of Electronic Health Records•International Engagement

Philip Yeske, PhDUMDF Science and Alliance Officer

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Mitochondrial Disease Sequence Data Resource (MSeqDR)

MSeqDR is an international mitochondrial disease community collaborative effort to create a unified genomic data resource that facilitates diagnosis andenables improved understanding of individual mitochondrial diseases

https://mseqdr.org is a central entry for clinicians, diagnostic labs, & researchers to enable genomic data sharing and analyses in suspected mitochondrial disease

– Flexible, updated suite of web-based and open access software tools accessible from your office/clinic desktop to securely mine all genetic & exome data in real-time

•Exploit collective information of variant allele frequencies in a large cohort of individuals with suspected mitochondrial disease (gem.app, G-browse, etcsg)•Will be linked to relevant phenotype & laboratory data•Accelerate pace and accuracy of known & novel gene discovery in mito disease

– Genomic data deposition for individual and/or community mining» Deposit aggregate-level deidentified exome or variant data to share at various

levels of comfort (BioDAS Server)» Patient-determined deposition & access to exome and phenotype data» Assist with data curtain & transfer to public resources (ClinVar, NCBI)

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https://mseqdr.org

Xiaowu Gai, PhD, Lishuang Shen, PhD

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https://mseqdr.org

Falk MJ et al, Mol Gen Metab, 2015

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MSeqDR GBrowse• Visualization of variants in both nuclear & mitochondrial (mtDNA) genomes• Hosts custom tracks for mitochondrial disease community

MSeqDR LOVD• Locus specific database for all mitochondrial disease genes and all genes that

encode mitochondrial proteins• Curates gene, transcript, variant, and disease data relevant to mitochondria

MSeqDR-GEM.app• Web-based repository and tool to readily enable analysis of sequence data from

gene panels, exomes, genomes, and mtDNA genomes• Supports analysis of data from individuals, families, or cohorts

MSeqDR Tools• Centralized host and link to public and custom tools that enable users to perform

dataset and variant level analyses in both nuclear & mtDNA genomes• Provides support to phenome and ontology tools for mitochondrial disease

HBCR: Human BP Codon Resources

Mitochondrial Disease Sequence Data Resource: Major Domainshttps://mseqdr.org

Falk MJ et al, Mol Gen Metab, 2015

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• MSeqDR tools technical optimization and response to community feedback

– GUID system implementation and assignment to all data types

– Phenotype data integration (existing vs new data)• Integrate NINDS Mito Disease Common Data Element Terms• HPO ontology tree-like structure

– Match degree of phenotype data shared to user access rights

– Further integration with GEM.app (GENESIS Project)

– Further integration with NIH ClinGen and NCBI (dbGAP, ClinVar)

• Ethical use and oversight– Data security protections (aggregate data, cloud computing)– Develop web portal to directly deposit deidentified phenotype data

• Translate access page into different languages– Develop data access and use oversight committee

• Clinical diagnostic labs, researchers, physicians, family support groups, etc.

MSeqDR “go-live” preparation underway

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MSeqDR Live Hands-On Tutorials

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MSeqDR User Analytics

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Acknowledgements

FUNDINGUnited Mitochondrial Disease Foundation

NAMDC Pilot Grant Award #NAMDC7407 (NINDS/NICHD, NIH)

U01-HG006546 (NHGRI, NIH)

U41-HG006834 (NHGRI, NIH)

[email protected]

MEEI/HarvardXiaowu Gai, PhD

Lishuang Shen, PhD

University of MiamiStephan Zuchner, MD

Michael Gonzalez, PhD

NICHD, NIHDanuta Krotoski, PhD

Melisa Parisi, MD, PhD

UMDFChuck Mohan, CEO

Dan Wright, PresidentPhilip Yeske, PhD

Janet OwensCliff Gorski

CHOPClaire Sheldon, MD, PhD

Elizabeth McCormick, MS, CGC

MSeqDR PROTOTYPE DEVELOPMENT PARTICIPANTS:• Doug Wallace, Michio Hirano, Doug Kerr, Curt

Scharfe, Li Dong, Hakon Hakonarson, Bruce Cohen, Amy Goldstein, Richard Haas, Russell Saneto (USA)

• Marcella Attimonelli, Mannis van Oven (Italy)• Holger Prokisch (Germany)• Mark Tarnopolsky, Isabella Thiffault (Canada)• Richard Rodenburg, Jan Smeitink, IFM de Coo, Bert

Smeets, Fons Stassen (The Netherlands)• Virginia Brilhante (Finland)• Yasushi Okazaki (Japan)• Donna Maglott, Wendy Rubinstein (NCBI)• Heidi Rehm (ClinGen)• Clinical diagnostic laboratories:

• Jeana DaRe, David Ralph (Transgenomics)• Renkui Bai, Sherri Bale (GeneDx)• Richard Boles, Christine Stanley (Courtagen)

Page 31: Marni Falk, Convegno Mitocon 2015

OUTLINE• Mitochondrial Disease Clinical Care & Research in US

– CHOP Mito-Genetics Diagnostic Clinic experience

• Crossing the line from clinical care to human research– Establishing local and/or national biospecimen repositories– Enrolling subjects in national registries and biobanks

• North American Mitochondrial Disease Consortium (NAMDC)• Mitochondrial Disease Community Registry (MDCR)• Mitochondrial Disease Sequence Data Resource (MSeqDR)

• Translational research with mito disease biospecimens– Etiology-based studies of genetics and metabolism– Subgroup specific pathophysiology and therapies

Page 32: Marni Falk, Convegno Mitocon 2015

VALIDATION OF GENETIC ETIOLOGY AND

UNDERLYING PATHOPHYSIOLOGY

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A lot of research is needed to validate novel mutations for suspected mito disease

• Traditional Sanger sequence validation– Confirm mutation presence and segregation with disease in family

• Functional analyses if novel mutation and/or disease gene– Is this clinical or research?

• What does it take to make variant “medically actionable”?

– Enzyme activity assay if known enzyme– Should other mito function(s) be assayed in tissue or cell lines?

• RC enzyme activity• Oxidant effects• Mitochondria content• mtDNA content• Mitochondrial membrane potential

– Gene rescue experiment in patient’s cells?– Transmitochondrial cybrids if novel mtDNA variant?

Page 34: Marni Falk, Convegno Mitocon 2015

149,953(130,948/19,005)

20,828(20,468/360)

10,936

Synonymous

11,179(10,819/360)

Non-Synonymous

CodingTotal(SNPs/Indels)

212 genes

816(797/19)

18(17/1)

4(4/0)

Gene Candidates

Novel

Family-based whole exome sequencing: Disease diagnosis becomes a computer game

Biparental compound

heterozygous

MitoCarta

8genes

2genes

1gene

2(2/0)

Predicted pathogenic

Sequence Variants

Case 1: Young girl with Leigh syndrome, chronic lactic acidosis (3-5 mM), complex I/III deficiency. POLG heterozygote. Normal SNP array. Normal sequence of mtDNA genome and 18 individual nuclear genes. Only child, no family history of disease.

Xiaowu Gai, PhD, Eric Pierce, MD, PhD

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Visualization of next generation sequencing mutationsMutation #2:

G>C transversion (p.N251K)112 of 231 reads

(maternally inherited)

Mutation #1: G>T transversion (p.P308Q)

29 of 63 total reads(paternally inherited)

Xiaowu Gai, Stephen Dingley

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ControlFCL

RED: Mito Marker(anti-CcO subunit IV)

GREEN: anti-FLAG (Tagged CcO, subunit Vb)

ProbandFCL

Satish Srinivasan, PhD

Case 1: Mito morphology and protein import is defective in the patient’s skin cells

YELLOW: overlay

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Page 38: Marni Falk, Convegno Mitocon 2015

Human Mito Disease Subject Fibroblasts

Zhang Z et al, PLOS ONE, 2013

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Cytosolic and mitochondrial translation are differentially affected in human RC disease

Zhang Z et al, PLOS ONE, 2013

Heterogeneous RC diseases,human muscle + FCLs

Public transcriptome datasets (GEO)

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Zhang and Falk, IJBCB, 2014

RC dysfunction dysregulates central nodesof the nutrient-sensing signaling network to mediate downstream cellular response

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DEVELOPMENT OF PERSONALIZED DISEASE

THERAPIES

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WT CI CIV CI/III

Primary RC disease patient fibroblasts have variable “phosphokinase” node profiles

Mai Tsukikawa, MS

( p38/ ERK1/2)

mTORC1(Raptor/GβL)

AKT1/2PKB

TSC1/2 Rheb

•BCAA•Excess nutrientsmTORC2

(Rictor/GβL)

•Protein Synthesis•ribosome biogenesis•mRNA translation

•Cell growth•Autophagy inhibition

4E-BP1S6K1

S6IRS-1

JNKFOXO1

MAPK

Insulin

•Growth Factors

NRF1/2

ERBB

PDK-1

PPARγ

AMPKACC

PGC-1α

TFAM

SIRT1•NAD+

•↑AMP or ↓ATP•Reductive stress•Hypoxia•Metformin•AICAR

•Rapamycin

•Nicotinic Acid

•mtDNA-encoded OXPHOS subunits

•MITO Biogenesis & OXPHOS•nDNA-encoded RC subunits

•Antioxidant defense •Glutathione synthesis

•P450 metabolism•Heme Biosynthesis

P

P

P

P

GSK-3

•Glycogen Synthesis

P

P

PP

PP

•Rosiglitazone•Fibrates

•Lipid synthesis

•Lipogenesis

•Resveratrol

•Longevity•Glycolysis

•Fatty acid oxidation•Stress Response

•Pathogen Resistance

•NORMAL MITOCHONDRIAL FUNCTION

P

•Glucose

PI3-K

P

P

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Directly inhibiting cytosolic translation rescues rotenone-induced cell death

in a variety of cell types

0

25

50

75

100

1 day 2 days 3 days 4 days 5 days 6 days

Control

ROT

ROT + CHX

Cell

Dea

th P

erce

nt(%

)

0

20

40

60

80

100

120

day 1 day 2 day 3 day 4 day 5 day 6

Control

ROT

ROT + CHX

ROT + ASM

ROT + ATM

Cell

Dea

thof

Per

cent

(%)

0

25

50

75

100

Control 12.5nM ROT

25nM ROT

50nM ROT

100nM ROT

Control + CHX

1.25nM ROT + CHX

25nM ROT + CHX

50nM ROT + CHX

100nM ROT + CHX

Cell

deat

h pe

rcen

t(%

)

A

C D

B

Podocytes11 mM glc

0

25

50

75

100

1 day 2 days 3 days 4 days 5 days 6 days

Control

ROT

ROT + CHX

Cell

Dea

th P

erce

nt(%

)Fibroblasts5.6 mM glc

Podocytes(48 hrs)

11 mM glc

(100 nM)

(1.8 uM)

HeLa5.6 mM glc

(125 nM)

(3.6 uM)

(1.8 uM)

(1.8 uM)

(1.8 uM)

(40 nM)

(50 nM)

Peng M et al, Human Molecular Genetics, In press

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Cycloheximide maintains total cellular respiratory capacity in direct RC inhibition

0

20

40

60

80

ROUTINE LEAK ETS

CONTROL

50nM ROT

ROT + CHX

Oxy

gen

flux

per m

ass (

pmol

.s-1

.mg-

1 )

******

******

**

**

0

20

40

60

80

ROUTINE LEAK ETS

control

50nM AA

AA + CHX

Oxy

gen

flux

per m

ass (

pmol

.s-1

.mg-

1)

*****

NS

** *

NS

0

20

40

60

80

ROUTINE LEAK ETS

control

0.25uM Oligo

Oligo + CHX

Oxy

gen

flu

x p

er m

ass

(pm

ol.s-

1.m

g-1 )

***NS

***

* *

NS

CI CIII

CVPeng M et al, Human Molecular Genetics, In press

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0

10,000

20,000

30,000

40,000

50,000 TMREM

ean C

ell F

luor

esce

nce

(RFU

)

Control 25 nM 50 nM 25 nM 50 nM CHXROT ROT ROT ROT

+ CHX + CHX

 

0.0E+00

4.0E+05

8.0E+05

1.2E+06

1.6E+06

2.0E+06

Control 50nMRotenone

ROT-0.9uMCHX

Mea

n Ce

ll Fl

uore

scen

ce(R

FU)

MitoTracker Green

# *

 

  1.0E+061.1E+061.2E+061.3E+061.4E+061.5E+061.6E+061.7E+061.8E+061.9E+062.0E+06

F35 Q1007 Q1007-0.9uM CHX

MitoTracker Green

Mea

n Ce

ll Fl

uore

scen

ce(R

FU)

Control FBXL4 FBXL4+ CHX

* ***

50,000

60,000

70,000

80,000

90,000

100,000

110,000

Q1007 control Q1007-0.9uM chx

Mea

n Ce

ll Fl

uore

scen

ce MitoSox

*

FBXL4 FBXL4+ CHX

Peng M et al, Human Molecular Genetics, In press

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H+NADH + H+NAD + 2+

H+FAD + 2+FADH

2

TC A CYCLE

Respiratory Chain Functions

III

V

Cyt C

III IVH+

H+

e- e-

H+2 + ½ O2

H O2

H+H+

H+H+

H+H+

H+

H+

H+H+H+ H+H+

CoQ

ADP + P ATP

IMM

Matrix

e-

e-

V

mTORC1S6

AMPKP

P

Mitochondrion

Lysosome

Ribosome

Ribosome

MITOPHAGY

ProteotoxicStress

CHX

RAPAProbucol

LiCl,3-MA

Probucol

[c]

[1]

[4]

[d]

[5]

[a]

[3]

[b]

[2]

Peng M et al, Human Molecular Genetics, In press

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Zhang Z et al, PLOS ONE, 2013

Nicotinic acid normalizes mTORC1 & AMPK activities, NADH/NAD+ levels, and total cellular respiratory

capacity in ND4/ND6 human fibroblasts

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CONCLUSIONCharacterizing and therapeutically targeting central alterations in the nutrient-sensing signaling network

may offer a personalized means to modify global sequelae downstream of OXPHOS dysfunction and

improve health outcomes in primary RC disease

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New Model for Getting to Effective Therapies for Mitochondrial Diseases

In Vitro Laboratory Drug testing in Mito Disease

-Patients’ cells (Fibroblasts vs Tissue-specific)-Genetic models of RC disease

-Integrated physiologic endpoints-Toxicity studies

Disease Definition

-Phenotype + Function-Biochemical

-Organelle-Genetic etiology

-Molecular Pathway

Outcome Prioritization

-Organ system-Pathophysiology

-Function-Biomarker

Treatment Options

-Off-purpose FDA drugs-Medical Foods

-Dietary Supplements-Vitamins

-New drugs from Pharma

Standard of Care

Clinical Trials

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Acknowledgements

University of PennsylvaniaRui Xiao, PhD

David L. Gasser, PhD Eiko Nakamaru-Ogiso, PhD

Joseph Baur, PhD

FUNDINGR01-HD065858 (NICHD, NIH)R03-DK082521 (NIDDK, NIH)R01-DK055852 (NIDDK, NIH)

IDDRC New Investigator Award, NICHDPhiladelphia Foundation

Tristan Mullen FundAngelina Maio Fund

Kelsey Wright FoundationJuliet’s Cure Mitochondrial Disease Research FundCenter for Mitochondrial & Epigenomic Medicine

CHOPJim (Zhe) Zhang, PhD

Marc Yudkoff, MDEric Rappaport, PhD Michael Bennett, PhDDouglas Wallace, PhD

Colleen Clarke, MS, CGC Arizona State UniversitySid Hecht, PhD

Omar Khdour, PhD

FALK LAB