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 completely 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 mutation 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 adolescent 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), Samsung 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; 8 Department 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

C 2015 American Neurological Association V 231

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istal myopathy is a clinically and genetically heterogeneous group of predominantly distal muscle degenerative diseases. Distal myopathy is classified into subgroups based on clinical aspects and genetic causes. To date, > 15 causative genes have been reported in different forms of distal myopathies.1 These genes encode proteins that have various functions in all areas of muscle cell biology, including sarcomeric integrity and function (MYOT, TTN, MYH7, NEB, LDB3, and FLNC), sialic acid biosynthesis (GNE), DNA/RNA-binding proteins (TIA1), membrane repair (DYSF), guanosine triphosphatase (DNM2), vesicular trafficking and signal transduction (CAV3), calcium-activated ionic channel (ANO5), mitotic progression (KLHL9), and chaperone proteins (VCP and DNAJB6).2–17 Recent genomic analysis has steadily updated novel genetic causes; however, considerable myopathy patients still wait for the genetic cause to be uncovered. Most types of distal myopathies are associated with autosomal dominant inheritance, but 4 types are associated with autosomal recessive inheritance: distal nebulin myopathy caused by NEB mutations; GNE myopathy caused by GNE; Miyoshi myopathy caused by

DYSF, and Miyoshi muscular dystrophy 3 caused by ANO5.2–5 Distal nebulin myopathy and GNE myopathy have childhood to early adult onset, and initially show ankle dorsiflexor weakness and a mild elevation in serum creatine kinase (CK).2,3 Conversely, Miyoshi myopathy or Miyoshi muscular dystrophy 3 have adult onset, predominant ankle plantar flexor weakness, no facial muscle involvement, and severe elevation in serum CK.4,5 Recent whole exome or targeted next generation sequencing has proven to be an efficient tool to identify rare genetic causes in distal myopathies. We have identified a couple of compound heterozygous mutations in ADSSL1 by exome sequencing, and suggest they are the underlying cause in 2 autosomal recessive distal myopathy families with adolescent onset. This suggestion was further confirmed with an in vitro assay using mouse myoblast cells and an in vivo assay using Adssl1 knockdown zebrafish.

Subjects and Methods Patients This study included a total of 10 members from 2 unrelated 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 adenylosuccinate 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 chromatograms 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 surrounding regions are highly conserved from human to yeast species.

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FC628 and FC630; Fig 1A). Three and 1 affected members were included from FC628 and FC630, respectively. The phenotype seemed to be inherited by the autosomal recessive mode because their parents were unaffected, and no other patient was identified from the close relatives of the 2 families. This study also included 500 healthy controls who had no clinical features or family history of distal myopathy, which was confirmed after careful clinical and electrophysiological examinations. Written informed consent was obtained from all participants according to the protocol approved by the institutional review board for Sungkyunkwan University, Samsung Medical Center.

Clinical and Electrophysiological Examinations Two independent neurologists did the clinical evaluation. Clinical information included assessments of age at onset, muscle impairments, sensory loss, deep tendon reflexes, and muscle atrophy. The muscle strength of the flexor and extensor muscles was assessed manually with the standard Medical Research Council scale.18 The age at onset was determined by asking patients for their ages when symptoms, including distal muscle weakness, first appeared. Neurophysiological studies were done on 4 affected individuals (3 men and 1 woman). Motor and sensory conduction studies of the median, ulnar, peroneal, tibial, and sural nerves were tested, and needle electromyography was performed in the bilateral upper and lower limb muscles. In all patients, an electrocardiography and echocardiography were done, and serum CK levels were measured.

Muscle Biopsy and Histological Examination Histopathological analyses including immunohistochemistry of the left vastus lateralis muscles were done in the 4 patients: the 3 patients (II-1, II-2, and II-4) in FC628 were 24, 25, and 17 years of age, respectively, and the other patient (II-2 in FC630) was 20 years of age. Frozen 10 mm sections were examined after staining with hematoxylin and eosin (H&E), modified Gomori trichrome (GT), nicotinamide adenine dinucleotide–tetrazolium reductase (NADH-tr), and myosin adenosine triphosphatase (ATPase) preincubated at pH 4.3, 4.6, and 9.4. Staining with Congo red, acid phosphatase, and periodic acid–Schiff (PAS) was performed on a frozen muscle specimen of Patient II-2 of FC628. Additionally, muscle specimens were analyzed by immunohistochemistry with antibodies against the C-terminus of dystrophin, the rod domain of dystrophin, the N-terminus of dystrophin, dysferlin, a-sarcoglycan, b-sarcoglycan, c-sarcoglycan, d-sarcoglycan (Leica Microsystems, Newcastle upon Tyne, UK), a-dystroglycan (Millipore, Billerica, MA), and caveolin (BD Biosciences, San Diego, CA).

Lower Limb Magnetic Resonance Imaging Two patients (II-2 in FC628 and II-2 in FC630) were examined by lower limb magnetic resonance imaging (MRI) of the hip, thigh, and calf muscles at 30 and 23 years, respectively. MRI was undertaken by a 1.5T system (Siemens Vision, Erlangen, Germany). The imaging was done in the axial (field of view [FOV] 5 24–32cm, slice thickness 5 10mm, slice gap 5 0.5–1.0mm) and coronal planes (FOV 5 38–40cm, slice

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thickness 5 4–5mm, slice gap 5 0.5–1.0 mm). The following protocol was used for all patients: T1-weighted spin-echo (SE) (repetition time [TR] 5 570–650 milliseconds, echo time [TE] 5 14–20 milliseconds, 512 matrixes), T2-weighted SE (TR 5 2,800–4,000 milliseconds, TE 5 96–99 milliseconds, 512 matrixes), and fat-suppressed T2-weighted SE (TR 5 3,090–4,900 milliseconds, TE 5 85–99 milliseconds, 512 matrixes).

Exome Sequencing and Filtering of Variants Exome sequencing was performed with the Human SeqCap EZ Human Exome Library v3.0 (Roche/NimbleGen, Madison, WI), and the HiSeq2000 and HiSeq2500 Genome Analyzer (Illumina, San Diego, CA) for 6 samples from the FC628 family (3 affected: II-1,II-2, II-4; 3 unaffected: I-1, I-2, II-3) and for 1 sample from the FC630 family (affected: II-2). The University of California, Santa Cruz assembly hg19 was the reference sequence. We selected functionally significant variants (missense, nonsense, exonic indel, and splicing site variants) from the whole exome sequencing data, and then variants registered as novel or uncommon variants (minor allele frequencies  0.01) in dbSNP142 (http://www.ncbi.nlm.nih. gov), the 1000 Genomes project database (http://www. 1000genomes.org/), and Exome Variant Server (http://evs.gs. washington.edu/EVS/) were further filtered. Functionally significant cosegregating variants were selected from the 6 samples of the FC628 family exome sequencing data.

In Silico Analysis and Determination of Causative Mutation The Sanger sequencing method confirmed the candidate variants using the genetic analyzer ABI3130XL (Life Technologies, Foster City, CA). The genomic evolutionary rate profiling (GERP) scores were determined by the GERP1 1 program (http://mendel.stanford.edu/SidowLab/downloads/gerp/index. html). We performed conservation analysis of the protein sequences using MEGA5, version 6.06 (http://www.megasoftware.net/). In silico analyses were done with the prediction algorithms SIFT (http://sift.jcvi.org), MUpro (http://www.ics. uci.edu/baldig/mutation), and PolyPhen-2 (http://genetics. bwh.harvard.edu/pph2/). The SMART program predicted domains for the ADSSL1 protein (http://smart.embl.de/).

Construction of Wild-Type and Mutant ADSSL1 Vectors A human ADSSL1 gene was obtained from Origene Technologies (Rockville, MD). The gene was transferred to pCR2.1TOPO vector (Invitrogen, Carlsbad, CA) by polymerase chain reaction (PCR) amplification using the following primers: ADSSL1-F, 50 -GCATGGTGGGGAGGAGCTGTGGGG-30 ; and ADSSL1-R, 50 -CTAAAACAGCTGGATCATCGACTCT30 . After construction of the wild-type ADSSL1, site-directed mutagenesis was performed to obtain c.910G>A (D304N) and c.1045delA (I350fs) mutations using a QuikChange SiteDirected Mutagenesis Kit (Stratagene, La Jolla, CA). The primers for the mutagenesis were as follows: ADSSL1-G910A-F,

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50 -CCGCCCTCCTCAACATTGACTTCG GG-30 ; ADSSL1G910A-R, 50 -AGTCAATGTTGAGGAGGG CGGCGTTG-30 ; ADSSL1-1048delA-F, 50 -ACGTGTGGGCTCGGGGCCTT CCCCA-30 ; and ADSSL1-1048delA-R, 50 AAGGCCCCGATG CCCACACGTGTGG-30 . All the constructed sequences were confirmed by Sanger’s capillary sequencing method.

Expression and Purification of ADSSL1 Wild-type and mutant ADSSL1 genes were cloned into pGEX4T vector (GE Healthcare, Piscataway, NJ), which encodes glutathione-S-transferase (GST) protein. GST-fused ADSSL1 proteins were expressed in the Escherichia coli strain BL21 (GE Healthcare). Overnight-grown E. coli was diluted 1/100 in Luria-Bertani broth and incubated at 37 8C for 6 hours, and then 1mM isopropyl b-D-1-thiogalactopyranoside was added. After 2 hours, the cells were harvested and underwent freezing and thawing 4 times. Protein purification was done with the GST Spin Trap system (GE Healthcare). Expression level of ADSSL1 in the patient (FC628, II-2) and controls was determined using muscle biopsied samples. Standard Western blotting was performed using antimyc antibody (Ab; Abcam, Cambridge, UK), anti-ADSSL1 Ab (Novus Biologicals, Littleton, CO), antiactin Ab, antimouse secondary Ab, and antirabbit secondary Ab (Sigma, St Louis, MO).

Measurement of ADSSL1 Enzyme Activity Enzymatic activity of the purified ADSSL1 proteins was measured according to previous reports.19,20 The formation of adenylosuccinate was determined at 280nm after incubation with assay buffer (HEPES, pH 8.0) containing 10mM MgCl2, 60 lM guanosine triphosphate (GTP), 150 lM inosine monophosphate (IMP), and 25mM aspartate (Sigma).

Muscle Cell Viability Assay Mouse myoblast cells (C2C12) were cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum and penicillin/streptomycin (Life Technologies). For knockdown of mouse Adssl1 in C2C12 cells, Adssl1-specific siRNAs were used: Adssl1#1, 50 -GGAAGUACAACGUCAAGCAdTdT-30 ; Adssl1#2, 50 -GGAGCCAUGUCAUAAUAAAdTdT-30 ; Adssl1#3, 50 -GUAUGAAGCCCUGCAUGGUdTdT-30 ; and 50 -CCUACG CCACCAAUUUCGUdTdT-30 as a control. Knockdown of Adssl1 was done with Lipofectamine 2000 reagent (Invitrogen). Human wild-type and mutant ADSSL1 were cloned into the pCMV-myc vector, and overexpression was performed with Lipofectamine 3000 reagent (Invitrogen). Cell viability was measured either with the siRNA knockdown after 5 days or with a combination of transfection with wild-type and mutant pCMV-myc ADSSL1 after 2 days. Cell viability was determined with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. After 2 hours of incubation in 10mM MTT solution, the cells were lysed with dimethylsulfoxide (DMSO). The relative numbers of viable cells were determined by measuring their absorbance at 560nm.

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Zebrafish Housing and Handling Zebrafish (AB strain) were raised, staged, and maintained according to standard protocols.21 Embryos were obtained by natural spawning and cultured in 1X E3 solution (360: 300mM NaCl, 10.2mM KCl, 19.8mM CaCl2, and 19.8mM MgSO4 [adjusted to pH 7.2]) plus 0.0001% methylene blue at 28.5 8. For bright field imaging, embryos were anesthetized with 0.04% tricaine (Sigma) in E3 solution.

Microinjection of mRNA and Morpholino Oligonucleotides into Zebrafish Embryos To transiently express the protein, human ADSSL1 (wild type, G910A, and 1048delA) was subcloned into the pCS21 vector. The mRNAs of the DNA constructs were synthesized in vitro using the mMessage mMachine SP6 kit (Ambion, Austin, TX). The synthesized mRNA was stored at 280 8C, and 100 to 200pg/nl of each mRNA were microinjected into 1- to 2-cell stage embryos. For the silencing of the zebrafish adssl1 expression, splicing-blocking antisense morpholino oligonucleotides (MOs), targeting intron 7 and exon 8 or exon 9 and intron 9, were designed and synthesized by GeenTools (Philomath, OR): MO1, 50 -TGTCTGTTTATCTACAGGAATACGC-30 ; and MO2, 50 -TCAATGTAGGACAAACACCCTTTAT-30 . The MOs were dissolved in nuclease-free water and microinjected into 1-cell stage embryos at 2ng per embryo. Embryos were incubated at 28.5 8C in 3 1 E3 media (5.0mM NaCl, 0.17mM KCl, 0.43mM CaCl2, 0.4mM MgCl2, pH 7.2) and 0.2mM 1-phenyl-2-thiourea (Sigma) was added for phenotype analysis.

Whole Mount Immunostaining of Zebrafish Larvae Dechorionated embryos were collected at 24 hours postfertilization (hpf ) and fixed in 4% paraformaldehyde for 1 hour at room temperature (RT). After washing 3 times with 3 1 phosphate-buffered saline (PBS), embryos were blocked with blocking solution (2% bovine serum albumin, 0.5% Triton X100, 0.05% DMSO, and 5% normal goat serum in 31 PBS) for 2 hours at RT. Embryos were incubated with primary rabbit antilaminin Ab (1:50, Sigma) and mouse monoclonal antimyosin Ab (1:25; Developmental Studies Hybridoma Bank, Boston, MA) at 4 8C overnight in blocking solution. After washing 4 times with PBST (0.5% Triton X-100 in 3 1 PBS) for 15 minutes per wash, embryos were incubated with Alexa Flour594–conjugated antimouse secondary Ab and Alexa Flour-488– conjugated antirabbit secondary Ab (1:250, Invitrogen) in 2% normal goat serum/PBST for 2 hours at RT. Embryos were washed with PBST 4 times and mounted in 70% glycerol/PBS. To analyze the muscle, a confocal microscope (LSM700; Zeiss, Oberkochen, Germany) was used, and images were processed and analyzed with Zeiss ZEN imaging software.

Results Clinical Manifestations The clinical presentations of the 4 patients are summarized in Table 1. All affected individuals were born at full term in healthy nonconsanguineous parents, and the Volume 79, No. 2

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TABLE 1. Clinical Features in 4 Patients with Compound Heterozygous Mutations in the ADSSL1 Gene

Characteristic

FC630

FC628

Sex

II-1 M

II-2 M

II-4 M

II-2 F

Age at examination, yr

31

30

28

23

Age at onset, yr

15

13

13

13

16

17

15

10

1

1

1

1

11

11

1

11

Mild (UG

RHOXF1, c.529G>C

MAP7D3, c.1682A>G

p.Q1568E

p.D177H

p.K561R

dbSNP142

rs140614802

rs559454746

rs77432041

rs369063193

—

rs138060880

—

1000Ga

—

—

—

0.0041

—

—

—

EVSdbb

0.00008

—

0.00008

0.00008

—

—

—

5.03

3.98

1.67

5.22

0.95

23.04

22.91

—

0.16

0.08

0.61

0.14

0.000

1.000e

20.875e

20.692e

GERP

c

In silico analysisd SIFT

0.00e e

PP2

1.000

MUpro

20.528e

0.08 e

—

0.297

0.991

—

20.575e

20.801e

0.936 0.865

e

a

Variant allele frequencies in the 1000 Genomes database (March 2014, http://www.1000genomes.org/). Variant allele frequencies in the Exome Variant Server database (December 2014, http://evs.gs.washington.edu/EVS/). c Genomic evolutionary rate profiling scores. d SIFT score < 0.05, PolyPhen-2 (PP2) score  1, and MUpro score < 0 indicate a prediction of pathogenicity. e Pathogenic prediction. b

from 2 families. The mean total sequencing yield of 7 whole exome sequences from 7 samples was approximately 10.6Gbp/sample with a 93.9% coverage rate for the targeted exon regions (10 3 read depth). The screening for variants in distal myopathy–related genes revealed > 50 functionally significant variants in the proband of the FC628 family (II-1). However, no variant was considered as the underlying cause of the myopathy phenotype. Most variants have been frequently reported in several public genome variant databases (dbSNP, 1000 Genomes project, and Exome Variant Server) as well as in-house exome data (n 5 280). In addition, several rare variants (allele frequencies of < 0.01) did not cosegregate with the affected individuals or did not fit the recessive inheritance mode in the FC628 family. We then filtered exome data from 6 members of the FC628 family (3 affected, 3 unaffected) with the principles of recessive inheritance and allele frequencies < 0.05 from functionally significant variants (single nucleotide polymorphism [SNP] quality  20). The filtering identified 2 pairs of compound heterozygous mutations in autosomal genes ADSSL1 and IGSF9B, and 3 hemizygous variants in X-linked genes IIAA1210, RHOXF1, and MAP7D3 (Table 2). Among them, we noticed the compound heterozygous mutations c.910G>A (p.D304N) and c.1048delA (p.I350fs) in ADSSL1 because ADSSL1, which is strongly expressed in skeletal muscle, encodes a muscle isozyme called adenylosuccinate synthase.7 Two ADSSL1 mutations were located 238

in the adenylosuccinate synthase domain, and in silico analyses with the SIFT, PolyPhen-2, and MUpro programs predicted the pathogenicity of the mutations (see Fig 1). Complete cosegregation of both mutations was confirmed by the Sanger sequencing method. Moreover, we identified the same compound mutations in another distal myopathy family (FC630) with similar clinical phenotypes. Both families showed that each unaffected parent transferred an ADSSL1 mutation to the affected progenies. Both mutations were not found in the Korean healthy controls (n 5 500). Although each mutation has been rarely reported in dbSNP142 and the Exome Variant Server with allele frequencies of 0.00008, no individual had both mutations. The mutation site is highly conserved among different species from yeast to human. Because both families had the same pairs of mutations, we determined whether the origin of the mutations was the same in both families. SNP analysis at the vicinity of the ADSSL1 locus exhibited the same haplotype blocks in both families within very narrowed regions: 1.9Mbp length for c.910G>A and 0.2Mbp length for c.1048delA. This result implies that each mutation in both families may have originated from each single founder of a distant ancestor. Loss of ADSSL1 Activity Reduced Muscle Cell Viability To determine the effect of the compound heterozygous mutations on cultured muscle cells, we measured the Volume 79, No. 2

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FIGURE 4: Effect on the enzymatic activity and cell viability. (A) Enzyme activity was determined for the wild-type and mutant ADSSL1 purified from Escherichia coli. (B) Influence of ADSSL1 on cell viability was measured after C2C12 cells were treated with control or Adssl1-specific siRNA. Reverse transcriptase polymerase chain reaction was performed on the knockdown of Adssl1. (C) Mouse fibroblasts, NIH3T3 cells, were treated with the control or Adssl1-specific siRNA, and then viability was measured. (D) Recovery of cell viability was determined after C2C12 cells were treated with the control or Adssl1-specific siRNA, and then the overexpression of wild-type or mutant ADSSL1 protein was performed. Western blotting confirms the expression of ADSSL1. Arrows indicate ADSSL1 protein. ns 5 nonspecific band. (E) Expression level of ADSSL1 in the patient (FC628, II-2) was compared to age- and sex-matched controls (Ctr1 and Ctr2). (F) Stability of mutant ADSSL1 protein was determined using Western blotting. After overexpression of protein, cycloheximide (CHX, 25ng/ml) was applied for the indicated time. (G) Residual protein level in the Western blotting was determined using ImageJ (NIH, Bethesda, MD).

enzymatic activity of the proteins with the D304N or I350f mutations. An enzyme activity assay showed that both mutations significantly affect the synthesis of adenylosuccinate (Fig 4). Next, we investigated the significance of ADSSL1 in skeletal muscle. We measured the change in cell viability in C2C12, a mouse myoblast cell line, after treatment with 3 different Adssl1-specific siRNAs. Knockdown of Adssl1 for 5 days resulted in the reduced viability of the C2C12 cells treated with the Adssl1-specific siRNAs compared to the control-siRNA–treated cells. Interestingly, the decrease in cell viability by the Adssl1-specific siRNA treatments was not observed in a mouse fibroblast cell line, NIH3T3. We then determined whether the reduced cell viability by the knockdown of Adssl1 could be rescued by the expression of human wild-type or mutant ADSSL1. Overall expression of the ADSSL1 proteins did not affect the viability of the control-siRNA–treated cells. However, overexpression of wild-type but not mutant ADSSL1 protein significantly increased the cell viability in Adssl1-specific siRNA-treated C2C12 cells. Collectively, these data show that the enzymatic activity of ADSSL1 is crucial for muscle cell viability, and the novel compound heterozygous mutations affect the viability due to reduced enzymatic activity. February 2016

Next, we determined the expression level of ADSSL1 in the patient sample. The biopsied muscle sample showed reduced expression of the mutant protein (see Fig 4E). In addition, the truncated mutant protein was not visible. Then we examined the stability of the mutant proteins by determination of protein half-life. Residual protein levels after treatment with cycloheximide, an inhibitor of protein synthesis, were compared after expression in HEK293 cells (see Fig 4F, G). Mutant proteins were degraded more rapidly than wild-type protein. The half-life of mutant proteins (D304N, 3.8 hours; I305fs, 2.3 hours) was significantly shorter than that of wild-type protein (13.4 hours). Therefore, the mutant proteins are structurally unstable, and so the residual level in the patient sample was less than in the controls. Depletion or Mutations of ADSSL1 Results in Muscle Defects in Zebrafish To determine whether ADSSL1 is required for normal muscle development in vivo, 2 different antisense MOs against zebrafish adssl1 were designed and injected into developing zebrafish embryos (Fig 5). We paid attention to 2 features, muscle birefringence and muscle myosin 239

FIGURE 5: Loss-of-function of zebrafish adssl1 phenocopies distal myopathy. (A) Diagram of designed morpholinos (MOs) for zebrafish adssl1. Rectangles, black lines, and red bars indicate exons, introns, and location of MOs, respectively. (B) About 5 somites in the red box were counted to quantify the muscle phenotype. A 5 anterior; P 5 posterior. (C) Class II phenotype in the adssl1 MO2-injected zebrafish larva at 24 hours postfertilization (hpf). MHC 5 myosin heavy chain. (D–D0 ) Muscle morphology of uninjected control zebrafish larva at 24 hpf. (E–E0 ) Defects in the muscle of the zebrafish larva injected with adssl1 MO. (F–F0 ) The morphology in the muscle of the zebrafish larva coinjected with adssl1 MO and the mRNA of human ADSSL1 D304N. (G–G0 ) Disruption in the muscle of the zebrafish larva coinjected with adssl1 MO and the mRNA of human ADSSL1 I350fs. (H–H0 ) Rescued muscle phenotype by coinjection of human ADSSL1 (wild-type [WT]) mRNA and adssl1 MO in zebrafish larva at 24 hpf. (I) The table shows the embryo numbers with the muscle phenotype in each genotype. (J) A graph shows the quantified data. Asterisks indicate a disorganized fiber pattern (C) and loosely packed myofibers with occasional prominent gaps (E–G). Arrows point out the breakage of the myosepta. (C) Cells detached from the myosepta (E0 –G0 ). Scale bars 5 40 lm.

Park et al: ADSSL1 and Distal Myopathy

expression, to quantify muscle damage because abnormal birefringence is a finding common to zebrafish models of muscular dystrophies.22,23 The majority of adssl1 MOinjected embryos (morphants) showed severe disruptions in the birefringence that could be due to the detachment of fibers from the myotendinous junctions. On immunostaining with antilaminin Ab, uninjected control embryos showed normal V-shaped chevron myosepta, whereas the adssl1 MO-injected embryos showed U-shaped myosepta at 24 hpf. In addition, the laminin of the morphants remained visibly localized to the free end of the detached cells, unlike the enrichment of the laminin in the terminal cytoplasm at the muscle attachments in the controls. Further analysis by labeling with anti–myosin heavy chain Ab showed that the adssl1 morphants had severe lesions in the muscle myofibers compared with the control embryos (about 50% of MO1 and about 80% of MO2): class I, loosely packed myofibers with occasional prominent gaps; and class II, overall disorganized fiber patterns due to abundant gaps between myofibers. Accordingly, these results suggest that ADSSL1 plays an important role in myofiber attachment. To understand the pathogenesis of the affected individuals with the ADSSL1 mutations (D304N and I350fs), we injected the mRNAs encoding the mutated human ADSSL1 into the adssl1 MO-injected zebrafish embryos and compared the phenotypes to those of the adssl1 morphants. We found that the majority of the embryos expressing mutated ADSSL1s showed skeletal muscle phenotypes similar to the adssl1 morphants, thereby suggesting a loss-of-function defect for the ADSSL1 mutant forms (see Fig 5F–F0 and G–G0 ). Because the loss of adssl1 induced a high frequency of zebrafish with muscle lesions, we wanted to test whether the wild-type human ADSSL1 gene could rescue the adssl1 morphant phenotype. Scoring both birefringence and myosin labeling, we found that the injection of RNA encoding wild-type human ADSSL1 largely rescued the muscle defects of the adssl1 morphants at 24 hpf (see Fig 5H–H0 , I, and J). The data suggest that the novel mutations of ADSSL1, identified from 2 different Korean families, are deleterious in that they cause myopathy, and a skeletal muscle–related role of ADSSL1 is evolutionarily conserved. Collectively, our findings suggest the importance of ADSSL1 in skeletal muscle development/ function, and its loss of function may be causative of human muscular dystrophies.

Discussion Our study has identified compound heterozygous ADSSL1 mutations (p.D304N and p.I350fs) from 2 unrelated families with adolescent onset autosomal recesFebruary 2016

sive distal myopathy. We believe that these compound heterozygous mutations are strongly implicated as the underlying cause of the myopathy phenotype in both families. These mutation pairs were completely cosegregated with the affected individuals in both families in an autosomal recessive manner. Both mutations were not found in 500 healthy Korean controls, although they have been rarely reported in dbSNP142 and the Exome Variant Server database. So far, no study has reported that ADSSL1 is implicated in any kind of human genetic disease. Thus, this study may be the first report of an inherited disease caused by the ADSSL1 defect. ADSSL1 (Mendelian Inheritance in Man #612498) mapped on chromosome 14q32.33 encodes the ADSSL1 (adenylosuccinate synthetase-like 1) protein, which is a muscle-specific enzyme with strong expression in skeletal muscles.19 This enzyme has a role in purine nucleotide interconversion by catalysis of the initial reaction in the conversion of IMP to adenosine monophosphate.19 Structurally, the 2 mutations were located in the adenylosuccinate synthase domain, which is highly conserved through species from yeast to mammals. The position of 304Asp is close to the conserved residue, 303Leu, which is characteristic of adenylosuccinate synthetase.19 Moreover, the frameshift mutation of I350fs results in a loss of GTP-binding sites (amino acids 405–409).19 In silico analyses also predicts that the D304N mutation might be pathogenic. Detailed MRI analysis revealed a distinct pattern of muscular involvement in ADSSL1 myopathy. Marked hyperintense signal changes were revealed in the calf muscles compared to the thigh muscles, which was well related with the distal dominancy. Additionally, we could observe a sequential pattern of muscle involvement associated with disease duration and severity. In the early disease stage, gastrocnemius muscles were initially involved, and revealed the most severe fatty replacements, and to a lesser degree the soleus, tibialis anterior, or peronei muscles. In the later stage, quadriceps muscles in the thigh showed fatty infiltrations and muscle atrophies. Clinically, the present patients had adolescent onset, predominant distal muscle involvement, facial muscle weakness, mild serum CK elevation, and few rimmed vacuoles. These clinical and pathological manifestations were both similar to and different from those of previously reported autosomal recessive distal myopathies. Miyoshi myopathy and Miyoshi muscular dystrophy 3 are clearly distinguished from the present ADSSL1 myopathy. Both of them showed adult onset, predominant ankle plantar flexor weakness, no facial muscle involvement, and marked CK elevation.4,5,24 Distal nebulin myopathy showed selective involvement of the tibialis 241

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of Neurology

anterior muscles and mild CK elevation.3,25 However, distal nebulin myopathy predominantly involves the finger extensors and axial muscles, and frequently accompanies craniofacial dysmorphisms including high arched palate, which were not found in our patients.3,26 The presence of nemaline rods and Z-line streaming in muscle specimens distinguishes distal nebulin myopathy from this myopathy also.3,25 GNE myopathy was similar to the ADSSL1 myopathy because of mild elevation of serum CK and rimmed vacuoles.2,27 MRI of GNE myopathy has indicated predominant involvement of tibialis anterior muscles, but the present patients showed initially severe involvement of gastrocnemius muscles.27,28 Experimentally, we showed that these mutations are harmful to the normal function of skeletal muscle. The present mutant proteins exhibited reduced enzymatic activity compared to the wild type. Because ADSSL1 is predominantly expressed in skeletal muscles,19 loss of activity might be detrimental to muscle cells. Accordingly, we also showed that abrogation of Adssl1 reduced the viability of mouse muscle cells, which was not observed in fibroblasts. In these conditions, the addition of wild-type human ADSSL1 but not mutant ADSSL1 rescued the cell viability, which was reduced by the abrogation of Adssl1. The data indicate that the ADSSL1 activity is crucial for muscle cell integrity. The significance of ADSSL1 in the integrity of muscle tissue was also shown in vivo. The adssl1 knockdown zebrafish showed muscle defects that resemble those of depletions in genes including APO2 and DAG1, which are essential in skeletal musculature.29,30 Especially, adssl1 morphants had cell-free spaces in the somites, implicating failure or rupture of myofiber attachment31 and disrupted myosepta, which are part of the connective tissue. These phenotypes suggest that ADSSL1 may be essential in the integrity of the myoseptum as well as the attachment of myofibrils to the myoseptum. It is thus conceivable that the impaired motility of the affected individuals with mutations in ADSSL1 result from myoseptal defects already in existence before the onset of contractions of the muscles. Purine nucleotides are essential to energy transfer, metabolic regulation, and synthesis of DNA and RNA. Previously, a defect in purine nucleotide synthesis was reported to result in decreased cell viability; a decrease in the adenylosuccinate synthetase (ADSS) level is associated with apoptosis, which was reversed by the addition of guanosine.28,32 In addition, deficiency of adenylosuccinase, which engages in the next step with ADSS/ADSSL1 in purine synthesis, leads to severe psychomotor delay and autism.33 In the upstream part of the pathway, the 242

abnormal activity of phosphoribosylpyrophosphate synthetase 1 (PRPS) also causes Arts syndrome, Charcot– Marie–Tooth disease, and PRPS1 superactivity, which are associated with mental retardation, peripheral neuropathy, hyperuricemia, and gout.34–36 Further investigation of ADSSL1 will be constructive from some perspectives. Because this is the first report that mutant ADSSL1 proteins cause a human disease, it will be informative to study its defined pathological features to develop therapeutic strategies. Because this disease might be due to an enzymatic defect in purine synthesis metabolism, several approaches, such as the addition or deprivation of metabolites, and induction of the ADSS protein in the skeletal muscle to rescue the defect, might be possible. In addition, elucidation of the pathological progression of ADSSL1, which is distally predominant, is also interesting in that the patients did not exhibit any cardiac symptoms. Although ADSSL1 is predominantly expressed in skeletal and cardiac muscle, several factors such as leaky expression of ADSS or location-specific dependency on ADSSL1 might determine the disease progression and characteristics. In conclusion, we suggest that compound heterozygous mutations in ADSSL1 cause a novel type of recessively inherited distal myopathy. It appears that the lossof-function mutations may develop distal myopathy by the defect of de novo purine nucleotide biosynthesis due to a deficiency in enzyme activity. This study is the first report that ADSSL1 is involved in a human genetic disease, and will be useful in performing exact molecular diagnostics of distal myopathy.

Acknowledgment This study was supported by the Korean Health Technology R&D Project, Ministry of Health and Welfare (HI12C0135, HI14C3484) and by National Research Foundation of Korea grants funded by Ministry of Science, ICT and Future Planning (MSIP) (NRF2014R1A2A2A01004240, 2013R1A1A1059056), Republic of Korea. We thank the patients and their families for their essential help with this work.

Author Contributions Concept and study design: H.J.P., J.E.L., K.W.C., B.O.C. Data acquisition and analysis: Y.B.H., Y.-C.C., J.L., J.-S.L., W.M.M., S.M.K., H.I.K., H.J.K., Y.S.H., H.D.H., K.N., S.C.J., S.-B.K., S.Ho.K., K.-W.O., S.Hy.K., J.H.Y. Drafting the manuscript and figures: H.J.P., Y.B.H., E.J.K., D.-H.K., J.E.L., K.W.C., B.-O.C. Volume 79, No. 2

Park et al: ADSSL1 and Distal Myopathy

Potential Conflicts of Interest

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