CARRIER DETECTION

1. CARRIER DETECTION NASSER A. ELHAWARY Professor of Medical Genetics

2. Some Definitions • Genetic locus: is a specific position or location on a chromosome. Locus usually refers to a specific gene. • Alleles are alternative forms of a gene at a given locus. • Homozygous: a subject in which both alleles on a locus are identical. • Heterozygous: a subject in which both alleles on a locus are different. • Compound heterozygote: a subject having 2 different mutant alleles on a given locus.

3. Screening for genetic disease • Screening those at high risk • Carrier testing for AR & XL disorders • Pre-symptomatic diagnosis of AD

4. How to Detect Carriers?.... through • Clinical manifestations in carriers. • Biochemical abnormalities in carriers. • Linkage between a disease locus and a polymorphic marker.

5. 1. Clinical manifestations in carriers… • Carriers for some disorders have mild clinical manifestations. • e.g., In X-linked ocular albinism, retinal pigmentation may be seen in a manifesting female carrier. • Sometimes, either there is no sign at all in AR- or XL-carriers or overlap with general variations (like aging).

6. 2. Biochemical abnormalities in carriers… • The biochemical abnormality seen is a direct product of the gene, and then carrier detection ensures. e.g. In carriers of Tay-Sachs disease, the range in enzyme activity ‘hexosaminidase’ is intermediate between the normal and patient. • Sometimes, the biochemical abnormality is not a direct result of action of the gene product, in DMD (CPK).

7. Creatine phosphokinase (CPK) in obligate carrier females of DMD

8. 3. Linkage between a disease locus & polymorphic marker… • DNA polymorphic markers: e.g. STR • Potential pitfall with linked polymorphic DNA markers: - Recombination - Polymorphic variation: e.g. (CGG)n - Locus heterogeneity: A disorder inherited in the same manner can be due to mutations in more than one gene (sensori-neural hearing impairment/deafness. e.g. 1ry AR microcephaly have 6 distinct loci. STR44 STR45 STR48 STR50 Elhawary et al., 2006 ClinBiochem

9. STR44 STR45 STR48 STR50 Elhawary et al., 2006 ClinBiochem A pedigree of X-linked DMD showing STRs

10. Presymptomaticdiagnosis of AD Due to either delayed age of onset or reduced penetrance, the following may help to diagnose the AD disorders: • Clinical examination • Specialist investigation • Biochemical testing • Linkage DNA markers

11. Examples of Autosomal dominant • Fam. Hypercholestrolemia (1 in 500): High serum levels of total cholesterol and LDL-cholesterol. • Polycyctickidenydisease (1 in 1,250): Large number of fluid filled cysts form within the kidneys and cause renal failure. • Marfan syndrome (1 in 4,000): Abnormal patient's eyes, cardiovascular system, musculoskeletal system (Spider-like fingers. • Huntington disease (1 in 15,000): neurodegenerative mutations in (CAG)n

12. Prenatal Screening Maternal serum screening • Neural tube defects (NTDs, 1/1000) (↑ in AFP). • Down syndrome & Chromosomal abnormalities ‘Triple test’ at 16 wks: • Maternal serum AFP (↓ in DS pregnant) • unconjugated estriol (μE3) (↓ in DS pregnant). • hCG (human chorionic gonadotropin) (↑). • Ultrasonography

13. ttt by Folic acid & vit B12 NTDs (open, closed) • Neural tube defects (NTDs) are one of the most common birth defects. • An NTD is an opening in the spinal cord or brain that occurs very early in human development. • The 3rd or 4thwkof pregnancy, specialized cells on the dorsal side of the fetus begin to fuse and form the neural tube (NT). When the NT does not close completely, an NTD develops.

14. hCG (human chorionic gonadotropin) • The hormone hCG is produced during pregnancy. • It is made by cells that form the placenta, which nourishes the Egg after it has been fertilized and becomes attached to the uterine wall. • hCG Levels can first be detected by a blood test about 11 days after conception and about 12-14 days after conception by a urine test. In general, the hCG levels will double every 72 h. • The level will reach its peak in the first 8-11 wkof pregnancy andthen will decline and level off for the remainder of the pregnancy.

15. μE3 (unconjugated estriol) • Estriol is one of the three mainestrogens produced by the human body (estroneE1 ‘menopause’, estradiol E2’during reproductive’, estriol E3 ‘during pregnancy’). • Estriol can be measured in maternal blood or urine and can be used as a marker of fetal health. • If levels of "unconjugated estriol" are abnormally low in a pregnant woman, this may indicate chromosomal or congenital anomalies like Down syndrome (47,+21) or Edward's syndrome (XX,47,+18). • It is included as part of the triple test & quadruple test for antenatal screening for fetal anomalies. • Because many pathological conditions in a pregnant woman can cause deviations in estriol levels, these screenings are often seen as less definitive of fetal-placental health. Conditions which can create false positives and false negatives in estriol testing for fetal distress include preeclampsia, anemia and impaired kidney function.

16. SummaryTriple screen of maternal serum • Triple test measures with a 70% sensitivity and means high risk of chrom. abnormalities and NTDs. Direct testing of fetal DNA in maternal blood simplify current screening programs. • Quad test (81% sensitivity by adding inhibin A ‘inhibits FSH secretion’

17. Quad screen of maternal serum: Quantification at gestational age 16-18 wks of • Inhibin A (increased) • Beta-hCG (increased) • AFP (decreased) • Unconjugated estriol (uE3) (decreased) suggest of a fetus with DS. • Also, inhibin A is used as a marker of ovarian cancer.

18. Preimplantation genetic diagnosis (PGD) Prenatal diagnosis is so difficult on an established pregnancy to view a possible termination. So, • The female is given hormones to induce hyper-ovulation. • Oocytes are harvested transcervically, under sedation, with US guidance. • Motile sperm from a semen sample are added to the oocytes in culture (in-vitro fertilization, IVF). • Incubation to allow fertilization

19. Preimplantation genetic diagnosis… • At the early embryo (blastocyst) at the eight-cell stage on the 3rd day, fertilization is achieved using intracytoplasmic sperm injection (ICSI). • At the 8-cell stage, the early embryo is biopsied and one or 2 cells are removed for analysis. The IVF is strictly regulated by Human Fertilization & Embryology Authority (HFEA). • PGD is used in CF, DMD, HD, β-thal, SMA, FraX & chromosomal abnormalities by FISH is performed.

20. Genetic Counseling Dr. NASSER A. ELHAWARY Professor of Medical Genetics

21. Genetic Counseling • The counselor should ensure that the consultand understands that: • The medical diagnosis, prognosis, and treatment. • Mode of inheritance of the disorder and Risk of developing and transmitting it. • Choices or options available for dealing with the risk.

22. Steps in genetic counseling… • Accurate Diagnosis based on.. • Take personal-family-medical history (pedigree, checklist), • Carry out an accurate examination, • Undertake appropriate investigations (include chromosome, molecular studies or referral to neurologist… etc).

23. Steps in genetic counseling… Problems arise with firm diagnosis… • If the disorder shows etiological heterogeneity (e.g., hearing loss & non-specific mental retardation both have genetic and environ-mental factors). • The GC can be extremely difficult when the heterogeneity extends to different modes of inheritance. • e.g., Ichthyosis.. AD, AR, XR • e.g., Ehlers-Danlos syndrome.. AD, AR, XL

24. Steps in genetic counseling… • Calculating & representing the risk • Discussing the options. After the last 2 steps, (e.g. prenatal diagnosis should be discussed with details of techniques, limitations, risks associated with various applied methods). • Communication and support adoption (between the counselor and the consultand due to psychlogical and emotional factors in infertile cases, AD) • Special problems in GC 1- Consanguinity: Kuwait (54%), KSA (54%), Pakistan (40-50%), Egypt (28%), Algeria (23%), Japan (2-4%), UK or USA (2%). 2- Incest (relationships occur betn. 1stdegree relatives; 3- Adoption 4- Disputed paternity

25. Treatment of Genetic Diseases Conventional Approaches Therapeutic Applications of Recombinant DNA tech. Gene Therapy Treatment of Genetic Diseases

26. Conventional Approaches treatment… 1- Protein/enzyme Replacement If a genetic disorder is found to be the result of a deficiency or an abnormality of an enzyme or a protein, ttt may involve replacement of the deficient enzyme or protein (e.g. use of factor VIII concentrate in ttt of hemophilia).

27. Conventional Approaches treatment… 2- Drug Treatment Statins can help to lower the cholesterol levels in FHC. Avoiding some drugs or foodsto prevent manifestations of some genetic diseases (Sulfonamides & fava beans in G6PD). Gentamicin (aminoglycoside antibiotics) was used as nasal drops to treat patients with CF.

28. Conventional Approaches treatment… 3- Tissue Transplantation • Replacement of diseased tissue has been replaced. e.g. Renal transplantation in adult poly-cyctic kidney (PCK) disease. or Lung transplantation in CF.

29. Therapeutic Applications of Recombinant DNA tech... Biosynthesis of Gene products • Insulin extracted from pig pancreas. • Using recDNA technology, insulin can be obtained from human insulin gene by introducing cDNA of insulin into vectors. • Other examples: HGH (dwarfism), Factor VIII (hemophilia A), Factor IX (hemophilia B), b-interferon (multiple sclerosis).

30. GENE THERAPY Gene therapy: defined by GTAC as the deliberate introduction of genetic materialsinto human somatic cells for therapeutics, prophylactic or diagnostic purposes. • Regulatory requirements: - All programs are focusing only on somatic cell gene therapy but not on germlinegene therapy.

31. Gene Therapy… • Technical aspects: we have to address: • Gene characterization • Target cells, tissue & organ:e.g. b-thal-assemia ttt involves removing bone marrow from affected individuals, treating it in vitro, and then returning it to the patient by transfusion. • Vector system

32. Gene Therapy… • Vector system: The vector by which the foreign gene is introduced, need to be both efficient and safe. • The treated tissue or cells has a reasonable lifespan, and the body doesn’t react adversely to the gene product (produce antibodies product). • Some vectors resulted in a malignancy or mutagenic effect on either the somatic cell or germ-cell lines through insertion of the gene or DNA sequence in the host DNA.

33. Gene Therapy… • Animal models:These should be present to assist the suitability of gene therapy trials in humans (DMD, FA*, CF, HD). • In-utero fetal gene therapy:Risk to use adeno-virus vector is overcome by using in-utero stem-cell transplantation (Krabbe disease or Hurler syndrome). • Target organs:liver, CNS, muscle, bone marrow. *Friedreich’s ataxia (1860): Genetic disease (AR) due to progressive damage of nervous system resulting in symptoms ranging from gait disturbance to speech problems

34. Enrichment info… • Krabbe disease (globoid cell leukodystrophy) is a rare AR (1/100,000 births) • Often fatal degenerative disorder that affects the myelinsheath of the nervous system. • Mutation by GALC gene causes a deficiency of an enzyme called galactocerebrosidase

35. Enrichment info… • Hurler syndrome, also known as muco-polysaccharidosis type I (MPS I). • It is an AR that results in the buildup of MPS due to a deficiency of α-L-iduronidase, an enzyme responsible for the degradation of MPS in lysosomes. • Without this enzyme, a buildup of heparan sulfate and dermatan sulfate occurs. • Symptoms appear during childhood and early death can occur due to organ damage.

36. Gene Therapy… • Gene Transfer:can be carried out either ex-vivo (ttt of cells or tissue from an affected individual in culture, and re-introduce into the affected one; or • in-vivo(if cells can’t be cultured or replaced in the affected individual). - Viral agents… - Non-viral agents…

37. In-vivo and ex-vivo gene therapy

38. Viral agents, Gene Therapy… 1- Viral agents: used to transport foreign genetic materials into cells. • Oncoretrovirus:RNA viruses integrate into the host DNA by making copy of their RNA molecule using RTase. If these can be integrated to dividing stem cells, all progeny cells will inherit a copy of the viral genome. It Introduces a relatively small <7kb DNA sequences. • Lentivirus:It includes HIV, they are complex viruses that infect macrophages & lymphocytes, integrated tonon-dividing cells, hence useful in neurological conditions.

39. Viral agents, Gene Therapy… • Adenovirus: used as vectors in gene therapy to infect a wide variety of cell types. • Unlike retroviruses, they can infect non-dividing cells & carry up to36 kb of foreign DNA. • They don’t integrate into the host genome, thus avoiding possible insertional mutagenesis. • Its potential effect in malignancy. • Expression of the introduced gene is unstable and often transient.

40. Viral agents, Gene Therapy… • Adeno-associated virus:are non-pathogenic viruses in humans that require a co-infection with helperadenoviruses or certain Herpes virus to infect. * In absence of helper, they integrates to chrom DNA (chrom 19q13.3-qter). * Carry and insert up to 5 kb into the cell.

41. Viral agents, Gene Therapy… • Herpes-virus: They are neurotropic (infect nervous tissue) and may target gene therapy in Parkinson disease. * Toxic effects on nerve cells, hence immune response * Don’t integrate to host genome and thus expression of the gene productis temporarily and unstable.

42. Non-viral methods, Gene Therapy… 2- Non-viral methods: It is harmful to immune system, safer, simple to use, enable large scale production, but efficacy is limited. • Naked DNA:via injection (mini-dystrophin). • Liposome-mediated DNA transfer: Liposomes can facilitate introduction of foreign DNA, but not efficient in gene transfer. • Receptor-mediated endocytosis: Glycoprotein containing galactose will be recognized by receptors on the surface of liver cells.

43. Diagrammatic representation of liposome-mediated gene therapy

44. Gene Therapy… • RNA modificationtargets mRNA either by suppressing mRNA levels or by correcting/ adding function to the mRNA. • Antisense oligonucleotides: The sequence-specific binding of an antisense oligo-nucleotide (18-30 mer) to a target mRNA to inhibit the expression of protein.

45. Gene Therapy… • RNA interference: Any gene may be a potential silencing by RNA interference. • In contrast to antisense oligo (inhibited mRNA), mRNA is cleaved into 1000-fold more active. • Hence, homologous sequences to synthetic double-stranded RNA molecules known as small interfering RNAs (siRNAs). • siRNAs can be delivered in drug form to stabilize antisense oligonucleotides.