Transcript Molecular genetic testing

分子诊断-2: Practical
张咸宁
[email protected]
Tel：13105819271; 88208367
Office: A705, Research Building
2013/09
AREAS OF APPLICATION OF MOLECULAR
DIAGNOSTICS
Infectious Disease
Neoplastic Disease
Genetic Disease
Identity Testing
HLA Typing
Pharmacogenetics
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
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
• 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
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
GeneReviews
Summary
One paragraph on:
Disease characteristics
Diagnosis/testing
Management
Genetic counseling
Robert Guthrie
The Father of Newborn Genetic Screening
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. β2Thienylalanine（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.
Standard Techniques Used in Prenatal Diagnosis
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)
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!
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Maternal serum screen: ?
Smear swab: ?
Saliva: ?
Hair: ?
1979：DNA diagnosis（Yuet Wai Kan）
Methods in MD: cytogenetic,
biochemical, DNA-based tests
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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)
Detector
Column Oven
Autosampler
Temperature
Rack
Interface
Pump
Degasser
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
Genetics and Molecular Pathology), et al.