증례 Lab Med Online Vol. 3, No. 1: 50-55, January 2013 http://dx.doi.org/10.3343/lmo.2013.3.1.50 진단유전학 Array Comparative Genomic Hybridization 을이용한남성태아 der(x)t(x;y)(p22.31;q11.22) 의산전진단 Prenatal Diagnosis of der(x)t(x;y)(p22.31;q11.22) in a Male Fetus by Using Array Comparative Genomic Hybridization 김혜원 1 서을주 1 이진옥 2 홍마리아 2 심재윤 3 이범희 4 Hyewon Kim, M.D. 1, Eul-Ju Seo, M.D. 1, Jin-Ok Lee, M.S. 2, Maria Hong, M.S. 2, Jae-Yoon Shim, M.D. 3, Beom Hee Lee, M.D. 4 울산대학교의과대학서울아산병원진단검사의학과 1, 아산생명과학연구원 2, 산부인과 3, 소아청소년과 4 Department of Laboratory Medicine 1, Asan Institute for Life Sciences 2, Departments of Obstetrics and Gynecology 3, Pediatrics 4, Ulsan University of Medical College and Asan Medical Center, Seoul, Korea Xp/Yq translocations are rare chromosomal rearrangements, and the phe-notype of male carriers varies according to the segment of the Xp region that is deleted. In this case report, we describe a der(x)t(x;y)(p22.31;q11.22) translocation, detected by conventional cytogenetic analysis, in a male fetus at a gestational age of 16 weeks. Chromosomal analysis of parental blood confirmed that this chromosomal aberration had been maternally inherited. Array comparative genomic hybridization (CGH) analysis of fetal blood further indicated a nullisomy of Xp22.31-pter and a breakpoint between the STS and KAL1 genes. The STS, NLGN4, ARSE, CSF2RA, and SHOX genes are present in the region that was deleted, and are known to be related to conditions such as X-linked ichthyosis, chondrodysplasia punctata, mental retardation, and facial dysmorphism in humans. Prenatal ultrasonographic findings and autopsy results were consistent with Xp22.31-pter deletion phenotypes. Genetic counseling was provided for the mother. The observations from this case study indicate that advanced molecular techniques can provide a more precise prenatal diagnosis of chromosomal anomalies than conventional cytogenetics can. Key Words: Array CGH, Prenatal diagnosis, X/Y translocation 서론 성염색체간의전좌는드문염색체이상으로서대개 X 염색체단 완원위부와 Y 염색체장완 Yq11.22 에서절단점을갖는다 [1, 2]. 그 결과로 X 염색체의일부가소실되어도여성의경우는저신장외에 정상표현형을보인다. 반면남성의경우는 X 염색체단완원위부가 Corresponding author: Eul-Ju Seo, M.D. Department of Laboratory Medicine, Ulsan University of Medical College and Asan Medical Center, 88 Olympic-ro 43-gil, Songpa-gu, Seoul 138-736, Korea Tel: +82-2-3010-4507, Fax: +82-2-478-0884, E-mail: ejseo@amc.seoul.kr Received: March 28, 2012 Revision received: August 3, 2012 Accepted: August 27, 2012 This article is available from http://www.labmedonline.org 2013, Laboratory Medicine Online This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/3.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. 영염색체 (nullisomy) 상태가되어표현형의이상을나타낼수있으나태아의생존능 (viability) 에영향을미치지는않는것으로알려져있다 [3]. 표현형의이상은소실된 X염색체단완의크기에따라스테로이드설파테이즈결핍 (steroid sulfatase deficiency) 에따른반성어린선 (X-linked ichthyosis, XLI), 점상연골형성장애 (chondrodysplasia punctata, CDPX1), 눈백색증 (ocular albinism), 정신지체, 저신장, 칼만증후군등이있다 [4, 5]. 그러므로정확한전좌위치를파악하고어떤유전자의결실이있는지를확인하는것은발현되는표현형을예측하여산전유전상담시정확한정보를제공하기위해매우중요하다. 그러나고전적인염색체핵형분석법으로는충분한정보를얻기힘들므로최근에는형광제자리부합법 (FISH), 어레이비교유전체보합법 (array comparative genomic hybridization, array CGH) 등분자유전학적기법이산전진단과유전상담을위하여이용되고있다. 발달장애로의뢰되어말초혈액에서 X와 Y염색체전좌를확인한남아의증례는국내에서 1예보고된바있다 [6]. 본증례는 FISH와 array CGH를이용하여산모로부터유래한태 50 www.labmedonline.org eissn 2093-6338
아의비정상 X 염색체를확인하였고염색체이상에따른유전자의 변화와표현형을예측하고산전유전상담을시행했던경우로서, array CGH 를이용하여 X 와 Y 염색체전좌를산전진단한것은국 내에서본증례가최초이다. 1. 임상경과및산과력 증례 31 세산모가임신 16 주산전모체혈청선별검사상알파태아단백 (alpha-fetoprotein, AFP) 0.836 MoM, 사람융모생식샘자극호르몬 (human chorionic gonadotropin, hcg) 0.769 MoM, 비결합에스트 리올 (unconjugated estriol, ue3) 0.070 MoM, 인히빈 A (inhibin A) 0.944 MoM 으로에드워드증후군의위험도가증가되어시행한양 수염색체검사에서 46,Y,der(X)t(X;Y)(p22.31;q11.22) 의비정상핵형 을보였다. 이에산전유전상담을위해본원으로내원하였다. 산전 초음파에서태아는위장이작아기관식도누공이의심되었고, 재 태연령에비해 2 주가량성장이지연되어있었으며, 비골이보이지 않고처진귀 (low-set ear) 의외형을보였다. 산모는초산부로서자 X X Y A Fig. 1. Partial karyograms of the mother (A) and fetus (B). They show the same aberration on the X chromosome, der(x)t(x;y)(p22.31;q11.22). B 연유산 1회, 자궁외임신 1회의산과력이있었으며신체검사에서약간키가작은것외에는이상소견을보이지않았다. 임신 21주 4 일에제대혈을채취하여고식적염색체핵형검사를시행하고 DNA 를추출하였다. 이후자궁내태아사망 (fetal death in utero) 으로유산하였으며부검결과성별은남성으로양안격리증 (hypertelorism), 비골이없는편평한코, 양쪽귀의처짐 (low set ear) 과같은두개안면기형과호상족 (rocker-bottom foot) 이있었고외에기타다른장기에는이상이없었다. 산모는다음임신에서착상전유전자진단 (preimplantation genetic diagnosis) 혹은임신초기융모막채취 (chorionic villi sampling) 를시행하기로하고퇴원하였다. 2. 염색체검사와 FISH 검사부모의핵형을확인하기위해말초혈액림프구를식물적혈구응집소 (phytohemagglutinin, PHA) 로자극하여 72시간배양한후 550 band의해상도를보이는 G-분염의분열중기세포 5개를분석하였고모든핵형은 International System for Human Cytogenetic Nomenclature (ISCN, 2009)[7] 에따라기술하였다. 핵형분석결과남편은 46,XY로정상이었으나산모는 46,X,der(X)t(X;Y)(p22.31; q11.22) 을보였고, 추후에확인검사로시행한제대혈염색체검사에서태아는산모와동일한비정상 X염색체를나타내었다 (Fig. 1). 비정상 X염색체의성상을동정하기위하여 CEPY (DZY1), LSI KAL1/CEP X (DXZ1), LSI STS/CEP X (DXZ1) 소식자 (Vysis, Downers Grove, IL, USA) 를이용하여산모의검체로 FISH를시행하였다. CEPY의신호가비정상 X염색체단완말단에서나타나고 STS 의신호는소실된반면 KAL1 신호는관찰되어 (Fig. 2), STS와 KAL1 유전자사이에서절단이발생하고 Yq의이형염색질 (heterochromatin) 을포함한장완말단이 Xp22.31 에전좌된 46,X,der(X)t(X;Y) (p22.31;q11.22) 핵형으로보고하였다. 따라서태아의경우산모로 A B C Fig. 2. FISH analysis of maternal chromosomes. (A) Two red signals of KAL1 were retained on normal and derivative X chromosomes. (B) FISH for STS shows a deletion in der(x)t(x;y). Green signals located on the centromere of X chromosomes were used as a control. (C) A red signal of CEPY (DYZ1) appears on the derivative X chromosome. http://dx.doi.org/10.3343/lmo.2013.3.1.50 www.labmedonline.org 51
부터유래한영염색체 Xp22.31-pter을가진 46,Y,der(X)t(X;Y) (p22.31;q11.22)mat 라고할수있었다. 3. Array CGH 분석제대혈및산모혈액에서추출한 DNA에대하여고해상도 oligonucleotide Whole Human Genome CGH microarray kit 244K (Agilent Technologies, Santa Clara, CA, USA) 로 array CGH를시행하였다. 참고용 DNA는정상남성 DNA (Promega Corporation, Madison, WI, USA) 를사용하였다. 태아의 array CGH 결과는 X염색체단완말단부의영염색체성으로 8.4 Mb 결실, arr Xp22.33p22.31 (1-8,472,556)x0 및 Y염색체장완의 12.6 Mb 중복, arr Yq11.221q11.23 (16,015,435-28,659,754)x2 을나타내었다. 산모의 array CGH에서 Xp22.33-p22.31 의 8.4 Mb 결실, Y염색체장완의획득을보여태아의 X염색체의유전체이상과동일하였다 (Fig. 3), 염색체검사 array CGH 결과를종합하여최종적으로태아의핵형은 46,Y,der(X)t(X;Y)(p22.31;q11.22)mat.arr Xp22.33p22.31 (1-8,472,556)x0 mat,yq11.221q11.23(16,015,435-28,659,754)x2 mat 로결정되었다. 4. 유전자검사동의서본증례에서시행한모든검사는산모및배우자의자필동의를얻어수행하였다. 고찰 Xp22;Yq11 전좌를가진여성의경우는 X 염색체원위부의결실 이있더라도정상 X염색체가존재하므로저신장이외에임상적인증상이없지만, 남성에서는 X염색체단완말단의영염색체성 (nullisomy) 과 Y염색체장완의이염색체성 (disomy) 을초래하여임상증상을나타내고성인남성의경우에는성염색체의전좌를보인증례에서대부분불임이동반된다 [8]. 이와같은구조적인염색체이상은 Xp와 Yq 사이의상동적인유전자서열때문에남성생식세포의감수분열에서재조합이일어나기쉽기때문이고 [9] 다양한인접유전자증후군 (contiguous gene syndrome) 의원인으로여겨지고있으며 [4, 10] 유전자형과표현형의관계를연구하기위한좋은사례가되고있다. 특히산전검사의영역에서는예상되는표현형에대한정보를얻기위해서절단점을분명히결정하는것이필요하며, FISH와 array CGH가이러한목적에부합하는검사로이용되고있다. 본증례에서는태아의 DNA와산모의 DNA를이용하여 array CGH를시행한결과, 동일한유전체이상을가진 der(x) t(x;y)(p22.31;q11.22) 를확인할수있었고, 이러한모계유전의비정상 X염색체에서 Xp22.31 의절단점을분명히결정할수있었다. 비정상 X염색체에서결실된유전자중 STS, NLGN4, ARSE, CS- F2RA, SHOX의 5가지가사람의질병과연관이있는것으로알려져있다 [11]. 염색체상에서이유전자들의상대적인위치는 Fig. 3 과같다. XLI (OMIM ID #308100) 는피부건조증과인설을주증상으로하는피부질환으로대부분보인자인어머니로부터유래된 STS 유전자의결실에의해미토콘드리아스테로이드설파테이즈활성이결여되어대사되지못한콜레스테롤설페이트 (cholesterol sulfate) 가각질층에축적되어나타나며, 반성열성유전양상을보인다 [4, 12]. 조직검사나효소활성도를측정함으로서확진할수있다. 여성 A B Fig. 3. Array CGH profile of the fetal DNA (A) and maternal DNA (B). The fetal X chromosome shows a nullisomy of Xp22.31-Xpter. The fetal Y chromosome shows gain of Yq11.221-Yqter. The maternal X chromosome shows a deletion in Xp22.31-Xpter containing SHOX, CSF2RA, ARSE, NLGN4, and STS. The Y chromosome reveals gain of Yq11.221-Yqter. 52 www.labmedonline.org http://dx.doi.org/10.3343/lmo.2013.3.1.50
Table 1. Comparison of the results of conventional karyotyping, FISH and array CGH obtained in the case Conventional karyotyping FISH Array CGH ISCN nomenclature 46,Y,der(X)t(X;Y) ish der(x)t(x;y) arr Xp22.33p22.31(1-8,463,190)x1 mat, (p22.31;q11.22)mat (DYZ1+,STS-,KAL1+,DXZ1+)mat Yq11.221q12(16,015,435-28,659,754)x2 mat Information about der(x) Loss at Xp22.31 pter Breakpoint of der(x) 8.4Mb loss at Xp22.33p22.31 Gain at Yq11.22 qter between STS and KAL1 12.6Mb gain at Yq11.221q12 Gain of Yq12 OMIM disease-causing genes in deleted region STS, NLGN4, ARSE, XG, CSF2RA, SHOX Related phenotypes Skin lesion Mental retardation Attention deficit hyperactivity disorder Craniofacial dysmorphism* Pulmonary symptom Skeletal anomaly* Short stature *Phenotypes presented in this case. 에서는피부증상이나타나지않는데구조적이상이있는 X염색체의비활성화때문일것으로여겨지고있다 [13]. ARSE는아릴설파테이즈 E (arylsulfatase E) 를발현하며 CDPX1 (OMIM ID #302950) 의원인유전자로알려져있다. 이질환은두개안면기형, 특히코의기형이특징적으로, 편평한콧대 (flattened nasal bridge), 짧은코기둥 (short nasal columella), 작은콧구멍등의외형을보인다. 그외에사지단축, 백내장, 골단부의석회침착등을동반하는데, 성염색체열성형은스테로이드설파테이즈결핍, 피부질환, 경도의지능저하, 주의력결핍과다행동증후군 (ADHD) 를동반하는경우가많다 [14, 15]. NLGN4는 neuroligin 4를발현하는유전자로자폐, 정신지체및주의력결핍과다행동장애와일부연관이있는것으로알려져있다 [10, 14, 16, 17]. Xp 인접유전자증후군에서정신지체나간질은 NLGN4 외에도 VCX 유전자그룹, ASMT 등과연관이있다는보고가있는데 [14, 18] 보고된증례중일부의환자에서는댄디-워커증후군 (Dandy-Walker malformation) 과같은대뇌의구조적인결함이관찰되기도했다 [14]. CSF2RA는 GM-CSF (granulocyte and macrophage colony-stimulating factor) 수용체의아단위로폐포단백증 (pulmonary alveolar proteinosis, OMIN ID #306250) 과관련이있으며 [19] SHOX는성장관련유전자로저신장과전완부의마델룽변형 (Madelung s deformity) 을특징으로하는 Leri-Weill dyschondrosteosis (OMIN ID #127300) 의원인유전자이다 [20, 21]. 본증례에서 array CGH상으로발견된유전정보와데이터베이스의질환정보를통해태아의표현형을예측할수있었으며 (Table 1), 부검소견은두개안면기형과호상족 (rocker-bottom foot) 의경미한형태이상을보였다. 외형적인이상외의가능한문제는정신지체나 ADHD와같은인지기능의장애, 성인이되었을때무정자 증및불임의가능성을고려할수있다. 증례와같이유전형과표현형과의관계를밝히는것은특히산전검사에서, 문헌고찰및결손된유전자에대한정보를통해예측이가능하다. 최근의 array CGH 기법은유전자복제수변이 (copy number variation, CNV) 에관한광범위한정보를줌으로써유전질환의연구및환자의진단에큰역할을할것으로기대되고있다 [22]. 한메타분석에의하면산전검사에있어서고식적인염색체핵형분석에비해 array CGH를이용하면 3.6-5.2% 의부가적인정보를더얻을수있다고하였다 [23]. 그러나검사기법의표준화와같은기술적문제나결과의해석및결과를어디까지고지해야하는지에관한윤리적문제등해결해야할과제가남아있다. 이에대해 2009년 The American College of Obstetricians and Gynecologists (ACOG) 에서는산전검사영역에서의 array CGH 이용에관한의견 (committee opinion) 을통해분명한이점에도불구하고적어도산전진단영역에서현재까지는고식적인세포유전학적방법을대치할수없다고밝힌바있다 [24]. 결론적으로고식적염색체검사에서구조적이상이관찰된다고하더라도정확한표현형을예측하기는어렵다. 그러나염색체이상에의한유전자의양적인변화를정확히알수있다면산전유전상담을하는데있어도움을받을수있으며 array CGH는그러한목적에부합하는정보를제공할수있다. 요약 X염색체단완과 Y염색체장완사이의전좌는드물게관찰되며, Xp/Yq 전좌를가진남성은결실된 Xp의범위에따라다양한임상증상을나타낸다. 임신 16주에시행한산전염색체검사에서 der(x)t(x;y)(p22.31;q11.22) 가검출된남자태아를보고하고자한 http://dx.doi.org/10.3343/lmo.2013.3.1.50 www.labmedonline.org 53
다. 태아의비정상염색체는산모로부터유래하였고, array CGH분석에서 STS와 KAL1 유전자의사이에절단점을가진 Xp22.31-pter 의 nullisomy 임을알수있었다. X염색체의결실부위에는반성어린선, 정신지체, 점상연골형성장애, 안면기형, 저신장의원인유전자인 STS, NLGN4, ARSE, CSF2RA, SHOX 등이존재한다. 태아의산전초음파소견과부검결과 Xp22.31-pter 결실과연관된표현형이관찰되었고, 보인자인산모에대한유전상담이실시되었다. 새로운분자유전학적진단기법은산전진단에있어염색체이상에관한보다정확한유전체정보를제공하는데도움이된다. 감사의글 본연구는보건복지부보건의료기술진흥사업희귀질환진단치료기술연구사업단과제 (A080588-2) 의지원으로이루어졌음. 참고문헌 1. Burnside RD, Mikhail FM, Cosper PC. A prenatally ascertained X;Y translocation characterized using conventional and molecular cytogenetics. Am J Med Genet A 2008;146A:1221-4. 2. Ballabio A and Andria G. Deletions and translocations involving the distal short arm of the human X chromosome: review and hypotheses. Hum Mol Genet 1992;1:221-7. 3. Bardoni B, Floridia G, Guioli S, Peverali G, Anichini C, Cisternino M, et al. Functional disomy of Xp22-pter in three males carrying a portion of Xp translocated to Yq. Hum Genet 1993;91:333-8. 4. Ballabio A, Bardoni B, Carrozzo R, Andria G, Bick D, Campbell L, et al. Contiguous gene syndromes due to deletions in the distal short arm of the human X chromosome. Proc Natl Acad Sci U S A 1989;86:10001-5. 5. Hsu LY. Phenotype/karyotype correlations of Y chromosome aneuploidy with emphasis on structural aberrations in postnatally diagnosed cases. Am J med Genet 1994;53:108-40. 6. Ha JS, Ryoo NH, Jeon DS, Kim JR, Cho YJ, Kim EJ, et al. A case of X;Y translocation with complex minor anomalies and mental retardation: 46,Y,der(X)t(X;Y)(p22.3;q11.2)mat. Korean J Clin Pathol 2002;22:125-9. 7. Shaffer LGSM, Campbell LJ, editor. ISCN 2009: An international system for human cytogenetic nomenclature. Bssel: S.Karger, 2009. 8. Morel F, Fellmann F, Roux C, Bresson JL. Meiotic segregation analysis by FISH investigation of spermatozoa of a 46,Y,der(X),t(X;Y)(qter-- >p22::q11-->qter) carrier. Cytogenet Cell Genet 2001;92:63-8. 9. Yen PH, Tsai SP, Wenger SL, Steele MW, Mohandas TK, Shapiro LJ. X/ Y translocations resulting from recombination between homologous sequences on Xp and Yq. Proc Natl Acad Sci U S A 1991;88:8944-8. 10. Bukvic N, Carri VD, Di Cosola ML, Pustorino G, Cesarano C, Chetta M, et al. Familial X;Y translocation with distinct phenotypic consequences: Characterization using FISH and array CGH. Am J Med Genet A 2010;152A:1730-4. 11. Fujita PA, Rhead B, Zweig AS, Hinrichs AS, Karolchik D, Cline MS, et al. The UCSC Genome Browser database: update 2011. Nucleic Acids Res 2011;39:D876-82. 12. Bonifas JM, Morley BJ, Oakey RE, Kan YW, Epstein EH Jr. Cloning of a cdna for steroid sulfatase: frequent occurrence of gene deletions in patients with recessive X chromosome-linked ichthyosis. Proc Natl Acad Sci U S A 1987;84:9248-51. 13. Doherty MJ, Glass IA, Bennett CL, Cotter PD, Watson NF, Mitchell AL, et al. An Xp; Yq translocation causing a novel contiguous gene syndrome in brothers with generalized epilepsy, ichthyosis, and attention deficits. Epilepsia 2003;44:1529-35. 14. van Steensel MA, Vreeburg M, Engelen J, Ghesquiere S, Stegmann AP, Herbergs J, et al. Contiguous gene syndrome due to a maternally inherited 8.41 Mb distal deletion of chromosome band Xp22.3 in a boy with short stature, ichthyosis, epilepsy, mental retardation, cerebral cortical heterotopias and Dandy-Walker malformation. Am J Med Genet A 2008;146A:2944-9. 15. Petit C, Melki J, Levilliers J, Serville F, Weissenbach J, Maroteaux P. An interstitial deletion in Xp22.3 in a family with X-linked recessive chondrodysplasia punctata and short stature. Hum Genet 1990;85:247-50. 16. Jamain S, Quach H, Betancur C, Råstam M, Colineaux C, Gillberg IC, et al. Mutations of the X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism. Nat Genet 2003;34:27-9. 17. Boycott KM, Parslow MI, Ross JL, Miller IP, Bech-Hansen NT, MacLeod PM. A familial contiguous gene deletion syndrome at Xp22.3 characterized by severe learning disabilities and ADHD. Am J Med Genet A 2003;122A:139-47. 18. Melichar VO, Guth S, Hellebrand H, Meindl A, von der Hardt K, Kraus C, et al. A male infant with a 9.6 Mb terminal Xp deletion including the OA1 locus: Limit of viability of Xp deletions in males. Am J Med Genet A 2007;143:135-41. 19. Suzuki T, Sakagami T, Rubin BK, Nogee LM, Wood RE, Zimmerman SL, et al. Familial pulmonary alveolar proteinosis caused by mutations in CSF2RA. J Exp Med 2008;205:2703-10. 20. Ross JL, Scott C Jr, Marttila P, Kowal K, Nass A, Papenhausen P, et al. Phenotypes Associated with SHOX Deficiency. J Clin Endocrinol Metab 2001;86:5674-80. 54 www.labmedonline.org http://dx.doi.org/10.3343/lmo.2013.3.1.50
21. Calabrese G, Fischetto R, Stuppia L, Capodiferro F, Mingarelli R, Causio F, et al. X/Y translocation in a family with Leri-Weill dyschondrosteosis. Hum Genet 1999;105:367-8. 22. Seo EJ. Clinical Applications of Chromosomal Microarray Analysis. J Genet Med 2010;7:111-8. 23. Hillman SC, Pretlove S, Coomarasamy A, McMullan DJ, Davison EV, Maher ER, et al. Additional information from array comparative genomic hybridization technology over conventional karyotyping in prenatal diagnosis: a systematic review and meta-analysis. Ultrasound Obstet Gynecol 2011;37:6-14. 24. ACOG Committee Opinion No. 446: array comparative genomic hybridization in prenatal diagnosis. Obstet Gynecol 2009;114:1161-3. http://dx.doi.org/10.3343/lmo.2013.3.1.50 www.labmedonline.org 55