GENETIC COUNSELING AND GENE THERAPY

MODERATOR: Dr. Uday KumarDr. Sahana Devadas Introduction: • Genetic diseases are ubiquitous, affecting all human beings where ever they live. They place considerable health and economic burdens not only on affected people and their families but also on the community. As more environmental diseases are successfully controlled, those that are wholly or partly genetically determined are becoming more important

The prevalence of various genetic diseases is given below:

GENETIC COUNSELINGDefinition • It has been defined as an educational process that seeks to assist affected and / or at risk individuals to understand the nature of a genetic disorder, its transmission, and the options available to them in management and family planning. Indications for genetic counseling: Advanced parental age:• Maternal age > 35 years• Paternal age > 50 years• Child with congenital anomalies or dysmorphology • Consanguinity or incest

Types of genetic diseases

Estimated prevalence per 100 population

Single gene:Autosomal dominantAutosomal recessivex-linked recessive chromosomal abnormalities common disorders with appreciable genetic componentcongenital malformations

2-1021-26-7   7-1020

Total 38-51

Family history of heritable disorders or diseases , including:• Adult onset• Complex/multi factorial inheritance• Chromosomal abnormality• Single gene disorders• Heterozygote screening based on ethinicity, including:• Sickle cell anemia (W.African, Mediterranean, Arab,Indo- Pakistani, Turkish , S.E Asian .• Tay-sachs , canavan (Ashkenazi - Jewish , French - Canadian) • Thalassemias (Mediterranean, Arab, Indo- Pakistani.)

Steps in genetic counseling:Steps in genetic counseling:

• Diagnosis- based on accurate family history, medical history,• Examination and investigations• Risk assessment• Communication• Discussion of options• Long-term contact and support Diagnosis: • A full and accurate family history is a corner stone in the genetic assessment and counseling process.• The 1st and most important step in the diagnosis of genetic disorders is construction of a family tree. • The pattern of inheritance can be shown from the pedigree . for eg: vertical transmission in autosomal dominant disorders, horizontal transmission in autosomal recessive disorders and oblique transmission in X-linked recessive disorders Examination• Anthropometry• Head to toe• Correct description of dysmorphology• Photographic record• Parental examinationInvestigations • Chromosome analysis• Biochemical analysis• DNA analysis

• Histopathology

RISK ESTIMATIONRISK ESTIMATION  

QUANTIFICATION

conditition Incidence/1000 Parents affected/not Recurrence risk

Congenital heart disease

3-8 Normal with 1 child affected 2diabetes 60 Normal with 1 child affected 5-10

epilepsy 5 One parent affected 5-15

Severe mental deficiency

30 One siblingaffected

5 Down syndrome 1.5 Tri 1, 1 child affected

T21/22 or 13-15/21 T21/21 1 33

Cleft lip 1 1 child affected , 2 children affected, 1 parent and child affected

3-5 10 17

Manic depression - One parent affected 15schizophrenia 8 One parent affected 9-20 Huntington chorea 0.06 One parent affected 50 Rh hemolytic disease - After 1 still birth, father

homozygous 100 galactosemia 0.025 One child affected 25

 phenylketonuria 0.1 Complex traits-Hemoglobinopathies

25

• The fact that the parents have just had a child with autosomal recessive disorder(recurrence risk equals 1 in 4) does not mean that their next 3 children will be unaffected.• A couple faced with a probability of 1 in 25 that their next baby will have a neutral tube defect should be reminded that there are 24 chances out of 25 that their next baby will not be affected.

Calculating and presenting the risk• Hardy- Weinberg law– By knowing the frequency of AR diseases , the frequency of carrier can be calculated– P2+2pq+q2=1 where p is the frequency of one of a pair of alleles and q is of others.– Gives the frequency of carrier in the population but not the recurrence risk.Baye’s theorem• Devised by Reverend Baye’s in 1763• Provides the overall probability of an event or outcome• Provides assessment of recurrence risks • Allows refinement of recurrence risk estimates.• Used in the interpretation of genetic screening and diagnostic test results.

QUALIFCATION –THE NATURE OF A RISK • A ‘high’ risk of 1 in 2 for a trivial problem such as an extra digit(polydactyly) will deter very few parents. In contrast a ‘low’risk of 1 in 25 for a disabling condition such as a neural tube defect can have a very significant deterrent effect. A woman who grew up watching her brother develop Duchenne muscular dystrophy and subsequently die from the condition aged 21 yrs, may not risk having children even if there is only a 1% chance that she is a carrier . other factors, such as whether it is associated with pain and suffering, and whether prenatal diagnosis is available , will all be relevant to the decision –making process. Placing risks in context• For placing risks in context• 1 in 10 is high risk, • 1 in 20 as intermediate • And 1 in 50 as low riskCOMMUNICATION• The ability to communicate is essential in genetic counseling. It is a 2 way process. Both parents should be

present for the discussion , the genetic basis for the problem should be described using simple language and visual aids.• Patients or the relatives should be encouraged to clear their doubts on the condition.• It is a good practice to record the communication and send a letter summarizing the issues discussed.• It should be non directive and non judgmental.Directive counseling• Directive counseling has a positive influence on the consultees decision . The non directive approach involves presentation of the facts in an unbiased manner leaving the entire responsibility of decision with the consultee.

Confidentiality

• Medical genetics team may learn many family secrets , such as previous abortions , previous abnormal births and occasional false paternity.• The team should observe high moral values , confidentiality and should respect the self esteem and moral values of the parents.OUTCOMES • Most consultands have a reasonable recall of the information given.• 30% of the consultands have difficulty in recalling the precise risk figure.• 50%have been influenced by the counseling in their reproductive behavior.Special problems• Consanguinity and incest : consanguineous marriage is one b/w blood relative who have at least one common ancestor no more remote than a great-great-grandparent • Union b/w 1st degree relatives (brother-sister)/ parent child is called incest.• Most of the children born to consanguineous marriage carries 2-6 lethal recessive mutations +1-2 AR mutations for harmful but viable traits.

3-5% 1/8Third degree

5-10% 1/4Second degree

50% 1/2First degree

Risk of abnormality in offspring

Proportion of shared genes

frequency

Genetic relationship of partners

small N NMosaic 1 N NTrisomy

21

100 N C 100 C N21/21 5 N C 10-15 C N21/22

translocation

Recurrence risk(%)

motherfatherComplex traits-Hemoglobinopathies

10Congenital malformation

10-15AR Disorder

25 35

Mental retardationSevereMild

frequencyComplex traits-Hemoglobinopathies

Genetic centers in India• 1. Center for genetic disorders , Department of Human Genetics, Guru Nanak Dev university,Amritsar, Punjab• 2. Dept of Human Genetics and Anatomy, St. Johns Medical college Bangalore.• 3. Dept of Pediatrics, K.E.M hospital. Mumbai.• 4. ICMR Immunohematology center, K.E.M hospital. Mumbai.• 5. ICMR Genetic research center, Wadia hospital for children,Mumbai.• 6. Dept of Genetics ,Ramakrishna Mission Hosp. ,Calcutta.• 7. Depts of Pediatrics and Hematology PGI, Chandigarh.• 8. Genetics unit, Dept of pediatrics, AIIMS, New Delhi.• 9. Dept of Medical genetics, Sanjay gandhi postgraduate institute of Medical science, Lucknow• 10. Dept of Genetic Medicine, Sri Gangaram Hosp. Rajinder Nagar, New Delhi.• 11. Dept of Genetics, ICH ,Chennai• 12. Genetic center, Dept of Pediatrics, BJMC, Pune.

Gene Therapy

• History• What is gene therapy?• How does it work?• Techniques of gene therapy• Candidate diseases• Factors for gene therapy to become effective treatment for genetic disease.• Recent developments in gene therapy.• Current status of gene therapy

• Arguments in favour of gene therapy• Arguments against gene therapy.History of Gene therapy• 1953: scientists Francis Crick & James Watson- determined double helical structure of DNA.• 1973: American doctor Stanfeild Rogers tried to treat sisters with Hyperargininemia using human pappiloma virus.• 1980: Dr.Martin Cline first attempted at human gene therapy in university of California,L.A.• 1984:The human gene therapy working group (HGTG) created.• 1999:Death of Jesse Gelsinger , the first casualty in gene therapy.

TREATMENT OF GENETIC DISEASESEnvironmental manipulation:• Restriction• Removal• Replacement Gene manipulation:Gene therapy

Environmental manipulationEXAMPLES OF METHODS FOR TREATING GENETIC DISEASE

MucopolysaccharidosesBMT

SCID due to adenosine deaminase deficiencyReplacement of deficient enzyme/protein Blood transfusion

Congenital non –hemolytic jaundiceEnzyme induction by drugs . Phenobarbitone

DisorderComplex traits-Hemoglobinopathies

What is gene therapy?• Genes are the basic physical and functional units of heredity.• Genes are specific sequences of bases that encode instructions on how to make proteins.• It’s these proteins that perform most life functions and even make up the majority of cellular structures, not the genes

TECHNICAL ASPECTS• Gene characterization • Target cells or organ identification.• Vector system• Incorporation of therapeutic gene into host genome• Production of desired protein

Approaches used for correcting faulty genes:• A normal gene, inserted into a non specific location within the genome to replace a non functional gene.

Gaucher disease-glucosidaseHemophilia ACryoprecipitate/Factor

VIII

1-antitrypsin deficiency1-antitrypsin Trypsinogen deficiencyTrypsin

Complex traits-Hemoglobinopathies

• Abnormal gene swapped for a normal gene through homologous recombination• Abnormal gene could be repaired through selective reverse mutation which returns the gene to its normal function.• The regulation of a particular gene could be altered.How does gene therapy work?• In most gene therapy studies-: a normal gene is inserted into the genome to replace an abnormal disease causing gene• A carrier molecule called a vector must be used to deliver the therapeutic gene to the patient’s target cells• Currently the most common vector is a virus, that has been genetically altered to carry normal human DNA.• Viruses have evolved a way of encapsulating and delivering their genes to human cells

• Scientists have tried to take advantage of this capability and manipulate the virus genome to remove disease causing genes and insert therapeutic genes.

TYPES OF GENE THERAPY• Somatic cell gene therapy :

Methods of somatic cell gene therapy• Exvivo – Isolate cells with a defective gene from an affected individual– Growing the isolated cells in culture– Correct the genetic defect by transforming cells with remedial gene– Transplanting back these cells into the patient. In order to transfer the remedial gene packaged retro viral method is employedExample:1.In adenosine deaminase (ADA) deficiency.2.In familial hypercholesterolemia,

• Invivo – Direct delivery of a remedial gene into cells of a particular tissue of an affected person– Isolation of cells from patients not required– In gene construct, the remedial gene represents a sequence that codes a protein that corrects the genetic defect

– Remedial gene is under the control of tissue specific strong promoter. Some of the viral vectors-(adeno, retro virus) used to deliver remedial gene inside the patient– Eg: cystic fibrosis,hemophilia b

-• Disease targets

Blood clottingFactor VIII & IX

Hemophilia A & B

Lungs,pancreasCFTRCystic fibrosis

Lungs,liver(cirrhosis)

-Antitrypsin-AT deficiency

Lymphoid tissueAdenosine deaminase

Severe combined immunodeficiency

Tissues Gene(s involved)

Single gene defect

LiverPhenylalanine hydroxylase

Phenylketonuria

Liver,vascular endothelial smooth muscle cells

LDL receptorHypercholestremia

Blood formed elements

-globinComplex traits-Hemoglobinopathies

• The cardiovascular system (including the peripheral vasculature) has become an important target for gene therapy.

• to inhibit smooth-muscle cell proliferation and PREVENT RESTENOSIS.

•• to promote the vascularization of tissues by intramuscular injection of naked DNA vectors encoding the vascular endothelial growth factor (VEGF) gene in patients with with critical LIMB ISCHAEMIA due to poor peripheral vascularization

CANCER. One approach uses gene therapy with cytokine or neoantigen

genes to INCREASE TUMOR IMMUNOGENICITY. The vector is usually injected directly into the tumor, and there is some evidence that once the immune system is stimulated, nontransduced tumor cells may also be eliminated by the immune system.

Genes that CONTROL TUMOR GROWTH when expressed in nontumor cells may also be effective in cancer gene therapy.

interfere with tumor ANGIOGENESIS.

Synovial cellsIL-1 recep .antagonist

RA

Immune system

Antisense constructs,immunoenhancers

HIV-1

VariousCytokine,HLA genes.P53

Cancer

TissueGenetic approach

Complex traits-Hemoglobinopathies

. Finally, lytic viral vectors that selectively replicate and kill malignant cells are being developed. One example is an adenovirus designed to replicate in cells deficient in p53, a tumor-suppressor protein that is mutated in many different cancers

Vectors used for gene therapy• Viral options for gene delivery – Retrovirus – Adenovirus – Adeno associated virus – Herpes simplex virus – Vaccinia – Influenza

Vectors used for gene therapy• Non viral options – Plasmid DNA• Naked• Liposome• Ligand-DNA complex– Transkaryotypic therapy– Calcium phosphate precipititationIdeal vector• Capable of direct in vivo administration• Targeted delivery to specific cell• Safely integrated into genome• Transferred to all daughter cells• Site of insertion should be specific and should include excision of defective gene and its replacement by normal gene• Integrated into non oncogenic sites• Infection should not cause cell lysis.

• Currently no vector satisfies most of these criteria

Gene Delivery Strategies for Gene Therapy

Vectors used for gene therapy• Viral options for gene therapy:• Retroviruses• Adenoviruses

• Adeno associated viruses• Herpes simplex viruses

Retro virus• RNA virus with reverse transcriptase• Moloneymurin leukemic virus & Gibbon leukemic virus are most widely used.

benefits:100% transduction Can infect variety of cell linesDoes not lead to cell lysis Precise integration * cellular DNA is possibleLong term expression – integration* chromosomal DNAlimitations• Cell receptors are required and most retro viral recepters are not identified.• Requires cell division• Potential for insertional mutagenesis• Limited size of DNA insert• Potential recombination of therapeutic virus with endogenous retro viruses that can be pathogeneic Adenovirus• Large double stranded DNA virus• Natural viral pathogen to human being

• Benefits:• Infects non dividing cells• Large segments of DNA can be transported • Low risk of insertional mutagenesis• Broad range of target cells.• Efficient in –vivo delivery• Low risk of oncogenesis Limitations• Can lead to cell lysis • Doesn’t stably integrate into the chromosome, but remains as episomes• Transient expression. Gradually lost• Immunogenic – major limiting factor• Can initiate inflammatory responseAdeno associated virus

• Small DNA containing parvovirus • Requires adeno virus for replication(co- infection)• Replicates as double stranded DNA but packed as single stranded DNA • Integrates into specific location on human chromosome 19 , which is linked to B- cell CLL• Less efficient & less precise• Does not require cell divisionOthers

• Herpes virus: large double stranded DNA virus• Exists as episomes in the target cells.• Can accommodate a large gene• Useful for the introduction of genes in CNS• Vaccinia and influenza are in experimental stage.Non- viral options• Direct injection of naked DNA • Plasmids are incorporated into liposomes( synthetic cationic lipid)• No specific receptors needed.• Ligand DNA complex:targeted gene delivery.• Plasmid DNA and specific polypeptide ligand complex are generated• Taken up by the process of endocytosis by cells.• Incorporated into the DNA• Limiting factor is escape of DNA from endosomes to nucleus.Trans karyotypic therapy

• A small sample of patients’ cells are removed ,• genetically modified with the gene of therapeutic by a process called electro poration.(using a brief electrical pulse in open pores in the cells)CaPo4 precipitation• Enters cell by endocytosis and incorporated into the nucleus.• Advantages:• No infection risk• Completely synthetic• No limitation of insert size.Disadvantages:

• Low efficacy • Limited target cell range• Transcient expressionCandidate diseases for gene therapy• Gene therapy is likely to have the greatest success with diseases that are caused by single gene defect• By the end of 1993, gene therapy had been approved for the use on diseases like: Severe combined immunodeficiency Familial hypercholesterolemia Cystic fibrosis Gaucher’s disease

Criteria for selection of disease candidate for human gene therapy- eve nicholas• The disease is incurable, life threatening• Organ, tissue & cell types affected by the disease have been identified• The normal counter part of the defective gene has been isolated & cloned• Normal gene can be introduced into a substantial sub- fraction of the cells from the affected tissue or that introduction of the gene into the available target tissue, such as bone marrow, will some how alter the disease process in the tissue affected by the disease.

Some protein products of recombinant DNA technology

Factors which have kept gene therapy from becoming an effective treatment• Short-lived nature of gene therapy• Immune response• Problems with viral vectors• Multi-gene disordersRECENT DEVELOPMENT IN GENE THERAPY• University of California, Losangeles,research team gets genes in to the brain using liposomes coated in a polymer –polyethylene glycol(PEG).• Transfer of genes in to the brain is a significant achievement because viral vectors are too big to get across

the “blood brain barrier” .This method has potential for treating Parkinsons disease . • RNA interference or genes silencing may be a new way to treat Huntingtons disease.• New gene therapy approach ,repairs errors in m-RNA derived from defective genes.• Techniques has potential to treat Thalassemia ,Cystic fibrosis & some cancers.

• Gene therapy for treating children with X-SCID or “BUBBLE BOY “ disease is stopped in France, when the treatment caused leukemia in one the patients.• Researcher`s at Western Reserve University & Copernicus Therapeutics are able to create Tiny Liposomes 15nm`s across that can carry therapeutic DNA through pores in the nuclear membrane.• Sickle cell is successfully treated in mice. CURRENT STATUS OF GENE THERAPY• FDA has not yet approved any human gene therapy product for sale.• Current gene therapy is experimental and has not proved very successful in clinical trials.• In 1999, gene therapy suffered a major set back with a death of 18yr old JESSE GELSINGER (OTC deficiency)

Researchers also are experimenting with introducing a 47th (artificial

human) chromosome into target cells. This chromosome would exist autonomously alongside the standard 46 --not affecting their workings or causing any mutations. It would be a large vector capable of carrying substantial amounts of genetic code, and scientists anticipate that, because of its construction and autonomy, the body's immune systems would not attack it. A problem with this potential method is the difficulty in delivering such a large molecule to the nucleus of a target cell.

Arguments in favor of gene therapy• Can be used to treat desperately ill patients or to prevent the on set of horrible illness.

• Conventional treatment has failed for the candidate diseases for gene therapy & for these patients gene therapy is the only hope for future• Eric Juengst summarized the Arguments in favor of and against human germ line gene therapy.• Germ line gene therapy offers a true cure & not simply palliative or symptomatic treatment

Arguments against the development of germ line gene therapy.• Germ line gene therapy experiments would involve too much scientific uncertainty & clinical risks & the long term effects of such therapy are unknown.• As germ line gene therapy involves research on early embryos and affects their offspring. Such research essentially creates generations of unconsenting research subjects.• Gene therapy is very expensive and will never be cost effective enough to merit high social priority.

Some questions to ponder

• When should gene therapy be used? Should it be used to treat critically ill patients? Should it be used to treat babies and children? • What effect would gene therapy have on future generations if germline (reproductive) cells were genetically altered? How might this alteration affect human variation? • Who should decide what are "good" or "bad" uses of genetic modifications? How do you define "normal" with regard to human beings? • What if we could alter human traits not associated with disease? Would it be okay to use gene therapy to improve or enhance a person's genetic profile? • Who will have access to gene therapy, treatments and long-term follow-ups? Will gene therapy and genetic enhancements create an advantage for those who can afford it?

• The questions raised here have no clear right or wrong answer. Your responses will depend on your values, as well as

on the opinions of those around you.References

• Elements of medical genetics;10th edn,Mueller &Young.• Essentials of medical genetics; 4th edn, Connor& Ferguson smith.

• Principles of medical genetics;2nd edn. Gleehrter,Collins& Ginsburg• Genetics counseling in pediatric practice: Phadke &Phadke,Ind. Ped.• Memorix pediatrics; Dieter Harms & Jochem Scharf• Nelson textbook of pediatrics 16 th edn• Harrison’s principles of internal medicine 15th edn• Textbook of pediatrics Forfar 5th edn• Genetic disorder by M L Kulkarni.

GENE ADDITION Cystic fibrosisFamilial hypercholesterolemiaHemophilia A and BThalassemiaImmunodeficiencyMetabolic disorderDuchene’s muscular dystrophyRetinitis pigmentosaExpress CFTR in pulmonary system and/or GI tractExpress low-density lipoprotein receptor in liverExpress factor VIII or IX and secrete in circulationExpress normal globin in red blood cellsExpress mutant genes, such as adenosine deaminaseExpress missing enzymes or transportersExpress mutant dystrophin protein in muscle cellExpress normal protein in retinaGene correctionLesch-NyhanRetinitis pigmentosa (dominant)Sickle cell diseaseCystic fibrosisModify hypoxanthine phosphoribosyl transferase locusCorrect missense mutationCorrect -globin mutation

Correct F508 mutation in pulmonary systemModify vascular biologyCardiovascular diseases

Coronary artery restenosisPeripheral vascular disease

HypertensionBlock cell proliferation in vessel wall

Induce angiogenesisExpress genes (e.g., tissue kallikrein) to induce vasodilation

Refrences:• Elements of medical genetics;10th edn,Mueller &Young.• Essentials of medical genetics; 4th edn, Connor& Ferguson

smith.• Principles of medical genetics;2nd edn. Gleehrter,Collins&

Ginsburg• Genetics counseling in pediatric practice: Phadke &Phadke,Ind.

Ped.• Memorix pediatrics; Dieter Harms & Jochem Scharf• Nelson textbook of pediatrics 16 th edn• Harrison’s principles of internal medicine 15th edn• Textbook of pediatrics Forfar 5th edn• Genetic disorder by M L Kulkarni.

Emery’s text book of genetics.