Principal mutation hotspot for central core disease and related myopathies in the C-terminal transmembrane region of the RYR1 gene
Introduction
The congenital myopathies are a group of disorders that are often difficult to distinguish clinically, and their classification has therefore been undertaken on the basis of specific histological characteristics [1]. However, the recent identification of genetic causes of some of these disorders has blurred the lines of demarcation. Central core disease (CCD) and nemaline myopathy (NEM) are examples of congenital myopathies that may have similar clinical presentations, show different predominant histological features in biopsied muscle, but are now being shown to have some overlap genetically.
CCD is characterised by hypotonia and proximal muscle weakness, and is generally considered to be autosomal dominant (AD) with incomplete penetrance, although sporadic cases are common, and autosomal recessive (AR) inheritance has been proposed [2]. Histological examination of affected muscle in CCD shows a predominance of type I fibres, containing amorphous-looking central areas (cores), that lack normal oxidative enzyme activity. Electron microscopy demonstrates a variable degree of disintegration of the contractile apparatus within this central core region, ranging from Z line streaming and absence of mitochondria to total loss of myofibrillar structure [3]. The cores may be single or multiple within each muscle fibre, and extend the length of the fibre.
Severity of the disease is highly variable, ranging from severe weakness, sometimes accompanied by additional musculo-skeletal features such as congenital hip dislocation and joint contractures, to the 40% of individuals demonstrating the disease histologically, but who are clinically asymptomatic [4]. The classification of these asymptomatic individuals has been problematic, but recently published guidelines, from the first ENMC workshop on CCD, indicate that they should not be referred to as having CCD as they do not have the disease per se [5].
An important feature of CCD is its association with malignant hyperthermia (MH), an AD hypermetabolic disorder of skeletal muscle that is triggered in susceptible individuals by commonly used inhalational anaesthetics and depolarising muscle relaxants. The presence of cores in clinically asymptomatic individuals might actually only come to light during investigations for MH [4], [5]. Mutations in the gene for the skeletal muscle calcium release channel (RYR1) were linked first to MH [6], [7], then, subsequently, to CCD [8], [9]. However, the extent of the association remains to be completely defined. One study, of 13 CCD patients, found that all were susceptible to MH when screened by the in vitro muscle contracture test [4], while a more recent study identified a number of families where the CCD phenotype segregated with markers around RYR1 but not with MH status [10].
As with MH, CCD demonstrates genetic heterogeneity. A further family has been reported by Curran in which CCD and MH do not segregate with each other or with RYR1 [10]. Cores have also been reported in the muscle of individuals with hypertrophic cardiomyopathy due to mutations in the beta-myosin heavy chain gene (MYH7) on chromosome 14 [11].
Until recently, seven missense mutations in RYR1 have been published as being causative of CCD, accounting for the disease in perhaps 12 families [12], and clustering in the N terminal (3) and the central region (4) of the gene. Cores have also been described in clinically asymptomatic individuals with elevated serum creatine kinase levels in two families with MH susceptibility due to the common gly341arg mutation in RYR1 [5], [13].
The 3′ transmembrane domain region of the gene has now been identified as the major mutation hotspot for CCD-causing mutations, with 13 mutations being identified in three studies and accounting for approximately one third of cases [14], [15], [16].
NEM is typically characterised by slowly progressive proximal muscle weakness and delayed motor milestones, and has both AD and AR forms. As with CCD, the severity of symptoms is highly variable, ranging from severe fatal neonatal disease to relatively benign forms. The main histological feature of NEM is the presence within the muscle fibres of rod shaped structures – the nemaline bodies. NEM has been found to be associated with mutations in a number of genes – TPM3 (alpha tropomyosin) [17], NEB (nebulin) [18], ACTA1 (sarcomeric actin) [19], TPM2 (beta tropomyosin) [20] and TNNT1 (troponin T1) [21].
Less commonly, patients have been reported with both cores and nemaline bodies [22], [23]. Two recent reports, each describing individual families with relatively mild AD early onset myopathy with both cores and rods, have identified mutations in the transmembrane domain of RYR1 – Thr4637Ala [24] and Tyr4796Cys [25].
Section snippets
Patients
The first patient (patient 1) to be examined in this study was a member of a large CCD family described previously [26] and known to link to RYR1 [8], [9]. Subsequently, a cohort comprising a total of 26 other unrelated cases was collected. All cases were referred on the basis of clinically evident myopathy with histological confirmation of central cores. Twenty one of the probands displayed a typical pattern of CCD, of which five had a family history indicative of AD inheritance, one indicated
Mutation screening of RYR1
The results are listed in Table 2. Twelve different sequence variations were identified that result in amino acid changes in 15 of the 27 patients. Eleven of the variations were single base changes. Of these, eight were novel and three have previously been published – G14582A (Arg4861His), T14693C (Ile4898Thr) and A14740G (Arg4914Gly) [14], [15], [16].
Screening of muscle cDNA in patient 1 identified nine silent polymorphisms (C1077T, C2979T, G7500A, G7527A, C7863T, G7977A, T8118C, T8190C and
Discussion
This report extends the results of two recent studies that identified the transmembrane domain region of the RYR1 gene as a common site for the occurrence of mutations causing CCD. Fourteen of 21 unrelated CCD patients analysed (67%) had mutations in this region. We also describe a novel mutation in one of six unrelated core/rod disease patients.
The mutations, found in this study, cluster in the same regions of the gene as those recently described – in exons 95, 100, 101 and 102 [15], [16]. In
Electronic database information
Accession numbers and URLs for data in this article are as follows:
Genbank – http://www.ncbi.nlm.nih.gov/Genbank/
For human RYR1 [J05200], human RYR3 [NM001036], pig RYR1 [M91452], rabbit RYR1 [X15750], rabbit RYR2 [U50465], fish RYR1 [U97329], Drosophila melanogaster RYR1 [D17389] and Caenorhabditis elegans RYR1 [D45899]
Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ for RYR1 [MIM 180901], CCD [MIM 117000], NEM [MIM 161800], minicore myopathy [MIM157550], and MH
Acknowledgements
N.G.L. was supported by Australian National Health and Medical Research Council project grant 139039 and Fellowship Grant 139170.
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