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임상세포유전학 CLINICAL CYTOGENETICS

cell

chromosome

DNA

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출생전 진단 : 34 세 이상 임산부 , 염색체 이상아 가족력 출생후 소아기 : 다발성 기형 설명하기 어려운 정신박약 , 성장지연 성염색체질환이 의심되는 경우

성장기 이후 : 외성기 이상 (ambiguous genitalia),

무월경 , 습관성 유산 , 불임 ( 배우자와 같이 검사 )

혈액종양 , 고형종양

염색체검사 대상자는 누구인가 ?

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Problems of early growth & development, failure to thrive, developmental delay, dysmorphic faces, multiple malformations, short stature, ambiguous genitalia, mental retardation

Stillbirth & neonatal death - amenorrhea, infertility, recurrent miscarriage (spontaneous abortion)

Fertility problems Family history Neoplasia Pregnancy in woman of advanced age

Clinical Indications for Chromosome Analysis

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● Definition : IQ score <70-75, 2.5-3% Mild (85%): IQ 50-75, Moderate (10%): IQ 35-55, Severe (3-4%): IQ 20-40, Profound (1-2%): IQ 20-25

● Causes ① Genetics : Fragile X syndrome, Single gene defect,

Chromosome disorders (Down syndrone, etc)② Prenatal illness : Fetal alcohol syndrome, Drug abuse

& cigarette smoking, Maternal infection, hypertension,

toxemia, Birth defect① Childhood illness: hyperthyroidism, whooping cough,

chickenpox, measles② Environmental factors: malnutrition, lead poisoning

Mental Retardation (MR)

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Incidence of Cytogenetic Ds

1% of live births2% of pregnancies in women older than 35 years50% of all spontaneous first-trimester abortions

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Peripheral Blood (PB); Congenital anomaly, Fragile X syndrome Bone marrow (BM); Hematologic malignancies (leukemia, MDS, MPN)

Lymph node; Malignant lymphoma Soft tissue; Solid tumor

Amniotic fluid (AF), CVS; Prenatal diagnosis Skin biopsy- fibroblast

염색체검사 검체 선택

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RPMI 1640FBS (15%)Pen-StrepL-GlutaminePHAPB/BM cells

MTX(10-7M)

Thymidine (10-5M)

1) PB/BM Culture

3-5 hrs 17 hrs

2) Harvest5 hrs1) Colcemid (50 g/ml) treatment

2) Hypotonic Solution (KCl;0.075M) treatment3) Fixation (methanol:Acetic acid=3:1)

3) Slide preparation & Staining (Giemsa-Trypsin)4) Microscopy and Karyotyping 5) Printing (Photography) and Reporting

Giemsa Banding (Giemsa Banding (G-BandingG-Banding) ) TechniqueTechnique

72 hrs

High-resolution Banding TechniqueHigh-resolution Banding TechniqueUsing Methotrexate Cell SynchronizationUsing Methotrexate Cell Synchronization

* PHA, Phytohemagglutinin (T cell mitogen)**MTX , Methotrexate

Back to Basic염색체검사 장비 및 재료

CO2 incubator at 37oC, 5%CO2

clean bench

Inverted Microscope

Slide preparation

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Conventional Cytogenetics in Hematologic Malignancies

검사자 숙련도에 따라 슬라이드의 질적 차이가 많다분열세포 적고 , 염색체 길이 짧고 , quality 는

불량하다 . 복잡하고 다양한 핵형을 보이는 경우가 많다

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CCD camera

Main ProgramMain ProgramMicroscope

Monitor PC Printer

Computerized Image Analyzer Cytovision®

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46,XY

Chromosome Identification ( 염색체 동정 )

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A

C

B

D

F G

E

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Back to BasicSchematic Diagram of Chromosomes

Metacentric Submetacentric Acrocentric

9q34.19q34.1

qterqter

pterpterpp

qq

cencen

ArmArm RegionRegion BandBand SubbandSubband

Back to BasicAbnormalities of Chromosome number

46,XY (XX) Normal male (female) karyotype

Heteroploid ( 이수성 , 異數性 ) – 염색체 수가 46 개가 아닌 경우

Euploid ( 정배수성 , 正倍數性 ) haploid (n) polyploid - diploid (2n), triploid (3n), tetraploid(4n),

Aneuploid ( 이수성 , 異數性 ) : state of not having euploidy Aneuploid karyotypes are given names with the suffix -somy (rather

than -ploidy, used for euploid karyotypes), such as trisomy and monosomy.

ex) Down syndrome = Trisomy 21 syndrome Turner syndrome = Monosomy X syndrome

Back to BasicExamples of Proper Nomenclature and Description of Structural Aberration

Nomenclature Explanation

46,XY (XX) Normal male (female) karyotype47,XY,+21 Female trisomy 21, Down syndrome

del(11)(q23) Terminal deletion ( 결실 )

del(5)(q22q33) Interstitial deletion dup(5)(p14p15.3) duplication ( 중복 )inv(9)(p11q13) Pericentric inversion ( 역위 )inv(3)(q22q26) Paracentric inversioni(17q) Isochromosome ( 동완염색체 )t(9;22)(q34;q11) Balanced translocation ( 전좌 , 전위 )der(13;21)(q10;q10) Robertsonian translocationder(1)t(1;7)(p11;q11) Derivative chromosome ( 파생염색체 )

add(7)(p11) Extra material of unknown origin ( 추가 )mar Marker chromosome ( 표지염색체 )

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Terminal deletion del(11)(q23) Interstitial deletion del(5)(q22q33) Ring chromosome r(7)(p22q36)

Back to BasicInsertion ins(22;3)(q12;q21q32)

Robertsonian translocation der(13;21)(q10;q10)

Reciprocal translocation t(2;5)(q21q31)

Duplication dup(1)(q22q25)

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der(13;15)der(13;15)

45,XX,der(13;15)(q10;q10)

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Paracentric inversion inv(3)(q21q26.2)

Isochromosome i(17)(q10)

Pericentric inversionInv(3)(p13q21)

Back to BasicISCN 2009

An International System for Human Cytogenetic Nomenclature (2009)

The complete citation for reference lists is:

ISCN (2009):An International System for Human Cytogenetic Nomenclature, Schaffer L.G., Tommerup N. (eds);S. Karger, Basel, 2009

ISCN (1985, 1991, 1995, 2005)

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Patient-Specific Embryonic Stem Cells Derived from Human SCNT Blastocysts. Science: 19 May 2005

- 체세포 복제 방식을 통한 환자 맞춤형 줄기세포 확립

21. F. Mitelman, An International System for Human Cytogenetic Nomenclature (S. Karger, Basel, Switzerland, 1995).

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Morphology (A) and karyotyping (B) of interspecies blastocysts derived from a human cord fibroblast transfer into enucleatedbovine oocyte. An interspecies blastocyst at hatching were obtained 144 hours after culture and subsequently provided forchromosome analysis using a cytovision. Karyotyping shows 46 pairs of autosome and sex chromosome of XY.

A.K. Tarkowski and J. Rossant, Haploid mouse blastocysts developed from bisected zygotes. Nature 259 (1976), pp. 663–665

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형광물질로 표지된 소식자를 이용하여 염색체의 전체 또는 일부분에 보합시키는 기법으로 염색체 이상의 규명과 유전자 위치 규명 (mapping) 에 이용

Fluorescence in situ Hybridization (FISH)

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1. Slide preparation2. Slide denaturation3. Probe preparation

4. Hybridization

5. Post-hybridization wash 6. Detection7. Count stain

형광동소보합법 (FISH) 검사과정

Back to BasicClinical Application of FISH

Microdeletion syndromes (=contiguous gene syndrome)

Prader-Willi syndrome (del 15q11.2-q12)Angelman syndrome (del 15q11-q13)DiGeorge/CATCH 22 syndrome (del 22q11)Williams syndrome (del 7q11.23)WAGR syndrome (Wilm's tumor, aniridia,

genitourinary malformation and mental retardation) (del 11p13)

Langer-Giedion syndrome (del 8q24)Miller Dieker syndrome (del 17p13.3)Smith-Magenis syndrome (del 17p11.2)Retinoblastoma (del 13q13)

Marker chromosome = Very small unidentified chromosome Supernumerary chromosome

Extra structurally abnormal chromosomes

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Multicolor FISH (mFISH, M-FISH, SKY) Cross-species color banding, RxFISH

indispensable for the precise characterization of complex chromosome rearrangements and numerical chromosome aberrations

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Comparison of conventional CGH and array-CGH

• CGH (comparative genomic hybridization) : survey DNA copy number variations of whole genome.

• With CGH, differentially labeled test (i.e. tumor) and reference (i.e. normal individual) genomic DNAs are cohybridized to normal metaphase chromosomes, and fluorescence ratios along the length of chromosomes provide a cytogenetic representation of the relative DNA copy-number variation.

• Chromosomal CGH resolution is limited to 10-20 Mb –therefore, anything smaller than that will not be detected.

Array based comparative genome hybridization

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세포유전학 검사정보

강동성심병원 진단검사의학과http://labmed.hallym.ac.kr

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Regenerative Medicine

Genetics, Genomics, Proteomics