RESEARCH ARTICLE ADSSL1 Mutation Relevant to Autosomal Recessive Adolescent Onset Distal Myopathy Hyung Jun Park, MD,1,2 Young Bin Hong, PhD,3 Young-Chul Choi, MD, PhD,2 Jinho Lee, MD,3 Eun Ja Kim, MD,3 Ji-Su Lee,4 Won Min Mo, MS,3 Soo Mi Ki, MS,4 Hyo In Kim,4 Hye Jin Kim,5 Young Se Hyun, PhD,5 Hyun Dae Hong,5 Kisoo Nam,6 Sung Chul Jung, MD, PhD,7 Sang-Beom Kim, MD, PhD,8 Se Hoon Kim, MD, PhD,9 Deok-Ho Kim, PhD,10 Ki-Wook Oh, MD,11 Seung Hyun Kim, MD, PhD,11 Jeong Hyun Yoo, MD, PhD,12 Ji Eun Lee, PhD,4,13 Ki Wha Chung, PhD,5 and Byung-Ok Choi, MD, PhD3,4,14 Objective: Distal myopathy is a heterogeneous group of muscle diseases characterized by predominant distal muscle weakness. A study was done to identify the underlying cause of autosomal recessive adolescent onset distal myopathy. Methods: Four patients from 2 unrelated Korean families were evaluated. To isolate the genetic cause, exome sequencing was performed. In vitro and in vivo assays using myoblast cells and zebrafish models were performed to examine the ADSSL1 mutation causing myopathy pathogenesis. Results: Patients had an adolescent onset distal myopathy phenotype that included distal dominant weakness, facial muscle weakness, rimmed vacuoles, and mild elevation of serum creatine kinase. Exome sequencing identified com- pletely cosegregating compound heterozygous mutations (p.D304N and p.I350fs) in ADSSL1, which encodes a muscle-specific adenylosuccinate synthase in both families. None of the controls had both mutations, and the muta- tion sites were located in well-conserved regions. Both the D304N and I350fs mutations in ADSSL1 led to decreased enzymatic activity. The knockdown of the Adssl1 gene significantly inhibited the proliferation of mouse myoblast cells, and the addition of human wild-type ADSSL1 reversed the reduced viability. In an adssl1 knockdown zebrafish model, muscle fibers were severely disrupted, which was evaluated by myosin expression and birefringence. In these conditions, supplementing wild-type ADSSL1 protein reversed the muscle defect. Interpretation: We suggest that mutations in ADSSL1 are the novel genetic cause of the autosomal recessive adoles- cent onset distal myopathy. This study broadens the genetic and clinical spectrum of distal myopathy and will be useful for exact molecular diagnostics. ANN NEUROL 2016;79:231–243 View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.24550 Received Jun 8, 2015, and in revised form Oct 7, 2015. Accepted for publication Oct 18, 2015. Address correspondence to Dr Byung-Ok Choi, Samsung Medical Center, Sungkyunkwan University School of Medicine, 81 Irwon-ro Gangnam-gu, Seoul 06351, Korea. E-mail: [email protected]; Dr Ki Wha Chung, Kongju National University, 56 Gongjudaehak-ro, Gongju, Chungnam 32588, Korea. E-mail: [email protected]; Dr Ji Eun Lee, Samsung Advanced Institute for Health Science & Tech., Samsung Genome Institute (SGI), Sam- sung Medical Center, Sungkyunkwan University, 81 Irwon-ro, Gangnam-gu, Seoul 06351, Korea. E-mail: [email protected] From the 1Department of Neurology, Mokdong Hospital, Ewha Womans University School of Medicine, Seoul, South Korea; 2Department of Neurology, Yonsei University College of Medicine, Seoul, South Korea; 3Department of Neurology, Samsung Medical Center, Sungkyunkwan University School of Medicine, Seoul, South Korea; 4Department of Health Sciences and Technology, Samsung Advanced Institute for Health Sciences and Technology, Sungkyunkwan University, Seoul, South Korea; 5Department of Biological Science, Kongju National University, Gongju, South Korea; 6Department of Chemistry, New York University, New York, NY; 7Department of Biochemistry, Ewha Womans University School of Medicine, Seoul, South Korea; 8Department of Neurology, Kyung Hee University College of Medicine, Kangdong Hospital, Seoul, South Korea; 9Department of Pathology, Yonsei University College of Medicine, Seoul, South Korea; 10Department of Bioengineering, University of Washington, Seattle, WA; 11Department of Neurology, College of Medicine, Hanyang University, Seoul, South Korea; 12Department of Radiology, Mokdong Hospital, Ewha Womans University School of Medicine, Seoul, South Korea; 13Samsung Genome Institute, Samsung Medical Center, Seoul, South Korea; and 14Neuroscience Center, Samsung Medical Center, Seoul, South Korea VC 2015 American Neurological Association 231 ANNALS of Neurology istal myopathy is a clinically and genetically hetero- DYSF, and Miyoshi muscular dystrophy 3 caused by Dgeneous group of predominantly distal muscle ANO5.2–5 Distal nebulin myopathy and GNE myopathy degenerative diseases. Distal myopathy is classified into have childhood to early adult onset, and initially show subgroups based on clinical aspects and genetic causes. ankle dorsiflexor weakness and a mild elevation in serum To date, > 15 causative genes have been reported in dif- creatine kinase (CK).2,3 Conversely, Miyoshi myopathy ferent forms of distal myopathies.1 These genes encode or Miyoshi muscular dystrophy 3 have adult onset, pre- proteins that have various functions in all areas of muscle dominant ankle plantar flexor weakness, no facial muscle cell biology, including sarcomeric integrity and function involvement, and severe elevation in serum CK.4,5 Recent (MYOT, TTN, MYH7, NEB, LDB3, and FLNC), sialic whole exome or targeted next generation sequencing has acid biosynthesis (GNE), DNA/RNA-binding proteins proven to be an efficient tool to identify rare genetic (TIA1), membrane repair (DYSF), guanosine triphospha- causes in distal myopathies. We have identified a couple tase (DNM2), vesicular trafficking and signal transduc- of compound heterozygous mutations in ADSSL1 by tion (CAV3), calcium-activated ionic channel (ANO5), exome sequencing, and suggest they are the underlying mitotic progression (KLHL9), and chaperone proteins cause in 2 autosomal recessive distal myopathy families (VCP and DNAJB6).2–17 Recent genomic analysis has with adolescent onset. This suggestion was further con- steadily updated novel genetic causes; however, consider- firmed with an in vitro assay using mouse myoblast cells able myopathy patients still wait for the genetic cause to and an in vivo assay using Adssl1 knockdown zebrafish. be uncovered. Most types of distal myopathies are associ- ated with autosomal dominant inheritance, but 4 types Subjects and Methods are associated with autosomal recessive inheritance: distal Patients nebulin myopathy caused by NEB mutations; GNE This study included a total of 10 members from 2 unrelated myopathy caused by GNE; Miyoshi myopathy caused by adolescent onset distal myopathy families in Korea (family IDs: FIGURE 1: Two autosomal recessive distal myopathy families with novel compound heterozygous mutations in ADSSL1. (A) Pedigrees of 2 distal myopathy families FC628 and FC630. Genotypes of 2 ADSSL1 mutations are indicated at the bottom of each examined individual. Arrows and asterisks indicate the proband of each family and individual whose genome was applied for exome sequencing (squares: male; circles: female; filled: affected; and nonfilled: unaffected). (B) Domain structure of adeny- losuccinate synthase-like 1 (ADSSL1) protein. ADSSL1 contains both a guanosine triphosphate–binding domain (GTP) and an adenylosuccinate synthase domain (ASS). Both identified mutations are located on the ASS domain. (C) Sequencing chromato- grams of c.910G>A (p.D304N) and c.1048delA (p.I350fs) mutations in ADSSL1. Arrows indicate mutation sites (Mut 5 mutant; WT 5 wild type). (D) Conservation analysis of amino acid sequences on the p.D304N mutation sites. The mutation site and sur- rounding regions are highly conserved from human to yeast species. 232 Volume 79, No. 2 Park et al: ADSSL1 and Distal Myopathy FC628 and FC630; Fig 1A). Three and 1 affected members thickness 5 4–5mm, slice gap 5 0.5–1.0 mm). The following were included from FC628 and FC630, respectively. The phe- protocol was used for all patients: T1-weighted spin-echo (SE) notype seemed to be inherited by the autosomal recessive mode (repetition time [TR] 5 570–650 milliseconds, echo time because their parents were unaffected, and no other patient was [TE] 5 14–20 milliseconds, 512 matrixes), T2-weighted SE identified from the close relatives of the 2 families. This study (TR 5 2,800–4,000 milliseconds, TE 5 96–99 milliseconds, also included 500 healthy controls who had no clinical features 512 matrixes), and fat-suppressed T2-weighted SE or family history of distal myopathy, which was confirmed after (TR 5 3,090–4,900 milliseconds, TE 5 85–99 milliseconds, careful clinical and electrophysiological examinations. Written 512 matrixes). informed consent was obtained from all participants according to the protocol approved by the institutional review board for Sung- Exome Sequencing and Filtering of Variants kyunkwan University, Samsung Medical Center. Exome sequencing was performed with the Human SeqCap EZ Human Exome Library v3.0 (Roche/NimbleGen, Madison, Clinical and Electrophysiological Examinations WI), and the HiSeq2000 and HiSeq2500 Genome Analyzer Two independent neurologists did the clinical evaluation. Clini- (Illumina, San Diego, CA) for 6 samples from the FC628 fam- cal information included assessments of age at onset, muscle ily (3 affected: II-1,II-2, II-4; 3 unaffected: I-1, I-2, II-3) and impairments, sensory loss, deep tendon reflexes, and muscle for 1 sample from the FC630 family (affected: II-2). The Uni- atrophy. The muscle strength of the flexor and extensor muscles versity of