종설 Journal of Genetic Medicine 2010;7: ) 염색체 Microarray 검사의임상적적용 울산의대서울아산병원진단검사의학과 서을주 Clinical Applications of Chromosomal Microarray Ana

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1 종설 Journal of Genetic Medicine 2010;7: ) 염색체 Microarray 검사의임상적적용 울산의대서울아산병원진단검사의학과 서을주 Clinical Applications of Chromosomal Microarray Analysis Eul-Ju Seo Department of Laboratory Medicine, University of Ulsan College of Medicine and Asan Medical Center, Seoul, Korea Chromosomal microarray analysis (CMA) enables the genome-wide detection of submicroscopic chromosomal imbalances with greater precision and accuracy. In most other countries, CMA is now a commonly used clinical diagnostic test, replacing conventional cytogenetics or targeted detection such as FISH or PCR-based methods. Recently, some consensus statements have proposed utilization of CMA as a first-line test in patients with multiple congenital anomalies not specific to a welldelineated genetic syndrome, developmental delay/intellectual disability, or autism spectrum disorders. CMA can be used as an adjunct to conventional cytogenetics to identify chromosomal abnormalities observed in G-banding analysis in constitutional or acquired cases, leading to a more accurate and comprehensive assessment of chromosomal aberrations. Although CMA has distinct advantages, there are several limitations, including its inability to detect balanced chromosomal rearrangements and low-level mosaicism, its interpretation of copy number variants of uncertain clinical significance, and significantly higher costs. For these reasons, CMA is not currently a replacement for conventional cytogenetics in prenatal diagnosis. In clinical applications of CMA, knowledge and experience based on genetics and cytogenetics are required for data analysis and interpretation, and appropriate follow-up with genetic counseling is recommended. Key Words : Microarray analysis, Chromosome aberrations, Genomic structural variation, Copy number variation 서론 접수 : 2010년 12월 3일수정본접수 : 2010년 12월 17일게재승인일 : 2010년 12월 24일게재일 : 2010년 12월 31일책임저자 : 서을주우 서울시송파구풍납동 울산의대서울아산병원진단검사의학과 Tel: 02) , Fax: 02) ejseo@amc.seoul.kr 본논문은 2010년 11월 18일제13차대한의학유전학회임상유전학추계학술대회로강연되었음. 1956년인간의염색체수가정확히 46개라는것이보고되었고 1), 1958년 Lejeune 에의해다운증후군에서 21번세염색체가밝혀진이후 2), 많은염색체이상질환들이전통적인염색체검사를통해알려졌다 3). 세포배양후분열중기세포의 G- banding 염색체를분석하는고식적인염색체검사는현재도임상세포유전검사실에서염색체의수적구조적이상을검출하기위해가장기본적으로사용하는검사법이다. 인간유전체의크기가 3,100 Mb (

112 서을주 nih.gov/projects/genome/assembly/grc/human/data/ind ex.shtml) 이므로, 고식적인염색체검사로얻어진 500-600 band 수준의분열중기염색체에서 band 당유전체의크기는 5-6 Mb 정도이다.

2 112 서을주 nih.gov/projects/genome/assembly/grc/human/data/ind ex.shtml) 이므로, 고식적인염색체검사로얻어진 band 수준의분열중기염색체에서 band 당유전체의크기는 5-6 Mb 정도이다 band 해상도의염색체검사로는 5 Mb 미만의염색체이상을검출하기어렵기때문에 5 Mb 미만의염색체결손을미세결손이라고한다. 특징적인표현형을나타내면서그원인이특정유전체부위의미세결손인질환을미세결손증후군이라고하며 4), 미세결손증후군을진단하기위해서원인유전체부위를표적한형광제자리부합법 (fluorescence in situ hybridization, FISH) 이나 PCR 기반의유전학적검사를시행한다. 이러한표적검사법은검출하고자하는유전체부위에대한결과만을알수있다 3, 5). 최근유전학적기법이급속히발전함에따라유전체전체의변화를한번에분석할수있는 microarray 기법이개발되었고, 특히염색체의미세한이상을검출할목적으로사용하는것이염색체 microarray 이다. 염색체 microarray 검사는고해상도 microarray 로염색체전체를한번에검색하여초현미경적인미세한변이를검출할수있기때문에외국의임상세포유전검사실에서는이미일상적검사로서시행되고있다. 염색체 microarray 검사를이해하기위하여 microarray 기법의개발과정과염색체 microarray 의종류, 검사방법, 임상적적응증, 검사결과의해석및유의사항에대하여고찰하고자한다. Microarray 기법의개발과염색체 microarray 종류유전체전체를스캔하여염색체의양적변이를분석한방법의시초는 1992년 Kallioniemi 등이발표한 comparative genomic hybridization (CGH) 기법이다 6). Test 세포와 reference 세포로부터추출한 DNA를각각다른두가지형광으로표지하여정상분열중기염색체에동시에부합한후에염색체지도상에나타나는형광강도비로 test 게놈과 reference 게놈의염색체양을상대적으로분석함으로써 test 세포에서염색체특정부위의양적변이를파악할수있다. 그러나분열중기 CGH는염색대수준의해상도를가지기때문에 5 Mb 이하의염색체양적변이를검출하기어렵다. 특정 DNA 조각들을슬라이드상에정렬한 microarray 가개발되기시작하면서 1997년에약 20종류의표적 DNA가고정된유리슬라이드상에서 CGH를시행한 matrix-based CGH가소개되었다 7). 1998년에는 20번염색체를표적한 3 Mb 간격의 array CGH 가보고되면서 array CGH라는용어가사용되었고보다고해상도에서염색체이상을분석할수있게되었다 8). Array CGH 기법의원리는 test DNA와 reference DNA를 Cy3와 Cy5로각각달리형광표지한후, 표적으로하는 DNA 조각들이일정하게정렬된슬라이드상에서동시에부합시키고, 각 DNA 조각에부합되어나타난형광강도비는두게놈간의 copy number 비를반영하는것이므로전체프로파일에서 genome-wide 하게 copy number variants (CNV) 를검출할수있다 9) (Fig. 1). Microarray 상에정렬된표적 DNA 조각을탐색자 (probe) 라고하며, BAC 클론 ( kb), fosmid/cosmid 클론 (40 kb), 작은삽입클론 ( kb), cdnas (0.5-2 kb), PCR 산물 (100 bp-1.5 kb), oligonucleotide (25-80 bp) 등을탐색자로올린다. Microarray 의해상도는탐색자의종류와전체개수및유전체상의분포특성에따라결정되고, 검출능력은탐색자의 signal-to-noise ratio와부합반응의효율성등플랫폼의특성에따라다르다 10). Fig. 1. Array-based comparative genomic hybridization for the identification of copy-number variants 9).

3 염색체 Microarray 검사의임상적적용 113 초기에는 BAC 클론 array 가주로사용되었는데, 2004 년에 Iafrate 등 11) 은 1 Mb 해상도의 BAC 클론 array CGH 기법을이용하여인간유전체상에 CNV라는유전체의구조적다형성이존재한다는사실을발견하였다. 그후인간유전체전체를포함하도록 26,000 개또는 32,000 개의 BAC 클론을올린 tiling-resolution BAC array가소개되었다 12-15). BAC array는표적 DNA 염기서열에특이성이높고 signal-tonoise ratio 가높아서민감도도좋지만, BAC 클론의크기때문에유전체의미세한양적변이를정확히측정하는데어려움이있다. 따라서 microarray 에올리는표적 DNA 조각을짧게만든 oligonucleotide 탐색자를사용하고탐색자개수를증가시켜서해상도를높이는방향으로 array CGH 기술이발전하게되었다 16). Oligonucleotide microarray 는탐색자디자인이용이하고원하는부위를고밀도로정렬할수있어서사용목적에맞추어다양한해상도를가진 CGH microarray 를제작할수있다. 44K 부터 1 million (Agilent Technologies, Santa Clara, CA, USA), 72K 부터 2.1 million (Roche NimbleGen, Madison, WI, USA) 등다양한플랫폼의 CGH oligonucleotide microarray 가유전체 CNV 연구와염색체이상을검출할목적으로사용되고있다. SNP 유전형분석을위해서고안된 SNP microarray 로 HapMap project 를수행하면서유전체의 CNV를검출할수있었고 17), 이후 SNP microarray 가염색체이상을검출하는데이용되기시작하였다 18, 19). Test와 reference 의두 DNA 를동시에부합시키는 array CGH와는달리, SNP microarray 는 test DNA만을부합시키고부합된탐색자의형광강도를 reference 에서얻어진 dataset 과비교하는것이다. SNP microarray 는 SNP 유전형의신호를통해 SNP 정보와유전체의 copy number 뿐만아니라 segmental uniparental disomy (UPD) 에대한정보도알수있다 19). 염색체 microarray 검사목적으로고안된 2.7 M array (Affymetrix Inc., Santa Clara, CA, USA) 와 CytoSNP (Illumina, San Diego, CA, USA) 의 SNP microarray 가시판되고있다. CGH oligonucleotide microarray 는 segmental UPD를검출할수없는단점이있는데, 최근에이을보완한 CGH microarray 가개발되었다. 이상과같이염색체 microarray 는여러플랫폼이있고각각장단점을가지고있으므로 microarray 선택시에각플랫폼의특성과한계점을잘파악하는것이중요하다. 염색체이상의검 출목적의 microarray 활용에관한 American College of Medical Genetics (ACMG) Practice guidelines 을보면 20), oligonucleotide microarray 가 BAC array에비해재현성이우수하고 batch-to-batch variation 이적다고하였다. 정신지체또는선천성기형이있는환자에서염색체 microarray 검사를시행할경우 microarray 플랫폼에대한권장사항으로서, 탐색자는염색체전체에걸쳐일정하게분포하고최소한 400 kb 이상을검출할수있어야하며표적부위에대해서는충분한탐색자밀도로고안되어야한다는내용이있다 21). 염색체 microarray 검사방법염색체 microarray 검사는적절한 microarray 의선정과검증, 검체에서 DNA 분리, reference DNA 준비, 형광표지, microarray 에부합, 데이터추출과분석, 결과해석, 비정상결과의검증과정, 결과보고의단계로이루어진다 22). ACMG 의 standards and guidelines for clinical genetics laboratories ( _E_2010.pdf, revised 01/2010) 는선천성염색체이상의검출을위한염색체 microarray 검사의각단계마다 standards 와 guideline 을제시하였다. 임상적적응증염색체 microarray 검사는고식적인핵형분석의해상도를극복할수있고표적한부위만검출하는 FISH 검사나분자유전학적방법대신유전체전체에서임상적으로중요한유전체변이를검출할수있기때문에, 외국의임상세포유전검사실은염색체 microarray 검사를염색체검사와병행하거나심지어염색체검사대신시행하기도한다. 발달지연및정신지체가있는환자에서염색체검사를시행하면 3-5% 에서비정상소견을검출하지만염색체 microarray 검사를시행할경우 9-15% 에서비정상소견을검출할수있고정확한위치와크기까지파악할수있다 23-25). 일부유전체질환들은특징적인임상증상을가진환자의표현형을보고일차적으로 FISH와같은표적검사를시행하여확진될수도있으나, 대부분의유전체질환들은비특이적인임상증상이많고, 임상적으로중요한미세한양의유전체변이가염색체의어떠한부위에서도존재할수있기때문에유전체전체를대상으로한염색체 microarray 검사가진단율을높일수

4 114 서을주 있는유용한검사법이다 23). 미국과유럽의세포유전검사실의책임자들그룹인 International Standard Cytogenomic Array (ISCA) Consortium 21) 에서는정신지체또는선천성기형이있는환자에대해염색체 microarray 검사가가장처음시행 (first-tier) 하는진단검사가될수있다고보고하였다. ISCA Consortium 은 21,698 명의환자를포함한 33개의연구를분석하였고, 정신지체또는선천성기형이있는환자대상으로염색체 microarray 검사는핵형분석의진단률 3% 보다훨씬높은 15-20% 의진단률을보였다. ACMG Practice guideline 20) 과 ISCA Consortium 21) 에서제시한염색체 microarray 검사의적용범위는거의비슷하다. 진단검사법으로서염색체 microarray 검사를가장먼저시행할수있는적응증으로는 1) 비특이적인다발성기형, 2) 비증후군성발달지연또는정신지체, 3) 자폐증상질환이있다. 성장지체과언어발달지연의경우염색체 microarray 검사를 추가로시행해볼수있다. 그외염색체 microarray 검사를염색체검사의보조적검사법으로활용할수있는데, 염색체검사에서검출된불균형재배열이나 marker 염색체를검증하기위해 26, 27) (Fig. 2), 또는복잡한유전체변이를보이는종양의정확한핵형분석을위해시행할수있다 28). 산전진단목적으로염색체 microarray 검사를적용한보고들이있지만 29, 30), 미국산부인과학회 31) 는염색체 microarray 검사가여러문제점을내포하고있기때문에현재로서는산전진단으로고식적인염색체검사를대신할수는없다는의견이다. 염색체 microarray 검사의한계점으로는균형재배열의염색체이상과 low-level 모자이시즘을검출하기어렵고, 임상적중요성이불명확한 CNV에대한해석, 고가의검사비용등이다. 다운증후군과같이임상증상이뚜렷한염색체질환, 염색체재배열의가족력, 습관성유산의기왕력이있는경우는염 A B Fig. 2. (A) Karyogram of the patient. The deletion in the long arm of chromosome 3 was apparently a terminal deletion, as shown by the arrow. The karyotype was initially designated 46,XX,del(3)(q29). (B) Chromosomal microarray profile of chromosome 3. The X-axis represents the probe index on chromosome 3, and the Y-axis represents the signal log2 ratio of the probe. The 3q29 region showed a 4.0-Mb heterozygous interstitial deletion in Chr3:195,007, ,085,431 26).

7% 를차지하고 2,600개이상의유전자가포함되어있다 32). 개인마다평균 11.3 Mb에 390개의유전자를가진 CNV가존재하므로염색체 microarray 검사의해석시유의하여야한다.

5 염색체 Microarray 검사의임상적적용 115 색체검사를, Williams 증후군과같이임상증상이뚜렷하고 FISH 검사가가능한질환은 FISH 검사를시행하는것이권장된다 20, 21). 염색체 microarray 검사의해석임상세포유전검사실에서염색체 microarray 검사의목적은환자의병적요인이되는유전체변이를검출하고자하는것이므로, 검출된 CNV에대한해석이매우중요하다. 인간유전체에는다형성을보이는 CNV, 즉질환의발병과는연관이없을것으로간주되는 benign CNV가존재하는데, 이러한 CNV 는전체유전체의 % 를차지하고 2,600개이상의유전자가포함되어있다 32). 개인마다평균 11.3 Mb에 390개의유전자를가진 CNV가존재하므로염색체 microarray 검사의해석시유의하여야한다. 염색체 microarray 에서검출된유전체변이는병적인변이인지다형성을나타내는 CNV인지감별이필수적이고 33), 환자와부모에서염색체검사 /FISH 검사등검증과정이뒤따라야하며, 적절한유전상담이제공되어야한다 20). 인간유전체의구조적다형성에대한데이터베이스 ( 에서다형성의 CNV를확인할수있고 (Fig. 3), 환자에서검출된염색체 microarray 데이터베이스는 DECIPHER ( 에서검색해볼수있다 (Fig. 4). 다음증례는염색체 microarray 검사의적용에서시작하여검출된유전체변이의해석과검증단계를거쳐최종진단을하고유전상담까지제공하는전체과정이다 (Fig. 5). 비특이적인다발성기형과발달지연을가진환자에서일차적으로시행한고식적인염색체검사는정상핵형을보였지만, 유전체변이가의심되므로염색체 microarray 검사를시행하였다. 4번염색체말단결손과 22번염색체말단중복의유전체변이를보이므로환자부모의균형전좌가능성을예상하고이를 FISH 검사로검증하였다. 환자의 FISH 검사에서 4p16 의결손이있고 22q13.3 의탐색자가비정상 4번에서추가로관찰되어염색체 microarray 결과와일치하는소견이며, 환자어머니의 FISH 검사에서 4p16 와 22q13.3 의균형전좌를나타내고있다. 따라서환자는 t(4;22)(p16.2;q13.31) 의균형전좌보인자인어머니로부터유래한불균형전좌형의 der(4)t(4;22)(p16.2;q13.31) 을가지고있고, 이로인한유전자의양적불균형이환자의임상표현형에영향을주었을가능성이높다. 이를기반으로환자와가족에서유전상담, 나아가서는환자어머니의가계에서균형전좌및이로인한불균형전좌의진단과유전상담이추가로진행될수있다. 결론염색체 microarray 검사는외국의임상세포유전검사실에서는이미선천성염색체이상의검출목적으로매우유용한진 Fig. 3. Genomic Variants Database, a curated database providing a comprehensive summary of structural variations in the human genome including benign CNVs. Fig. 4. DECIPHER, Database of Chromosomal Imbalance and Phenotype in Humans Using Ensemble Resources.

116 서을주 A B 염색체 microarray 검사는검사적분석적과정의정확성뿐만아니라, 분석에서얻어지는수많은유전체변이에대한해석과정이중요한데, 이를위해서임상정보, 임상의학적및유전학적 ( 세포유전, 유전학, 유전체학 ) 지식, 적절한검증단계등총체적지식과검사적능력이요구된다.

6 116 서을주 A B 염색체 microarray 검사는검사적분석적과정의정확성뿐만아니라, 분석에서얻어지는수많은유전체변이에대한해석과정이중요한데, 이를위해서임상정보, 임상의학적및유전학적 ( 세포유전, 유전학, 유전체학 ) 지식, 적절한검증단계등총체적지식과검사적능력이요구된다. 염색체 microarray 검사가임상적유용성과타당성을가진다는사실이입증된바, 국내임상검사실에서도염색체 microarray 검사의제도적도입이시급하고, 적절한임상적응증에따라유용하게활용될수있도록노력이필요할것이다. 국문초록 C D Fig. 5. (A) The patient was found to have a normal karyotype. (B) The chromosomal microarray showed a 3.5Mb deletion in 4p16 (left) and a 3.1-Mb duplication in 22q13.3 (right). (C) The patient showed loss of the red signal for the Wolf-Hirschhorn probe (left) and an extra green signal for ARSA in 4p16 (right). (D) The mother had a balanced translocation, t(4;22)(p16.2;q13.31), according to the red signal of WHS in 22q13 (left) and the green signal of ARSA in 4p16 (right). 단검사법으로서자리잡고있다. 우리나라는염색체이상검출 목적의 microarray 방법이아직건강보험요양급여종목으로 고시되어있지않지만, 국내여러연구자들이임상적유용성에 대한논문을많이발표하고있고, 세포유전검사실무에서는환 자의정확한진단과유전상담을위해염색체 microarray 분 석이필요한경우를많이접하고있다. 염색체 microarray 검사는유전체전체를한번에검색하여초현미경적인염색체이상을매우정밀하고정확하게검출할수있다. 외국에서는현재자주활용되는임상진단검사로자리잡았고, 염색체검사또는표적부위를검출하는 FISH 검사나 PCR 기반의분자유전학적방법을대체하고있다. 최근발표된 consensus 들은염색체 microarray 검사를비특이적인다발성기형, 발달지연또는정신지체, 자폐증상질환의환자에서는염색체검사보다먼저시행할수있는검사로제안하였다. 염색체 microarray 검사는핵형분석에서검출된염색체불균형을검증하기위해염색체검사에보조적으로활용할수있고, 염색체이상에대한보다정확하고종합적인분석이가능하다. 그러나염색체 microarray 검사는균형재배열의염색체이상과 low-level 모자이시즘을검출하기어렵고, 임상적중요성이불명확한 CNV에대한해석과검사비용이고가라는한계점이있다. 이러한이유로인해현재로서는염색체 microarray 검사가산전진단목적으로는고식적인염색체검사를대신할수는없다는의견이다. 임상검사실에서염색체 microarray 검사시행시, 유전학적및세포유전학적지식과경험이결과분석과해석과정에서요구되며, 적절한검증과정단계와유전상담이동반되어야한다. 감사의글본연구는보건복지부보건의료기술진흥사업희귀질환진단치료기술연구사업단과제 (A ) 의지원으로이루어졌음.

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Can032.hwp

Can032.hwp Chromosomal Alterations in Hepatocellular Carcinoma Cell Lines Detected by Comparative Genomic Hybridization Sang Jin Park 1, Mahn Joon Ha, Ph.D. 1, Hugh Chul Kim, M.D. 2 and Hyon Ju Kim, M.D. 1 1 Laboratory