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.