分子诊断 -2: Practical

1. 分子诊断-2: Practical 张咸宁 zhangxianning@zju.edu.cn Tel：13105819271; 88208367 Office: C303, Teaching Building 2014/09

2. Molecular Classification of Genetic Disease • Disorders for which both the gene and mutation are known • Disorders for which the gene is known, but not the mutation • Disorders for which neither the gene nor the mutation is known • Polygenic disorders

3. APPLICATIONS OF MOLECULAR GENETIC TESTING  Clinical diagnosis/confirmation  Carrier screening  Prenatal diagnosis  Presymptomatic/predisposition diagnosis

4. Resources • Gene Tests: www.genetests.org • American College of Medical Genetics: www.acmg.net • National Society of Genetic Counselors: www.nsgc.org • OMIM: http://www.omim.org • The journals:MolecularDiagnosis,DiagnosticMolecularPathology, Journal ofMolecularDiagnosis,Genetic Testing and Molecular Biomarkers,Prenatal Diagnosis, …

7. GeneReviews Content Summary Diagnosis Clinical Description Differential Diagnosis Management Genetic Counseling Molecular Genetics Resources References

9. Testing Timeline Amniocentesis for chromosome & other genetic testing CVS for chromosome & other genetic testing Blood for maternal serum screening Blood for Thalassemia & sickle cell Newborn Screening PGD Blood for NIPT http://www.geneagenetics.com.au/Genetic-testing

10. The Father of Newborn Genetic Screening Robert Guthrie

11. Robert Guthrie (1916-1995) Microbiologist, SUNY Buffalo Son with MR/DD and niece with PKU Devised “Guthrie test” originally to monitor PKU therapy Conceptualized NBS for PKU and the “Guthrie spot”

12. Guthrie Test • is a bacterial inhibition assay. β2-Thienylalanine（噻吩丙氨酸）is placed in the medium and normally causes the inhibition of Bacillus subtilis（枯草杆菌）growth. However, in the presence of excess of phenylalanine, this inhibition is overridden and bacterial growth occurs. This test is the least expensive screening method available for determining excess phenylalanine in the blood, but other tests are used to confirm findings.

13. Disease Targets of Newborn Screening Phenylketonuria（苯酮尿症） Galactosemia（半乳糖血症） Congenital hypothyroidism （先天性甲状腺功能减退症） Sickle cell/hemoglobinopathies Cystic fibrosis Metabolic screen (TMS。串联质谱筛查法) others?

14. National NBS Status: 2006

16. ACMG NBS Expert Group, 2006 Recommended screening for Core panel of 29 diseases Secondary targets of 25 diseases Total of 54 diseases should be included in NBS test panels Watson et al. Genet. Med. 2006; 8:1S-11S

17. Prenatal Diagnosis • The use of tests during a pregnancy to determine whether an unborn child is affected with a particular disorder. • Began in 1966.

18. The Principal Indications for Prenatal Diagnosis by Invasive Testing 1. Advanced maternal age. 2. Previous child with a de novo chromosome abnormality. 3. Presence of structural chromosome abnormality in one of the parents. 4. Family history of a genetic disorder that may be diagnosed or ruled out by biochemical or DNA analysis. 5. Family history of an X-linked disorder for which there is no specific prenatal diagnostic test. 6. Risk of a neural tube defect (NTD). 7. Maternal serum screening and ultrasound.

19. Standard Techniques Used in Prenatal Diagnosis

20. Methods of NoninvasiveTesting in Prenatal Diagnosis ● Maternal serum alpha-fetoprotein ● Maternal serum screen (MSS) ● Ultrasonography ● Isolation of fetal cells from maternal circulation

21. Screening and Diagnostic Tests for Down Syndrome The triple screen is a noninvasive screening test to determine whether there is an increased risk for Down syndrome. It is only a screening test and not a diagnostic test. Increased risk is associated with the following: • Low maternal serum α-fetoprotein (MSAFP) • Low unconjugated estriol (uE3) • Elevated human chorionic gonadotropin (hCG) Diagnostic tests include amniocentesis羊膜穿刺, chorionic villus sampling (CVS)绒毛吸取术, and percutaneous umbilical blood sampling (PUBS)脐穿刺.

22. Ultrasound result for a fetus with a meningomyelocele脊髓脊膜膨出, visible as fluid-filled sacs (arrow) located toward the base of the spinal column.

24. Methods of InvasiveTesting in Prenatal Diagnosis ● Amniocentesis:15th to 16th weeks ●Chorionic villus sampling (CVS):10th to 12th weeks ●Cordocentesis (PUBS):19th to 21th weeks ●Preimplantation genetic diagnosis (PGD):before the fertilization

25. Amniocentesis & CVS

26. Amniocentesis:15th to 16th weeks

27. PGD (preimplantation genetic diagnosis)

28. ICSI (intracytoplasmic sperm injection)

30. Noninvasive Testing! • Maternal serum screen: ? • Smear swab: ? • Saliva: ? • Hair: ?

31. Commonly Utilized Prenatal Screens Week NT=nuchal translucency 颈项透明层厚度 PAPP-A=妊娠相关蛋白A DIA= 抑制素A二聚体 AFP= 甲胎蛋白 hCG=人绒毛膜促性腺激素 uE3= 游离雌三醇

32. Noninvasive Prenatal Testing (NIPT) estimate risks based-on DNA

33. Sources of Fetal DNA in Maternal Blood cell-free fetal DNA（cffDNA。游离胎儿DNA）在妊娠第4周开始经胎盘进入母体血液循环，约占孕妇血浆游离DNA的10%。cffDNA片段可代表胎儿的全基因组。胎儿出生之后，cffDNA迅速在母体内被清除。

35. Performance Comparison

37. 1976：1st DNA diagnosis(Prof. Yuet Wai Kan, UCSF)

38. The 1st Genetic Testing in China • Prof. 吴冠芸 (1924-, PUMC): α-thalassemia, 1981.

40. Acknowledge（PPT特别鸣谢！） • UCLA David Geffen School of Medicine • www.medsch.ucla.edu/ANGEL/ • Prof. Grody WW (Divisions of Medical Genetics and Molecular Pathology), et al.