A novel mutation in the DYSF gene in a patient with a presumed inflammatory myopathy

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1 2018; 38, doi: /neup Case Report A novel mutation in the DYSF gene in a patient with a presumed inflammatory myopathy Jin Tang, Xueqin Song, Guang Ji, Hongran Wu, Shuyan Sun, Shan Lu, Yuan Li, Chi Zhang and Huiqing Zhang Department of Neurology, The Second Hospital of Hebei Medical University, Shijiahzuang, China Dysferlinopathy, a progressive muscular dystrophy, results from mutations in the Dysferlin gene (DYSF, MIM*603009). Traditional diagnosis relies on the reduction or absence of dysferlin. However, altered dysferlin has been observed in other myopathies, leading to a precise diagnosis through molecular genetics. In this study, we report a patient who was previously misdiagnosed as inflammatory myopathy based on routine clinicopathological examinations alone. However, muscle biopsy specimens were analyzed further by immunohistochemistry of muscular dystrophy-related proteins, and gene-targeted next generation sequencing (NGS) was used to correctly identify muscular dystrophy. DNA was sequenced with NGS and the detected mutation was verified by Sanger sequencing. Our targeted NGS found a novel missense mutation (c.5392g > A) in the DYSF gene, allowing correct diagnosis of LGMD2B in our patient. We discovered of a novel missense mutation in the DYSF gene and have broadened the DYSF mutation spectrum, which may be correlated in patients with presumed dysferlinopathy, especially when lymphocytic infiltration is observed. Key words: DYSF, inflammatory myopathy, LGMD2B, muscle biopsy, next generation sequencing. INTRODUCTION Dysferlin, encoded by the DYSF gene, is a calciumdependent transmembrane protein and is expressed mainly in skeletal and cardiac muscles. Dysferlin enhances calcium-mediated membrane fusion and sarcolemmal Correspondence: Xueqin Song, MD, Department of Neurology, The Second Hospital of Hebei Medical University, 215 Heping West Road, Xinhua District, Shijiahzuang , Hebei, China. sxq5679@163.com Received 06 February 2018; Revised 05 April 2018; Accepted 09 April 2018; Published online 25 May repair. 1 Mutation in Dysferlin gene (DYSF, MIM*603009) is associated with autosomal recessive disease characterized by a spectrum of clinically heterogeneous muscular dystrophies. 2 Predominantly, proximal and distal muscles are implicated in dysferlinopathy which is most pronounced in the pelvic and shoulder girdle muscles (Limbgirdle muscular dystrophy type 2B, LGMD2B), or in distal parts of the legs involving either the gastrocnemius and soleus muscles in cases of Miyoshi myopathy (MM), or the anterior tibial muscles, as in distal anterior compartment myopathy (DACM). Other atypical clinical characteristics of dysferlinopathy include scapuloperoneal syndrome, elevated serum creatinine kinase (CK) concentration, and congenital muscular dystrophy. Traditional diagnostic criteria relies on immunohistochemical analysis based on the reduction or complete absence of dysferlin protein in muscle biopsies. However, altered immunolocalization of dysferlin is observed not only in primary dysferlinopathy, but also in other myopathies with normal protein content, including calpainopathy, sarcoglycanopathy and dystrophinopathy. 3 Targeted sequencing remains the gold standard in the precise diagnosis of dysferlinopathy. 4 In humans, DYSF maps to chromosome 2p13 and encompasses 55 exons spanning over 150 kb of genomic DNA. Over 517 DYSF variants have been described to date in the Human Genome Mutation Database ( wwwkl0.hgmd.cf.ac.uk/ac), with no hotspot widespread across the gene. 5 A study by Jin et al. found that in Chinese patients, DYSF mutations were clustered in the N- terminal region with exonic rearrangements in 23% of the patients. Furthermore, sequencing identified one pathogenic mutation in these patients. 6 Sanger sequencing, considered a time-consuming, labor-intensive method for DYSF genetic testing, has been gradually replaced by next generation sequencing (NGS). 7 In this study, we applied NGS for a precise diagnosis in a patient with muscular dystrophy who was misdiagnosed 2018 The Authors. published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of This is an open access article under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is noncommercial and no modifications or adaptations are made.

2 434 J Tang et al. earlier as inflammatory myopathy based on routine clinicopathological examinations alone. This study was approved by the ethics committee of the Second Hospital of Hebei Medicine University and informed consent was obtained from the patient involved in the study. CLINICAL SUMMARY A 22-year-old Chinese woman presented with a progressive proximal muscle weakness in pelvic girdles and bilateral low limbs. Six months later after the onset of the symptoms, the patient visited a local hospital and received a careful examination. Serum CK was found to be elevated ( U/L). A neurological examination and evaluation including electromyography and nerve conduction testing revealed myogenic damage. She underwent muscle biopsy from the right quadricep that showed lymphocytic infiltration, which led to the diagnosis of inflammatory myopathy. She was treated with methylprednisolone and methotrexate for 3 months. However, she did not show any clinical improvement and the inflammatory marker serum CK remained elevated (CK U/L) after treatment. She came to our hospital for further consultation. Her muscle weakness progressed up to the shoulder and she had difficulty raising her arms and climbing stairs, as well as rising independently after squatting down. She had a wadding gait and bilateral calf atrophy. A detailed neurological examination showed a weak tendon reflex and inability to stand on her tiptoes. Neither cardiac nor respiratory complications were found and no family member was found to have similar conditions. PATHOLOGICAL FINDINGS After informed consent, further muscle biopsy specimens were obtained from the patient s left biceps brachii muscle. These specimens were used for histopathology detection, including hematoxylin and eosin (HE), modified Gomori trichrome (MGT), nicotinamide adenine dinucleotide phosphate-tetrazolium reductase (NADH-TR), adenosine triphosphatase (ATPase), periodic acid-schiff (PAS) and oil red O (ORO) according to traditional methods. 8 Immunohistochemistry on muscle biopsies was performed as previously described 9 using monoclonal antibodies against dysferlin, NH 2 -terminal, COOH-terminal and mid-rod-region of dystrophin and alpha-,beta-, gamma-,delta-sarcoglycan for differential diagnosis and CD4+, CD8+, CD68+ and CD20+ as markers of immunocytes 10 (Table 1). Histopathology exhibited dystrophic features with variation in fiber size, increased numbers of central nuclei, and slight connective tissue hyperplasia. Sight degeneration, necrosis, phagocytosis, fiber splitting, ring fiber and regeneration were also noted (Fig. 1A). Some small vacuoles in fibers or sarcoplasma were observed; however, absence of lipid droplet formation on ORO excluded lipid myopathy. Lymphocytic infiltration was seen in perivascular spaces and the epimysium (Fig. 1A), which may have led to the confused diagnosis of inflammatory myopathy. Immunohistochemistry results on muscle biopsies indicated a strong reduction in dysferlin expression and a mild decrease in dystrophin-n expression (Fig. 1B D). Meanwhile, a small number of CD8+ T lymphocytes and a moderate number of CD68+ macrophagocytes infiltrated were found around the endomysium (Fig. 1E, F). Based on these findings, we suspected that our patient may have harbored LGMD2B. Targeted NGS was performed on the patient s DNA for approximately 430 genes currently related to muscular dystrophy, which produced a set of data with mean depth of folds and which covered 99.0% of the target sequence at 20 or greater. Overall, 3100 variants were generated and a homozygous mutation in DYSF (chr ) was identified (Fig. 2). Both the maternal and paternal alleles harbored a c.5392g > A (NM_ ) resulting in a missense mutation (p. Glu1798Lys) in exon 48 of the gene which was further confirmed by Sanger sequencing. This mutation was neither found in the 1000 genome project nor in our in-house database consisting of 2000 Chinese healthy individuals. It was described in one of the alleles in the ExAC Browser database but was not previously observed in the East Asian population. To our knowledge, this site was previously reported in a different variation form (chr , NM_ , c.5296g > T, p.glu1766term). Thus, our case presents a novel amino acid change which reveals a broadened mutation spectrum of the DYSF gene. On the basis of consistency with an autosomal recessive model, extremely low frequency and damaging nature of the mutation (missense), as well as previous reports of DYSF mutations in dysferlinopathy which may present as proximal pelvic and shoulder girdle muscle weakness (LGMD2B), we concluded that the homozygous mutation we found was responsible for the observed pathophysiology in our patients. DISCUSSION Muscular dystrophy is a clinically heterogeneous group of diseases with broadly overlapping characteristics among different subtypes, mainly muscle weakness or atrophy, abnormal posture and high serum CK ( units/l or more), 11 which can make the diagnosis challenging even for an experienced clinician. Clinical phenotypes could vary from inter-and intra-familial members even in a specific subtype like dysferlinopathy. 12 Thus, 2018 The Authors. published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of

3 DYSF gene mutation and presumed inflammatory myopathy 435 Table 1 The details of each monoclonal antibody used in immunohistochemistry Protein Monoclonal antibodies Dilution Source of antibody Dysferlin JAI , rabbit 1:100 Novocastra Laboratiories NH2-terminal region of dystrophin Dy10/12B2, mouse 1:20 Novocastra Laboratiories COOH-terminal region of dystrophin Dy8/6C5, mouse 1:50 Novocastra Laboratiories Large central rod-domain of dystrophin Dy4/6D3, mouse 1:50 Novocastra Laboratiories Alpha-sarcoglycan Ad1/20A6, mouse 1:50 Vector Laboratories Beta-sarcoglycan βsarc/5b1, mouse 1:50 Vector Laboratories Gamma-sarcoglycan 35DAG/21B5, mouse 1:50 Vector Laboratories Delta-sarcoglycan δsarc3/12c1, mouse 1:50 Vector Laboratories NGS should be considered to make it possible to reach a specific diagnosis. Sequencing analysis in our study corrected the previous misdiagnosis in our patient. The patient suffered from chronic progressive weakness in proximal limbs and pelvic girdle muscles, manifesting as high serum CK. Based on the combination of myogenic damages in electromyography and muscle histological pathology, absence of dysferlin proteins on immunohistochemical analysis and a homozygous missense mutation in DYSF, an accurate diagnosis of LGMD2B was eventually made. Compared to the first muscle biospy, we have seen an active dystrophic process, such as increased fiber size variability, segmental necrosis, regeneration, fibrosis and inflammatory reaction, 13 but inflammatory infiltrates were observed in both. Macrophages and T cells are common in LGMD2B but cytotoxic T cells and B cells are absent or rare, and this may help in making the differential Fig. 1 Muscle biopsy from left biceps brachii. (A) Hematoxylin and eosin (HE) staining. Lymphocytic infiltration is seen in perivascular spaces and epimysium (arrow). Variation in fiber size, sight degeneration, necrosis and fiber splitting are noted. (B F) Immunohistochemical staining with anti-dysferlin (B), antidystrophin (C) and lymphocytes (E, F). (B) Dysferlin expression; (C) a mild decrease in the NH 2 -terminal region of dystrophin expression; (D) normal control of dysferlin; CD8 + T lymphocyte (E) and CD68+ macrophagocyte (F). Scale bar: 50 μm The Authors. published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of

4 436 J Tang et al. Fig. 2 A missense mutation (p. lu1798lys) c.5392g > A (NM_ ) in DYSF (chr ) in the patient as revealed by next generation sequencing. Red oval represents the site of mutation. diagnosis. In general, 60 70% of patients manifest inflammatory infiltrates even before clinical manifestations. 8 Thus it is worth emphasizing that ruling out dysferlin deficiency is important before initiating a course of corticosteroid to avoid a non-recoverable loss of strength. 14 Muscular dystrophy-related tests on both protein and genetic levels should be carried out to further clarify the specific disease. Immunohistochemistry in LGMD2B patients conventionally shows reduction or absence of dysferlin protein. However, a secondary dysferlin deficiency may be observed in some non-dysferlinopathy LGMD patients, such as those with LGMD1C and LGMD2A with primary caveolin-3 and calpain-3 protein deficiency, respectively. Further genetic testing to identify the pathogenic mutation and rule out other muscular dystrophies are very important for accurate diagnosis. 3 A mild dystrophin reduction also appeared in our patient potentially leading to dystrophinopathy, which may have disrupted the interdependence of sarcolemmal and associated proteins, 15 but this was excluded because no pathogenic variations were detected in Becker muscular dystrophy (BMD) and Duchenne muscular dystrophy (DMD) genes. The clinical application of NGS has initiated a new era of accurate genetic diagnosis which may not only avoid excessive medical interventions but also aids in providing appropriate genetic counseling. 16 Our case highlights the significance of recognizing clinical clues in conjunction with applications of genetic tests to avoid misdiagnosis in hereditary muscular dystrophy. Furthermore, we report a novel missense mutation (c.5392g > A) which broadens the DYSF mutation spectrum and helps diagnosis in any patient with presumed dysferlinopathy. ACKNOWLEDGMENTS Thanks to Hong Yan, Wenkui Zhan and Yuchen Song (Beijing JinZhun Gene Technology Co., Ltd) for their assistance with acquisition and analysis of genetic data. DISCLOSURE The authors declare no conflict of interest. REFERENCES 1. Norwood F, Visser MD, Eymard B et al. EFNS guideline on diagnosis and management of limb girdle muscular dystrophies. Eur J Neurol 2010; 14: Vernengo L, Oliveira J, Krahn M et al. Novel ancestral Dysferlin splicing mutation which migrated from the Iberian peninsula to South America. Neuromuscul Disord 2011; 21: Fanin M, Angelini C. Progress and challenges in diagnosis of dysferlinopathy. Muscle Nerve 2016; 54: Rehm HL. Disease-targeted sequencing: A cornerstone in the clinic. Nat Rev Genet 2013; 14: Krahn M, Béroud C, Labelle V et al. Analysis of the DYSF mutational spectrum in a large cohort of patients. Hum Mutat 2009; 30: E345 E Jin SQ, Meng Y, Wei Z, Lyu H, Yuan Y, Wang ZX. Dysferlin gene mutation Spectrum in a large cohort of Chinese patients with Dysferlinopathy. Chin Med J (Engl) 2016; 129: Shin HY, Jang H, Han JH et al. Targeted nextgeneration sequencing for the genetic diagnosis of dysferlinopathy. Neuromuscul Disord 2015; 25: Fanin M, Angelini C. Muscle pathology in dysferlin deficiency. Neuropathol Appl Neurobiol 2010; 28: Hayashi YK, Ogawa M, Tagawa K et al. Selective deficiency of α-dystroglycan in Fukuyama-type congenital muscular dystrophy. Neurology 2001; 57: Gallardo E, Rojas-García R, De LN et al. Inflammation in dysferlin myopathy: Immunohistochemical characterization of 13 patients. Neurology 2001; 57: Takahashi T, Aoki M, Suzuki N et al. Research paper: Clinical features and a mutation with late onset of limb girdle muscular dystrophy 2B. J Neurol Neurosurg Psychiatry 2013; 84: Ueyama H, Kumamoto T, Nagao S, Masuda T, Horinouchi H, Fujimoto S. A new dysferlin gene mutation in two Japanese families with limbgirdle muscular dystrophy 2B and Miyoshi myopathy. Neuromuscul Disord 2001; 11: Rosales XQ, Gastier-Foster JM, Lewis S et al. Novel diagnostic features of dysferlinopathies. Muscle Nerve 2010; 42: Hoffman EP, Rao D, Pachman LM. Clarifying the boundaries between the inflammatory and dystrophic 2018 The Authors. published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of

5 DYSF gene mutation and presumed inflammatory myopathy 437 myopathies: Insights from molecular diagnostics and microarrays. Rheum Dis Clin North Am 2002; 28: Piccolo F, Moore SA, Ford GC, Campbell KP. Intracellular accumulation and reduced sarcolemmal expression of dysferlin in limb girdle muscular dystrophies. Ann Neurol 2000; 48: Facio FM, Lee K, O Daniel JM. A genetic counselor s guide to using next-generation sequencing in clinical practice. J Genet Couns 2014; 23: The Authors. published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of