1. Using Genetics to Understand Diseases & Tailor Treatment

2. Dr. Ameet Patki Medical Director
M.D., D.N.B., F.C.P.S., F.R.C.O.G. (U.K.)
Medical Director
ReGenesis, Reliance Life Sciences
Centre for Assisted Reproduction, Endoscopy
and Fetal Medicine Mumbai
Consultant Obstetrician & Gynecologist
Sir Harkisondas Hospital & Research Centre, Mumbai
Hon. Assoc. Prof.
K.J. Somaiya Medical College and Hospital, Mumbai

3. Genetics - Scientific Karma?
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!
A concoction of the family that came before us
Hopefully a little more than just the sum of their parts
While we all make decisions that define our future, we always make them based on who we were born.

4. Understanding Genetics
The foundation level is the molecule called DNA
The information in DNA is organized into Genes
Genes, in turn, make up Chromosomes - Genome
Every cell in an individual contains the genome
In reality, of course, companies cannot (and in my opinion should not) remain sitting in the lap of their academic parents. They must move to their own premises where they can address their own operational needs and develop their own cultures. (POINT TO SLIDE) This represents one of the biggest challenges for a new company – where to go, how far away from mother and at what stage? London has a visible dearth of suitable, affordable incubator space and Science Park clustering capability. If I were to leave one plea today, it would be that serious efforts be made to address this matter – by provision of funds and co-ordination for schemes aimed at providing serviced accommodation for new companies. If this is not provided within the London area, these companies will go further afield and the potential clustering benefits will be lost. I am quite sure that other regions will be only to happy to receive our companies and benefit from the local wealth and employment they will create.
(CLICK)
Genetics is the science of hereditary & variation.

5. The genome is a genetic instruction book for human biology
What is the Human Genome?
The genome is a genetic instruction book for human biology
There are twenty-three chapters, called chromosomes
Each chapter contains several thousand stories, called genes
Each paragraph is made up of words, written in letters called bases
There are 1 billion words and 3 billion letters in the book which makes it longer than… 800 Bibles."
The order of these letters will dictate whether an organism is a human or another species, such as a fruit fly or mouse.

6. What is the Human Genome?
This book of 3 billion DNA letters is actually three books in one
It is a history book that contains a record of our relatedness to other members of our species and to all other living things on the planet
It is a parts list for developing a human body from sperm and egg
It is a medical textbook that contains the clues for the understanding, prevention and cure of disease   
All the information in every book ever written if translated into DNA, could fit easily in a teaspoon.

7. DNA Unlocked the Key to Life
The discovery of the double helix by James Watson(25) and Francis Crick (36) on Feb. 28, 1953, unveiled “The Secret of Life."
DNA is the molecule that makes and maintains all life
It enables life to re-create itself
It contains the blueprints and the toolbox for understanding how humans work
DNA is both bricks and blueprint - an engineer's dream.

8. 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)

9. 50 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 upto 100,000 times longer than the chromosome itself.

10. 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)

11. 50 Years on : The Double Helix Twists & Turns
The strands of life are like a sticky zip.

12. What The Double Helix Cannot Tell You
DNA may be the script of life, but some scenes are largely improvised
A 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 sequence

13. 9 of the WHO’s 10 leading causes of global deaths have genetic components
Heart disease (13.7%)
Stroke (9.5%)
Pneumonia (6.4%)
HIV/AIDS (4.2%)
COPD (4.2%)
Diarrhea (4.1%)
Perinatal (4.0%)
Tuberculosis (2.8%)
Trachea/bronchus/lung cancer (2.3%)
? Traffic accidents (2.2%)

14. Nearly 4,000 genetic diseases afflict human beings
How to Conquer a Genetic Disease
Nearly 4,000 genetic diseases afflict human beings
Which altered gene causes the disease?
What protein does this gene normally produce?
Can the altered protein or gene be fixed or replaced?
In reality, of course, companies cannot (and in my opinion should not) remain sitting in the lap of their academic parents. They must move to their own premises where they can address their own operational needs and develop their own cultures. (POINT TO SLIDE) This represents one of the biggest challenges for a new company – where to go, how far away from mother and at what stage? London has a visible dearth of suitable, affordable incubator space and Science Park clustering capability. If I were to leave one plea today, it would be that serious efforts be made to address this matter – by provision of funds and co-ordination for schemes aimed at providing serviced accommodation for new companies. If this is not provided within the London area, these companies will go further afield and the potential clustering benefits will be lost. I am quite sure that other regions will be only to happy to receive our companies and benefit from the local wealth and employment they will create.
(CLICK)

15. Was about conditions wholly caused by
The Old Genetics
Was about conditions wholly caused by
An extra or missing complete chromosome or part of a chromosome
Down Syndrome (Trisomy -21)
Turner Syndrome (XO)
A mutation in a single gene
Cystic Fibrosis (mutations in CFTR gene)
Thalassemia
Sickle cell disease
Down’s Syndrome
Karyotyping is used for studying changes in number and structure of chromosomes.

16. The New Genetics
A “New Genetics" has emerged driven by knowledge gained at the DNA level
Diagnostic DNA testing
Prenatal DNA testing
Predictive DNA testing
Diagnostic tools- Fluorescence In Situ Hybridization (FISH), Polymerase Chain Reaction(PCR)

17. Genetic Diagnostic Tests
Karyotyping
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.

18. Diagnostic DNA testing
A patient presents with clinical features of a disorder, and a DNA test is undertaken to confirm a diagnosis
- Infectious Disease Testing -
Human Immunodeficiency Virus (HIV)
Hepatitis C Virus (HCV)
Hepatitis B Virus (HBV)
Cytomegalo Virus (CMV)
Tuberculosis (TB)
- Chronic Myeloid Leukemia

19. CML Philadelphia chromosome : t(9;22)(q34;q11) Orange = Chr. 9
Green = Chr. 22
Philadelphia chromosome : t(9;22)(q34;q11)

20. Newer Diagnostic testing
Comparative Genomic Hybridization (CGH)
Used in cancer research
In solid tumors chromosome quality is poor
DNA of tumor & normal DNA labeled with 2 fluorescent colors are co-hybridized on a normal metaphase
Detects amplification & deletions
Spectral Karyotyping (SKY)
Multicolor FISH (m-FISH)
Used to detect complex translocations
CGH
(Developed by Kallioneimi et al., 1992) can accurately determine total or partial aneusomy by loss or gain of DNA using a combination of PCR and FISH technology.
Presently it takes 2-3 days to obtain the diagnosis.
However, in future it is possible to apply CGH to single cells by developing strategies to make it compatible with the time frame with regular IVF. can detect any abnormality in a blastomere with the exception of balanced translocation.
Quantitative Fluorescent Multiplex PCR
QF-PCR has been successfully applied for detection of chromosomal abnormalities in a sample having 10 cells or more.
QF-PCR is based on the observation that within the exponential phase of PCR amplification, the amount of specific DNA product is proportional to the quantity of initial target. That, coupled with the detection of fluorescently labeled PCR product by automated laser DNA analyser makes the quantification of the product obtained from small DNA sample possible. The labeling of the PCR product is by labeling the specific primer.
Spectral Imaging
An alternative to conventional FISH – Use of 24 painting probes, one for each chromosome type, labeled in the ration of 5 different fluorochromes and observed by spectral imagining.
Has been successfully used for polar bodies which are at metaphase stage.

21. Prenatal DNA Testing Amniocentesis Chorionic villus sampling
(15-17 wks)
Chorionic villus sampling
(9-11 wks)
Fetal blood sampling
(18-20 wks)
CVS
Most genetic testing presently is done through amniocentesis when the fetus is 12 to 16 weeks old. This consists of drawing a sample of the amniotic fluid from around the fetus and examining a floating cell from the fetus. If the analysis shows that the fetus is genetically defective, then the parents have the option of aborting the fetus. Essentially all couples do elect to have an abortion. This is far more distressing to most couples, because the fetus is so fully developed. Screening techniques and and prenatal diagnostic techniques thus diagnose an abnormality or defect post implantation and thus patient has to opt for termination of pregnancy.The Pre-Implantation Genetic Diagnosis technique is performed before pregnancy begins. This avoids “the stress, emotional trauma and subsequent moral dilemma” of termination of pregnancy.

22. Preimplantation Genetic Diagnosis
Early form of prenatal diagnosis bringing the hope of healthy babies to couples at risk of transmitting genetic disorders to their offspring by NEGATIVE SELECTION of the affected embryos prior to implantation.
No longer a boutique medicine (Simpson).
Embryology met Genetics and so was born this new adjunct to IVF.
This is a procedure that is intended to weed out genetically defective embryos before they have a chance to develop. It is usually requested by prospective parents who are concerned about passing an incurable genetically based disease or disorder to their child where one or both partners have been genetically screened and found to be a carrier or are affected by a disease.

23. Present Day Scope for PGD/PND
Wide variety of disorders
Chromosomal abnormality
Structural (Translocation, Inversion, Deletion)
Numerical (Trisomy, Monosomy)
Single gene disorders
Autosomal Disorders
Dominant (Neuroblastoma)
Recessive (-Thalassemia, Cystic Fibrosis)
Sex linked disorders (Haemophilia, Muscular Dystrophy)
Some genetic diseases are sex-linked and thus only the males are affected. Molecular diagnosis may not be available for all these disorders and thus they cannot be detected directly. By sexing the embryos and doing a selective transfer of female embryos we can eliminate all of the male embryos thus preventing the transmission of the disease.
From single gene abnormality to extra chromosomes.

24. PCR - Cystic Fibrosis  F 508 Mutation Cleavage stage Embryo Biopsy
PGD Facility
78bp
250bp
100bp
50bp
The Micromanipulator
FISH -Trisomy 18, X, Y
PCR - Cystic Fibrosis  F 508 Mutation
Cleavage stage Embryo Biopsy
250bp
50bp
861bp
242bp
285bp
FISH - Polyploidy
Polar Body Biopsy
PCR -  Thalassemia
PGD help patients “From Infertility to healthy family”.

25. Indication for PGD/PND
Advanced Maternal Age
Familial Association to specific diseases (Down’s syndrome, Muscular dystrophy, Cytic fibrosis, Hemophilia)
Medical history/ conditions associated with genetic conditions
Multiple miscarriages
History of previously affected baby
Some genetic diseases are sex-linked and thus only the males are affected. Molecular diagnosis may not be available for all these disorders and thus they cannot be detected directly. By sexing the embryos and doing a selective transfer of female embryos we can eliminate all of the male embryos thus preventing the transmission of the disease.

26. Predictive DNA testing
Predictive or presymptomatic DNA testing allows genetic disorders to be detected in advance of clinical presentation
HNPCC (Colon cancer)
Hereditary Nonpolyposis Colorectal Cancer
BRCA1 and 2 (Breast & Ovarian Ca)
MODY 1,2,3 (Diabetes)
Maturity Onset Type Diabetes in Young
Alpha-synuclein (Parkinson’s disease)
An ounce of testing could mean a pound of prevention.

27. The Human Genome Project (HGP)
The HGP was an international research effort to decode the human genome
Initiated in 1990 & completed two years prior to deadline in 2003
The human genome consists of over three billion chemical base pairs
Approximately 30,000-40,000 genes

28. Beyond the Human Genome Project
Now that all genes are known, we will start understanding their function ® PATHWAYS
We will then be able to correlate disease states to certain genes (Pathobiology)
DISEASE ® GENE (S) GENE (S) ® DISEASE
We will find ways for rational treatment, rational prevention & rational diagnostics.

29. Promise of the Human Genome Project
Improve diagnosis & treatment through the application of genetic information & Technologies
Predictive medicine
Pharmacogenomics
Population screening
Genomic Medicine is here…………..

30. If genetics has been misunderstood, genomics is even more mysterious
Transition from Genetics to Genomics
If genetics has been misunderstood, genomics is even more mysterious
Genetics is the study of hereditary & variation
“Genomics," a term coined only 15 years ago, is the study of the functions and interactions of all the genes in the genome
Genomics is the study and identification of genes and gene function.

31. Genomic Medicine
The science of genomics rests on direct experimental access to the entire genome & applies to conditions like:
Colon cancer
Breast cancer
Alzheimer disease
HIV infection
Tuberculosis
Atherosclerosis
Inflammatory bowel disease
Diabetes
Parkinson’s disease
These disorders are due to the interactions of multiple genes and environmental factors. They are thus known as multifactorial disorders.

32. Impact of Genomic Medicine
Genomic medicine will change health care by :
Providing knowledge of individual genetic predispositions
Creating pharmacogenomics
Allowing population based screening for certain Mendelian disorders
The focus is not the treatment of disease but the eradication of the genetic problems that cause disease.

33. Impact of Genomic Medicine
Knowledge of individual genetic predisposition will allow :
Individualized screening
Individualized lifestyle changes
Presymptomatic medical therapies
Anti-colon cancer agents before colon cancer develops
Antihypertensives before hypertension develops
Tamoxifen as a prophylactic for CA breast

34. Impact of Genomic Medicine
Pharmacogenomics will allow:
Use of Individualized medication based on genetically determined variation in effects and side effects
More powerful/safer medicines the first time
More accurate methods for determining medication doses
New medications for specific genotypic disease subtypes
Drugs tailored to our individual genetic profiles would reduce overall medical costs.

35. How Does Society Prepare for Genomics?
Education to achieve understanding of :
The basics of the science of genetics
The eventual use of genetics in health care
How to deal with risk & predisposition
The personal impact of genetic information
The social impact of genetics
In reality, of course, companies cannot (and in my opinion should not) remain sitting in the lap of their academic parents. They must move to their own premises where they can address their own operational needs and develop their own cultures. (POINT TO SLIDE) This represents one of the biggest challenges for a new company – where to go, how far away from mother and at what stage? London has a visible dearth of suitable, affordable incubator space and Science Park clustering capability. If I were to leave one plea today, it would be that serious efforts be made to address this matter – by provision of funds and co-ordination for schemes aimed at providing serviced accommodation for new companies. If this is not provided within the London area, these companies will go further afield and the potential clustering benefits will be lost. I am quite sure that other regions will be only to happy to receive our companies and benefit from the local wealth and employment they will create.
(CLICK)

36. Concerns with Genomics
Discrimination against individuals
Discrimination against groups
Genes run in families
Confidentiality/privacy
In reality, of course, companies cannot (and in my opinion should not) remain sitting in the lap of their academic parents. They must move to their own premises where they can address their own operational needs and develop their own cultures. (POINT TO SLIDE) This represents one of the biggest challenges for a new company – where to go, how far away from mother and at what stage? London has a visible dearth of suitable, affordable incubator space and Science Park clustering capability. If I were to leave one plea today, it would be that serious efforts be made to address this matter – by provision of funds and co-ordination for schemes aimed at providing serviced accommodation for new companies. If this is not provided within the London area, these companies will go further afield and the potential clustering benefits will be lost. I am quite sure that other regions will be only to happy to receive our companies and benefit from the local wealth and employment they will create.
(CLICK)

37. Tomorrow’s Medicine Genomics opens up new therapeutic options:
Drugs derived from genetic engineering
When genes acts as drugs : gene therapy
Medicine made to measure : pharmacogenomics
Predicting susceptibility to disease : diagnostic tests
Vaccines & the treatment of infectious diseases
Designing babies
The aim is to detect & target specific diseases & develop more efficient and selective treatment.
In reality, of course, companies cannot (and in my opinion should not) remain sitting in the lap of their academic parents. They must move to their own premises where they can address their own operational needs and develop their own cultures. (POINT TO SLIDE) This represents one of the biggest challenges for a new company – where to go, how far away from mother and at what stage? London has a visible dearth of suitable, affordable incubator space and Science Park clustering capability. If I were to leave one plea today, it would be that serious efforts be made to address this matter – by provision of funds and co-ordination for schemes aimed at providing serviced accommodation for new companies. If this is not provided within the London area, these companies will go further afield and the potential clustering benefits will be lost. I am quite sure that other regions will be only to happy to receive our companies and benefit from the local wealth and employment they will create.
(CLICK)

38. Genetics 25 Years Hence
By the double helix's 75th birthday, your genome might be as familiar as your shoe size
In 25 years babies might have genetic identity cards
This would include information from an individual's genome sequence
Genetic ID cards could offer benefits for medical diagnosis, treatment and prevention
The complete genetic makeup of individuals could soon be scanned and recorded on a smart card.

39. There's no room for technophobes in the medical profession
The Year 2028
There's no room for technophobes in the medical profession
As a doctor swipes her patient's genetic ID card, the information downloads in seconds
A string of letters scrolls down her computer screen
These are the raw data of DNA, the code of life
It seems reasonable to imagine genome sequencing happening as a matter of routine in 25 years time.

40. The Year 2028
For the patient, waiting anxiously, the news will be mixed
The results reveal that he carries a gene that increases his risk of cancer
But should he fall ill, his genes also predict the best therapy -
A new tailor-made drug that brings with it an 80% chance of remission
Could this be the future of medicine?

41. Better Drugs in The Year 2028?
The drug is designed using genetic knowledge
It targets an abnormal piece of DNA in cancerous cells, killing them but sparing healthy cells
After a year's treatment, 90% of patients are free of disease, and 50% show a complete or near disappearance of the abnormal gene that triggers the disease
Mapping our DNA may help doctors to prescribe the safest, most effective medicine.

42. Microarrays/ DNA Biochips in Medical Practice
The DNA microchip/ microarray is a revolutionary new tool used to identify mutations in genes
The chip, which consists of a small glass plate encased in plastic, is manufactured somewhat like a computer microchip
On the surface, each chip contains thousands of immobilized DNA sequences
Microarrays allow thousands or tens of thousands of specific DNA or RNA sequences to be detected simultaneously
Anticipated Developments –
DNA microarrays offer unprecedented opportunities for analysing gene expression, understanding gene function, and detecting gene dysfunction.
Microarray expression profiles will define new prognostic subgroups in cancer and other diseases.
The potential clinical benefit brought by microarrays will have to be rigorously appraised and weighed against their costs before their introduction into routine clinical use.
The increased diagnostic and prognostic information provided by microarrays should assure their entry into clinical practice in specialist centres within 3-5 years and in most large hospitals within 5-10 years.
Monitoring the Genome on a Chip.

43. Applications of Microarray Technology
Gene discovery
Disease diagnosis
Drug discovery: Pharmacogenomics
Toxicological research: Toxicogenomics
Gene expression profiling
Genotyping
DNA sequencing
Spotted DNA microarray
Currently only used
as a research tool.
Gene expression profiling RNA extracted from a complex sample (such as body tissues or fluids or bacterial isolates) is applied to the microarray. The result reveals the level of expression of tens of thousands of genes, effectively all the genes in the genome, in that complex sample. This result is known as a gene expression "profile" or "signature."
Genotyping Genomic DNA, extracted from an individual's blood or saliva, is amplified by the polymerase chain reaction and applied to the microarray. The genotype for hundreds or thousands of genetic markers across the genome can be determined in a single hybridisation. This approach has considerable potential in risk assessment, both in research and clinical practice.
DNA sequencing DNA extracted from an individual's blood is amplified and applied to specific "re-sequencing" microarrays. Thousands of base pairs of DNA can be screened on a single microarray for mutations in specific genes whose normal sequence is already known. This greatly increases the scope for precise molecular diagnosis in single gene and genetically complex diseases.
Spotted DNA microarray:
RNA extracted from samples 1 and 2 is labelled with red or green fluorescent dyes. The dye labelled RNA populations are mixed and hybridised to the microarray, on which has been spotted cDNA from thousands of genes, each spot representing one gene. The RNA from each sample hybridises to each spot in proportion to the level of expression of that gene in the sample. After hybridisation, the red and green fluorescent signal from each spot is determined, and the ratio of red to green reflects the relative expression of each gene in the two samples. For example, the gene TEP1 is shown to be expressed at a higher level in sample 2 than in sample 1. 
Oligonucleotide Microarray:
Up to half a million distinct oligonucleotides are synthesised on the microarray by photolithography and act as probes in individual "features" on the microarray surface. About 30 distinct oligonucleotides, printed as individual features, represent the partial sequence of one gene. Fluorescent labelled cDNA derived from a single test sample is hybridised to the microarray, allowing the expression level of up to 15 000 genes to be measured in the test sample

44. The Future for Alzehimer Disease - 2010
5 or 6 genetic variations identified that strongly predispose for Alzheimer disease; another 10 or 12 with weaker association
Chip-based genetic test gives personal likelihood of developing the condition
Chip-based genetic test identifies the drug most likely to be effective for given individual
Chip-based genetic test determines individual likelihood of drug side effects
                           

45. Gene Therapy-The Next Twist in the Genome Tale
Put most simply, it introduces a "good" gene into a person who has a disease caused by a "bad" gene.
Gene therapy is a novel approach to treating diseases based on modifying the expression of a person's genes toward a therapeutic goal. Gene therapy is most often been discussed in the context of treating lethal and disabling diseases although it also has a potential for disease prevention.
The premise of gene therapy is based on correcting disease at the level of DNA molecules and thus compensating for the abnormal genes. There are essentially two forms of gene therapy, one of which is called somatic gene therapy. Somatic gene therapy involves the manipulation of gene expression in cells so as to be corrective for the patient, but this correction is not inherited by the next generation. This is the type of gene therapy that is currently being investigated at the Institute for Human Gene Therapy, as well as at other laboratories around the world. The other form of gene therapy is called germline gene therapy; this involves the genetic modification of germ cells that will pass the selected change on to the next generation. Research on germline intervention is strictly limited to animal model systems, and there is no intent to pursue this type of approach in humans at any time in the near future; this is because of significant technical and ethical challenges.

46. Gene Therapy for Parkinson's disease
Genetically modified virus carries the DNA into the body
It passes into the nucleii of some brain cells
These cells can then produce dopamine
Gene therapy can be used to treat cancer, heart disease, Alzheimer's & Parkinson's disease.

47. DNA Based Vaccines The DNA vaccines are an offshoot of gene therapy.
Technique involves the direct injection of plasmids-loops of DNA that contain genes for proteins produced by the organism being targeted for immunity
Once injected into the host's muscle tissue, the DNA is taken up by host cells, which then start expressing the foreign protein
The protein serves as an antigen that stimulates immune responses
If the body later encounters the organism carrying this antigen, its defenses are ready to launch a protective attack
In reality, of course, companies cannot (and in my opinion should not) remain sitting in the lap of their academic parents. They must move to their own premises where they can address their own operational needs and develop their own cultures. (POINT TO SLIDE) This represents one of the biggest challenges for a new company – where to go, how far away from mother and at what stage? London has a visible dearth of suitable, affordable incubator space and Science Park clustering capability. If I were to leave one plea today, it would be that serious efforts be made to address this matter – by provision of funds and co-ordination for schemes aimed at providing serviced accommodation for new companies. If this is not provided within the London area, these companies will go further afield and the potential clustering benefits will be lost. I am quite sure that other regions will be only to happy to receive our companies and benefit from the local wealth and employment they will create.
(CLICK)
The DNA vaccines are an offshoot of gene therapy.

48. Clinical Trials with DNA Based Vaccines
The first clinical trials using injections of DNA began for HIV in 1995
Four other clinical trials using DNA vaccines against influenza, herpes simplex virus, T-cell lymphoma, and an additional trial for HIV were started in 1996
A DNA vaccine for Malaria is being developed
Expected to be particularly useful to prevent and treat infectious diseases such as Herpes, Hepatitis, and AIDS.
In reality, of course, companies cannot (and in my opinion should not) remain sitting in the lap of their academic parents. They must move to their own premises where they can address their own operational needs and develop their own cultures. (POINT TO SLIDE) This represents one of the biggest challenges for a new company – where to go, how far away from mother and at what stage? London has a visible dearth of suitable, affordable incubator space and Science Park clustering capability. If I were to leave one plea today, it would be that serious efforts be made to address this matter – by provision of funds and co-ordination for schemes aimed at providing serviced accommodation for new companies. If this is not provided within the London area, these companies will go further afield and the potential clustering benefits will be lost. I am quite sure that other regions will be only to happy to receive our companies and benefit from the local wealth and employment they will create.
(CLICK)

49. Benefits of DNA Based Vaccines
Traditional vaccines are expensive and take a long time to produce
DNA is relatively inexpensive and is easier to produce
DNA vaccines are much more stable, allowing them to be easily transported
With a live vaccine (Polio vaccine), there is always a danger of it reverting and becoming infective. DNA cannot become infective
In reality, of course, companies cannot (and in my opinion should not) remain sitting in the lap of their academic parents. They must move to their own premises where they can address their own operational needs and develop their own cultures. (POINT TO SLIDE) This represents one of the biggest challenges for a new company – where to go, how far away from mother and at what stage? London has a visible dearth of suitable, affordable incubator space and Science Park clustering capability. If I were to leave one plea today, it would be that serious efforts be made to address this matter – by provision of funds and co-ordination for schemes aimed at providing serviced accommodation for new companies. If this is not provided within the London area, these companies will go further afield and the potential clustering benefits will be lost. I am quite sure that other regions will be only to happy to receive our companies and benefit from the local wealth and employment they will create.
(CLICK)

50. Genetically Engineered Drugs
Protein
Indication
Year
Insulin
Diabetes
1982
Human growth hormone
Growth deficiency
1985
alpha-interferon
Viral infections Cancer
Hepatitis B vaccine
Vaccine against Hepatitis
1986
Tissue plasminogen activator
Cardiovascular disease
1987
Erythro-poietin
Anaemia
1988
Factor VIII
Haemophilia
1993
Herceptin
Breast Cancer
1999 )
In reality, of course, companies cannot (and in my opinion should not) remain sitting in the lap of their academic parents. They must move to their own premises where they can address their own operational needs and develop their own cultures. (POINT TO SLIDE) This represents one of the biggest challenges for a new company – where to go, how far away from mother and at what stage? London has a visible dearth of suitable, affordable incubator space and Science Park clustering capability. If I were to leave one plea today, it would be that serious efforts be made to address this matter – by provision of funds and co-ordination for schemes aimed at providing serviced accommodation for new companies. If this is not provided within the London area, these companies will go further afield and the potential clustering benefits will be lost. I am quite sure that other regions will be only to happy to receive our companies and benefit from the local wealth and employment they will create.
(CLICK)

51. Designer Babies ?
In the Brave New World predicted, scientists will do more than screen for disease free embryos
Parents will be able to reject their own genetic heritage, and have instead of plump -
Beautiful, Clever or Sporty genes implanted into their embryonic children
The babies of the future could be protected from diseases.
In reality, of course, companies cannot (and in my opinion should not) remain sitting in the lap of their academic parents. They must move to their own premises where they can address their own operational needs and develop their own cultures. (POINT TO SLIDE) This represents one of the biggest challenges for a new company – where to go, how far away from mother and at what stage? London has a visible dearth of suitable, affordable incubator space and Science Park clustering capability. If I were to leave one plea today, it would be that serious efforts be made to address this matter – by provision of funds and co-ordination for schemes aimed at providing serviced accommodation for new companies. If this is not provided within the London area, these companies will go further afield and the potential clustering benefits will be lost. I am quite sure that other regions will be only to happy to receive our companies and benefit from the local wealth and employment they will create.
(CLICK)

52. Que Sera Sera ………… Whatever will be will be,
The future’s not ours to see,
Que Sera Sera,
What will be will be…………

53. The Past 25 Years………
Significant advances in ART techniques have revolutionized both male & female factor infertility

54. History of ICSI
1992 – Palmero et al., responsible for the world’s first baby conceived with ICSI.

55. ICSI Bypasses the effective biologic mechanism of sperm selection.
Application to human reproduction has not been preceded by extensive research trials in mammals.
Human experience with ICSI is the experimental record.
Cause of concern with ICSI:
Promotion of transgenerational transmission of genetic defects to the offspring causing gametogenic failure.

56. Male factor infertility should be considered as a
ICSI
Male factor infertility
should be considered
as a
potentially heritable
condition
(Meschede et al., 2000)

57. Incidence of Chromosomal Aberration in Infertile oligozoospermic and azoospermic males (Liebaers et al., In: Textbook of Assited Reproductive Techniques – Chapter 24; 2001)
Aberrations
Infertile Males
(n = 7876)
Oligozoosermia
(n = 1701)
Azoospermia
(n = 1151)
Autosomes
1.3%
3.0%
1.1%
Sex Chromosomes
3.8%
1.6%
12.6%
Total
5.1%
4.6%
13.7%
Chromosome aberrations increase as sperm counts decrease

58. 2.2 % had variety of major congenital malformations.
First trimester losses/major congenital malformations in ICSI derived pregnancies
Ejaculate sperms
Epididymal sperms
Testicular Sperms
First trimester loss
24.6%
31.2%
33.3%
2.2 % had variety of major congenital malformations.
Wisano et al., Hum. Reprod
Borduelle, Devroey et al., Hum. Reprod
Survey of 1455 children
Largest American and European study.

59. Areas of Concern
13.7% Azoospermic males and 4.6% oligozoospermic males have abnormal karyotypes.
Further, meiotic disturbances limited to spermatogenic cells are found in 6% of males with severe OAT
Newer research indicates that children born with ICSI for male infertility have twice the rate of major congenital malformations.
Hansen et al., N.Engl. J. Med. 2002; 346:

60. Genetic concern associated with Male Factor Infertility
Genetic Abnormality
Resultant Reproductive Abnormality
Y Chromosome Microdeletion
(Multiplex PCR)
Azoospermia, severe oligozoospermia
Cystic Fibrosis Gene Mutation
(PCR)
Congenital absence of Vas Deferens
Structural defects or Aneuploidies
(Blood Karyotype and Sperm FISH)
Variable (Klinefeleter’s syndrome, XYY Males)

61. Genetic concern associated with Male Factor Infertility
Genetic Abnormality
Resultant Reproductive Abnormality
Y Chromosome Microdeletion
(Multiplex PCR)
Azoospermia, severe oligozoospermia

62. Y Microdeletion The how and Y of Male infertility Incidence
Y chromosome microdeletion common cause of spermatogenic failure.
Incidence
15-20% of men with idiopathic azoopermia
7-10% of men with idiopathic severe oligozoospermia
Y chromosome microdeletion first described in 1976 by Tiepolo and Zuffardi.

63. Y Chr is paternally inherited from father to son

64. Y Chr DNA Y Chr is a genetic junkyard as well as a gold mine
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 mine

65. Y Microdeletion Multiplex PCR
Simple, powerful and fast tool ideal for screening of idiopathic infertile male
When used with rigorous procedures reaches the accuracy of other sophisticated techniques such as Southern blot.

66. Y Chr microdeletions MDS are clustered in 3 main regions:
AZFa, AZFb, AZFc
15 novel genes identified on Y Chr
DFFRY, RBM, DAZ genes associated with male infertility

67. Effect of deletions on extent 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, 1999

68. Del at DYS240 at AZFc Region
PCR for Y microdeletions
M +A K V -A B K V -B C K V -C D K V -D
M = Molecular weight marker
A,B,C,D = Multiplex MMX
+ = Normal control
- = Negative control D/W
06 & 07 = Test patients
Promega Y chromosome Deletion Detection System, Version 1.1
Del at DYS240 at AZFc Region

69. PCR for Y microdeletions
038A
038B
038C
038D M
M = Molecular weight marker
A,B,C,D = Multiplex MMX
038 = Test patient
The absence of bands (for 038) in MMX A, B, C and D indicates deletion of loci DYS237, DYS236 at AZFd region alongwith deletion of entire DAZ gene and locus DYS240 at AZFc region.
Promega Y chromosome Deletion Detection System, Version 1.1

70. Y-13 Translocation-FISH,Karyotyping
45,X Metaphase
Azoospermic male
Normal features
Karyotype 13 X 18
FISH
Green - X
Orange - Y
Blue

71. M = Molecular weight marker A,B,C,D = Multiplex MMX 085 = Test patient
The absence of bands (for 085) in MMX A, B, C and D indicates deletion of loci
DYS271, KALY at AZFa region;
DYS212, SMCY, DYS218, DYS219, DYS221 at AZFb region;
DYF51S1, DYS237, DYS236 at AZFd region and
Entire DAZ gene and locus DYS240 at AZFc region.
Promega Y chromosome Deletion Detection System, Version 1.1

72. Our Data on Y chromosome Microdeletions
Indication
Total
Normal
Deletion present in
Abnormal %
Azoospermia 25 19 7
28%
Severe OAT 35 31 4
11.43%
OAT 13 11 1
7.7% 73 61 12
16.44%
Our data coincides with the data reported by clinics worldwide

73. Y Microdeletion
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.
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.

74. Genetic concern associated with Male factor Infertility
Genetic Abnormality
Resultant Reproductive Abnormality
Cystic Fibrosis - CFTR Gene Mutation (PCR)
Congenital absence of Vas Deferens

75. Cystic Fibrosis (CF) & male infertility
CF is a autosomal recessive disorder
Caused due to mutations in CFTR gene
Current medical support has improved life expectancy
These individuals
have delayed puberty
are generally infertile
have Azoospermia
Congenital absence of vas deferens (CAVD) .
PCR detects 85% of all mutations

76. Cystic Fibrosis
Product of cystic fibrosis gene is known as cystic fibrosis transmembrane conductance regulator (CFTR) protein.
 F 508 prevents normal maturation of the CFTR protein and causes failure of its normal localization to the cell membrane.

77. CBAVD and Cystic Fibrosis
Before performing ICSI with sperm from CBAVD males with CFTR mutations, their partners should also be tested to avoid the risk of having a child affected with Cystic Fibrosis.

78. Detection of Cystic Fibrosis mutation
Common gene mutations in CAVD are:
F508
W1282 X
N1303 K
G542 X
1717G-A
R553X
R117H

79. 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

80. Genetic concern associated with Male factor Infertility
Genetic Abnormality
Resultant Reproductive Abnormality
Structural defects or Aneuploidies
(Blood Karyotype and Sperm FISH)
Variable (Klinefeleter’s syndrome, XXY Males)

81. Abnormal karyotype in men with severe male factor infertility

82. Karyotyping - Male contribution - 200 cases
Primary/Sec. infertility 71 (35.5%) : BOH/RSA in wife 129 (64.5%)
Chromosome Abnormalities : (4.5%)
Numerical abnormalities : (1.5%)
47,XXY 2 cases Primary infertility
47,XYY 1 case CAVD
Structural Abnormalities - Reciprocal translocations : (3%)
46,XY,t(7;13)(p11;q34) OAT
46,XY,t(8;10)(p23;q24) BOH
46,XY,t(10;13)(q23.2;q34) "
46,XY,t(11;16)(q21;p13.3) "
45,X,t(Y;13)(q23;q11) "
45,XY,t(13;14)(p11;q11) "

83. Partial karyotypes showing balanced reciprocal translocations in 3 male partners of BOH cases.
t(8;10)(p23;q24)
t(10;13)(q23.2;q34)
t(7;13)(p11;q34)

84. Chromosome Variants (10%)
Variants n Indication
46,X,inv(Y) OAT / BOH
46,XY,inv(9) 2 Azoospermia / BOH
46,X,Yqh+ 3 Polytailed sperm / BOH (2)
46,X,Yqh- 3 Primary Infertility (2)/ BOH
46,XY,9qh+ 5 BOH
46,XY,21ps+ 2 Primary infertility
46,XY,22ps+ 2 Primary infertility
Single cell mosaicism (2%)
46,XY (1/50 cell with 47,XXY) BOH (2 cases)
46,XY {1 cell with t(7;14)(p12;q12)} OAT
46,XY{ 1cell with trisomy 21} BOH (2 cases)

85. FISH on Sperm Normal 13, 21 Disomy 18 Disomy 18 XY Sperm XY Sperm
X and Y Sperm
X and Y Sperm
Y bearing Sperm (Normal)

86. Should Y chromosome analysis and genetic counselling be offered to infertile male?
96% of couples choose Y chromosome testing when offered
Transmission of infertility to offspring is weighed carefully by infertile couples
- Rucker et al., 1998

87. Genetic testing should become an integral part of patient assessment and treatment planning

88. Genetic Counseling
All couples considering ICSI are offered counseling to inform of the potential genetic risks to their possible offspring's.

89. Concerns on doing ICSI in infertile males
Y Chr microdeletions are passed to Sons
As a result the Sons are also infertile since they have inherited the same genetic defect that rendered their father infertile
These couples need to be counseled & may opt for
Using donor sperm
Adoption
To have only daughters through PGD
Such male babies may produce normal amounts of
sperm during puberty and as youths - their sperms can
be harvested for future use

90. Package for workup of male infertility
Karyotyping
Fluorescence in-situ hybridization (FISH) on sperm
Y Chr microdeletions of 18 loci
Cystic Fibrosis mutation analysis

91. 3. AZF Microdeletion: Possibility of transmitting from father to son.
Genetic Counseling
Karyotyping: Strongly recommended. In addition testicular biopsy and chromosome content of individual spermatozoa.
CF Mutation Screening: Screen for large number of mutations and the new intron 857 variant.
3. AZF Microdeletion: Possibility of transmitting from father to son.
4. Newborn Screening: Karyotyping or DNA mutation analysis.

92. Thank You