分子诊断分子诊断 -2: Practical-2: Practical

张咸宁zhangxianning@zju.edu.cn

Tel ： 13105819271; 88208367 Office: A705, Research Building

2013/09

Infectious Disease 

Neoplastic Disease 

Genetic Disease 

Identity Testing 

HLA Typing

Pharmacogenetics

AREAS OF APPLICATION OF MOLECULAR DIAGNOSTICS

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

APPLICATIONS OF MOLECULAR GENETIC TESTING

Clinical diagnosis/confirmation

Carrier screening

Prenatal diagnosis

Presymptomatic/predisposition diagnosis

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: Molecular Diagnosis, Diagnostic Molecular Pathology, Journal of Molecular Diagnosis, Genetic Testing and Molecular Biomarkers, Prenatal Diagnosis, …

www.genetests.org

Funded by National Institutes of Health

Located atUniversity of WashingtonSeattle, WA

Information resource for healthcare providers to help integrate genetic services into patient care

1160 clinics

• GeneReviews: “User manual” for genetic testing for specific diseases

405 GeneReviews One new Review added each week

• Laboratory Directory: “Yellow Pages” of genetics labs

• Illustrated Glossary: Genetic counseling and testing terms

~610 Clinical and research laboratories ~1460 Inherited diseases ~1180 clinical tests ~280 research only

• Clinic Directory: “Yellow Pages” of genetic services

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

GeneReviews Content

Summary

Diagnosis

Clinical Description

Differential Diagnosis

Management

Genetic Counseling

Molecular Genetics

Resources

References

GeneReviewsSummary

Disease characteristics

Diagnosis/testing

Management

Genetic counseling

One paragraph on:

The Father of Newborn Genetic Screening

Robert Guthrie

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”

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.

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?

National NBS Status: 2006

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

Prenatal Diagnosis

• The use of tests during a pregnancy to determine whether an unborn child is affected with a particular disorder.

• Began in 1966.

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.

Technique Time (Weeks) Disorders Diagnosed

Non-Invasive

maternal serum screening

α-Fetoprotein 16 Neural tube defects

16 18 Down syndrome

Ultrasound 18 Structural abnormalities (e.g., central nervous system, heart, kidneys, limbs)

Invasive

Amniocentesis 16

Fluid Neural tube defects

Cells Chromosome abnormalities, metabolic disorders, molecular defects

Chorionic villus sampling 10-12 Chromosome abnormalities, metabolic disorders, molecular defects

Fetoscopy

Blood (cordocentesis) Chromosome abnormalities, hematological disorders, congenital infection

Liver Metabolic disorders (e.g., ornithine transcarbamylase deficiency)

Skin Hereditary skin disorders (e.g., epidermolysis bullosa)

Standard Techniques Used in Prenatal Diagnosis

Methods of Noninvasive Testing in Prenatal Diagnosis

● Maternal serum alpha-fetoprotein● Maternal serum screen (MSS)● Ultrasonography● Isolation of fetal cells from maternal

circulation

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 .

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.

Methods of Invasive Testing 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

Amniocentesis & CVS

Amniocentesis: 15th to 16th weeks

PGD (preimplantation genetic diagnosis)

ICSI (intracytoplasmic sperm injection)

Noninvasive Testing!

• Maternal serum screen: ?

• Smear swab: ?

• Saliva: ?

• Hair: ?

1979 ： DNA diagnosis （ Yuet Wai Kan）

Methods in MD: cytogenetic, biochemical, DNA-based tests

• 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

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

SSCP

WAVE Transgenomic (USA)

Interface Pump Degasser

Detector

Column Oven

Autosampler

Temperature Rack

DHPLC

高分辨率熔解曲线分析技术（ high resolution

melting ， HRM）

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

ASO (allele-specific oligonucleotide) hybridization to detect an known

mutation

ASO (allele-specific oligonucleotide) hybridization to detect an known

mutation

Array Comparative Genomic Hybridization (aCGH)

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

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

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

Petricoin EF, et al. Trends Mol Med, 2004, 10:59–64

Acknowledge （ PPT特别鸣谢！）

• UCLA David Geffen School of Medicine

• www.medsch.ucla.edu/ANGEL/

• Prof. Grody WW (Divisions of Medical Prof. Grody WW (Divisions of Medical Genetics and Molecular Pathology), et al.Genetics and Molecular Pathology), et al.