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1 HJ Park, et al. DYSF mutations in Korean ISSN Korean Journal of Neuromuscular Disorders Vol. 7 No. 2, December 2015 Orginal Article 산성알파글루코시다아제가감소한근육병에서 Whole Exome Sequencing 을이용한 DYSF 유전자복합이형접합돌연변이의규명 이화여자대학교의과대학목동병원신경과 a, 연세대학교의과대학신경과학교실 b 박형준 a, 최지현 a, 최영철 b, 박기덕 a Identification of Compound Heterozygous DYSF Mutations Using Whole Exome Sequencing in a Myopathy with Decreased Acid-Alpha Glucosidase Activity Hyung Jun Park, MD a, Ji-Hyun Choi, MD a, Young-Chul Choi, MD, PhD b, Kee Duk Park, MD, PhD a a Department of Neurology, Mokdong Hospital, Ewha Womans University School of Medicine; b Department of Neurology, Yonsei University College of Medicine, Seoul, Korea KEYWORDS Limb-girdle muscular dystrophy, DYSF, Glycogen storage disease type II, Whole exome sequencing Background: This study was designed to identify the genetic cause in myopathy family with decreased acid-alpha glucosidase activity. Methods: Clinical and laboratory features of two affected family members were analyzed. Then, whole exome sequencing (WES) was performed. Results: The proband (a 54-year-old woman) and her sister (a 57-year-old woman) presented to our neurologic clinic with proximal muscle weakness. They recalled very active and sporty life since adolescence. At the late teens, they first noticed difficulty in climbing stairs. Neurological examination revealed muscle weakness and atrophies of proximal muscles, predominantly at lower limbs. Electromyography revealed chronic myopathic finding and serum creatine kinase level was elevated in the proband. In addition, serum acid-alpha glucosidase activities were decreased in two patients. WES identified compound heterozygous mutations (c.5713c>t and c.937+1g>a) in DYSF, which were previously reported to be an underlying cause of limb-girdle muscular dystrophy 2B. Conclusions: We identified compound heterozygous DYSF mutations in a myopathy family with decreased acid-alpha glucosidase activity. This result demonstrated the usefulness of WES for the diagnosis of limb-girdle muscular dystrophy. 서론 팔다리이음근디스트로피 (limb-girdle muscular dystrophy) 는골반과어깨근육의점진적인약화와위축을특징으로 하는유전성근육질환이다. 팔다리이음근디스트로피는유전양상으로상염색체우성유전을하는 1형과상염색체열성유전을하는 2형으로구분되며현재까지각각 8개와 22개의원인유전자가밝혀져있다. 1 고전적인진단방법은 Received: August 28, 2015 / Accepted: November 2, 2015 Address for correspondence: Kee Duk Park, MD Department of Neurology, Ewha Womans University School of Medicine, 1071 Anyangcheon-ro, Yangcheon-gu, Seoul 07985, Korea Tel: , Fax: , pkd1165@ewha.ac.kr Korean Journal of Neuromuscular Disorders

2 Korean Journal of Neuromuscular Disorders Vol. 7 No. 2, 2015 병력청취및신경학적검사, 검사실검사, 근육병리, 근육조직내의단백분석으로원인유전자를추정하고해당유전자에대한염기서열분석으로확인하는순차적인방법이다. 2,3 그러나이러한방법만으로는다양한임상양상및병리소견을보이는팔다리이음근디스트로피의특징때문에원인유전자를선별하지못하거나잘못된원인유전자를추정하는경우가빈번하였다. 다만, 차세대염기서열분석의발전으로적은노동및낮은비용으로많은유전자에대한검사가가능해져서이러한문제점들이보완되고있다. 4 저자들은혈중산성알파글루코시다아제가감소되어폼페병을의심했던상염색체열성의근육병자매에서전체엑솜염기서열분석 (whole exome sequencing, WES) 으로원인유전자를규명하였다. 대상과방법 1. 환자및임상분석 연구는근육병환자인발단자 (II-3, Fig. 1A) 와언니 (II-2, Fig. 1A) 를대상으로진행하였다. 두자매에서병력을청취하고, 인지기능, 뇌신경기능, 근력, 감각, 관절의구축, 및건반사의변화를확인하였다. 발단자는신경전도검사및근전도검사의전기생리학적검사, 혈청크레아틴카나아제, 심전도및흉부 X선검사를받았다. 또한두자매모두에서혈청내산성알파글루코시다아제를측정하고 WES를시행하였다. 2. 유전자분석다음의순서로두자매의 WES를시행하였다. QuickGene DNA whole blood kit S (Kurabo, QuickGene, Kurabo, Japan) 를이용하여환자혈액에서 DNA를추출하였다. 유전체염기서열중단백질을코딩하는엑손부위만을선별하기위한라이브러리로 Agilent Sureselect Target Enrichment Ver. 5 (Agilent Technologies, Santa Clara, CA, USA) 를사용하였다. DNA 시료의구분을위해서 6 bp index sequence (Illumina, San Diego, CA, USA) 를사용하였다. 그리고차세대염기서열분석은 NextSeq500 platform (Illumina, San Diego, CA, USA) 으로시행하였다. 3. 변이분석생산된염기서열정보는 6 bp index sequence의정보로 A B Figure 1. Pedigree and sequencing chromatograms. (A) Pedigree of Korean patients with compound heterozygous DYSF mutations (square: male; circle: female; filled: affected; and non-filled: unaffected). (B) Sequencing chromato-grams of DYSF mutations c.937+1g>a and c.5713c>t (p.r1905x). Arrows indicate mutation sites. 80 대한신경근육질환학회지 2015

3 HJ Park, et al. DYSF mutations in Korean 일차적으로분류하였다. 분류된 fastq 파일을표준염기서열인 hg19 참조서열에정렬 (alignment) 하기위해서 Burrows- Wheeler Aligner (BWA) algorithm (ver ) 을이용하였다. 5 파일의형식전환을위해서 SAMtools (ver ) 로분류하고 Picard tools (ver.1.102) 로중복을제거한후 the genome analysis toolkit (GATK; ver ) 로알려진삽입 -결실부위 (indel site) 의재배열과 base quality score recalibration (BQSR) 을시행하였다. 그리고변이확인을위해서 SAMtools 와 GATK Haplotype Caller, freebayes (ver ) 알고리즘을이용하였다. 6,7 변이분석을위해서최소두개이상의알고리즘에서확인된변이를대상으로 loci depth 가 10 이상이고 variant frequency가 0.5 이상인경우또는 loci depth가 20 이상이고 variant frequency 0.25 이상인변이만을선택하였다. 변이의 in-silico 분석정보 (SIFT, Polyphen2, LRT, MutationTaster) 와데이터베이스의대립유전자의빈도정보 (1000 Genomes Project phase 3 data, UK10K cohorts data, ExAC consortium data and the NHLBI Exome Sequencing Project ESP6500 data) 의확인을위해서 dbnsfp (ver. 2.4) 을사용하여 snpeff (ver. 4.0c) 로주석을달았다. 다음두환자에서공통적으로발견되는근육병원인유전자의변이를선별하였다. 미국의학유전학및유전체학회 (the American college of medical genetics and genomics, ACMG) 와분자병리학회 (the association for molecular pathology, AMP) 의진단지침으로병원성을판단하였다. 8 마지막으로원인돌연변이에대해서고전적염기서열분석 (Sanger sequencing) 으로재확인하였다. 결과 1. 임상양상 는팔다리의근력약화로방문하였다. 두자매에서발단자가우측발의기형으로 3세에수술을받은것을제외한다른병력은없었다. 두자매의아버지와어머니는치매로투병후 83세와 86세에사망하였으나, 치매가악화되기전까지팔다리의근력약화는없었다고하였다. 두자매는 10대중반까지운동을잘하는편이었으나 10대후반부터양하지의근력약화와근위축을느꼈다고하였다. 30대부터난간을잡고계단을올랐고, 40대부터팔의힘이약해지고보행기를사용하였으며, 50세부터휠체어로이동하게되었다. 신경학적검사상인지기능과뇌신경의기능은정상이었다. 근력약화와근위축은팔다리의몸통쪽근육에서심했고허벅지근육이가장약했다. 감각은정상이었고건반사는저하되었다. 발단자의신경전도검사및근전도검사는전신형근육병에합당한소견이었으나근긴장성방전 (myotonic discharge) 은없었다. 혈청크레아틴키나아제는 34세에 2,752 IU/L ( 정상값 : 185 IU/L) 와 54 세에 789 IU/L로증가되어있었다. 두자매모두심장기능및호흡기능저하와관련된증상호소는없었고, 발단자의심전도및흉부 X선검사는정상이었다. 전체산성알파글루코시다아제의혈중농도는발단자에서 17.0 nmol/hr/mg protein ( 정상값 : 30 nmol/hr/mg protein) 과발단자의언니에서 19.6 nmol/hr/mg protein으로낮았고, 아카보오스를처리한값도발단자에서 8.4 nmol/ hr/mg protein ( 정상값 : 10 nmol/hr/mg protein) 과발단자의언니에서 5.5 nmol/hr/mg protein으로낮았다. 그러나아카보오스를처리한값과전체의산성알파글루코시다아제의비율은발단자에서 49.4% ( 정상값 : 25%) 와발단자의언니에서 49.4% 로정상범위였다. 2. 유전자검사 발단자 (54 세여자, II-3) 와발단자의언니 (57 세여자, II-2) 두자매의혈액검사상산성알파글루코시다아제의혈 Table 1. Whole exome sequencing analysis for two individuals Samples II-2 II-3 Total yields (Gbp) Mappable reads of total reads (%) Coverage of target region ( 1X) Coverage of target region ( 20X) Total number of SNPs 67,788 68,088 Total number of indels 4,348 4,248 Number of coding SNPs 22,783 22,876 Number of nonsynonymous variants 12,160 12,208 Number of functionally significant variants in myopathy related genes Cosegregating variants in myopathy related genes 18 SNP, single nucleotide polymorphism. Korean Journal of Neuromuscular Disorders

4 Korean Journal of Neuromuscular Disorders Vol. 7 No. 2, 2015 Table 2. Functionally significant variants in myopathy-related genes Gene Map Ref Seq a Variants 1000G dbsnp138 SIFT b Poly- Nucleotides Amino acids (2014 Sep) Phen2 c Description CPT2 1p32.3 NM_ c.1102g>a p.v368i rs Benign CRYAB 11q23.1 NM_ c.324+4t>g - rs Benign PFKM 12q13.11 NM_ c.512g>a p.r171q rs Benign GAA 17q25.2-q25.3 NM_ c.668g>a p.r223h rs Benign c.1726g>a p.g576s rs Benign c.2065g>a p.q576s rs Benign NEB 2q22 NM_ c.3081a>t p.k1027n rs Uncertain significance c.10809g>c p.w3603c rs Uncertain significance COL6A3 2q37 NM_ c.3287c>t p.r1096c rs Uncertain significance DYSF 2p13.3-p13.1 NM_ c.937+1g>a - rs Pathogenic c.5713c>t p.r1905x rs Pathogenic COL6A2 21q22.3 NM_ c.2039g>a p.r680h rs Benign TMEM43 3p25 NM_ c.504a>t p.k168n rs Benign c.536t>c p.m179t rs Benign GMPPB 3p21.31 NM_ c.551a>g p.q184r rs Benign DOK7 4p16.2 NM_ c.887a>g p.q296r rs Benign MYOT 5q31 NM_ c.220a>c p.k74q rs Benign DPM2 9q34.13 NM_ c.227c>g p.t76s rs Benign Sep, september; Ref Seq, reference sequence; SIFT, sorting intolerant from tolerant; PolyPhen2, prediction of functional effects of human non-synonymous single nucleotide polymorphisms (nssnps). a GenBank registration number of reference sequence; b SIFT: <0.05 predicted to be deleterious; c PolyPhen2: ~1 indicates prediction of pathogenicity. 중농도는정상값보다낮았지만폼페병의대표적인임상양상인호흡기능장애가없어서다음검사로 WES를시행하였다. 두환자의엑솜분석데이터는 Table 1에요약하였다. 자매에서공통된근육병원인유전자의변이는총 18개로 Table 2에정리하였다. ACMG/AMP 진단기준을바탕으로 DYSF 유전자의 c.937+1g>a와 c.5713c>t (p.arg1905x) 두변이가병적변이 (pathologic variant) 로분류되었고, 실제로두변이모두팔다리이음근디스트로피2B의원인유전자로여러차례보고된것이었다. 9,10 이돌연변이들은고전적염기서열분석으로재확인할수있었다 (Fig. 1B). 혈중산성알파글루코시다아제혈중농도의감소와관계하여 GAA유전자의 c.1726g>a 변이와 c.2065g>a 변이를확인하였다. 11 이 GAA유전자의변이들은산성알파글루코시다아제의거짓결핍과연관되지만이전보고와는달리동형변이 (homozygous variant) 가아닌이형변이 (heterozygous variant) 로존재하였다. 고찰 본연구에서산성알파글루코시다아제의혈중농도가감소로폼페병이의심된근육병가족에서 DYSF 유전자 의복합이형접합돌연변이 (c.937+1g>a, c.5713c>t) 를확인하였다. 이두돌연변이는 ACMG/AMP 진단기준으로병적변이로분류되었고이돌연변이들은스플라이싱돌연변이 (splicing site mutation) 와무의미돌연변이 (nonsense mutation) 로디스펄린 (dysferlin) 단백형성에심각한영향이예상되고, 실제팔다리이음근디스트로피 2B형의원인돌연변이로보고되었기에두자매근육병의원인으로생각하였다. 팔다리이음근디스트로피 2B형의원인유전자인 DYSF 유전자 (MIM *603009) 는염색체 2p13.3-p13.1에위치하고디스펄린단백을부호화한다. 12 디스펄린단백은근육원섬유마디 (sarcomere) 에존재하는막단백 (membrane protein) 으로정확한기능은모르나, 세포막의수리, T-세관의합성및근육재생, 근육모세포 (myoblast) 의분화에관계할것으로추측하고있다 현재까지 DYSF 유전자의 2,300여개의원인돌연변이가보고되었고 ( 한국인에서는 c.2494c>t와 c.663+1g>c가가장흔한돌연변이로알려져있다. 16,17 두자매는유년기에는좋은운동능력을보이다가 10대후반부터하지의근력약화와근위축이나타나고, 40대부터팔의힘이약해졌다. 또한혈중크레아틴키나아제수 82 대한신경근육질환학회지 2015

5 HJ Park, et al. DYSF mutations in Korean 치가보행가능시기에는정상값보다 15배이고, 발병 30 년후에도 4배이상으로증가되어있었다. 이것은팔다리이음근디스트로피 2B형의전형적인임상양상이다. 18 반면, 두자매에서모두산성알파글루코시다아제의혈중농도는감소되었지만호흡기능부전과근육긴장증 (myotonia) 없는근긴장성방전 (myotonic discharge) 과같은폼페병을시사하는임상특징은없었다. 19 폼페병에서치료제가실용화됨에따라서유전진단이되지않은근육병환자에서폼페병을찾기위한노력들이활발히이루어지고있고, 선별검사로산성알파글루코시다아제혈중농도검사가가장널리사용되고있다. 20,21 그러나효소의혈중농도의분석만으로는위양성과위음성의위험이있어서확진을위한근육조직검사, 섬유모세포 (fibroblast) 에서의재측정또는유전자검사가필요하다. 22 실제로, 두자매의전체산성알파글루코시다아제농도와아카보오스를처리후농도는정상값보다낮았으나, 아카보오스를처리한값과전체산성알파글루코시다아제값의비율은정상이었다. 이것은산성알파글루코시다아제의거짓결핍을보이는환자에서나타날수있는소견이다. 23 비록본연구의두환자모두에서거짓결핍의원인으로알려진 GAA유전자의 c.1726g>a 변이와 c.2065g>a 변이를확인하였지만, 이두변이는현재까지연구에서는항상같은대립유전자에존재해서동형접합변이 (homozygous variant) 상태로만보고되어왔다. 24,25 따라서두자매의산성알파글루코시다아제의감소한원인에대해서는추가적인연구가필요하다. 그러나두자매의부모님은사망하였고정상인형제들의검사는거부하여서연구를더이상진행할수없었다. WES는차세대유전자염기서열분석 (next generation sequencing) 을기반으로전체엑손영역을분석하여유전학적원인을찾는매우경제적이고효율적인분석방법이다. 4 그러나 WES도몇몇제한점을갖고있다. 26 첫째, 현재까지알려진엑손의위치정보가완벽하지는않다. 둘째, 한번에읽을수있는염기서열이 개염기쌍 (base pair) 정도로제한적이다. 셋째, DNA 염기서열중구아닌 (guanine) 과시토신 (cytosine) 의함량이높거나반복염기서열로된경우는정확도가감소한다. 넷째, 유전체의구조변화와유전자발현을조절하는유전자조절부위에대한정보는확인하기어렵다. 본연구에서폼페병의특징적인임상양상은관찰되지는않았으나산성알파글루코시다아제혈중농도가감소되어있었던유전성근육병자매에서 WES를이용하여 DYSF유전자의복합이형접합돌연변이 (compound heterozygous mutation) 와 GAA 유전자의변이를확인하였다. 유전성근육병은매우다양한임상표현형을보일수있고, 이것을설명할수있는다양한유전학적원인규명을위해서본연구에서와같이 WES가유용할수있기에이를보고하는바이다. REFERENCES 1. Kaplan JC, Hamroun D. The 2015 version of the gene table of monogenic neuromuscular disorders (nuclear genome). Neuromuscul Disord 2014;24: Norwood F, de Visser M, Eymard B, Lochmuller H, Bushby K. EFNS guideline on diagnosis and management of limb girdle muscular dystrophies. Eur J Neurol 2007;14: Bushby K. Diagnosis and management of the limb girdle muscular dystrophies. Pract Neurol 2009;9: Bamshad MJ, Ng SB, Bigham AW, Tabor HK, Emond MJ, Nickerson DA, et al. Exome sequencing as a tool for Mendelian disease gene discovery. Nat Rev Genet 2011;12: Li H, Durbin R. Fast and accurate short read alignment with Burrows-Wheeler transform. Bioinformatics 2009;25: DePristo MA, Banks E, Poplin R, Garimella KV, Maguire JR, Hartl C, et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat Genet 2011;43: Van der Auwera GA, Carneiro MO, Hartl C, Poplin R, Del Angel G, Levy-Moonshine A, et al. From FastQ data to high-confidence variant calls: the Genome Analysis Toolkit best practices pipeline. Current Protoc Bioinformatics 2013;11: Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet Med 2015;17: Takahashi T, Aoki M, Tateyama M, Kondo E, Mizuno T, Onodera Y, et al. Dysferlin mutations in Japanese Miyoshi myopathy: relationship to phenotype. Neurology 2003;60: Vilchez JJ, Gallano P, Gallardo E, Lasa A, Rojas-Garcia R, Freixas A, et al. Identification of a novel founder mutation in the DYSF gene causing clinical variability in the Spanish population. Arch Neurol 2005;62: Labrousse P, Chien YH, Pomponio RJ, Keutzer J, Lee NC, Akmaev VR, et al. Genetic heterozygosity and pseudodeficiency in the Pompe disease newborn screening pilot program. Mol Genet Metab 2010;99: Liu J, Aoki M, Illa I, Wu C, Fardeau M, Angelini C, et al. Dysferlin, a novel skeletal muscle gene, is mutated in Miyoshi myopathy and limb girdle muscular dystrophy. Nat Genet 1998;20: Bansal D, Miyake K, Vogel SS, Groh S, Chen CC, Williamson R, et al. Defective membrane repair in dysferlin-deficient muscular dystrophy. Nature 2003;423: Han R. Muscle membrane repair and inflammatory attack in Korean Journal of Neuromuscular Disorders

6 Korean Journal of Neuromuscular Disorders Vol. 7 No. 2, 2015 dysferlinopathy. Skelet Muscle 2011;1: Klinge L, Harris J, Sewry C, Charlton R, Anderson L, Laval S, et al. Dysferlin associates with the developing T-tubule system in rodent and human skeletal muscle. Muscle Nerve 2010;41: Park YE, Kim HS, Lee CH, Nam TS, Choi YC, Kim DS. Two common mutations (p.gln832x and c.663+1g>c) account for about a third of the DYSF mutations in Korean patients with dysferlinopathy. Neuromuscul Disord 2012;22: Shin HY, Jang H, Han JH, Park HJ, Lee JH, Kim SW, et al. Targeted next-generation sequencing for the genetic diagnosis of dysferlinopathy. Neuromuscul Disord 2015;25: Klinge L, Aboumousa A, Eagle M, Hudson J, Sarkozy A, Vita G, et al. New aspects on patients affected by dysferlin deficient muscular dystrophy. J Neurol Neurosurg Psychiatry 2010;81: Teener JW. Late-onset Pompe's disease. Semin Neurol 2012;32: Gutiérrez-Rivas E, Bautista J, Vílchez J, Muelas N, Díaz- Manera J, Illa I, et al. Targeted screening for the detection of Pompe disease in patients with unclassified limb-girdle muscular dystrophy or asymptomatic hyperckemia using dried blood: a Spanish cohort. Neuromuscul Disord 2015;25: Musumeci O, la Marca G, Spada M, Mondello S, Danesino C, Comi GP, et al. LOPED study: looking for an early diagnosis in a late-onset Pompe disease high-risk population. J Neurol Neurosurg Psychiatry 2015 Mar 17. pii: jnnp American Association of Neuromuscular & Electrodiagnostic Medicine. Diagnostic criteria for late-onset (childhood and adult) Pompe disease. Muscle Nerve 2009;40: Okumiya T, Keulemans JL, Kroos MA, Van der Beek NM, Boer MA, Takeuchi H, et al. A new diagnostic assay for glycogen storage disease type II in mixed leukocytes. Mol Genet Metab 2006;88: Kroos MA, Mullaart RA, Van Vliet L, Pomponio RJ, Amartino H, Kolodny EH, et al. p.[g576s; E689K]: pathogenic combination or polymorphism in Pompe disease? Eur J Hum Genet 2008;16: Tajima Y, Matsuzawa F, Aikawa S, Okumiya T, Yoshimizu M, Tsukimura T, et al. Structural and biochemical studies on Pompe disease and a "pseudodeficiency of acid alpha-glucosidase". J Hum Genet 2007;52: Ng SB, Turner EH, Robertson PD, Flygare SD, Bigham AW, Lee C, et al. Targeted capture and massively parallel sequencing of 12 human exomes. Nature 2009;461: 대한신경근육질환학회지 2015

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<30342EC1F5B7CA2DB1E8B3B2C8F D E687770> 대한임상신경생리학회지 11(2):59~63, 2009 ISSN 1229-6414 Case Report 미요시근육병환자에서밝혀진 Dysferlin 유전자돌연변이 동국대학교의과대학신경과학교실 1, 부산대학교의과대학신경과학교실 2, 연세대학교의과대학신경과학교실 3 지명구 1 김남희 1 김대성 2 최영철 3 Identification of a Dysferlin Gene