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GENETIC COUNSELING AND GENE
THERAPY
MODERATOR: Dr. Uday Kumar
Dr. 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:
Estimated prevalence per
100 population
Types of genetic
diseases
Single gene:
Autosomal dominant
Autosomal recessive
x-linked recessive
chromosomal abnormalities
common disorders with appreciable
genetic component
congenital malformations
2-10
2
1-2
6-7
7-10
20
Total
38-51
GENETIC COUNSELING
Definition
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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:
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Maternal age > 35 years
Paternal age > 50 years
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Child with congenital anomalies or dysmorphology
Consanguinity or incest
Family history of heritable disorders or diseases , including:
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Adult onset
Complex/multi factorial inheritance
Chromosomal abnormality
Single gene disorders
Heterozygote screening based on ethinicity, including:
Sickle cell anemia (W.African, Mediterranean, Arab,IndoPakistani, Turkish , S.E Asian .
• Tay-sachs , canavan (Ashkenazi - Jewish , French - Canadian)
• Thalassemias (Mediterranean, Arab, Indo- Pakistani.)
Steps in genetic counseling:
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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
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Anthropometry
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Chromosome analysis
Head to toe
Correct description of dysmorphology
Photographic record
Parental examination
Investigations
Biochemical analysis
DNA analysis
Histopathology
RISK ESTIMATION
conditition
Congenital heart
disease
diabetes
epilepsy
Severe mental
deficiency
Down syndrome
Cleft lip
Incidence/1000
3-8
60
Parents
affected/not
Normal with 1 child
affected
Normal with 1 child
affected
One parent affected
5
30
1.5
One sibling
affected
Tri 1, 1 child affected
T21/22 or 13-15/21 T21/21
1 child affected , 2
children affected, 1 parent
and child affected
1
Manic depression
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One parent affected
schizophrenia
8
0.06
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One parent affected
Huntington chorea
Rh hemolytic disease
galactosemia
phenylketonuria
0.025
0.1
One parent affected
After 1 still birth, father
homozygous
One child affected
Complex traits
-Hemoglobinopathies
Recurrence
risk
2
5-10
5-15
5
1
33
3-5
10
17
15
9-20
50
100
25
25
QUANTIFICATION
• 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
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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
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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
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For placing risks in context
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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.
1 in 10 is high risk,
1 in 20 as intermediate
And 1 in 50 as low risk
COMMUNICATION
• 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
Confidentiality
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Medical genetics team may learn many family secrets , such as
previous abortions , previous abnormal births and occasional false
paternity.
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The team should observe high moral values , confidentiality and
should respect the self esteem and moral values of the parents.
OUTCOMES
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Most consultands have a reasonable recall of the information given.
30% of the consultands have difficulty in recalling the precise risk
figure.
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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.
Genetic relationship
frequency
of partners
Proportion of
shared genes
Risk of
abnormality in
offspring
First degree
1/2
50%
Second degree
1/4
5-10%
Third degree
1/8
3-5%
Mental retardation
Severe
Mild
AR Disorder
Congenital
malformation
25
35
10-15
10
father
mother Recurrence
risk(%)
translocation
21/22
21/21
Trisomy
21
Mosaic
N
C
N
C
N
N
C
N
C
N
N
N
10-15
5
100
100
1
small
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.
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Gene Therapy
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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 DISEASES
Environmental manipulation:
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Restriction
Removal
Replacement
Gene manipulation:
Gene therapy
Environmental manipulation
EXAMPLES OF METHODS FOR TREATING GENETIC DISEASE
Disorder
Enzyme induction by drugs .
Phenobarbitone
Congenital non –hemolytic jaundice
Replacement of deficient enzyme/protein
Blood transfusion
SCID due to adenosine deaminase deficiency
BMT
Mucopolysaccharidoses
Trypsin
Trypsinogen deficiency
1-antitrypsin
1-antitrypsin deficiency
Cryoprecipitate/Factor
VIII
-glucosidase
Hemophilia A
Gaucher disease
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
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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.
• 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
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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
employed
Example:
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
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Disease targets
Single gene defect
Gene(s
Severe combined involved)
Adenosine
immunodeficiency deaminase
-AT deficiency
-Antitrypsin
Cystic fibrosis
CFTR
Hemophilia A & B Factor VIII &
IX
Tissues
Lymphoid tissue
Lungs,liver(cirr
hosis)
Lungs,pancreas
Blood clotting
-globin
Hyperchol LDL receptor
estremia
Phenylketo Phenylalanine
nuria
hydroxylase
Cancer
HIV-1
RA
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Blood formed
elements
Liver,vascular
endothelial smooth
muscle cells
Liver
Genetic
approach
Cytokine,HLA
genes.P53
Antisense
constructs,im
munoenhancer
sIL-1 recep
.antagonist
Tissue
Various
Immune
system
Synovial cells
The cardiovascular system (including the peripheral vasculature) has
become an important target for gene therapy.
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to inhibit smooth-muscle cell proliferation and PREVENT
RESTENOSIS.
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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.
. 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
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Viral options for gene delivery
– Retrovirus
– Adenovirus
– Adeno associated virus
– Herpes simplex virus
– Vaccinia
– Influenza
Vectors used for gene therapy
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Non viral options
– Plasmid DNA
• Naked
• Liposome
• Ligand-DNA complex
– Transkaryotypic therapy
– Calcium phosphate precipititation
Ideal 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
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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 lines
Does not lead to cell lysis
Precise integration * cellular DNA is possible
Long term expression – integration* chromosomal DNA
limitations
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Cell receptors are required and most retro viral recepters are not
identified.
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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
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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
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Can lead to cell lysis
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Transient expression. Gradually lost
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Herpes virus: large double stranded DNA virus
Doesn’t stably integrate into the chromosome, but remains as
episomes
Immunogenic – major limiting factor
Can initiate inflammatory response
Adeno 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 division
Others
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
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A small sample of patients’ cells are removed ,
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Enters cell by endocytosis and incorporated into the nucleus.
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Low efficacy
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
Advantages:
No infection risk
Completely synthetic
No limitation of insert size.
Disadvantages:
Limited target cell range
Transcient expression
Candidate 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
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Short-lived nature of gene therapy
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RNA interference or genes silencing may be a new way to treat
Huntingtons disease.
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New gene therapy approach ,repairs errors in m-RNA derived from
defective genes.
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Techniques has potential to treat Thalassemia ,Cystic fibrosis &
some cancers.
Immune response
Problems with viral vectors
Multi-gene disorders
RECENT 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 .
• 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
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FDA has not yet approved any human gene therapy product for sale.
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In 1999, gene therapy suffered a major set back with a death of
18yr old JESSE GELSINGER (OTC deficiency)
Current gene therapy is experimental and has not proved very
successful in clinical trials.
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
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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?
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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 fibrosis
Familial hypercholesterolemia
Hemophilia A and B
Thalassemia
Immunodeficiency
Metabolic disorder
Duchene’s muscular dystrophyRetinitis pigmentosa
Express CFTR in pulmonary system and/or GI tract
Express low-density lipoprotein receptor in liver
Express factor VIII or IX and secrete in circulation
Express normal globin in red blood cells
Express mutant genes, such as adenosine deaminase
Express missing enzymes or transporters
Express mutant dystrophin protein in muscle cell
Express normal protein in retina
Gene correction
Lesch-Nyhan
Retinitis pigmentosa (dominant)
Sickle cell disease
Cystic fibrosis
Modify hypoxanthine phosphoribosyl transferase locus
Correct missense mutation
Correct -globin mutation
Correct F508 mutation in pulmonary system
Modify vascular biology
Cardiovascular diseases
Coronary artery restenosis
Peripheral vascular disease
Hypertension
Block cell proliferation in vessel wall
Induce angiogenesis
Express 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.