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Molecular Mechanism of Rigid Spine with Muscular Dystrophy Type 1 Caused by Novel Mutations of Selenoprotein N Gene

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Molecular Mechanism of Rigid Spine with Muscular Dystrophy Type 1 Caused by Novel Mutations of Selenoprotein N Gene Neurogenetics (2006) 7: 175–183 DOI 10.1007/s10048-006-0046-0 ORIGINAL ARTICLE Yuji Okamoto . Hiroshi Takashima . Itsuro Higuchi . Wataru Matsuyama . Masahito Suehara . Yasushi Nishihira . Akihiro Hashiguchi . Ryuki Hirano . Arlene R. Ng . Masanori Nakagawa . Shuji Izumo . Mitsuhiro Osame . Kimiyoshi Arimura Molecular mechanism of rigid spine with muscular dystrophy type 1 caused by novel mutations of selenoprotein N gene Received: 24 December 2005 / Accepted: 13 April 2006 / Published online: 15 June 2006 # Springer-Verlag 2006 Abstract Mutations of selenoprotein N, 1 gene (SEPN1) nonsense mutations including TGA. These nonsense cause rigid spine with muscular dystrophy type 1 (RSMD1), mutations are premature translation termination codons multiminicore disease, and desmin-related myopathy. We and they degrade immediately by the process of nonsense- found two novel SEPN1 mutations in two Japanese patients mediated decay (NMD). However, truncated selenoprotein with RSMD1. To clarify the pathomechanism of RSMD1, N was also expressed. A possible mechanism behind this we performed immunohistochemical studies using a newly observation is that SEPN1 mRNAs may be resistant to developed antibody for selenoprotein N. Selenoprotein N NMD. We report on the possible molecular mechanism was diffusely distributed in the cytoplasm of the control behind these mutations in SEPN1. Our study clarifies muscle, but was reduced and irregularly expressed in the molecular mechanisms of this muscular disorder. cytoplasm of a patient with RSMD1. The expression pattern was very similar to that of calnexin, a transmembrane Keywords SEPN1 . Rigid spine syndrome . protein of the endoplasmic reticulum. Selenoprotein N Anti-selenoprotein N antibody . RSMD1 . SEPN-RM seems to be an endoplasmic reticulum glycoprotein, and loss of this protein leads to disturbance of muscular function. One of the families had the SEPN1 homozygous Introduction mutation in the initiation codon 1_2 ins T in exon 1 and showed truncated protein expression. The other had a The peculiar phenotype caused by the combination of homozygous 20-base duplication mutation at 80 (80_99dup, muscle wasting and prominent spinal rigidity was first frameshift at R27) which, in theory, should generate many proposed by Dubowitz and termed “rigid spine syndrome (RSS)” [1, 2]. The spinal rigidity is a nonspecific feature; however, it is Y. Okamoto . H. Takashima (*) . I. Higuchi . W. Matsuyama . A. Hashiguchi . R. Hirano . A. R. Ng . M. Osame . K. Arimura quite prominent in the X-linked, autosomal dominant, and Department of Neurology and Geriatrics, autosomal recessive forms of Emery–Dreifuss muscular Kagoshima University, dystrophy [3, 4] and in some congenital myopathies [5, 6]. Graduate School of Medical and Dental Sciences, Several authors have pointed out distinctions between the 8-35-1 Sakuragaoka, Kagoshima City, Kagoshima 890-8520, Japan spinal rigidity in other myopathies and that found in RSS e-mail: [email protected] [7, 8]. Although the syndrome has a good prognosis, some Tel.: +81-99-2755332 patients die of respiratory failure in the first or second Fax: +81-99-2657164 decade. Nocturnal hypoventilation may be an early and characteristic symptom [9, 10]. In 1997, a subgroup of Y. Okamoto . S. Izumo Department of Molecular Pathology, merosin-positive congenital muscular dystrophy character- Center for Chronic Vial Disease, ized by slowly progressive weakness, rigid spine, and early Kagoshima University School of Medicine, respiratory failure was described [11]. Not long after, a Kagoshima, Japan gene locus on chromosome 1p35–36 was identified in M. Suehara . Y. Nishihira consanguineous Moroccan, Iranian, and Turkish families Department of Neurology, Okinawa Hospital, affected by congenital muscular dystrophy with spinal Okinawa, Japan rigidity and reduced vital capacity [12]. After linkage analysis, two frameshift, one nonsense, and three missense M. Nakagawa Department of Neurology and Gerontology, mutations in the selenoprotein N, 1 gene (SEPN1, OMIM Kyoto Prefectural University of Medicine, 606210) were identified as the cause of rigid spine with Kyoto, Japan muscular dystrophy type 1 (RSMD1) in the same families 176 [13]. SEPN1 codes for selenoprotein N, a novel seleno- of the upper and lower limbs were relatively preserved protein with an unknown function. Subsequent studies (MMT 4–5). He showed waddling gait and Gower’s sign. revealed that selenoprotein N is an endoplasmic reticulum Serum creatine kinase (CK) level was elevated at 590 IU/l, glycoprotein, highly expressed in several human fetal which is almost three times higher than the normal limit. tissues, but only slightly expressed in adult tissues Electromyographic findings showed myopathic change. including muscle based on Western blot analysis. However, Electrocardiogram showed negative T waves, but no con- immunostaining of selenoprotein N in human skeletal duction block. He has severe restrictive respiratory failure. muscle has not yet been reported due to problems in Chest X-ray study showed an elevated diaphragm. He is sensitivity of reported antibodies [14]. on a respirator during sleep but is mostly off it during In this study, we report two Japanese RSS patients with daytime. He was still able to walk albeit with support until two novel SEPN1 mutations. We examined their muscle the age of 34. pathology using a developed antibody directed against selenoprotein N. We detected selenoprotein N in biopsied Patient 2 This 31-year-old woman is the child of healthy, human muscle tissues from these patients and from unrelated parents, born after a normal pregnancy and controls and describe its distribution. We also provide uncomplicated delivery. Both parents and four siblings additional evidence to support the correlation between were normal. Her motor and intellectual milestones were selenoprotein N localization and endoplasmic reticulum in normal during infancy. She began to walk at the age of muscle. One of our patients had the mutation in the 11 months and to run at the age of 7, although her running initiation codon, which usually leads to absence of protein speed was relatively slow. At the age of 12, she felt production. Our patient, however, showed truncated pro- nocturnal dyspnea and palpitations. X-ray examination tein expression. We report on the possible molecular revealed limitation of diaphragmatic movement. Serum mechanism behind the mutation in the initiation codon and CK level was elevated at 130 IU/l. At the age of 14, she consequent protein expression. The other patient had a had severe respiratory failure secondary to pneumonia and homozygous 20-base duplication mutation. In theory, this she underwent tracheotomy. Thereafter, she has been on a would generate many nonsense mutations. These nonsense respirator during sleep but mostly off it during daytime. It mutations are premature translation termination codons was during this time that she visited our hospital for that degrade immediately by the process of nonsense- consultation. She had limitation of spine flexion, scoliosis, mediated decay (NMD). However, truncated selenoprotein and restricted forward flexion of the neck. She showed a N was also expressed. A possible mechanism behind this waddling gait and Gower’s sign. Her face was myopathic. observation is that SEPN1 mRNAs may be resistant to Diffuse and mild muscle atrophy and weakness (MMT 4) NMD. Our studies not only shed light on a new diagnostic were seen in the shoulder and axial muscles. The muscle method for RSS caused by SEPN 1 mutation, but also strength of the upper and lower limbs was relatively clarify molecular mechanisms of this muscular disorder. preserved (MMT 4–5). Neck flexion and extension, however, were particularly weak (MMT 3). She was diagnosed with muscular dystrophy based on muscle Materials and methods biopsy findings. Her muscle weakness progressed slowly, and she was still able to walk with support until the age of Patients 30 years. The patients, who were referred to us by their attending Patient 1 This 41-year-old man, whose parents were first neurologists, and family members included in the study cousins, was born after a normal pregnancy and uncom- signed an informed consent approved by the Institutional plicated delivery. His parents, one brother, and three sisters Review Board of Kagoshima University. DNA was had no known muscle disease. An elder sister, who had isolated from peripheral blood and skeletal muscle of all delayed motor milestones, died at the age of 3 because of subjects. measles. It was, therefore, not possible to confirm whether she had the same disease as the patient. His intellectual milestones were normal, but he had Mutation screening delayed motor milestones and did not walk unsupported until the age of 3. At the age of 7, he had abnormal gait By aligning the human genomic sequence with the and he was never able to run or jump. There was an SEPN1cDNA, we were able to identify all coding exons. apparent rigidity of the spine and restricted forward Using the Primer v3 program, we designed primers to flexion of the neck. At the age of 16, he began to have amplify exons and intronic splice junctions and then dyspnea during sleep. His dyspnea progressed to the point screened the amplified PCR products from patient genomic that a tracheotomy was performed at the age of 24. At the DNA for mutations using ABI Prism 377 sequencer age of 27, he had limitation of spine flexion, scoliosis, and (Applied Biosystems, Foster City, CA, USA). Briefly, we mild contracture of the ankle joints, but no limitation of amplified the coding region of SEPN1 from 50 ng of elbow and wrist joint movement. Diffuse and mild muscle patient genomic DNA using the primers and the hot start atrophy and weakness (MMT 4) were seen in the shoulder, PCR method [15]. Using the presequencing kit (USB, sternocleidomastoideus, and axial muscles. Muscle strength Cleveland, OH, USA), we purified patient PCR products 177 from relatives and control chromosomes and sequenced transcriptase-polymerase chain reaction (RT-PCR) was them with dye-terminator chemistry using an ABI377 carried out using SuperScript III First Strand Synthesis automated sequencer.
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