Human Molecular Genetics IV. Genetics of common diseases/ Multifactorial genetics
1 Using Genetics to Understand Diseases & Tailor Treatment.
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Transcript of 1 Using Genetics to Understand Diseases & Tailor Treatment.
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Using Genetics toUsing Genetics toUnderstand Diseases & Understand Diseases &
Tailor TreatmentTailor Treatment
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Dr. Ameet PatkiDr. Ameet Patki M.D., D.N.B., F.C.P.S., F.R.C.O.G. (U.K.)M.D., D.N.B., F.C.P.S., F.R.C.O.G. (U.K.)
Medical DirectorMedical DirectorReReGenesis, Genesis, Reliance Life Sciences Reliance Life Sciences
Centre for Assisted Reproduction, Endoscopy Centre for Assisted Reproduction, Endoscopy
and Fetal Medicine Mumbaiand Fetal Medicine Mumbai
Consultant Obstetrician & Gynecologist Consultant Obstetrician & Gynecologist Sir Harkisondas Hospital & Research Centre, MumbaiSir Harkisondas Hospital & Research Centre, Mumbai
Hon. Assoc. Prof. Hon. Assoc. Prof. K.J. Somaiya Medical College and Hospital, Mumbai K.J. Somaiya Medical College and Hospital, Mumbai
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Genetics - Scientific Karma?Genetics - Scientific Karma?
While we all make decisions that define our future, While we all make decisions that define our future, we always make them based on who we were born.we always make them based on who we were born.
I have my father's laugh but my mother's sense of humor!
My mother's temperament and my father's temper!
My father's mouth, with my mother's taste!
We are who we are when we're born!We are who we are when we're born! A concoction of the family that came before us
Hopefully a little more than just the sum of their parts
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Understanding GeneticsUnderstanding Genetics
Genetics is the science of hereditary & variation.Genetics is the science of hereditary & variation.
The foundation level is the The foundation level is the molecule called DNA molecule called DNA
The information in DNA is The information in DNA is organized into Genes organized into Genes
Genes, in turn, make up Genes, in turn, make up Chromosomes - GenomeChromosomes - Genome
Every cell in an individual contains Every cell in an individual contains the genomethe genome
What is the Human Genome?What is the Human Genome?
There are twenty-three chapters, called There are twenty-three chapters, called chromosomeschromosomes
Each chapter contains several thousand stories, Each chapter contains several thousand stories, called genescalled genes
Each paragraph is made up of words, written in Each paragraph is made up of words, written in letters called basesletters called bases
There are 1 billion words and 3 billion letters in the There are 1 billion words and 3 billion letters in the book which makes it longer than… 800 Bibles."book which makes it longer than… 800 Bibles."
The genome is a genetic instruction book for human biology
The order of these letters will dictate whether an organism is a The order of these letters will dictate whether an organism is a human or another species, such as a fruit fly or mouse.human or another species, such as a fruit fly or mouse.
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What is the Human Genome?What is the Human Genome?
It is a history book that contains a record It is a history book that contains a record of our relatedness to other members of of our relatedness to other members of our species and to all other living things our species and to all other living things on the planeton the planet
It is a parts list for developing a human It is a parts list for developing a human body from sperm and eggbody from sperm and egg
It is a medical textbook that contains the It is a medical textbook that contains the clues for the understanding, prevention clues for the understanding, prevention and cure of diseaseand cure of disease
This book of 3 billion DNA letters is actually This book of 3 billion DNA letters is actually three books in one three books in one
All the information in every book ever written if translated All the information in every book ever written if translated into DNA, could fit easily in a teaspoon.into DNA, could fit easily in a teaspoon.
DNA Unlocked thethe Key to LifeThe discovery of the double helix by James Watson(25) The discovery of the double helix by James Watson(25)
and Francis Crick (36) on Feb. 28, 1953, unveiled and Francis Crick (36) on Feb. 28, 1953, unveiled “The Secret of Life." “The Secret of Life."
DNA is the molecule that makes and maintains all lifeDNA is the molecule that makes and maintains all life It enables life to re-create itselfIt enables life to re-create itself It contains the blueprints and the toolbox for It contains the blueprints and the toolbox for
understanding how humans workunderstanding how humans work
DNA is both bricks and blueprint - an engineer's dream.DNA is both bricks and blueprint - an engineer's dream.
50 Years on : The Double Helix Twists & Turns50 Years on : The Double Helix Twists & Turns The three billion rungs are made up of The three billion rungs are made up of
chemical units, called "base pairs,“chemical units, called "base pairs,“ A, T, C and GA, T, C and G Particular combinations of these DNA Particular combinations of these DNA
base pairs (or genes) constitute coded base pairs (or genes) constitute coded instructions for the formation of proteins, instructions for the formation of proteins, which make up the body and govern its which make up the body and govern its biological functioning (examples of biological functioning (examples of proteins include insulin, collagen, proteins include insulin, collagen, digestive enzymes)digestive enzymes)
50 Years on : The Double Helix Twists & Turns50 Years on : The Double Helix Twists & Turns
Human DNA looks like a twisted ladder with three billion rungs
If unwound, your DNA would stretch over five feet, but it is only 50 trillionths of an inch wide
The total amount of DNA in the 100 trillion or so cells in the average‑sized human body laid end to end would run to the sun and back some twenty times
The DNA in a chromosome is so densely packed that it can be The DNA in a chromosome is so densely packed that it can be upto 100,000 times longer than the chromosome itself.upto 100,000 times longer than the chromosome itself.
50 Years on : The Double Helix Twists & Turns
The three billion rungs are made up of chemical units, called "base pairs,“
A, T, C and G Particular combinations of these DNA base
pairs (or genes) constitute coded instructions for the formation of proteins, which make up the body and govern its biological functioning (examples of proteins include insulin, collagen, digestive enzymes)
50 Years on : The Double Helix Twists & Turns50 Years on : The Double Helix Twists & Turns
The strands of life are like a sticky zip.
What The Double Helix Cannot Tell YouDNA may be the script of life, but some scenes are
largely improvisedA pregnant mother's influence on her
baby
The differences between identical twins
The fleeting liaisons of proteins in a cell
All seem to arise without direction from DNA's All seem to arise without direction from DNA's sequencesequence
Heart disease (13.7%) Heart disease (13.7%) Stroke (9.5%) Stroke (9.5%) Pneumonia (6.4%)Pneumonia (6.4%) HIV/AIDS (4.2%)HIV/AIDS (4.2%) COPD (4.2%)COPD (4.2%) Diarrhea (4.1%)Diarrhea (4.1%) Perinatal (4.0%)Perinatal (4.0%) Tuberculosis (2.8%)Tuberculosis (2.8%) Trachea/bronchus/lung cancer (2.3%)Trachea/bronchus/lung cancer (2.3%) ? Traffic accidents (2.2%)? Traffic accidents (2.2%)
9 of the WHO’s 10 leading causes of 9 of the WHO’s 10 leading causes of global deaths have genetic componentsglobal deaths have genetic components
How to Conquer a Genetic DiseaseNearly 4,000 genetic diseases afflict human Nearly 4,000 genetic diseases afflict human
beingsbeings Which altered gene causes Which altered gene causes
the disease? the disease?
What protein does this gene What protein does this gene normally produce? normally produce?
Can the altered protein or Can the altered protein or gene be fixed or replaced? gene be fixed or replaced?
The Old GeneticsThe Old GeneticsWas about conditions wholly caused by
An extra or missing complete An extra or missing complete chromosome or part of a chromosomechromosome or part of a chromosome
Down Syndrome Down Syndrome (Trisomy -21)(Trisomy -21)
Turner Syndrome Turner Syndrome (XO)(XO)A mutation in a single geneA mutation in a single gene
Cystic Fibrosis Cystic Fibrosis ((mutations in CFTR gene)mutations in CFTR gene)
ThalassemiaThalassemia
Sickle cell diseaseSickle cell diseaseKaryotyping is used for studying changes in number and Karyotyping is used for studying changes in number and structure of chromosomes.structure of chromosomes.
Down’s Syndrome
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The New GeneticsThe New GeneticsA A “New Genetics" has emerged driven by “New Genetics" has emerged driven by
knowledge gained at the DNA levelknowledge gained at the DNA level
Diagnostic DNA testing
Prenatal DNA testing
Predictive DNA testingDiagnostic tools- Fluorescence In Situ Hybridization (FISH), Diagnostic tools- Fluorescence In Situ Hybridization (FISH), Polymerase Chain Reaction(PCR)Polymerase Chain Reaction(PCR)
Genetic Diagnostic TestsKaryotyping Detection of chromosome abnormalities after culture
FISH Rapid test for detection of certain chromosomal abnormalities
PCR Detection of Gene defects at the DNA level
Three techniques for diagnosis of genetic abnormalities.Three techniques for diagnosis of genetic abnormalities.
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Diagnostic DNA testingA patient presents with clinical features of a disorder, and A patient presents with clinical features of a disorder, and
a DNA test is undertaken to confirm a diagnosisa DNA test is undertaken to confirm a diagnosis
Human Immunodeficiency Virus Human Immunodeficiency Virus (HIV)(HIV) Hepatitis C Virus (HCV)Hepatitis C Virus (HCV) Hepatitis B Virus (HBV)Hepatitis B Virus (HBV) Cytomegalo Virus (CMV)Cytomegalo Virus (CMV) Tuberculosis (TB)Tuberculosis (TB)
- Infectious Disease Testing -- Infectious Disease Testing -
- Chronic Myeloid Leukemia- Chronic Myeloid Leukemia
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CMLCML
Philadelphia chromosome : t(9;22)(q34;q11)
Orange = Chr. 9
Green = Chr. 22
Comparative Genomic Hybridization (CGH)Comparative Genomic Hybridization (CGH)• Used in cancer research Used in cancer research • In solid tumors chromosome quality is poorIn solid tumors chromosome quality is poor• DNA of tumor & normal DNA labeled with 2 fluorescent DNA of tumor & normal DNA labeled with 2 fluorescent
colors are co-hybridized on a normal metaphasecolors are co-hybridized on a normal metaphase• Detects amplification & deletionsDetects amplification & deletions
Spectral Karyotyping (SKY)Spectral Karyotyping (SKY)
Multicolor FISH (m-FISH)Multicolor FISH (m-FISH)• Used to detect complex translocationsUsed to detect complex translocations
Newer Diagnostic testingNewer Diagnostic testing
Prenatal DNA TestingPrenatal DNA Testing
AmniocentesisAmniocentesis (15-17 wks)(15-17 wks)
Chorionic villus Chorionic villus sampling sampling (9-11 wks)(9-11 wks)
Fetal blood samplingFetal blood sampling (18-20 wks)(18-20 wks)
Amniocentesis
CVS
Preimplantation Genetic DiagnosisPreimplantation Genetic Diagnosis Early form of prenatal diagnosis Early form of prenatal diagnosis
bringing the hope of healthy babies bringing the hope of healthy babies to couples at risk of transmitting to couples at risk of transmitting genetic disorders to their offspring genetic disorders to their offspring by by NEGATIVE SELECTIONNEGATIVE SELECTION of the of the affected embryos prior to affected embryos prior to implantation. implantation.
No longer a boutique medicine No longer a boutique medicine (Simpson).(Simpson).
Embryology met Genetics and so was born this new Embryology met Genetics and so was born this new adjunct to IVF. adjunct to IVF.
Wide variety of disorders Chromosomal abnormalityChromosomal abnormality• Structural Structural (Translocation, Inversion, Deletion)(Translocation, Inversion, Deletion)
• NumericalNumerical (Trisomy, Monosomy)(Trisomy, Monosomy)
Single gene disorders Single gene disorders
Autosomal DisordersAutosomal Disorders• Dominant Dominant (Neuroblastoma) (Neuroblastoma)
• RecessiveRecessive ((-Thalassemia, Cystic Fibrosis)-Thalassemia, Cystic Fibrosis)
Sex linked disorders (Haemophilia, Muscular Dystrophy)
From single gene abnormality to extra chromosomes.From single gene abnormality to extra chromosomes.
Present Day Scope for PGD/PNDPresent Day Scope for PGD/PND
78bp250bp100bp50bp
1 2 3 4 5 6 7 8 9
250bp
50bp
861bp
242bp
285bp
PGD Facility
The Micromanipulator
Cleavage stage Embryo Biopsy
Polar Body Biopsy
FISH -Trisomy 18, X, Y
FISH - Polyploidy
PCR - Cystic Fibrosis F 508 Mutation
PCR - Thalassemia
PGD help patients “From Infertility to healthy family”.PGD help patients “From Infertility to healthy family”.
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Indication for PGD/PNDIndication for PGD/PND Advanced Maternal AgeAdvanced Maternal Age
Familial Association to specific Familial Association to specific diseases (Down’s syndrome, diseases (Down’s syndrome, Muscular dystrophy, Cytic fibrosis, Muscular dystrophy, Cytic fibrosis, Hemophilia)Hemophilia)
Medical history/ conditions Medical history/ conditions associated with genetic conditionsassociated with genetic conditions
Multiple miscarriagesMultiple miscarriages
History of previously affected babyHistory of previously affected baby
Predictive DNA testingPredictive DNA testingPredictive or presymptomatic DNA testing allows Predictive or presymptomatic DNA testing allows
genetic disorders to be detected in advance of genetic disorders to be detected in advance of clinical presentationclinical presentation
HNPCC HNPCC (Colon cancer)(Colon cancer)
Hereditary Nonpolyposis Colorectal CancerHereditary Nonpolyposis Colorectal Cancer
BRCA1 and 2 BRCA1 and 2 (Breast & Ovarian Ca)(Breast & Ovarian Ca)
MODY 1,2,3 MODY 1,2,3 (Diabetes)(Diabetes) Maturity Onset Type Diabetes in YoungMaturity Onset Type Diabetes in Young
Alpha-synuclein Alpha-synuclein (Parkinson’s disease)(Parkinson’s disease)
An ounce of testing could mean a pound of prevention.An ounce of testing could mean a pound of prevention.
The Human Genome Project (HGP)The HGP was an international research effort to The HGP was an international research effort to
decode the human genomedecode the human genome
Initiated in 1990 & completed Initiated in 1990 & completed two years prior to deadline in two years prior to deadline in 20032003
The human genome consists of over three billion chemical base pairs
Approximately 30,000-40,000 genes
Beyond the Human Genome Project
We will find ways for rational treatment, rational We will find ways for rational treatment, rational prevention & rational diagnostics.prevention & rational diagnostics.
Now that all genes are known, we will start Now that all genes are known, we will start understanding their function understanding their function PATHWAYSPATHWAYS
We will then be able to correlate disease We will then be able to correlate disease states to certain genes (Pathobiology)states to certain genes (Pathobiology)
DISEASEDISEASE GENE (S)GENE (S)GENE (S)GENE (S) DISEASEDISEASE
Promise of the Human Genome Project
Improve diagnosis & treatment through the application of Improve diagnosis & treatment through the application of genetic information & Technologiesgenetic information & Technologies
Predictive medicinePredictive medicine
PharmacogenomicsPharmacogenomics
Population screeningPopulation screeningGenomic Medicine is here…………..Genomic Medicine is here…………..
Transition from Genetics to GenomicsTransition from Genetics to Genomics
If genetics has been misunderstood, genomics is If genetics has been misunderstood, genomics is even more mysteriouseven more mysterious
Genetics is the studyGenetics is the study of hereditary & of hereditary & variationvariation““Genomics," a term coinedGenomics," a term coined only only 15 years ago, is the study of the 15 years ago, is the study of the functions and interactions of all functions and interactions of all the genes in the genomethe genes in the genome
Genomics is the study and identification of genes and Genomics is the study and identification of genes and gene function.gene function.
Genomic MedicineGenomic MedicineThe science of genomics rests on direct experimental The science of genomics rests on direct experimental
accessaccess to the entire genome & applies to conditions like:to the entire genome & applies to conditions like: Colon cancerColon cancer Breast cancerBreast cancer
Alzheimer diseaseAlzheimer disease
HIV infectionHIV infection
TuberculosisTuberculosis
AtherosclerosisAtherosclerosis
Inflammatory bowel Inflammatory bowel diseasedisease
DiabetesDiabetes
Parkinson’s diseaseParkinson’s disease
These disorders are due to the interactions of multiple genes and These disorders are due to the interactions of multiple genes and environmentalenvironmental factors. They are thus known as multifactorial factors. They are thus known as multifactorial disorders.disorders.
Impact of Genomic MedicineImpact of Genomic Medicine
Genomic medicine will change health care by : Genomic medicine will change health care by : Providing knowledge of individual Providing knowledge of individual
genetic predispositionsgenetic predispositions
Creating pharmacogenomicsCreating pharmacogenomics
Allowing population based screening Allowing population based screening for certain Mendelian disordersfor certain Mendelian disorders
The focus is not the treatment of disease but the The focus is not the treatment of disease but the eradication of the genetic problems that cause disease. eradication of the genetic problems that cause disease.
Knowledge of individual genetic predisposition will allow :
Individualized screeningIndividualized screening
Individualized lifestyle changesIndividualized lifestyle changes
Presymptomatic medical therapiesPresymptomatic medical therapies Anti-colon cancer agents before colon Anti-colon cancer agents before colon
cancer developscancer develops Antihypertensives before hypertension Antihypertensives before hypertension
developsdevelops Tamoxifen as a prophylactic for CA breastTamoxifen as a prophylactic for CA breast
Impact of Genomic MedicineImpact of Genomic Medicine
Pharmacogenomics will allow: Use of Individualized medication based on Use of Individualized medication based on
genetically determined variation in effects and genetically determined variation in effects and side effectsside effects
More powerful/safer medicines the first timeMore powerful/safer medicines the first time
More accurate methods for determining More accurate methods for determining medication dosesmedication doses
New medications for specific genotypic New medications for specific genotypic disease subtypesdisease subtypes
Drugs tailored to our individual genetic profiles would Drugs tailored to our individual genetic profiles would reduce overall medical costs.reduce overall medical costs.
Impact of Genomic MedicineImpact of Genomic Medicine
How Does Society Prepare for Genomics?How Does Society Prepare for Genomics?
Education to achieve understanding of :Education to achieve understanding of :
The basics of the science of geneticsThe basics of the science of genetics
The eventual use of genetics in health careThe eventual use of genetics in health care
How to deal with risk & predispositionHow to deal with risk & predisposition
The personal impact of genetic informationThe personal impact of genetic information
The social impact of geneticsThe social impact of genetics
Concerns with GenomicsConcerns with Genomics
Discrimination against individualsDiscrimination against individuals
Discrimination against groupsDiscrimination against groups
Genes run in familiesGenes run in families
Confidentiality/privacyConfidentiality/privacy
Tomorrow’s MedicineTomorrow’s Medicine
Genomics opens up new therapeutic options:Genomics opens up new therapeutic options:
The aim is to detect & target specific diseases & develop The aim is to detect & target specific diseases & develop more efficient and selective treatment.more efficient and selective treatment.
Drugs derived from genetic engineeringDrugs derived from genetic engineering When genes acts as drugs : gene therapyWhen genes acts as drugs : gene therapy Medicine made to measure : Medicine made to measure :
pharmacogenomicspharmacogenomics Predicting susceptibility to disease : diagnostic Predicting susceptibility to disease : diagnostic
teststests Vaccines & the treatment of infectious diseasesVaccines & the treatment of infectious diseases Designing babiesDesigning babies
By the double helix's 75th birthday, your genome By the double helix's 75th birthday, your genome might be as familiar as your shoe sizemight be as familiar as your shoe size
• In 25 years babies might have In 25 years babies might have genetic identity cardsgenetic identity cards
• This would include information from This would include information from an individual's genome sequencean individual's genome sequence
• Genetic ID cards could offer benefits Genetic ID cards could offer benefits for medical diagnosis, treatment and for medical diagnosis, treatment and preventionprevention
Genetics 25 Years HenceGenetics 25 Years Hence
The complete genetic makeup of individuals could soon The complete genetic makeup of individuals could soon be scanned and recorded on a smart card.be scanned and recorded on a smart card.
The Year 2028The Year 2028
As a doctor swipes her patient's As a doctor swipes her patient's genetic ID card, the information genetic ID card, the information downloads in secondsdownloads in seconds
A string of letters scrolls down A string of letters scrolls down her computer screenher computer screen
These are the raw data of DNA, the code of lifeThese are the raw data of DNA, the code of lifeIt seems reasonable to imagine genome sequencing It seems reasonable to imagine genome sequencing happening as a matter of routine in 25 years time.happening as a matter of routine in 25 years time.
There's no room for technophobes in the medical There's no room for technophobes in the medical professionprofession
For the patient, waiting anxiously, the news will be For the patient, waiting anxiously, the news will be mixedmixed
The results reveal that he carries a The results reveal that he carries a gene that increases his risk of cancergene that increases his risk of cancer
But should he fall ill, his genes also But should he fall ill, his genes also predict the best therapy -predict the best therapy -
A new tailor-made drug that brings with it an 80% A new tailor-made drug that brings with it an 80% chance of remission chance of remission
Could this be the future of medicine?Could this be the future of medicine?
The Year 2028The Year 2028
The drug is designed using genetic The drug is designed using genetic knowledgeknowledge
It targets an abnormal piece of DNA in It targets an abnormal piece of DNA in cancerous cells, killing them but sparing cancerous cells, killing them but sparing healthy cellshealthy cells
After a year's treatment, 90% of After a year's treatment, 90% of patients are free of disease, and 50% patients are free of disease, and 50% show a complete or near disappearance show a complete or near disappearance of the abnormal gene that triggers the of the abnormal gene that triggers the diseasedisease
Better Drugs in The Year 2028?Better Drugs in The Year 2028?
Mapping our DNA may help doctors to prescribe the Mapping our DNA may help doctors to prescribe the safest, most effective medicine.safest, most effective medicine.
Microarrays/ DNA Biochips in Microarrays/ DNA Biochips in Medical PracticeMedical Practice
Monitoring the Genome on a Chip.Monitoring the Genome on a Chip.
The chip, which consists of a small glass plate encased in The chip, which consists of a small glass plate encased in plastic, is manufactured somewhat like a computer plastic, is manufactured somewhat like a computer microchipmicrochip
On the surface, each chip contains thousands of On the surface, each chip contains thousands of immobilized DNA sequencesimmobilized DNA sequences
Microarrays allow thousands or tens of thousands of specific Microarrays allow thousands or tens of thousands of specific DNA or RNA sequences to be detected simultaneouslyDNA or RNA sequences to be detected simultaneously
The DNA microchip/ microarray is a revolutionary new The DNA microchip/ microarray is a revolutionary new tool used to identify mutations in genestool used to identify mutations in genes
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Applications of Microarray TechnologyApplications of Microarray Technology Gene discovery Disease diagnosis Drug discovery:
Pharmacogenomics Toxicological research:
Toxicogenomics Gene expression
profiling Genotyping DNA sequencing
Spotted DNA microarraySpotted DNA microarray
Currently only used Currently only used as a research tool.as a research tool.
The Future for Alzehimer Disease - 2010The Future for Alzehimer Disease - 2010 5 or 6 genetic variations identified that 5 or 6 genetic variations identified that
strongly predispose for Alzheimer disease; strongly predispose for Alzheimer disease; another 10 or 12 with weaker associationanother 10 or 12 with weaker association
Chip-based genetic test gives personal Chip-based genetic test gives personal likelihood of developing the conditionlikelihood of developing the condition
Chip-based genetic test identifies the drug Chip-based genetic test identifies the drug most likely to be effective for given most likely to be effective for given individualindividual
Chip-based genetic test determines Chip-based genetic test determines individual likelihood of drug side effectsindividual likelihood of drug side effects
Put most simply, it introduces a "good" gene into a Put most simply, it introduces a "good" gene into a person who has a disease caused by a "bad" gene.person who has a disease caused by a "bad" gene.
Gene Therapy-The Next Twist in the Genome TaleGene Therapy-The Next Twist in the Genome Tale
Gene Therapy for Parkinson's diseaseGene Therapy for Parkinson's disease Genetically modified Genetically modified
virus carries the DNA virus carries the DNA into the body into the body
It passes into the It passes into the nucleii of some brain nucleii of some brain cellscells
These cells can then These cells can then produce dopamineproduce dopamine
Gene therapy can be used to treat cancer, Gene therapy can be used to treat cancer, heart disease, Alzheimer's & Parkinson's disease.heart disease, Alzheimer's & Parkinson's disease.
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DNA Based Vaccines
The DNA vaccines are an offshoot of gene therapy.The DNA vaccines are an offshoot of gene therapy.
Technique involves the direct injection of Technique involves the direct injection of plasmids-loops of DNA that contain genes for plasmids-loops of DNA that contain genes for proteins produced by the organism being proteins produced by the organism being targeted for immunitytargeted for immunity
Once injected into the host's muscle tissue, Once injected into the host's muscle tissue, the DNA is taken up by host cells, which then the DNA is taken up by host cells, which then start expressing the foreign proteinstart expressing the foreign protein
The protein serves as an antigen that The protein serves as an antigen that stimulates immune responsesstimulates immune responses
If the body later encounters the organism If the body later encounters the organism carrying this antigen, its defenses are ready carrying this antigen, its defenses are ready to launch a protective attackto launch a protective attack
Clinical Trials with DNA Based VaccinesClinical Trials with DNA Based Vaccines
Expected to be particularly useful to prevent and treat Expected to be particularly useful to prevent and treat infectious diseases such as Herpes, Hepatitis, and AIDS. infectious diseases such as Herpes, Hepatitis, and AIDS.
The first clinical trials using injections The first clinical trials using injections of DNA began for HIV in 1995of DNA began for HIV in 1995
Four other clinical trials using DNA Four other clinical trials using DNA vaccines against influenza, herpes vaccines against influenza, herpes simplex virus, T-cell lymphoma, and an simplex virus, T-cell lymphoma, and an additional trial for HIV were started in additional trial for HIV were started in 19961996
A DNA vaccine for Malaria is being A DNA vaccine for Malaria is being developeddeveloped
Benefits of DNA Based VaccinesBenefits of DNA Based Vaccines
Traditional vaccines are expensive and take a long Traditional vaccines are expensive and take a long time to producetime to produce
DNA is relatively inexpensive and is easier to produce DNA is relatively inexpensive and is easier to produce
DNA vaccines are much more stable, allowing them to DNA vaccines are much more stable, allowing them to be easily transportedbe easily transported
With a live vaccine (Polio vaccine), there is always a With a live vaccine (Polio vaccine), there is always a danger of it reverting and becoming infective. DNA danger of it reverting and becoming infective. DNA cannot become infectivecannot become infective
Genetically Engineered Drugs Genetically Engineered Drugs
)
ProteinProtein IndicationIndication YearYear
InsulinInsulin DiabetesDiabetes 19821982
Human growth hormoneHuman growth hormone Growth deficiencyGrowth deficiency 19851985
alpha-interferonalpha-interferon Viral infectionsViral infectionsCancerCancer
19851985
Hepatitis B vaccineHepatitis B vaccine Vaccine against HepatitisVaccine against Hepatitis 19861986
Tissue plasminogen Tissue plasminogen activatoractivator
Cardiovascular diseaseCardiovascular disease 19871987
Erythro-poietinErythro-poietin AnaemiaAnaemia 19881988
Factor VIIIFactor VIII HaemophiliaHaemophilia 19931993
HerceptinHerceptin Breast CancerBreast Cancer 19991999
Designer Babies ? Designer Babies ?
The babies of the future could be protected from diseases.The babies of the future could be protected from diseases.
In the Brave New World predicted, scientists will do In the Brave New World predicted, scientists will do more than screen for disease free embryosmore than screen for disease free embryos
Parents will be able to reject their own genetic Parents will be able to reject their own genetic heritage, and have instead of plump -heritage, and have instead of plump -
Beautiful, Clever or Sporty genes Beautiful, Clever or Sporty genes implanted into their embryonic childrenimplanted into their embryonic children
Que Sera Sera …………
Whatever will be will be,
The future’s not ours to see,
Que Sera Sera,
What will be will be…………
The Past 25 Years………The Past 25 Years………
Significant advances in ART techniques
have revolutionized both male & female
factor infertility
History of ICSIHistory of ICSI
• 1992 1992 – – Palmero et al.,Palmero et al., responsible for the world’s responsible for the world’s first baby conceived with first baby conceived with ICSI. ICSI.
ICSIICSI• Bypasses the effective biologic mechanism of sperm Bypasses the effective biologic mechanism of sperm
selection. selection.
• Application to human reproduction has not been preceded Application to human reproduction has not been preceded by extensive research trials in mammals. by extensive research trials in mammals.
• Human experience with ICSI is the experimental record.Human experience with ICSI is the experimental record.
Cause of concern with ICSI:Cause of concern with ICSI:
Promotion of Promotion of transgenerational transgenerational transmissiontransmission of genetic defects to the of genetic defects to the offspring causing gametogenic failure.offspring causing gametogenic failure.
Male factor infertility Male factor infertility should be considered should be considered
as a as a potentially heritable potentially heritable
conditioncondition(Meschede et al., 2000)(Meschede et al., 2000)
ICSIICSI
Incidence of Chromosomal Aberration in Infertile Incidence of Chromosomal Aberration in Infertile oligozoospermic and azoospermic malesoligozoospermic and azoospermic males (Liebaers et al., In: Textbook of Assited Reproductive Techniques –
Chapter 24; 2001)AberrationsAberrations Infertile MalesInfertile Males
(n = 7876)(n = 7876)
Oligozoosermia Oligozoosermia
(n = 1701)(n = 1701)
AzoospermiaAzoospermia
(n = 1151)(n = 1151)
AutosomesAutosomes 1.3%1.3% 3.0%3.0% 1.1%1.1%
Sex Sex ChromosomesChromosomes 3.8%3.8% 1.6%1.6% 12.6%12.6%
TotalTotal 5.1%5.1% 4.6%4.6% 13.7%13.7%
Chromosome aberrations increase as sperm counts decreaseChromosome aberrations increase as sperm counts decrease
First trimester losses/major congenital First trimester losses/major congenital malformations in ICSI derived pregnanciesmalformations in ICSI derived pregnancies
Wisano et al., Hum. Reprod. 1996.Wisano et al., Hum. Reprod. 1996.
Borduelle, Devroey et al., Hum. Reprod. 1996. Borduelle, Devroey et al., Hum. Reprod. 1996.
Survey of 1455 children Survey of 1455 children
Largest American and European study. Largest American and European study.
Ejaculate sperms Epididymal sperms Testicular Sperms
First trimester loss 24.6% 31.2% 33.3%
2.2 % had variety of major congenital malformations.2.2 % had variety of major congenital malformations.
Areas of ConcernAreas of Concern• 13.7% Azoospermic males and 4.6% 13.7% Azoospermic males and 4.6%
oligozoospermic males have abnormal karyotypes. oligozoospermic males have abnormal karyotypes.
• Further, meiotic disturbances limited to Further, meiotic disturbances limited to spermatogenic cells are found in 6% of males with spermatogenic cells are found in 6% of males with severe OATsevere OAT
• Newer research indicates that children born with Newer research indicates that children born with ICSI for male infertility have twice the rate of major ICSI for male infertility have twice the rate of major congenital malformations. congenital malformations.
Hansen et al., N.Engl. J. Med. 2002; 346: 725-30. Hansen et al., N.Engl. J. Med. 2002; 346: 725-30.
Genetic concern associated with Genetic concern associated with Male Factor InfertilityMale Factor Infertility
Genetic AbnormalityGenetic Abnormality Resultant Reproductive Resultant Reproductive AbnormalityAbnormality
Y Chromosome MicrodeletionY Chromosome Microdeletion
(Multiplex PCR)(Multiplex PCR)
Azoospermia, severe oligozoospermiaAzoospermia, severe oligozoospermia
Cystic Fibrosis Gene MutationCystic Fibrosis Gene Mutation
(PCR)(PCR)
Congenital absence of Vas DeferensCongenital absence of Vas Deferens
Structural defects or AneuploidiesStructural defects or Aneuploidies
(Blood Karyotype and Sperm FISH)(Blood Karyotype and Sperm FISH)
Variable (Klinefeleter’s syndrome, XYY Variable (Klinefeleter’s syndrome, XYY Males)Males)
Genetic concern associated with Genetic concern associated with Male Factor InfertilityMale Factor Infertility
Genetic Abnormality Resultant Reproductive Abnormality
Y Chromosome Microdeletion
(Multiplex PCR)
Azoospermia, severe oligozoospermia
Y MicrodeletionThe how and Y of Male infertilityThe how and Y of Male infertility Y chromosome microdeletion common cause of Y chromosome microdeletion common cause of
spermatogenic failure.spermatogenic failure.
Y chromosome microdeletion first described in 1976 by Y chromosome microdeletion first described in 1976 by Tiepolo and Zuffardi. Tiepolo and Zuffardi.
IncidenceIncidence 15-20% of men with idiopathic 15-20% of men with idiopathic
azoopermiaazoopermia
7-10% of men with idiopathic severe 7-10% of men with idiopathic severe oligozoospermia oligozoospermia
Y Chr is paternally inherited from father to sonY Chr is paternally inherited from father to son
Y Chr DNADNA
Genic DNA : production of proteins Junk DNA : has little apparent
function 98% of Y Chr is Junk DNA 50-70% of Junk DNA on Y Chr
contain highly repetitive sequences.
Y Chr is a genetic junkyard as well as a gold mineY Chr is a genetic junkyard as well as a gold mine
Y Microdeletion
Multiplex PCRMultiplex PCR
• Simple, powerful and fast tool ideal for Simple, powerful and fast tool ideal for
screening of idiopathic infertile malescreening of idiopathic infertile male
• When used with rigorous procedures When used with rigorous procedures
reaches the accuracy of other reaches the accuracy of other
sophisticated techniques such as sophisticated techniques such as
Southern blot.Southern blot.
Y Chr microdeletionsY Chr microdeletions
MDS are clustered in 3 MDS are clustered in 3 main regions:main regions:
AZFa, AZFb, AZFcAZFa, AZFb, AZFc
15 novel genes identified 15 novel genes identified on Y Chron Y Chr
DFFRY, RBM, DAZ genes DFFRY, RBM, DAZ genes associated with male associated with male infertilityinfertility
Effect of deletions on Effect of deletions on extent of spermatogenesisextent of spermatogenesis
AZF a Lack of germ cells or sertoli cells only syndrome (SOCS)
AZF b Spermatogenic Arrest
AZF c Maturation arrest of post meiotic germ cells
Krausz and McElreavey, 1999Krausz and McElreavey, 1999
PCR for Y microdeletionsPCR for Y microdeletions
M = Molecular weight marker
A,B,C,D = Multiplex MMX
+ = Normal control
- = Negative control D/W
06 & 07 = Test patients
M +A K V -A +B K V -B +C K V -C +D K V -D
Del at DYS240 at AZFc RegionPromega Y chromosome Deletion Detection System, Version 1.1
038
A 038
B
038
C 038
D
MM
M = Molecular weight M = Molecular weight markermarker
A,B,C,D = Multiplex A,B,C,D = Multiplex MMXMMX
038 = Test patient038 = Test patient
Promega Y chromosome Promega Y chromosome Deletion Detection System, Deletion Detection System, Version 1.1Version 1.1
The absence of bands (for 038) in MMX The absence of bands (for 038) in MMX A, B, C and D indicates deletion of loci A, B, C and D indicates deletion of loci DYS237, DYS236 at AZFdDYS237, DYS236 at AZFd region region alongwith deletion alongwith deletion of entire DAZ gene of entire DAZ gene and locus DYS240 at AZFcand locus DYS240 at AZFc region. region.
PCR for Y microdeletionsPCR for Y microdeletions
Y-13 Translocation-FISH,KaryotypingY-13 Translocation-FISH,Karyotyping
FISHFISH
Green - XGreen - X Orange - YOrange - Y Blue - 18Blue - 18
45,X MetaphaseAzoospermic male
Normal features
Karyotype
13
X
18
13
18
08
5A 0
85
B
08
5C 0
85
D
The absence of bands (for 085) in MMX The absence of bands (for 085) in MMX A, B, C and D indicates deletion of loci A, B, C and D indicates deletion of loci
• DYS271, KALY DYS271, KALY at at AZFaAZFa region; region;
• DYS212, SMCY, DYS218, DYS219, DYS212, SMCY, DYS218, DYS219, DYS221 DYS221 atat AZFb AZFb region; region;
• DYF51S1, DYS237, DYS236 DYF51S1, DYS237, DYS236 atat AZFdAZFd region and region and
• Entire DAZ gene and locus Entire DAZ gene and locus DYS240 DYS240 atat AZFc AZFc region. region.
M = Molecular weight M = Molecular weight markermarker
A,B,C,D = Multiplex MMXA,B,C,D = Multiplex MMX
085 = Test patient085 = Test patient
Promega Y chromosome Deletion Detection System, Version 1.1
Our Data on Y chromosome MicrodeletionsOur Data on Y chromosome Microdeletions
IndicationIndication TotalTotal NormalNormal Deletion Deletion present in present in
Abnormal Abnormal %%
AzoospermiaAzoospermia 2525 1919 77 28%28%
Severe OATSevere OAT 3535 3131 44 11.43%11.43%
OATOAT 1313 1111 11 7.7%7.7%
TotalTotal 7373 6161 1212 16.44%16.44%
Our data coincides with the data Our data coincides with the data reported reported by clinics worldwideby clinics worldwide
Y Microdeletion
• Hence other genes outside the AZF and/or environmental factors may modulate the effects of AZF deletions.
• Possibility that affected males can produce normal counts during puberty and young adulthood. Hence sperms could be harvested for future use.
• Newer research indicates no correlation between the severity of spermatogenic defect and the localization and extent of the Yq deletions. Genotype-phenotype correlations difficult to establish.
Genetic concern associated with Genetic concern associated with Male factor InfertilityMale factor Infertility
Genetic AbnormalityGenetic Abnormality Resultant Reproductive Resultant Reproductive AbnormalityAbnormality
Cystic Fibrosis - Cystic Fibrosis - CFTR Gene CFTR Gene Mutation Mutation (PCR)(PCR)
Congenital absence of Vas Congenital absence of Vas DeferensDeferens
CF is a CF is a autosomal recessiveautosomal recessive disorder disorder Caused due to Caused due to mutations in CFTR genemutations in CFTR gene Current medical support has Current medical support has improved life improved life
expectancyexpectancy
.
Cystic Fibrosis (CF) & male infertilityCystic Fibrosis (CF) & male infertility
These individualsThese individuals
have have delayed pubertydelayed pubertyare generally are generally infertileinfertilehave have AzoospermiaAzoospermiaCongenital absence of vas deferens (CAVD)Congenital absence of vas deferens (CAVD)
PCR detects 85% of all mutationsPCR detects 85% of all mutations
Cystic FibrosisCystic Fibrosis
Product of cystic fibrosis gene is known Product of cystic fibrosis gene is known as as cystic fibrosis transmembrane cystic fibrosis transmembrane conductance regulator (CFTR) conductance regulator (CFTR) protein.protein.
F 508 prevents normal maturation of F 508 prevents normal maturation of the CFTR protein and causes the CFTR protein and causes failure of failure of its normal localization to the cell its normal localization to the cell membrane. membrane.
CBAVD and Cystic FibrosisCBAVD and Cystic Fibrosis
Before performing ICSI with sperm from Before performing ICSI with sperm from
CBAVD males with CFTR mutations, CBAVD males with CFTR mutations,
their partners should also be tested to their partners should also be tested to
avoid the risk of having a child avoid the risk of having a child
affected with Cystic Fibrosis.affected with Cystic Fibrosis.
Detection of Cystic Fibrosis mutationDetection of Cystic Fibrosis mutation
Common gene mutations in CAVD are: Common gene mutations in CAVD are: F508F508
• W1282 XW1282 X
• N1303 KN1303 K
• G542 X G542 X
• 1717G-A1717G-A
• R553XR553X
• R117HR117H
Our Data on C.F. Our Data on C.F. Number Other Abnormalities
No. of Patients analyzed (Male Infertility with CAVD)
8
Normal 7 One patient – Normal Karyotype, but presence of Y microdeletion
Carrier of F 508 mutation 1 XYY karyotype
Genetic concern associated with Genetic concern associated with Male factor InfertilityMale factor Infertility
Genetic AbnormalityGenetic Abnormality Resultant Reproductive Resultant Reproductive AbnormalityAbnormality
Structural defects or AneuploidiesStructural defects or Aneuploidies
(Blood Karyotype and Sperm FISH)(Blood Karyotype and Sperm FISH)
Variable (Klinefeleter’s syndrome, Variable (Klinefeleter’s syndrome, XXY Males)XXY Males)
Abnormal karyotype in men with Abnormal karyotype in men with severe male factor infertilitysevere male factor infertility
Series Men screened
Abnormal karyotype rate (%)
Baschat et al (1996) 32 6.2
Peschka et al (1996) 200 3.0
Testart et al (1996) 261 4.2
Pandiyan and Jequier (1996)
1201 3.6
Mau et al (1996) 150 12.0
Meschede et al (1998) 432 2.1
Karyotyping - Male contribution - 200 casesKaryotyping - Male contribution - 200 casesPrimary/Sec. infertility 71 (35.5%) : BOH/RSA in wife 129 (64.5%)Primary/Sec. infertility 71 (35.5%) : BOH/RSA in wife 129 (64.5%)
Chromosome Abnormalities : (4.5%)Chromosome Abnormalities : (4.5%)
Numerical abnormalities : (1.5%)Numerical abnormalities : (1.5%)
47,XXY47,XXY 2 cases 2 cases Primary infertilityPrimary infertility
47,XYY47,XYY 1 case1 case CAVDCAVD
Structural Abnormalities - Reciprocal translocations : (3%)Structural Abnormalities - Reciprocal translocations : (3%)
46,XY,t(7;13)(p11;q34)46,XY,t(7;13)(p11;q34) OATOAT
46,XY,t(8;10)(p23;q24)46,XY,t(8;10)(p23;q24) BOHBOH
46,XY,t(10;13)(q23.2;q34)46,XY,t(10;13)(q23.2;q34) " "
46,XY,t(11;16)(q21;p13.3) "46,XY,t(11;16)(q21;p13.3) "
45,X,t(Y;13)(q23;q11) "45,X,t(Y;13)(q23;q11) "
45,XY,t(13;14)(p11;q11)45,XY,t(13;14)(p11;q11) " "
Partial karyotypes showing balanced reciprocal Partial karyotypes showing balanced reciprocal translocations in 3 male partners of BOH cases.translocations in 3 male partners of BOH cases.
t(8;10)(p23;q24) t(10;13)(q23.2;q34)
t(7;13)(p11;q34)
Chromosome Variants (10%)VariantsVariants nn IndicationIndication
46,X,inv(Y)46,X,inv(Y) 3 3 OAT / BOHOAT / BOH
46,XY,inv(9)46,XY,inv(9) 22 Azoospermia / BOHAzoospermia / BOH
46,X,Yqh+46,X,Yqh+ 33 Polytailed sperm / BOH (2)Polytailed sperm / BOH (2)
46,X,Yqh-46,X,Yqh- 33 Primary Infertility (2)/ BOHPrimary Infertility (2)/ BOH
46,XY,9qh+46,XY,9qh+ 55 BOHBOH
46,XY,21ps+46,XY,21ps+ 22 Primary infertilityPrimary infertility
46,XY,22ps+46,XY,22ps+ 22 Primary infertilityPrimary infertility
Single cell mosaicism (2%)Single cell mosaicism (2%)
46,XY (1/50 cell with 47,XXY)46,XY (1/50 cell with 47,XXY) BOH (2 cases)BOH (2 cases)
46,XY {1 cell with t(7;14)(p12;q12)}46,XY {1 cell with t(7;14)(p12;q12)} OATOAT
46,XY{ 1cell with trisomy 21} BOH (2 cases)46,XY{ 1cell with trisomy 21} BOH (2 cases)
X and Y Sperm
Normal 13, 21 Disomy 18 XY Sperm
X and Y Sperm
Disomy 18 XY Sperm
Y bearing Sperm (Normal)
FISH on Sperm
Should Y chromosome analysis and genetic Should Y chromosome analysis and genetic counselling be offered to infertile male?counselling be offered to infertile male?
• 96% of couples choose Y chromosome 96% of couples choose Y chromosome testing when offeredtesting when offered
• Transmission of infertility to offspring is Transmission of infertility to offspring is weighed carefully by infertile couplesweighed carefully by infertile couples
- Rucker et al., 1998- Rucker et al., 1998
Genetic testing should become an Genetic testing should become an integral part of patient assessment integral part of patient assessment
and treatment planningand treatment planning
Genetic Counseling
All couples considering ICSI are offered All couples considering ICSI are offered counseling to inform of the potential counseling to inform of the potential
genetic risks to their possible genetic risks to their possible offspring's. offspring's.
Concerns on doing ICSI in infertile malesConcerns on doing ICSI in infertile malesY Chr microdeletions are passed to SonsY Chr microdeletions are passed to SonsAs a result the Sons are also infertile since they have As a result the Sons are also infertile since they have inherited the same genetic defect that rendered their father inherited the same genetic defect that rendered their father infertileinfertile
These couples need to be counseled & may opt forThese couples need to be counseled & may opt for• Using donor spermUsing donor sperm• AdoptionAdoption• To have only daughters through PGDTo have only daughters through PGD• Such male babies may produce normal amounts of Such male babies may produce normal amounts of
sperm during puberty and as youths - their sperms cansperm during puberty and as youths - their sperms can
be harvested for future usebe harvested for future use
KaryotypingKaryotyping
Fluorescence in-situ hybridization (FISH) on spermFluorescence in-situ hybridization (FISH) on sperm
Y Chr microdeletions of 18 lociY Chr microdeletions of 18 loci
Cystic Fibrosis mutation analysisCystic Fibrosis mutation analysis
Package for workup of male infertilityPackage for workup of male infertility
Genetic Counseling Genetic Counseling 1.1. Karyotyping:Karyotyping: Strongly recommended. In addition Strongly recommended. In addition
testicular biopsy and chromosome content of individual testicular biopsy and chromosome content of individual
spermatozoa.spermatozoa.
2.2. CF Mutation Screening: CF Mutation Screening: Screen for large number of Screen for large number of mutations and the new intron 857 variant. mutations and the new intron 857 variant.
3. 3. AZF Microdeletion: AZF Microdeletion: Possibility of transmitting from Possibility of transmitting from father to son. father to son.
4. 4. Newborn Screening: Newborn Screening: Karyotyping or DNA mutation Karyotyping or DNA mutation analysis. analysis.
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