A novel missense mutation of RYR1 in familial idiopathic hyper CK-emia

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Highlights

  • We ascertained idiopathic hyper CK-emia with autosomal dominant inheritance.

  • We identified a novel nonsynonymous SNV in RYR1 in the pedigree.

  • The protein expression level of RYR1 was significantly reduced in the IHCK patient.

  • The mRNA expression level of RYR1 was also significantly reduced in the patient.

  • We conclude that the RYR1 gene is one of the causes of IHCK.

Abstract

Persistent elevation of serum creatine kinase (CK) without any symptoms has been called idiopathic hyper CK-emia (IHCK). We examined a four-generation Japanese pedigree of familial IHCK. The multipoint linkage analysis of the pedigree showed seven clear peaks of logarithm of odds (LOD) scores (> 1.4). By the exome sequencing followed by multiple filtering processes, we identified one novel heterozygous nonsynonymous single nucleotide variant (SNV), c.7034G>C, p.S2345T in the ryanodine receptor 1 gene, RYR1 cosegregated with IHCK in the pedigree. Mutation Taster predicted this substitution as “disease causing” (p = 0.999). The PolyPhen-2 and PANTHER subPSEC scores for the substitution are 0.911 (possibly damaging) and − 3.56 (probably damaging), respectively. We confirmed the absence of the SNV in 511 healthy Japanese individuals excluding the possibility of a normal variant with a very low frequency. Immunohistochemistry and Western blotting of biopsy samples consistently showed the expression level of RYR1 reduced in the patient. In real-time RT-PCR, the mRNA expression level of RYR1 was also significantly reduced in the patient (p = 0.009). These results suggest that the novel nonsynonymous SNV contribute to the vulnerability of the RYR1 protein through the dominant negative effect. We conclude that the SNV in the RYR1 gene is one of the responsible genes of IHCK.

Introduction

Persistent elevation of serum creatine kinase (CK) usually accompanies muscle weakness in patients with myopathies. However, it is also found in occasional subjects with a normal neurological examination. This condition may be due to subclinical or preclinical neuromuscular disorders such as malignant hyperthermia (MH), dystrophinopathy, hypothyroidism, hypoparathyroidism, alcoholism, or intake of statins and/or other drugs [1], [2], [3], [4], [5], [6], [7], [8]. When the cause of high level of serum CK in individuals is unknown even after extensive investigations, the condition is diagnosed as IHCK [9], [10], [11], [12].

Although more than 170 subjects with IHCK have been reported, most of the cases are described to be sporadic [1], [6], [9], [10], [11], [12], [13]. Online Mendelian Inheritance in Man describes familial IHCK as a probable autosomal dominant inherited condition (OMIM # 123320). So far CAV3 is the only gene in which mutations have been reported in four sporadic cases and one familial case of IHCK [14], [15], [16]. However, the molecular basis for the vast of IHCK is still unknown. Here we report a Japanese pedigree of IHCK with the autosomal-dominant mode of inheritance. To identify the causative mutation underlying the condition, we conducted the exome sequencing assisted with the linkage analysis.

Section snippets

Human subjects

Diagnosis of IHCK was done on the basis of the following criteria [17], [18]: (1) persistent serum CK elevation at rest; (2) normal neurological examination; (3) no intake of drugs known to cause hyper-CK-emia; (4) no history of exercise-related cramps or myoglobinuria; (5) no family history of neuromuscular disorders or MH; and (6) no recent muscle trauma. We ascertained a Japanese pedigree with IHCK consisted with 13 family members including 7 patients affected with IHCK in four generations (

Family study

We deduced the inheritance mode of IHCK in the pedigree, as the autosomal dominant transmission because of the presence of affected members in each generation (Fig. 1). Affected members have no clear abnormalities except for the high levels of serum CK (1005 ± 302 U/L) excluding one aged affected member, II-3, due to aging. Two affected members, III-3 and IV-3 experienced continuous mild hyperthermia. The proband, IV-3 was hospitalized for further evaluations. Slightly muscular weakness in

Discussion

The skeletal muscle ryanodine receptor 1, RYR1, encoded by the RYR1 gene acts as a calcium release channel in the homotetrameric structure on the sarcoplasmic reticulum. RYR1 enhances the Ca2 + channel activity of dihydropyridine receptor (DHPR) which is critical for excitation–contraction coupling [20]. In the skeletal muscle, RYR1 composes a well-ordered tetramer array with the group of four DHPRs allocated to one of every two RYR1 tetramer units [21]. The p.S2345T mutation is located in the

Conflict of interest/disclosures

We declare that we have no financial or other conflicts of interest in relation to this research and its publication.

Acknowledgment

We thank all family members for their participation. We also thank Chitoshi Oki and Mayumi Yamamoto for their technical assistance. This work was supported by the Grant-in-Aid for Scientific Research on Innovative Areas “Exploring molecular basis for brain diseases based on personal genomics” (# 23129504) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. This work was partly performed in the Cooperative Research Project Program of the Medical Institute of

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    These authors contributed equally to this work.

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