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1 基因體在生殖醫學的應用陳明醫師博士私立復興中小學台北建國高級中學台大電機系肄業台大醫學系畢業教育部全國高中數學競試優等 1987 教育部公費留學臨床遺傳諮詢學門 2000 英國格拉斯哥大學醫學遺傳學碩士英國文化部獎學金 2001 台大生命科學博士彰基基因醫學部部主任台大醫學院婦產科助理教授台大醫院基因醫學部主治醫師

2 孟德爾遺傳定律 ( 孟德爾 ) 去氧核糖核酸 : 雙股螺旋 ( 華生 and 克里克 ) 醫學遺傳學 ( 馬庫希克 )

2003, President Clinton announced the finish of the first draft of human genome with two leading scientists Francis Collins ( 現任美國國家衛生院院長 ) and John Craig

3 2003, President Clinton announced the finish of the first draft of human genome with two leading scientists Francis Collins ( 現任美國國家衛生院院長 ) and John Craig Venter (Celera Genomics 創辦人 ) 於 1990 年十月一日正式展開, 最終目的在解讀基因體核苷酸序列, 並鑑別所有人類基因的功能 2003 年, 生物學家慶祝 DNA 雙螺旋結構發現 50 週年, 同時人類基因組計畫也宣佈完成了現代智人 (Homo sapiens)dna 序列的最終草稿

4 DNA 結構分子生物學的搖籃 : 英國劍橋大學物理系卡文迪西實驗室

5 從染色體到基因解析度和診斷工具的演進 Submicroscopic defects (microdeletion/microduplication) Genome-wide Cytogenetic analyses SKY CGH mband 10 Mb 3Mb

haploid chromosome number of Homo sapiens and characterized the human karyotype in 1952 "Mammalian Chromosomes In Vitro - The

6 細胞遺傳學的歷史 Barbara McClintock : (1902~1992) Maize cytogeneticist Studying the mechanics and inheritance of broken and ring chromosomes of maize Finding transposons (Nobel Prize in 1983) Tao-Chiuh Hsu 徐道爵博士 : (1917~2003 祖籍浙江美籍華人 ) Finding the accurate haploid chromosome number of Homo sapiens and characterized the human karyotype in 1952 "Mammalian Chromosomes In Vitro - The Karyotype of Man", in The Journal of Heredity 43: ((1952)). Tjio ( 蔣有興博士, 係印尼華僑 )and Levan 1956 in Lund, Sweden confirmed 2N=46 for human

7 Conventional cytogenetics

8 Conventional staining PRINCIPLE: Chromosomes are stained with Giemsa stain to reveal their size and shape. USEFUL FOR: 1) Fra(x) and autosomal fragile site studies 2) Chromosome breakage study e.g., R/O Fanconi's anemia

9 Fragile X:

10 Fanconi anemia: Nonhomologous rearrangement

11 Breaks, Gaps

12 G-banding (Trypsin-Giemsa Banding) PRINCIPLE: 1) A brief treatment of chromosomes with trypsin followed by Giemsa staining reveals light and dark bands, which are consistent for each homolog. 2) G(+) bands reflect AT-rich, late replicating heterochromatic regions. USEFUL FOR: Routine chromosome analysis.

14 C-BANDING (Centromeric Heterochromatic Banding) PRINCIPLE: This procedure uses alkaline treatment such as Ba(OH) 2 or NaOH to extract most of the DNA from every chromosome, leaving only the more resistant repetitive sequence satellite DNA around centromeres and the distal end of the Y. USEFUL FOR: 1) Study for centromeric heterochromatic regions 2) R/O variants of D- and G- groups. 3) Study for aberrations involving dicentric chromosomes. 4) Identification of the Y.

16 Ag-NOR STAINING (Silver Staining) PRINCIPLE: The nucleolus organizing regions (NORs) contain DNA coding for 18s and 28s ribosomal RNA. Silver staining of the NORs marks the sites of transcriptionally active rrna genes, which are usually present on the acrocentric chromosomes. USEFUL FOR: 1) Identification of the NORs or double NORs. 2) Study for rearrangement involving the NORs.

18 Molecular cytogenetics (Combination of molecular biology and cytogenetics) 分子細胞遺傳學

19 Types of FISH probes

20 Direct: FISH method Indirect: from

21 Human Telomere Structure Telomere has two major functions: 1. End-replication: maintain telomere length 2. End-protection: protect end-joining

23 1p /1q 2p /2q 3p /3q 4p /4q 5p /5q 6p /6q 16p /16q 17p /17q 18p /18q 7p /7q 8p /8q 9p /9q 10p /10q 11p /11q 12p /12q 19p /19q 20p /20q 21q 13q 14q 15q 22q Xp /Xq, Yp /Yq

24 Wolf-Hirschhorn Syndrome Profound developmental delay, seizure, Greek-Helmet facial appearance

25 Chromoprobe Multiprobe-T System: G-banding: 4pter deletion ToTelVysion Multi-color DNA Probes: 4pter deletion 4p /4q 4p /4q /21q 4pter deletion

26 Spectral karyotyping (SKY) 螢光全頻譜核型分析

28 SKY analysis for structural chromosome aberrations

29 Comparative Genomic Hybridization (CGH) A FISH technique to measure DNA gains or losses throughout an entire genome. 競爭性全基因組雜合反應

32 Array-CGH 基因晶片染色體微陣列

33 Principle of array CGH: From

34 From

35 Array-based comparative genomic hybridization (acgh) 7q11.22q21.1 deletion (10.9 Mb) 11q23.2q23.3 deletion (7.2 Mb) Miller et al., 2010

36 Array Comparative Genomic Hybridization (acgh) 利用多種遺傳檢測方式 ( 如 :A. G-banding, B. SKY, C. 基因晶片 ) 檢測不明原因之遺疾病

Trisomy 13 (Patau syndrome) 47,XX,+13 巴陶氏症

Correlated with maternal age Features: #

abnormally shaped eyes # Heart defect # An

37 Trisomy 13 (Patau syndrome) 47,XX,+13 巴陶氏症 1 in 10,000 births ~95% is miscarriage Correlated with maternal age Features: # Central nervous system defects # Severe mental retardation # Posterior scalp lesions # Oral-facial clefts # Small, abnormally shaped eyes # Heart defect # An extra pinky finger (polydactyly) # Additional organ anomalies

Severe mental retardation # Characteristic facial features # Short sternum #

38 Trisomy 18 (Edwards syndrome) 47,XX,+18 愛德華氏症 1 in 6,000 births ~95% is miscarriage Correlated with maternal age Features: # Low birth weight # Severe mental retardation # Characteristic facial features # Short sternum # Heart defects # Clenched hands with overlapping fingers # Additional organ anomalies

with maternal age Features: # Low birth weight # Distinctive facial

39 Trisomy 21 (Down syndrome) 47,XX,+21 唐氏症蒙古症 1 in 700 live-born children Most common cause of mental retardation ~75% is miscarriage Correlated with maternal age Features: # Low birth weight # Distinctive facial features # Developmental delay/mental retardation # Congenital heart anomalies

40 Monosomy X (Turner syndrome) 45,X 透納氏症 1 in 2,500-5,000 births Short stature Absence of ovaries and failure to proceed through puberty Characteristic facial features and a webbed neck Congenital heart defects

49 單基因疾病的遺傳模式 Autosomal dominant inheritance (AD) 體染色體顯性 One copy of a gene is dominant over the other Autosomal recessive inheritance (AR) 體染色體隱性 A gene is expressed only when both copies are the same X-Linked inheritance 性連隱性 A genetic feature is carried by the X chromosome (females XX, males XY) Mitochondrial inheritance 粒線體遺傳 A genetic feature is carried by the mitochondrial DNA

50 分子診斷最重要的兩大發明 PCR ( 聚合脢連鎖反應 ) Kary Mullis invented it in 1983, won Nobel prize in 1993 Sanger sequencing ( 定序 )Frederik Sanger invented it in 1977, and won another Nobel prize in 1980.

51 PCR Denature (heat to 95 o C) Lower temperature to 56 o C Anneal with primers Increase temperature to 72 o C DNA polymerase + dntps

52 PCR

53 Autosomal dominant inheritance (AD) Paroxysmal kinesigenic dyskinesia (PKD; OMIM #128200) A heterozygous mutation (c.649dupc; p.arg217fs) in PRRT2. Wild-type The patient

54 Autosomal dominant inheritance (AD) Spinocerebellar ataxia (SCA) An abnormal expansion of the CAG repeats (>400 bp) in MJD1 M P N C P C M:100bp DNA ladder P:the patient Nc:Normal control Pc:Positive control

55 Autosomal recessive inheritance (AR) Beta-thalassemia (MIM #141900) A heterozygous mutation (c.125_128deltctt) in HBB. Wild-type The patient

56 Autosomal recessive inheritance (AR) Aromatic L-amino acid decarboxylase deficiency (AADC) Father and mother: a heterozygous IVS6+4A>T in AADC heterozygous IVS6+4A>T homozygous IVS6+4A>T

57 X-Linked inheritance Lowe syndrome (MIM #309000) A hemizygous mutation (c.1987c>t; p.r663*) of OCRL gene was found. Wild-type The patient

58 X-Linked inheritance Hemophilia A heterozygous c.3780c>g (p.d1260e) (mother: carrier) hemizygous c.3780c>g (p.d1260e)

59 Mitochondrial inheritance Leber hereditary optic neuropathy (LHON; OMIM#535000) The common LHON mutation, m.11778g>a, was found. Wild-type The patient

60 Geographic distribution of alpha thalassemias 甲型海洋性貧血分布

61 Geographic distribution of beta thalassemias 乙型海洋性貧血分布

62 Dated Back to Its Birth 胚胎著床前基因診斷的誕生 1990 年 Alan Handyside and colleagues reported the first ever PGD in 1990 by fetal sex determination to exclude adrenoleukodystrophy and X-linked mental retardation by successful birth of two girls. Handyside A et al., Nature 1990

63 Preimplantation Genetic Diagnosis ( PGD ) 胚胎著床前基因診斷 A non-invasive procedure to the mother Genetic diagnosis at its earliest form PGD is an option for couples at risk, to avoid termination of an affected fetus following prenatal diagnosis.

64 Early human preimplantation development in vitro Nature Reviews Genetics 3, , 2002

65 PGD of chromosomal translocation Case 1: maternal t(2;22) 2qTEL 22qTEL 22q deletion qTEL 22qTEL PGD:

66 2. PCR-based PGD: Simple PCR method: eg. α-thalassemia SEA typing ARMS PCR method: feasible in most monogenic diseases Linkage assay: eg. Fragile X syndrome Single cell acgh: ongoing project

67 PGD for α-thalassemia SEA typing ~10 cases/year For example: M M 287 bp 194 bp Lane 1~3:sample 1 Lane 4~6:sample 2 Lane 7~9:sample 3 Lane 10~12:negative control Lane 13~15:positive control (carrier) M:size marker Results: Sample 1: major Sample 2: normal Sample 3: major

68 PGD for monogenic disorders: 44 cycles since 2008, including alpha- and beta-thalassemias, hemophilia-a, Fragile-X, Acute Intermittenet Porphyria, CMT IIE, hearing loss, and AADC.

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<4D6963726F736F667420506F776572506F696E74202D20A5FEB279ACECA7DEADB2A95243322DA5CDA4C6ACECA7DEA4A7BDC4C0BB> 全球科技革命生化科技之衝擊劉承揚 http://mail.tku.edu.tw/cyliu/ 生化科技之衝擊基因解碼基因工程的產物基因療法微生物與複製人奈米生醫技術晶片科技生化科技的衝擊 1 細胞典型動物細胞中各個胞器 :⑴ 核仁 ;⑵ 細胞核 ;⑶