1、遺傳病的診斷,Prof. Cunyou ZHAO Ph.D. 趙存友 教授、博士生導(dǎo)師 電郵： 2019-12-02,第一節(jié) 臨床診斷,依據(jù)患者的表型，并結(jié)合家系分析，判定是否患有某種遺傳病，并確定遺傳方式和遺傳規(guī)律,第二節(jié) 遺傳學(xué)診斷,通過檢測(cè)遺傳物質(zhì)、蛋白產(chǎn)物和代謝產(chǎn)物的異常類型，對(duì)遺傳性疾病進(jìn)行診斷。 依診斷方法分為：細(xì)胞遺傳學(xué)和分子遺傳學(xué)診斷 依據(jù)臨床目的分為：診斷性檢測(cè)，癥狀前檢測(cè)，攜帶者檢測(cè)和產(chǎn)前檢測(cè) 依不同的遺傳病分為：染色體病診斷、基因組病診斷和單基因病診斷,Types of genetic abnormality遺傳異常類型,Three levels Genome mutat

2、ion Chromosome mutation Gene mutation,Genome Mutation基因組突變,Abnormalities of Chromosome number （染色體數(shù)量異常）,Heteroploid: chromosome number other than 46 Euploidy （整倍體） a chromosome number that is a multiple of the normal haploid set染色體數(shù)目是正常單倍體的多倍(2n, 3n, 4n): 92,XXXX or 92,XXYY Aneuploidy（非整倍體） Monosomy

3、 having only one member of a homologous pair: Turner syndrome (45,X; 45,XY,-22) Trisomy having three copies of a single chromosome: (47,XX,+21; 47,XY,+18 and 47,XY,+13),Chromosome mutation染色體突變,Abnormalities of Chromosome structure,Unbalanced rearrangement Deletions/Duplications Marker/Ring Chromoso

4、me Isochromosome Balanced rearrangement Translocations Robertsonian translocation Insertions Inversions,Gene Mutation 基因突變,Abnormalities of individual gene,Approaches of Tests,-Cytogenetics(細(xì)胞遺傳學(xué)） Conventional Cytogenetics Karyotype（核型) Molecular Cytogenetics Fluorescence in situ Hybridization (FISH

5、) Chromosomal microarray analysis (CMA) or Cytogenomic microarray,-Molecular Genetics (分子遺傳學(xué)） PCR Sequencing,Banding 5-10 Mb,FISH 150 kb,aCGH5-10 Kb,Sequencing 1 bp,Chromosomes photographed during metaphase or prometaphase and arranged in a standard sequence G-banding (G顯帶） Standard 550 bands (5-10

6、Mb) High Resolution 850 bands (2-3 Mb) Whole genome level of Chromosome number and structure variations,Karyotyping（核型分析）,Metaphase, prometaphase, prophase,Karyotype,Banding patterns are important for identification of chromosomes in karyotype analysis（顯色帶在核型分析的染色體鑒別中是非常重要的）,Triploidy三倍體(69,XXX),1/9

7、00 live births（出生率） Leading cause of mental retardation and heart defects（智力障礙和心臟缺陷） Wide flat skulls, 面寬平 40% congenital heart defects先天性心臟病,Trisomy 21:Down Syndrome (47,XX,+21),Trisomy 13: Patau Syndrome (47,XY,+13),1/15,000 births Lethal; mean survival time 1 month Facial malformations, eye defec

8、ts, extra fingers or toes, and large protruding heels Severe malformations of brain, nervous system, and heart Parental age only known risk factor,Trisomy 18: Edwards Syndrome (47,+18),1/11,000 births Average survival time 24 months Affected infants small at birth grow slowly and are mentally retard

9、ed Malformation of heart, hands, and feet For unknown reasons 80% of all trisomy 18 are female Advanced maternal age is a risk factor,Turner and Klinefelter Syndromes,45,X 1/10,000 births Females; short, wide chest; wide neck; rudimentary ovaries and abnormal sexual development No mental dysfunction

10、,47,XXY 1/1000 Features do not develop until after puberty 青春期后顯現(xiàn)癥狀 male with low fertility and mental dysfunction,Chromosome structure variations （染色體結(jié)構(gòu)變異）,Fluorescence In Situ Hybridization (FISH),Detect a specific genomic region (Not for whole) Probes with fluorescent molecules labelling Probe us

11、ing individual chromosomes or portions of chromosomes: detection of existence of an abnormal chromosome Repetitive DNA probes: detection of the number of copies of a particular chromosome Identifies chromosomal abnormalities (150kb),Applications,Mapping,Micro-deletion,Count,Translocation,Microarrays

12、,Array technology is the placement of a series of DNA/RNA probes on a glass slide and hybridization with genomic material from a patient of interest.,Microarray design customized and computerized,to identify genomic copy number variations (CNVs) Comparative genomic hybridization array (aCGH) (100 kb

13、) Single nucleotide polymorphism array (SNP) (510kb),Chromosomal microarray analysis (CMA) or Cytogenomic microarray （染色體微陣列分析）,aCGH Comparative genomic hybridization array,Unlike Sanger where each allele is only covered 2x at the most, NGS covers each allele tens to thousands of times depending on

14、assay Multiple different types of platforms Gene panels or whole exome Different amounts of coverage,Next Generation Sequencing (NGS),NGS sequencing,Roche/454 FLX: 2004 Illumina Solexa Genome Analyzer: 2006 Applied Biosystems SOLiDTM System: 2007 Helicos HeliscopeTM : recently available Pacific Bios

15、ciencies SMRT: launching 2010,NGS Platform,Trisomy 21,Karyotyping,aCGH,NGS,FISH,PCR for single gene detection,Polymerase Chain Reaction Quantitative: copy number Qualitative: presence or absence,Endpoint qPCR:,Real-time qPCR:,Sanger sequencing,X-Linked Mental Retardation XLMR,Am J Hum Genet. 2007 Feb;80(2):345-52.,CUL4B,Deletion、frame-shift and non-sense mutations,Silent and mis-sense mutations,Approaches