分子诊断 -2: Practical

1. 分子诊断-2: Practical 张咸宁 zhangxianning@zju.edu.cn Tel：13105819271; 88208367 Office: A705, Research Building 2013/09

2. AREAS OF APPLICATION OF MOLECULAR DIAGNOSTICS Infectious Disease  Neoplastic Disease  Genetic Disease Identity Testing HLA Typing Pharmacogenetics

3. 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

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

5. 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, …

6. www.genetests.org Information resource for healthcare providers to help integrate genetic services into patient care Located at University of Washington Seattle, WA Funded by National Institutes of Health

9. GeneReviews: “User manual” for genetic testing for • specific diseases • 405 GeneReviews • One new Review added each week • Laboratory Directory: “Yellow Pages” of genetics labs • ~610 Clinical and research laboratories • ~1460 Inherited diseases • ~1180 clinical tests ~280 research only • Clinic Directory: “Yellow Pages” of genetic services • 1160 clinics • Illustrated Glossary:Genetic counseling and testing terms

10. Molecular genetic testing:United States • Testing used in patient care must be done in “clinical” laboratories, not research laboratories • Clinical laboratories have to meet standards set by federal law (“CLIA”) • Non-US laboratories are used when testing is not available in the US

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

12. GeneReviewsSummary Disease characteristics Diagnosis/testing Management Genetic counseling One paragraph on:

15. The Father of Newborn Genetic Screening Robert Guthrie

16. 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”

17. 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.

18. 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?

19. National NBS Status: 2006

21. 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

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

23. 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.

24. Standard Techniques Used in Prenatal Diagnosis

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

26. 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.

27. 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.

29. 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

30. Amniocentesis & CVS

31. Amniocentesis:15th to 16th weeks

32. PGD (preimplantation genetic diagnosis)

33. ICSI (intracytoplasmic sperm injection)

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

36. 1979：DNA diagnosis（Yuet Wai Kan）

37. Chromosome banding FISH CGH PCR RFLP SSCP RT-PCR Quantitative PCR Real-time PCR CFLP Invader assay DHPLC CGGE TGGE ASO Protein truncating test (PTT) Chemical mismatch cleavage (CMC) RNase A cleavage Dideoxy fingerprinting (ddF) SAGE Mass spectrometry (MS) LOH DNA sequencing DNA microarray SELDI MALDI aCGH Methods in MD:cytogenetic, biochemical, DNA-based tests

39. FISH of interphase nuclei with a chromosome 21 centromeric probe (CAMBIO) showing three signals consistent with trisomy 21.

40. SSCP

42. WAVE Transgenomic (USA) Detector Column Oven Autosampler Temperature Rack Interface Pump Degasser DHPLC

43. 高分辨率熔解曲线分析技术（high resolution melting，HRM）

44. 荧光条形码标记的单分子检测技术（nCounter Analysis System）：一种高通量检测基因表达谱、miRNA等分子的技术 ● Gene Expression •  800个基因分析通量 •  具有弹性的样品需求，适用于血液样本与FFPE样本 •  只需要15分钟的手动操作时间 ● miRNA Analysis • 人类，小鼠 及大鼠的miRNA分析皆适用  •  完整包含miRBase资料库的miRNA（人类、小鼠、大鼠） ● Copy Number Variation • 同时检视人类基因组中800个区域 •  准确的标记感兴趣的区域 ● 帮助二代测序进行后期验证 NGS Validation

45. ASO (allele-specific oligonucleotide) hybridizationto detect an known mutation

46. ASO (allele-specific oligonucleotide) hybridizationto detect an known mutation

47. Array Comparative Genomic Hybridization (aCGH)

48. Mass Spectrometry-based Diagnostics • MALDI-TOF MS：matrix-assisted laser desorption/ionization-time of flight mass spectrometry • SELDI -TOF MS：surface-enhanced laser desorption/ionization- time of flight mass spectrometry

49. Rosenblatt KP, et al. Annu Rev Med, 2004, 55:97–112

50. Rosenblatt KP, et al. Annu Rev Med, 2004, 55:97–112