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


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.


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


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


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.

Thank You

1 Using Genetics to Understand Diseases & Tailor Treatment.

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Transcript of 1 Using Genetics to Understand Diseases & Tailor Treatment.