基因體在生殖醫學的應用 - cctmf.org.t · preimplantation genetic diagnosis ( pgd )...
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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
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)
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。
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)
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
SKY mBANDCGH
Submicroscopic defects(microdeletion/microduplication)
Cytogenetic analyses
Genome-wide
10 Mb 3Mb
從染色體到基因 解析度和診斷工具的演進
細胞遺傳學的歷史Barbara McClintock : (1902~1992)
Tao-Chiuh Hsu徐道爵博士: (1917~2003 祖籍浙江 美籍華人)
Maize cytogeneticist
Studying the mechanics and inheritance of broken and
ring chromosomes of maize
Finding transposons (Nobel Prize in 1983)
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 43:167-172 ((1952)).
Tjio (蔣有興博士,係印尼華僑)and Levan 1956 in Lund,
Sweden confirmed 2N=46 for human
Conventional cytogenetics
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
Fragile X:
Fanconi anemia:
Nonhomologous rearrangement
Breaks, Gaps
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.
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.
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.
Molecular cytogenetics
(Combination of molecular biology and cytogenetics)
分子細胞遺傳學
Types of FISH probes
from http://en.wikipedia.org/
FISH methodDirect:
Indirect:
Human Telomere Structure
Telomere has two major
functions:
1. End-replication: maintain
telomere length
2. End-protection: protect
end-joining
1p
/1q
2p
/2q
3p /3q
9p
/9q
7p /7q 8p
/8q
10p /10q 11p /11q 12p /12q
13q 14q 15q
6p /6q4p /4q 5p /5q
21q
22q
19p
/19q
20p /20q
Xp /Xq, Yp
/Yq
18p
/18q
16p /16q 17p /17q
Wolf-Hirschhorn Syndrome
Profound developmental delay, seizure, Greek-Helmet facial appearance
4pter deletion
4p /4q
Chromoprobe Multiprobe-T System:
4pter deletion4p /4q /21q
ToTelVysion Multi-color DNA Probes:
4pter deletion
G-banding:
Spectral karyotyping (SKY)
螢光全頻譜核型分析
SKY analysis for structural chromosome aberrations
Comparative Genomic Hybridization
(CGH)
競爭性全基因組雜合反應
A FISH technique to measure DNA gains or losses throughout an entire genome.
Array-CGH
基因晶片
染色體微陣列
Principle of array CGH:
From www.molecular-cancer.com
From www.implen.de
Miller et al., 2010
7q11.22q21.1 deletion (10.9 Mb)
11q23.2q23.3 deletion (7.2 Mb)
Array-based 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
Array Comparative Genomic Hybridization (aCGH)
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
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
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
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
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
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.
Denature (heat to
95oC)
Lower temperature to
56oC Anneal with primers
Increase temperature to
72oC DNA polymerase +
dNTPs
PCR
PCR
Wild-type
The patient
• Paroxysmal kinesigenic dyskinesia (PKD; OMIM #128200)
• A heterozygous mutation (c.649dupC; p.Arg217fs) in PRRT2.
Autosomal dominant inheritance (AD)
Autosomal dominant inheritance (AD)
• Spinocerebellar ataxia (SCA)
• An abnormal expansion of the CAG repeats (>400 bp) in MJD1
M P NC PC
M:100bp DNA ladder
P:the patient
Nc:Normal control
Pc:Positive control
Wild-type
The patient
• Beta-thalassemia (MIM #141900)
• A heterozygous mutation (c.125_128delTCTT) in HBB.
Autosomal recessive inheritance (AR)
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
Wild-type
The patient
• Lowe syndrome (MIM #309000)
• A hemizygous mutation (c.1987C>T; p.R663*) of OCRL gene
was found.
X-Linked inheritance
• Hemophilia A
X-Linked inheritance
hemizygous
c.3780C>G (p.D1260E)
heterozygous
c.3780C>G (p.D1260E)
(mother: carrier)
Wild-type
The patient
Mitochondrial inheritance
• Leber hereditary optic neuropathy (LHON; OMIM#535000)
• The common LHON mutation, m.11778G>A, was found.
Geographic distribution of alphathalassemias 甲型海洋性貧血分布
Geographic distribution of beta
thalassemias 乙型海洋性貧血分布
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
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.
Early human preimplantation development in vitro
Nature Reviews Genetics 3, 941-955, 2002
PGD of chromosomal translocation
Case 1: maternal t(2;22)
2 22
2qTEL
2qTEL
22qTEL
22qTEL
22q deletion
PGD:
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
PGD for α-thalassemia SEA typing
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
M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 M
Results:
Sample 1: major
Sample 2: normal
Sample 3: major
• ~10 cases/year
• For example:
287 bp
194 bp
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.