1. بسم الله الرحمن الرحيم

2. Molecular Prenatal Diagnosis By Dr. Ashraf El Harouni Professor of Pediatrics & Clinical Genetics Consultant Neurogenetics Division of Human Genetics & Genome Research National Research Center, Cairo, Egypt

3. Prenatal Diagnosis A new field has developed offering *prenatal counseling *prenatal counseling *prenatal screening *prenatal screening *prenatal diagnosis *prenatal diagnosis *genetic counseling *genetic counseling

4. Attributes of prenatal diagnosis The changing trends in diagnosis The changing trends in diagnosis New technology New technology Advanced techniques Advanced techniques Overwhelming information resources Overwhelming information resources Molecular databases Molecular databases Identification of disease-causing mutations Identification of disease-causing mutations

5. Prenatal Diagnosis National Research Center Prenatal counseling Prenatal counseling Genetic Ultrasound Genetic Ultrasound Fetal tissue sampling Fetal tissue sampling (amniocentesis, fetal blood sampling,...)

6. Prenatal Diagnosis Clinic Detailed genetic history Detailed genetic history Prenatal counseling Prenatal counseling Genetic ultrasound (2D, 3&4 D) Genetic ultrasound (2D, 3&4 D) Prenatal screening (Ultrasound/biochemical) Prenatal screening (Ultrasound/biochemical) Fetal tissue sampling Fetal tissue sampling

7. Experience of prenatal molecular diagnosis at NRC This presentation reports the molecular analysis of fetal amniotic fluid cells from pregnant mothers with a family history of Phenylketonuria (PKU) Duchenne Muscular Dystrophy (DMD) Spinal Muscular Atrophy (SMA)

8. What is PKU? PKU is one of the most common inborn errors of metabolism leading to mental retardation. PKU is one of the most common inborn errors of metabolism leading to mental retardation. Prevention of severe mental retardation can be avoided with early diagnosis and implementation of a diet low in phenylalanine. Prevention of severe mental retardation can be avoided with early diagnosis and implementation of a diet low in phenylalanine.

9. Clinical Features Affected children are normal at birth and would not be detected in the absence of compulsory mass screening. In newborns with classic PKU hyper- phenylalaninemia develops 48 – 72 hours after initiation of milk feeding. When blood phenylalanine concentration reach 15 mg/dl. phenylalanine spills over into the urine.

10. Untreated infants present with: Developmental delay Developmental delay Behavioral disturbance Behavioral disturbance Mental retardation Mental retardation Seizures (25%). Seizures (25%). Characteristic musty odor (sweat, urine) Characteristic musty odor (sweat, urine) Lighter pigmentation (hair, skin, eyes) Lighter pigmentation (hair, skin, eyes) Eczema (common) Eczema (common)

12. Molecular basis of PKU An autosomal recessive disorder An autosomal recessive disorder An average incidence of 1/10000 An average incidence of 1/10000 Different mutations in the phenylalanine hydroxylase (PAH) geneDifferent mutations in the phenylalanine hydroxylase (PAH) gene

13. The Phenylalanine Hydroxylase (PAH) enzyme These different mutations cause deficiency or absence of the activity of PAH enzyme  HPA  classical PKU.These different mutations cause deficiency or absence of the activity of PAH enzyme  HPA  classical PKU. The hallmark of PKU is The hallmark of PKU is the elevated phenylalanine level in blood. level in blood.

14. What is DMD? It is an inherited disease marked by progressive weakness & degeneration of skeletal muscles. It is an inherited disease marked by progressive weakness & degeneration of skeletal muscles. It is the most common lethal X-linked recessive disease (1/3500 male live births). It is the most common lethal X-linked recessive disease (1/3500 male live births). Full blown symptoms appears by 4-6 yrs; death occurs usually in the teens. Full blown symptoms appears by 4-6 yrs; death occurs usually in the teens.

16. In DMD, the disease is present in infancy, with muscle fiber necrosis and high serum CK enzyme, however the clinical manifestations are typically not recognized until 3 years of age or later. In BMD, the onset of the disease is usually after age 7 years and often in the second decade, with muscle cramps.

17. Molecular basis of DMD Is caused by mutations in dystrophin gene located in XP21. Is caused by mutations in dystrophin gene located in XP21. Mutated dystrophin leads to degeneration of skeletal muscles. Mutated dystrophin leads to degeneration of skeletal muscles.

19. Molecular diagnosis: DMD genotype is determined by size and location of the mutation. Up to 70% of DMD are associated with deletions, and 5-10% with duplications, while more than 90% of cases of BMD have deletions. The remainder are due to point mutations.

20. Mutations that disrupt the reading frame (frameshift) of dystrophin gene result in absence of dystrophin protein as in severe DMD phenotype, while mutations that do not disrupt the reading frame (inframe) of the gene result in abnormal but partially functioning dystrophin as in milder BMD phenotype

21. What is SMA? The second most common AR disorder The second most common AR disorder Common genetic cause of infant mortality and disability Common genetic cause of infant mortality and disability Incidence ~ 1:10,000 live births Incidence ~ 1:10,000 live births Carrier frequency ~ 1:50 individuals Carrier frequency ~ 1:50 individuals

22. Hereditary Spinal Muscular Atrophies

23. Three different forms of childhood SMA have been recognized on the basis of age of onset and progression of the disease. TypeI, Acute infantile SMA, Werding- Hoffmann Disease (WHD). TypeII, Chronic childhood SMA, Intermediate SMA. TypeIII, Mild juvenile SMA, Kugelberg- Welander Disease (KWD).

24. Type I SMA (WHD): It is the most severe, with onset in utero. Patients never sit, and die from respiratory failure before the age of 2 years. Type II SMA: It is of intermediate severity. Patients are able to sit unsupported, but cannot stand or walk.Bone deformities is a great problem. Survival usually greater than 4 years. Type III SMA (KWD): Mild, has an onset after the age of two. Patients are able to walk unaided, although they do so late. Slow deterioration results in scoliosis and wheel chair dependence.

25. Pathogenesis of SMA Are caused by degeneration of α-MN in the anterior horn cells of the spinal cord Are caused by degeneration of α-MN in the anterior horn cells of the spinal cord Affects voluntary muscles causing proximal symmetrical weakness and atrophy of limbs and trunk Affects voluntary muscles causing proximal symmetrical weakness and atrophy of limbs and trunk

26. Molecular basis of SMA Genes involved in SMA (types I, II, III) were mapped to chromosome 5q13 by linkage analysis. Genes involved in SMA (types I, II, III) were mapped to chromosome 5q13 by linkage analysis. There are at least 4-genes involved in SMA. There are at least 4-genes involved in SMA. SMN1 gene was established to be the SMA causative gene. SMN1 gene was established to be the SMA causative gene. The centromeric copy The telomeric copy BTFp44c NAIP SMNc H4F5c H4F5t SMN1 NAIP BTFp44t

27. Prenatal out clinic patient 69 mothers 15 with family history of PKU 20 with family history of DMD 34 with family history of SMA

28. The service started at the NRC In 1998 for PKU and DMD In 2002 for SMA

29. Molecular Prenatal Diagnosis (PKU) Families with known mutations Families with known mutations (ex: IVS10, R261Q, Y277D, L48S) (ex: IVS10, R261Q, Y277D, L48S) PCR amplification followed by PCR amplification followed by restriction digestion restriction digestion Families with unidentified mutation Families with unidentified mutation VNTR and STR VNTR and STR

30. DdeI restriction results of IVS10 mutation in PAH Wild type Mutant type 1 2 3 4 Lane1: homozygous mutant Lane2: heterozygous mutant Lane3: fetus is homozygous for the mutation

31. Amplification of VNTR in exon 13 of PAH gene F M P1 P2 100 bp S AF Fetus inherited only one affected allele from his parents.

32. Amplification of STR in intron 3 of PAH gene F M P AF Fetus did not inherit the affected allele of the father and it’s either normal or carrier since the mother is homozygote.

33. Molecular Prenatal Diagnosis of DMD Targeted multiplex PCR Targeted multiplex PCR for detection of deleted exons according to mutation in proband

34. Multiplex PCR amplification of major and minor hotspots of dystrophin gene 1 2 3 øX 4 5 6 7 8 9 Fetus has deletion of 3 exons in lane 3 and 6

35. Molecular prenatal diagnosis of SMA A prerequisite is the identification of a homozygous absence in an index proband A prerequisite is the identification of a homozygous absence in an index proband Is carried out by direct mutation detection of SMN1 exon 7 by PCR amplification followed by restriction enzyme digestion (DraI) Is carried out by direct mutation detection of SMN1 exon 7 by PCR amplification followed by restriction enzyme digestion (DraI)

36. Amplified exon 7 digested with Dra enzyme 1 2 3 4 5 1 2 3 4 5 161 bp 188 bp P P A A A P P A A A A : A : homozygous absence. P : P : presence.

37. Molecular results of PKU The 15 fetuses of mothers with history of PKU: The 15 fetuses of mothers with history of PKU: Ten were apparently normal (carrier or normal) Ten were apparently normal (carrier or normal) Five were affected Five were affected

38. Molecular results of DMD The 20 fetuses of mothers with history of DMD: The 20 fetuses of mothers with history of DMD: Ten had the same deletions of the index cases Ten had the same deletions of the index cases Ten were normal with no deletion Ten were normal with no deletion

39. Molecular results of SMA The 34 fetuses of mothers with history of SMA: 28 were normal with no homozygous absence of exon 7 28 were normal with no homozygous absence of exon 7 6 had homozygous absence of exon 7 6 had homozygous absence of exon 7

40. Conclusion This study showed reliable technical results (no false positives or negatives). This study showed reliable technical results (no false positives or negatives). Prenatal diagnosis is an important tool for preventive medicine. Prenatal diagnosis is an important tool for preventive medicine. Prenatal diagnosis is important for proper genetic counseling. Prenatal diagnosis is important for proper genetic counseling.

41. The Team Clinical Prof. Dr. Ashraf El HarouniMolecular Dr. Laila Effat Dr. Laila Effat Dr. Mona Essawi Dr. Mona Essawi Dr. Khalda Amr Dr. Khalda Amr Ghada Al-Etribi Ghada Al-Etribi Mohamed Abd Al-Hamid Mohamed Abd Al-Hamid Prenatal Dr. Khaled Gaber

42. THANK YOU