원저 Lab Med Online Vol. 5, No. 3: 121-126, July 2015 진단유전학 디스트로핀유전자에새로이발견된불연속적엑손중복을보이는 Duchenne 형근디스트로피환자 5 예 Novel Non-contiguous Duplications in the DMD Gene in Five Patients with Duchenne Muscular Dystrophy 임정훈 1 조선미 1 유내 1 이경아 1,2 John Hoon Rim, M.D. 1, Sun-Mi Cho, M.D. 1, Nae Yu, M.D. 1, Kyung-A Lee, M.D. 1,2 연세대학교의과대학진단검사의학교실 1, 연세대학교의과대학신경근육병재활연구소 2 Department of Laboratory Medicine 1, Yonsei University College of Medicine, Seoul; Department of Rehabilitation 2, Institute of Neuromuscular Disease, Gangnam Severance Hospital, Yonsei University College of Medicine, Seoul, Korea Background: Muscular dystrophy is an X-linked recessive disorder caused by mutations in the DMD gene. Muscular dystrophy is classified into 2 types; Duchenne muscular dystrophy (DMD), which has severe clinical symptoms, and Becker muscular dystrophy (BMD), which has much milder clinical symptoms. Phenotypic progression to either DMD or BMD can be predicted by analyzing mutations in DMD by using the reading frame rule. Methods: Of 88 patients with mutations in DMD, which were detected using Multiplex Ligation-dependent Probe Amplification DMD test kit (MRC-Holland, The Netherlands), medical records of 5 patients with non-contiguous duplications were reviewed. These rare non-contiguous duplications in DMD were compared with those reported previously. Results: We identified 3 novel non-contiguous duplications in DMD that included exons 2-7 and 45-51, exons 5-37 and 50-59, and exons 52-53 and 56-61. The 5 patients with these non-contiguous duplications showed the phenotypic features of DMD. Especially, duplication of exons 52-53 and 56-61 was observed in a family, i.e., 2 DMD-affected brothers and their carrier mother. Conclusions: Prediction of phenotypes associated with complex non-contiguous duplications by using the reading frame rule is difficult because the duplications affect the expression of DMD together. Because most patients with non-contiguous duplications showed the phenotypic features of DMD, the reading frame rule should be interpreted cautiously. This study provides important insights on the non-contiguous duplications in DMD for understanding genotype-phenotype correlations and for developing dystrophin for therapeutic purposes. Key Words: Duchenne muscular dystrophy, DMD gene, Non-contiguous duplication, Multiplex ligation-dependent probe amplification (MLPA) 서론 근디스트로피 ( 근이영양증, muscular dystrophy) 는디스트로핀 (dystrophin, DMD) 유전자의돌연변이로인해발병하는질환으로 [1], 듀센형 (Duchenne muscular dystrophy, DMD) 은유년기에하 Corresponding author: Kyung-A Lee Department of Laboratory Medicine, Yonsei University College of Medicine, 211 Eonju-ro, Gangnam-gu, Seoul 135-720, Korea Tel: +82-2-2019-3531, Fax: +82-2-2019-4822, E-mail: KAL1119@yuhs.ac Received: September 5, 2014 Revision received: December 3, 2014 Accepted: February 13, 2015 This article is available from http://www.labmedonline.org 2015, 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. 지근위부의근력저하로독립보행이어려워지며, 대부분 30대이전에호흡부전이나심부전으로사망하게되는중증형이고, 베커형 (Becker muscular dystrophy, BMD) 은 DMD보다증상이경미하여상대적으로늦은나이에발현되며, 진행양상이완만하다 [2]. 따라서 DMD/BMD가의심되는환자에게 DMD 유전자검사는진단및예후예측에필수적인역할을한다 [3]. DMD 유전자의돌연변이종류로는결손 (deletion) 이전체환자의 55-65% 로가장흔하며 [4], 중복 (duplication) 은약 5-10% [5], 나머지 20-30% 는점돌연변이 (point mutation), 미세결손 (microdeletion), 미세삽입 (microinsertion) 등이있다 [6]. 이러한 DMD 유전자돌연변이를검출하는유전자검사법으로는다중중합효소연쇄반응 (multiplex PCR), 정량서던블롯 (quantitative Southern blotting), 형광제자리부합법 (FISH), 직접염기서열분석 (sequence analysis) 등이있는데, 최근 Multiplex Ligation-dependent Probe Amplification (MLPA) 방법이 DMD/BMD 환자및여성보인자진단에우선 eissn 2093-6338 www.labmedonline.org 121
적으로추천되고있다 [7, 8]. DMD와 BMD에서는유전자돌연변이의종류및부위를바탕으로임상양상의차이와중증도예측이가능하다. Monaco 등은비교적간단하게표현형을예측할수있는해독틀법칙 (reading frame rule) 을제시하였고, 이는현재까지결과분석에유용하게사용되고있다 [9]. 기본원리는결손또는중복부위가 mrna의번역해독틀 (translational reading frame) 을이동시킬경우 (out-offrame) 에는전사가조기에종식하게되는등디스트로핀단백질의형성에문제가발생하여증상이심한 DMD가발현하게되지만, 반대로번역해독틀이보존 (in-frame) 되는결손, 중복의경우에는분자량이작거나비정상적인단백질이생성되어증상이경미한 BMD가발현한다는것이다. 이해독틀법칙은현재까지주요한 Leiden 근디스트로피돌연변이데이터베이스 (http://www.dmd. nl/) 와비교하였을때, 92% 의정확도를보이며, 특히 RNA 발현까지고려하면정확도가 99.5% 까지높아진다 [10]. DMD 유전자의결손돌연변이의경우에는해독틀법칙결과와임상경과와의상관성이정립되어있지만, 중복돌연변이의경우에는결손에비해 RNA 수준의변화를예측하기어려운경우가많음에도불구하고해독틀법칙결과와임상적인상관성에대한보고는현재까지많지않다 [11]. 특히단일부위의중복이아닌불연속적엑손부위들의중복에대한연구는매우드문실정이다. 본연구는 MLPA 방법을사용하여진단된 DMD 유전자변이중불연속적엑손중복에의한근디스트로피증례들을보고하고, 환자들의질병양상과유전자변이결과와의상관성에대해고찰하고자한다. 증례및방법 1. 증례 2007 년 8 월부터 2011 년 7 월까지단일대학병원에서 DMD/BMD 진단을위해시행한 MLPA 검사결과중양성환자는총 88명이었다. 이중연속적부위의결손돌연변이는 67명 (76.1%), 연속적부위의중복돌연변이는 15명 (17.0%) 이었다. 이외점돌연변이를보인환자 1명을제외하고, 기존데이터베이스에보고되어있지않으며, 불연속적으로두부위에서중복돌연변이를보인 5명의환자에대해임상증상및검사결과를후향적으로분석하였다. 2. MLPA 검사방법환자의말초혈액으로부터 QIAamp DNA Blood Mini Kit (QIA- GEN Inc, Hildern, Germany) 를이용하여 DNA를추출하였다. MLPA는 MLPA DMD test kit (MRC-Holland, Amsterdam, The Netherlands) 를이용하였다. 환자의 DNA를프로브혼합물 034 (DMD 엑손 1-10, 21-30, 41-50, 61-70), 035 (DMD 엑손 11-20, 31-40, 51-60, 71-79) 와교잡하여결합시킨후 PCR 증폭을하였으며, PCR 반응은 95ºC에서 30초, 60ºC에서 30초, 72ºC에서 1분씩 35회증폭하고, 마지막에 72ºC에서 20분간항온시켰다. PCR 증폭산물은 Applied Biosystems 3,500 L system (Applied Biosystems, Foster City, CA, USA) 으로분석하였다. DMD 유전자변이의분석은 Softgenetics MLPA 프로그램 (GeneMarker 1.70) 을이용하여매실험에서의대조군반응과비교하여분석하였으며, 상대적인최댓값이 0.75 이하이면결손, 1.3 이상이면중복으로보았다. 결과 각환자의임상소견 (Table 1) 및 MLPA 검사결과는다음과같다. 환자 1은 18세남자환자로보행장애를평가하기위해 3세에시행한근육조직검사상근디스트로피로진단되었다. 이후상지및하지근력약화로독립적인보행이어려워지며호흡재활이필요한상태가되어임상적으로 DMD 진단을받았으며, 확정적진단을위해시행한 MLPA 검사상 DMD 유전자엑손 2-7 부위와엑손 45-51 Table 1. Clinical features of the 5 patients examined in this study Patient Sex Symptoms of onset/age 1* Male Gait disturbance/ 3 yr old 2* Male Delayed development/ 5 yr old 3 Male Delayed language development/ 4 yr old 4 Male Delayed language development/ 2 yr old 5 Female Lower limb weakness/ 24 yr old Age at the last follow-up Ambulation Cardiac involvement Scoliosis Chronic respiratory rehabilitation Creatine kinase (U/L) 20 yr old Impossible Normal Yes Yes 3,063 24 yr old Impossible Dilated cardiomyopathy Yes Yes 1,829 16 yr old Impossible Normal Yes Yes 1,016 14 yr old Impossible Mild cardiomyopathy Yes Yes 1,486 38 yr old Possible Normal No Yes 1,350 *Family study was not performed; Family members; Level of creatine kinase was measured at the time of diagnosis. 122 www.labmedonline.org
부위의불연속적중복이관찰되었다 (Fig. 1). 각각의중복은모두독립적으로 in-frame으로예측되었다. 환자 2는 5세에발달지연으로시행한근전도검사에서근디스트로피를진단받았으며, 10세부터독립적인일상생활이어려워휠체어에서생활하였다. 이후 20세에호흡약화로호흡재활이필요하여정확한진단을위해 MLPA 검사를시행하였다. 임상적으로 DMD가의심되어시행한검사결과상 DMD 유전자엑손 5-37 부위와엑손 50-59 부위가중복되었다. 이후주기적인통원재활치료를시행하였으나척주측만증 (scoliosis) 이진행되며확장형심근병증 (dilated cardiomyopathy) 소견이관찰되었다. 또한골다공증소견도보여전반적으로질병이진행되는양상이관찰되었다. 각각의중복은모두독립적으로 in-frame으로예측되었다. 환자 3과환자 4는형제이며언어발달지연으로타병원을방문하여시행한근육조직검사상근육병 (myopathy) 을진단받은후특별한치료없이지내다가각각 9세, 7세부터하지근육약화로인해자발적인생활이불가능할정도로질병이진행되어 6년후근디스 트로피의확정적진단을위해유전자검사를시행하였다. MLPA 검사상환자 3과환자 4에서모두 DMD 유전자의엑손 52-53 부위와엑손 56-61 부위의불연속적중복이관찰되었다. 환자 3과환자 4의어머니인환자 5는 24세에둘째아들 ( 환자 4) 을출산한후부터하지근력약화가시작되었으며 gower 증후가나타나고익상견갑골증 (winged scapula) 을보여근디스트로피로진단되었다. 환자 5에서도역시 MLPA 검사상 DMD 유전자엑손 52-53 부위와엑손 56-61 부위가중복된양상이관찰되어한가계내형제와어머니에서동일한불연속적엑손의중복이검출되었다. 각각의중복은 in-frame, out-of-frame 중복으로예측되었다. 고찰 현재까지 DMD 유전자두부위의불연속적중복에대한증례들은 5개연구에서 8명의환자가보고되었고드물게세부위에대한불연속적중복에대한증례보고도있다 (Table 2) [12]. Fig. 1. Results of MLPA analysis for patient 1 (non-contiguous duplication of exons 2-7 and 45-51). www.labmedonline.org 123
Table 2. A summary of the identified non-contiguous duplications in DMD Patient Phenotype Frame of the first duplication Frame of the second duplication Country Reference P1 DMD Exon 2-7/In frame Exon 45-51/In frame Korea This study P2 DMD Exon 5-37/In frame Exon 50-59/In frame Korea This study P3* DMD Exon 52-53/In frame Exon 56-61/Out of frame Korea This study P4* DMD Exon 52-53/In frame Exon 56-61/Out of frame Korea This study P5* DMD carrier Exon 52-53/In frame Exon 56-61/Out of frame Korea This study p1 DMD Exon 45-48/In frame Exon 54-55/In frame Germany Neurogenetics (2005) p2 NA Exon 5-19/Out of frame Exon 38-41/In frame Multicenter Human Mutation (2006) p3 NA Exon 45-55/In frame Exon 65-79/Out of frame Multicenter Human Mutation (2006) p4 DMD Exon 45-48/In frame Exon 55-63/Out of frame Japan Journal of Human Genetics (2008) p5 DMD Exon 2-7/In frame Exon 50-55/In frame Spain Journal of Neurogenetics (2008) p6 DMD Exon 2-7/In frame Exon 50-55/In frame Spain Journal of Neurogenetics (2008) p7 DMD carrier Exon 2-7/In frame Exon 50-55/In frame Spain Journal of Neurogenetics (2008) p8 IMD Exon 1/Undetermined Exon 42-43/Out of frame Mexico Journal of Genetics (2014) *, Family members. Abbreviations: DMD, Duchenne muscular dystrophy; IMD, intermediate muscular dystrophy; NA, not available. 불연속적중복의첫보고로서 2005년에 Janssen 등 [13] 이발표한 14명의 DMD 유전자중복또는결손환자들중 1명에서엑손 45-48 부위와엑손 54-55 부위의중복이관찰되었으며, 각부분의중복은 in-frame으로예측되지만, 환자는 DMD의표현형을나타냈다. 본연구에서나타난환자 1, 환자 2의경우도임상적으로 DMD 표현형을나타내는공통점을보였다. 환자 3, 환자 4, 환자 5는가족관계로공통적으로엑손 52-53 부위와엑손 56-61 부위가중복된양상을보였으며각각의중복은 in-frame, out-of-frame으로예측되어있다. 그외환자 1과환자 2 에서발견되었던엑손 2-7 부위의중복과엑손 45-51 부위의중복, 엑손 5-37 부위의중복과엑손 50-59 부위의중복은모두독립적으로 in-frame으로예측되어있다. 각각의중복은 in-frame 중복으로경미한증상을나타내는 BMD로예측되지만, 불연속적으로중복이조합되는대부분의증례들이임상증상이심한 DMD로진행하였다. 불연속적인중복의경우복합적인유전자재배열이동반될수있기때문에일반적인해독틀법칙에의한통상적인표현형예측이어려울수있다. 최근보고된 Lopez-Hernandez 등 [12] 의연구에서는엑손 1 부위와엑손 42-43 부위의불연속적중복이관찰된 14세남자환자증례가보고되었는데, 엑손 42-43 부위의독립적인중복은 out-of-frame으로 DMD가예측되지만, 해독틀법칙의예외적인경우로 DMD와 BMD의중간 (intermediate) 중증도의표현형을보였음을밝혀, 본증례에서불연속적 in-frame 중복이중증 DMD와연관되었던것과는대조적으로이질적인임상양상을보였다. 본증례와이전보고들을종합하여 DMD 유전자에서불연속적중복이호발하는부위를살펴보면, 대부분엑손 40-60 부위의중복을포함하고있다. DMD 유전자결손은민감점 (hot spot) 을가지 고있다 [10]. DMD 유전자결손의민감점은엑손 2-20 부위와엑손 44-53 부위로이두부위가결손의 50-70% 를차지하는것으로알려져있으며, 특히엑손 44-53 부위가가장높은빈도를보인다 [14]. 하지만아직까지중복에대한민감점은특별히없으며, 기존데이터베이스를바탕으로분석해보면엑손 2 부위가가장흔하게중복이관찰되는부위라고할수있다 [15]. 그러나본증례들에서는한명의환자에서만엑손 2 부위의중복이관찰되었다. 불연속적엑손중복의경우발생기전이다양하며, 이에대한여러가지가설들이제시되고있다 [12,16,17]. 우선, 불연속적중복은환자어머니의감수분열 (meiosis) 단계에서새로운 (de novo) 유전자재배열을통해중복이일어날수있는데, 환자 3, 환자 4, 환자 5 의가족처럼어머니가보인자로서다음세대에돌연변이가그대로전달되는경우이다. 어머니는보인자로서증상이나타나지않거나 [17], 본증례처럼경미한증상이나타날수있다. 기전으로서자매염색분체 (sister chromatid) 간의불균형적인교차로인한가능성이보고된바있으며 [18], 유사한기전이 Charcot-Marie-Tooth병 1A 형의원인유전자인 HNPP의발병과정에서보고되었다 [19]. 감수분열단계에서불연속적엑손중복이재발할위험도를 DMD 유전자변이의위치에따라예측하는것도가능하다 [20]. 또다른기전으로, 체세포분열 (mitosis) 과정에서이중고리 (double-loop) 가발생함으로써하나의연속적인엑손중복이일어난후중복내에서일부결손이일어나는과정을통해불연속적엑손중복이발생할수있다 [17]. 이는 B형혈우병의특정돌연변이발생과정과유사하다 [21]. 실제로 DMD 유전자 MLPA 검사에서섞임증 (mosaicism) 이관찰되는경우가이러한기전과연관되어있다. DMD 유전자에서다양한불연속적엑손중복이가능한원인은 DMD 유전자가인체유전자중가장큰유전자이기때문에 [10] 상 124 www.labmedonline.org
대적으로복잡한재배열 (rearrangement) 에취약하기때문이다. 특히전사 (transcription) 와복제 (replication) 과정에관여하는인자들이충돌할수있는가능성이높아지며 [22], 감수분열과정에영향을주어다양한조합의불연속적엑손중복을새롭게 (de novo) 발생시킬수있다. mrna 및단백질단계에서분석해보면, DMD 유전자의엑손중기능적인역할을하는엑손부위의중복이표현형과연관되어있다. DMD 유전자 mrna 단계의안정성 (stability) 에따라중복된엑손의단백질발현여부가달라질수있으며 [12], 이는해독틀법칙의중요한인자로생각되고있다. 디스트로핀단백질은크게액틴결합부위 (actin-binding domain), 중앙막대부위 (central rod domain), 시스테인이풍부한부위 (cysteine-rich domain) 와 C-말단부위 (C-terminal domain) 로나눌수있으며, 4개의경첩부위 (hinge region) 에의해연결되어있다 [23]. 이중제3경첩부위 (H3) 에결손또는중복이있는경우임상증상이경미하여대부분 BMD로진행된다고보고되어있다 [10]. 하지만본연구에서나타난 3가지양상의불연속적중복의경우모두공통적으로제3경첩부위에서절단점 (breakpoint) 이발생하였음에도불구하고임상증상이심각한 DMD로진행되었다. 이는추가적으로액틴결합부위또는중앙막대부위에절단점이발생함으로인해경미한증상이나타날수있는가능성이상쇄되었다고해석할수있다 (dominant negative effect). 결론적으로 DMD 유전자의불연속적엑손중복은각각의중복이상호복합적으로작용하여유전자발현에영향을줄수있기때문에해독틀법칙에의한표현형예측이어렵다. 또한환자증례마다표현형과분자유전학적검사결과와의연관성에대한개별적인해석이매우중요하며, 이는다른가족에대한유전상담측면에서도중요하다. 본연구의증례분석결과대부분중증표현형을발현하는경우가많았다. 불연속적엑손중복은유전형-표현형상관성에대한연구및유전자치료목적의디스트로핀을개발하는데있어중요성을가지므로향후더많은증례를통한병태생리학적연구가이루어져야할것이다. 요약 배경 : 근디스트로피는 DMD 유전자의돌연변이로인해발병하는성염색체열성유전질환으로심한임상양상을나타내는 DMD와경미한임상양상을나타내는 BMD가있다. DMD 유전자의돌연변이를검출하여분석결과를바탕으로해독틀법칙을적용하면 DMD 또는 BMD로진행하는표현형예측이가능하다. 방법 : DMD/BMD 진단을위한 MLPA DMD test kit (MRC-Holland, The Netherlands) 를사용하여검출한돌연변이양성환자총 88명 중 DMD 유전자의불연속적엑손중복이나타난 5예의의무기록을분석하였다. 비교적드물게발생하는두부위의불연속적엑손중복돌연변이 5예의유전자검사결과및임상표현형을이전보고되었던불연속적엑손중복돌연변이증례들과비교분석하였다. 결과 : 본연구에서기존보고된바없는세가지형태의불연속적엑손중복돌연변이가관찰되었으며, 각각 DMD 유전자엑손 2-7 부위와엑손 45-51 부위, 엑손 5-37 부위와엑손 50-59 부위, 엑손 52-53 부위와엑손 56-61 부위의중복이었다. 본연구에서제시된 DMD 유전자의불연속적엑손중복 5예는모두 DMD 표현형을나타냈다. 결론 : DMD 유전자의불연속적엑손중복돌연변이에서각각의중복이상호복합적으로작용하여유전자발현에영향을주기때문에일반적인해독틀법칙에의한표현형예측이어렵다. 대부분의불연속적엑손중복돌연변이는 DMD 표현형을나타냈으며, 이에대한분별력있는해독틀법칙적용이필요하다. 본증례고찰은근디스트로피의유전형-표현형예측또는치료목적의디스트로핀개발과관련한기전연구에있어중요할것으로생각된다. 감사의글 본연구는보건복지부보건의료연구개발사업의지원에의하여이루어진것임 (A120030). REFERENCES 1. Emery AE. Population frequencies of inherited neuromuscular diseases--a world survey. Neuromuscul Disord 1991;1:19-29. 2. Forrest SM, Cross GS, Flint T, Speer A, Robson KJ, Davies KE. Further studies of gene deletions that cause Duchenne and Becker muscular dystrophies. Genomics 1988;2:109-14. 3. Prior TW and Bridgeman SJ. Experience and strategy for the molecular testing of Duchenne muscular dystrophy. J Mol Diagn 2005;7:317-26. 4. Koenig M, Hoffman EP, Bertelson CJ, Monaco AP, Feener C, Kunkel LM. Complete cloning of the Duchenne muscular dystrophy (DMD) cdna and preliminary genomic organization of the DMD gene in normal and affected individuals. Cell 1987;50:509-17. 5. Hu XY, Ray PN, Murphy EG, Thompson MW, Worton RG. Duplicational mutation at the Duchenne muscular dystrophy locus: its frequency, distribution, origin, and phenotypegenotype correlation. Am J Hum Genet 1990;46:682-95. 6. Roberts RG, Bobrow M, Bentley DR. Point mutations in the dystrophin gene. Proc Natl Acad Sci U S A 1992;89:2331-5. www.labmedonline.org 125
7. Lai KK, Lo IF, Tong TM, Cheng LY, Lam ST. Detecting exon deletions and duplications of the DMD gene using Multiplex Ligation-dependent Probe Amplification (MLPA). Clin Biochem 2006;39:367-72. 8. Gatta V, Scarciolla O, Gaspari AR, Palka C, De Angelis MV, Di Muzio A, et al. Identification of deletions and duplications of the DMD gene in affected males and carrier females by multiple ligation probe amplification (MLPA). Hum Genet 2005;117:92-8. 9. Monaco AP, Bertelson CJ, Liechti-Gallati S, Moser H, Kunkel LM. An explanation for the phenotypic differences between patients bearing partial deletions of the DMD locus. Genomics 1988;2:90-5. 10. Aartsma-Rus A, Van Deutekom JC, Fokkema IF, Van Ommen GJ, Den Dunnen JT. Entries in the Leiden Duchenne muscular dystrophy mutation database: an overview of mutation types and paradoxical cases that confirm the reading-frame rule. Muscle Nerve 2006;34:135-44. 11. Tuffery-Giraud S, Béroud C, Leturcq F, Yaou RB, Hamroun D, Michel- Calemard L, et al. Genotype-phenotype analysis in 2,405 patients with a dystrophinopathy using the UMD-DMD database: a model of nationwide knowledgebase. Hum Mutat 2009;30:934-45. 12. López-Hernández LB, Gómez-Díaz B, Bahena-Martínez E, Neri-Gómez T, Camacho-Molina A, Ruano-Calderón LA, et al. A novel noncontiguous duplication in the DMD gene escapes the reading-frame rule. J Genet 2014;93:225-9. 13. Janssen B, Hartmann C, Scholz V, Jauch A, Zschocke J. MLPA analysis for the detection of deletions, duplications and complex rearrangements in the dystrophin gene: potential and pitfalls. Neurogenetics 2005;6:29-35. 14. Imoto N, Arinami T, Hamano K, Matsumura K, Yamada H, Hamaguchi H, et al. Topographic pattern of the rearrangement of the dystrophin gene in Japanese Duchenne muscular dystrophy. Hum Genet 1993;92:533-6. 15. White SJ, Aartsma-Rus A, Flanigan KM, Weiss RB, Kneppers AL, Lalic T, et al. Duplications in the DMD gene. Hum Mutat 2006;27:938-45. 16. Zhang Z, Takeshima Y, Awano H, Nishiyama A, Okizuka Y, Yagi M, et al. Tandem duplications of two separate fragments of the dystrophin gene in a patient with Duchenne muscular dystrophy. J Hum Genet 2008;53:215-9. 17. Fenollar-Cortés M, Gallego-Merlo J, Trujillo-Tiebas MJ, Lorda-Sánchez I, Ayuso C. Two non-contiguous duplications in the DMD gene in a Spanish family. J Neurogenet 2008;22:93-101. 18. Hu XY, Ray PN, Worton RG. Mechanisms of tandem duplication in the Duchenne muscular dystrophy gene include both homologous and nonhomologous intrachromosomal recombination. EMBO J 1991;10: 2471-7. 19. Lopes J, Tardieu S, Silander K, Blair I, Vandenberghe A, Palau F, et al. Homologous DNA exchanges in humans can be explained by the yeast double-strand break repair model: a study of 17p11.2 rearrangements associated with CMT1A and HNPP. Hum Mol Genet 1999;8: 2285-92. 20. Helderman-van den Enden AT, de Jong R, den Dunnen JT, Houwing- Duistermaat JJ, Kneppers AL, Ginjaar HB, et al. Recurrence risk due to germ line mosaicism: Duchenne and Becker muscular dystrophy. Clin Genet 2009;75:465-72. 21. Sommer SS. Recent human germ-line mutation: inferences from patients with hemophilia B. Trends Genet 1995;11:141-7. 22. Helmrich A, Ballarino M, Tora L. Collisions between replication and transcription complexes cause common fragile site instability at the longest human genes. Mol Cell 2011;44:966-77. 23. Hoffman EP, Brown RH Jr, Kunkel LM. Dystrophin: the protein product of the Duchenne muscular dystrophy locus. Cell 1987;51:919-28. 126 www.labmedonline.org