Clinical and genetic findings in a large cohort of patients with ryanodine receptor 1 gene-associated myopathies

Ryanodine receptor 1 (RYR1) mutations are a common cause of congenital myopathies associated with both dominant and recessive inheritance. Histopathological findings frequently feature central cores or multi-minicores, more rarely, type 1 predominance/uniformity, fiber-type disproportion, increased internal nucleation, and fatty and connective tissue. We describe 71 families, 35 associated with dominant RYR1 mutations and 36 with recessive inheritance. Five of the dominant mutations and 35 of the 55 recessive mutations have not been previously reported. Dominant mutations, typically missense, were frequently located in recognized mutational hotspot regions, while recessive mutations were distributed throughout the entire coding sequence. Recessive mutations included nonsense and splice mutations expected to result in reduced RyR1 protein. There was wide clinical variability. As a group, dominant mutations were associated with milder phenotypes; patients with recessive inheritance had earlier onset, more weakness, and functional limitations. Extraocular and bulbar muscle involvement was almost exclusively observed in the recessive group. In conclusion, our study reports a large number of novel RYR1 mutations and indicates that recessive variants are at least as frequent as the dominant ones. Assigning pathogenicity to novel mutations is often difficult, and interpretation of genetic results in the context of clinical, histological, and muscle magnetic resonance imaging findings is essential.     

Homozygous/compound heterozygote RYR1 gene variants: Expanding the clinical spectrum

The ryanodine receptor 1 (RYR1) is a calcium release channel essential for excitation‐contraction coupling in the sarcoplasmic reticulum of skeletal muscles. Dominant variants in the RYR1 have been well associated with the known pharmacogenetic ryanodinopathy and malignant hyperthermia. With the era of next‐generation gene sequencing and growing number of causative variants, the spectrum of ryanodinopathies has been evolving with dominant and recessive variants presenting with RYR1‐related congenital myopathies such as central core disease, minicore myopathy with external ophthalmoplegia, core‐rod myopathy, and congenital neuromuscular disease. Lately, the spectrum was broadened to include fetal manifestations, causing a rare recessive and lethal form of fetal akinesia deformation sequence syndrome (FADS)/arthrogryposis multiplex congenita (AMC) and lethal multiple pterygium syndrome. Here we broaden the spectrum of clinical manifestations associated with homozygous/compound heterozygous RYR1 gene variants to include a wide range of manifestations from FADS through neonatal hypotonia to a 35‐year‐old male with AMC and PhD degree. We report five unrelated families in which three presented with FADS. One of these families was consanguineous and had three affected fetuses with FADS, one patient with neonatal hypotonia who is alive, and one individual with AMC who is 35 years old with normal intellectual development and uses a wheelchair. Muscle biopsies on these cases demonstrated a variety of histopathological abnormalities, which did not assist with the diagnostic process. Neither the affected living individuals nor the parents who are obligate heterozygotes had history of malignant hyperthermia.     

An autosomal dominant congenital myopathy with cores and rods is associated with a neomutation in the RYR1 gene encoding the skeletal muscle ryanodine receptor

Central core disease (CCD) and nemaline myopathy (NM) are congenital myopathies for which differential diagnosis is often based on the presence either of cores or rods. Missense mutations in the skeletal muscle ryanodine receptor gene (RYR1) have been identified in some families with CCD. Mutations in the alpha-tropomyosin and alpha-actin genes have been associated with most dominant forms of NM. Analysis of the RYR1 cDNA in a French family identified a novel Y4796C mutation that lies in the C-terminal channel-forming domain of the RyR1 protein. This mutation was linked not only to a severe and penetrant form of CCD, but also to the presence of rods in the muscle fibres and to the malignant hyperthermia susceptibility (MHS) phenotype. The Y4796C mutation was introduced into a rabbit RYR1 cDNA and expressed in HEK-293 cells. Expression of the mutant RYR1 cDNA produced channels with increased caffeine sensitivity and a significantly reduced maximal level of Ca(2+) release. Single-cell Ca(2+) analysis showed that the resting cytoplasmic level was increased by 60% in cells expressing the mutant channel. These data support the view that the rate of Ca(2+) leakage is increased in the mutant channel. The resulting chronic elevation in myoplasmic concentration is likely to be responsible for the severe expression of the disease. Haplotyping analysis indicated that the mutation arose as a neomutation in the proband. This first report of a neomutation in the RYR1 gene has strong implications for genetic linkage studies of MHS or CCD, two diseases characterized by a genetic heterogeneity.     

Characterization of recessive RYR1 mutations in core myopathies

We have characterized at the molecular level, three families with core myopathies carrying apparent recessive mutations in their RYR1 gene and studied the pharmacological properties of myotubes carrying endogenous mutations as well as the properties of mutant channels expressed in HEK293 cells. The proband of family 1 carried p.Ala1577Thr+p.Gly2060Cys in trans, having inherited a mutation from each parent. Immunoblot analysis of proteins from the patient's skeletal muscle revealed low levels of ryanodine receptor (RyR1) but neither substitution alone or in combination affected the functional properties of RyR1 channels in a discernable way. Two affected siblings in family 2 carried p.Arg109Trp+p.Met485Val substitutions in cis, inherited from the unaffected father. Interestingly, both affected siblings only transcribed the mutated paternal allele in skeletal muscle, whereas the maternal allele was silent. Single-channel measurements showed that recombinant, mutant RyR1 channels carrying both substitutions lost the ability to conduct Ca2+. In this case as well, low levels of RyR1 were present in skeletal muscle extracts. The proband of family 3 carried p.Ser71Tyr+p.Asn2283His substitutions in trans. Recombinant channels with Asn2283His substitution showed an increased activity, whereas recombinant channels with p.Ser71Tyr+p.Asn2283His substitution lost activity upon isolation. Taken together, our data suggest major differences in the ways RYR1 mutations may affect patients with core myopathies, by compromising RyR1 protein expression, stability and/or activity.     

Detection of a novel mutation in the ryanodine receptor gene in an Irish malignant hyperthermia pedigree: correlation of the IVCT response with the affected and unaffected haplotypes.

Defects in the ryanodine receptor (RYR1) gene are associated with malignant hyperthermia (MH), an autosomal dominant disorder of skeletal muscle and one of the main causes of death resulting from anaesthesia. Susceptibility to MH (MHS) is determined by the level of tension generated in an in vitro muscle contracture test (IVCT) in response to caffeine and halothane. To date, mutation screening of the RYR1 gene in MH families has led to the identification of eight mutations. We describe here the identification of a novel mutation, Arg552Trp, in the RYR1 gene, which is clearly linked to the MHS phenotype in a large, well characterised Irish pedigree. Considering that the RYR1 protein functions as a tetramer, correlation of the IVCT with the affected and unaffected haplotypes was performed on the pedigree to investigate if the normal RYR1 allele in affected subjects contributes to the variation in the IVCT. The results show that the normal RYR1 allele is unlikely to play a role in IVCT variation.     

The clinical spectrum of missense mutations of the first aspartic acid of cbEGF-like domains in fibrillin-1 including a recessive family

Marfan syndrome (MFS) is a dominant disorder with a recognizable phenotype. In most patients with the classical phenotype mutations are found in the fibrillin-1 gene (FBN1) on chromosome 15q21. It is thought that most mutations act in a dominant negative way or through haploinsufficiency. In 9 index cases referred for MFS we detected heterozygous missense mutations in FBN1 predicted to substitute the first aspartic acid of different calcium-binding Epidermal Growth Factor-like (cbEGF) fibrillin-1 domains. A similar mutation was found in homozygous state in 3 cases in a large consanguineous family. Heterozygous carriers of this mutation had no major skeletal, cardiovascular or ophthalmological features of MFS. In the literature 14 other heterozygous missense mutations are described leading to the substitution of the first aspartic acid of a cbEGF domain and resulting in a Marfan phenotype. Our data show that the phenotypic effect of aspartic acid substitutions in the first position of a cbEGF domain can range from asymptomatic to a severe neonatal phenotype. The recessive nature with reduced expression of FBN1 in one of the families suggests a threshold model combined with a mild functional defect of this specific mutation.     

Familial and sporadic forms of central core disease are associated with mutations in the C-terminal domain of the skeletal muscle ryanodine receptor.

Central core disease (CCD) is an autosomal dominant congenital myopathy. Diagnosis is based on the presence of cores in skeletal muscles. CCD has been linked to the gene encoding the ryanodine receptor (RYR1) and is considered to be an allelic disease of malignant hyperthermia susceptibility. However, the report of a recessive form of transmission together with a variable clinical presentation has raised the question of the genetic heterogeneity of the disease. Analyzing a panel of 34 families exclusively recruited on the basis of both clinically and morphologically expressed CCD, 12 different mutations of the C-terminal domain of RYR1 have been identified in 16 unrelated families. Morphological analysis of the patients' muscles showed different aspects of cores, all of them associated with mutations in the C-terminal region of RYR1. Furthermore, we characterized the presence of neomutations in the RyR1 gene in four families. This indicates that neomutations into the RyR1 gene are not a rare event and must be taken into account for genetic studies of families that present with congenital myopathies type 'central core disease'. Three mutations led to the deletion in frame of amino acids. This is the first report of amino acid deletions in RYR1 associated with CCD. According to a four-transmembrane domain model, the mutations concentrated mostly in the myoplasmic and luminal loops linking, respectively, transmembrane domains T1 and T2 or T3 and T4 of RYR1.     

RYR mutation G1021A (Gly341Arg) is not frequent in Danish and Swedish families with malignant hyperthermia susceptibility

Malignant hyperthermia (MH) is a pharmacogenetic disorder. Susceptibility to MH (MHS) is presumed to be inherited in an autosomal dominant way. MH crises are triggered by halogenated inhalational anaesthetics and suxamethonium, and may be lethal if not treated early and adequately. Until now, eight mutations in the RYR1 gene have been described as causes of MHS phenotype in various MH families The mutation RYR1 G1021A (Gly341Arg) has been reported to account for approximately 10% of Caucasian MHS cases. However, in our study this mutation was discovered in only 1 out of 89 Scandinavian families, indicating that this mutation may be the cause of MHS in only about 1% of MHS families in those populations. The mutation may have been brought to Scandinavia by an immigrant.     

Difference in allelic expression of the CLCN1 gene and the possible influence on the myotonia congenita phenotype

Mutations in the CLCN1 gene, encoding a muscle-specific chloride channel, can cause either recessive or dominant myotonia congenita (MC). The recessive form, Becker's myotonia, is believed to be caused by two loss-of-function mutations, whereas the dominant form, Thomsen's myotonia, is assumed to be a consequence of a dominant-negative effect. However, a subset of CLCN1 mutations can cause both recessive and dominant MC. We have identified two recessive and two dominant MC families segregating the common R894X mutation. Real-time quantitative RT-PCR did not reveal any obvious association between the total CLCN1 mRNA level in muscle and the mode of inheritance, but the dominant family with the most severe phenotype expressed twice the expected amount of the R894X mRNA allele. Variation in allelic expression has not previously been described for CLCN1, and our finding suggests that allelic variation may be an important modifier of disease progression in myotonia congenita.     

Identification of a novel mutation in the ryanodine receptor gene (RYR1) in a malignant hyperthermia Italian family

Malignant hyperthermia (MH) is an inherited autosomal dominant pharmacogenetic disorder and is one of the main causes of death subsequent to anaesthesia. Around 50% of affected families are linked to the ryanodine receptor (RYR1) gene. To date, 19 mutations have been identified in the coding region of this gene and appear to be associated with the MH-susceptible phenotype. Here we report the identification by two independent methods of a novel mutation associated with the MH-susceptible phenotype in the RYR1 gene: the 6488G-->C transversion, resulting in the replacement of the Arg2163 with a proline residue.     

A novel RYR1 variant in an infant with a unique fetal presentation of central core disease

Ryanodine receptor type 1-related disorder (RYR1-RD) is the most common subgroup of congenital myopathies with a wide phenotypic spectrum ranging from mild hypotonia to lethal fetal akinesia. Genetic testing for myopathies is imperative as the diagnosis informs counseling regarding prognosis and recurrence risk, treatment options, monitoring, and clinical management. However, diagnostic challenges exist as current options are limited to clinical suspicion prompting testing including: single gene sequencing or familial variant testing, multi-gene panels, exome, genome sequencing, and invasive testing including muscle biopsy. The timing of diagnosis is of great importance due to the association of RYR1-RD with malignant hyperthermia (MH). MH is a hypermetabolic crisis that occurs secondary to excessive calcium release in muscles, leading to systemic effects that can progress to shock and death if unrecognized. Given the association of MH with pathogenic variants in RYR1, a diagnosis of RYR1-RD necessitates an awareness of medical team to avoid potentially triggering agents. We describe a case of a unique fetal presentation with bilateral diaphragmatic eventrations who had respiratory failure, dysmorphic facial features, and profound global hypotonia in the neonatal period. The diagnosis was made at several months of age, had direct implications on her clinical care related to anticipated need to long-term ventilator support, and ultimately death secondary an arrhythmia as a result of suspected MH. Our report reinforces the importance of having high suspicion for a genetic syndrome and pursuing early, rapid exome or genome sequencing as first line testing in critically ill neonatal intensive care unit patients and further evaluating the pathogenicity of a variant of uncertain significance in the setting of a myopathic phenotype.     

Novel KCNQ1 mutations associated with recessive and dominant congenital long QT syndromes: evidence for variable hearing phenotype associated with R518X

Congenital long QT syndrome may be transmitted as either an autosomal dominant or recessive trait. Two families with the autosomal recessive Jervell and Lange-Nielsen syndrome (JLNS), and one family with the autosomal dominant Romano-Ward syndrome (RWS) were evaluated for mutations in KCNQ1. Two different novel frameshift mutations were discovered in one of the JLNS families (1188delC) and in the RWS family (504delG). A third allele (R518X) was observed in the second JLNS family. The R518X allele was previously associated with recessive long QT syndrome without deafness, but was present in a congenitally deaf proband in our study. These data extend the range of known KCNQ1 mutations associated with both recessive and dominant forms of congenital long QT syndrome, and demonstrate that the R518X allele may be associated with or without congenital deafness.     

A case of discordance between genotype and phenotype in a malignant hyperthermia family.

Malignant hyperthermia (MH) is an inherited autosomal dominant pharmacogenetic disorder and is the major cause of anaesthesia-induced death. Malignant hyperthermia susceptibility is usually diagnosed by the in vitro contracture test (IVCT) performed on fresh muscle biopsies exposed to caffeine and halothane, respectively. Around 50% of affected families are linked to the ryanodine receptor (RYR1) gene. The human RYR1 gene maps to chromosome 19q13.1 and encodes a protein that associates as a homotetramer and acts as a calcium-release channel from the sarcoplasmic reticulum. To date, 17 mutations have been identified in the coding region of the RYR1 gene and appear to be associated to the MH-susceptible phenotype. Here we describe a rare case of discordance between genotype (characterised by the presence of the Arg614Cys mutation in the RYR1 gene) and MH-normal typed phenotype. Although the IVCT remains a very reliable procedure for the assessment of MH status, genetic data can provide in some cases an additional aid to clinical diagnosis.     

Absence of triadin, a protein of the calcium release complex, is responsible for cardiac arrhythmia with sudden death in human

Catecholaminergic polymorphic ventricular tachycardia (CPVT) is an inherited arrhythmogenic disease so far related to mutations in the cardiac ryanodine receptor (RYR2) or the cardiac calsequestrin (CASQ2) genes. Because mutations in RYR2 or in CASQ2 are not retrieved in all CPVT cases, we searched for mutations in the physiological protein partners of RyR2 and CSQ2 in a large cohort of CPVT patients with no detected mutation in these two genes. Based on a candidate gene approach, we focused our investigations on triadin and junctin, two proteins that link RyR2 and CSQ2. Mutations in the triadin (TRDN) and in the junctin (ASPH) genes were searched in a cohort of 97 CPVT patients. We identified three mutations in triadin which cosegregated with the disease on a recessive mode of transmission in two families, but no mutation was found in junctin. Two TRDN mutations, a 4 bp deletion and a nonsense mutation, resulted in premature stop codons; the third mutation, a p.T59R missense mutation, was further studied. Expression of the p.T59R mutant in COS-7 cells resulted in intracellular retention and degradation of the mutant protein. This was confirmed after in vivo expression of the mutant triadin in triadin knock-out mice by viral transduction. In this work, we identified TRDN as a new gene responsible for an autosomal recessive form of CPVT. The mutations identified in the two families lead to the absence of the protein, thereby demonstrating the importance of triadin for the normal function of the cardiac calcium release complex in humans.     

Frequency of the recessive 30delG mutation in the GJB2 gene in Northeast-Hungarian individuals and patients with hearing impairment.

Mutations in the GJB2 gene, which encodes a gap junction protein (connexin 26) account for up to 50% of cases of congenital autosomal recessive non-syndromic hearing impairment. A single mutation, 30delG, is responsible for 70% of this autosomal recessive hearing loss in Europe. This study describes the 30delG mutation analysis of 23 Hungarian families (64 individuals) with at least two subjects with congenital non-syndromic hearing defect and of 52 unrelated individuals from the Northeastern population of Hungary. In all patients, non-progressive hearing impairment varied from moderate to profound involving all frequencies. DNA was tested by PCR based restriction enzyme assay (BSiYI). Sixty-four percent of the patients displayed this one base deletion in GJB2. Out of these, 65.9% were homozygous for this mutation and 34.1% were heterozygotes. The latter showed compound heterozygosity since in these 14 patients, eight previously reported different nucleotide changes were observed on the second allele. The carrier frequency of the 30delG mutation among control group was one in 10.4 (9.6%). This high frequency of 30delG corresponds more to frequencies reported in Southern than in North Europeans.     

Mutation screening of the Ectodysplasin-A receptor gene EDAR in hypohidrotic ectodermal dysplasia

Hypohidrotic ectodermal dysplasia (HED) can be caused by mutations in the X-linked ectodysplasin A (ED1) gene or the autosomal ectodysplasin A-receptor (EDAR) and EDAR-associated death domain (EDARADD) genes. X-linked and autosomal forms are sometimes clinically indistinguishable. For genetic counseling in families, it is therefore important to know the gene involved. In 24 of 42 unrelated patients with features of HED, we found a mutation in ED1. ED1-negative patients were screened for mutations in EDAR and EDARADD. We found mutations in EDAR in 5 of these 18 patients. One mutation, p.Glu354X, is novel. In EDARADD, a novel variant p.Ser93Phe, probably a neutral polymorphism, was also found. Clinically, there was a difference between autosomal dominant and autosomal recessive HED patients. The phenotype in patients with mutations in both EDAR alleles was comparable to males with X-linked HED. Patients with autosomal dominant HED had features comparable to those of female carriers of X-linked HED. The teeth of these patients were quite severely affected. Hypohidrosis and sparse hair were also evident, but less severe. This study confirms Chassaing et al's earlier finding that mutations in EDAR account for approximately 25% of non-ED1-related HED. Mutations leading to a premature stop codon have a recessive effect except when the stop codon is in the last exon. Heterozygous missense mutations in the functional domains of the gene may have a dominant-negative effect with much variation in expression. Patients with homozygous or compound heterozygous mutations in the EDAR gene have a more severe phenotype than those with a heterozygous missense, nonsense or frame-shift mutation.     

Two sisters with Silver-Russell phenotype

Silver-Russell syndrome (SRS) is a well recognizable syndrome, but the etiology of SRS seems to be heterogeneous. SRS is listed in Mendelian Inheritance in Man as an autosomal dominant disorder because most described cases have been of sporadic occurrence, and most likely were caused by de novo autosomal dominant mutation, and because families with apparent dominant transmission of a SRS phenotype have been described. Still, in a few families, autosomal recessive inheritance has been suggested. We describe two sisters who meet the criteria for SRS proposed by Price et al. [1999]. The parents had normal facial features, normal height, and normal post-natal growth. This is the second well-documented case of familial recurrence of SRS that resembles an autosomal recessive inheritance pattern. Since sib recurrence is so rare in SRS, other modes of inheritance should be considered. The finding of maternal uniparental disomy 7 (mUPD7) in 10% of SRS cases suggests that lack of paternally expressed imprinted gene(s) or overexpression of maternal imprinted gene(s) on chromosome 7 cause SRS. The recurrence in sibs could be caused by a mutation in the imprinted gene or imprinting center carried by one parent. Alternatively, recurrence in sibs could represent germ line mosaicism for a dominant mutation in one of the parents.     

Novel missense mutations and unexpected multiple changes of RYR1 gene in 75 malignant hyperthermia families

Tammaro A, Di Martino A, Bracco A, Cozzolino S, Savoia G, Andria B, Cannavo A, Spagnuolo M, Piluso G, Aurino S, Nigro V. Novel missense mutations and unexpected multiple changes of RYR1 gene in 75 malignant hyperthermia families. Malignant hyperthermia (MH) is an autosomal dominant pharmacogenetic disorder of skeletal muscle characterized by disturbance of intracellular calcium homeostasis in the sarcoplasmic reticulum. Mutations of the ryanodine receptor 1 (RYR1) gene account for most cases, with some studies claiming up to 86% of mutations in this locus. However, RYR1 gene is large and variants are common even in the normal population. We examined 54 families with MH susceptibility and 21 diagnosed with equivocal MH. Thirty‐five were selected for an anesthetic reaction, whereas the remainder for hyperCKemia. In these, we studied all 106 exons of the RYR1 gene. When no mutation was found, we also screened: sodium channel voltage‐gated, type IV alpha subunit (SCN4A), calcium channel voltage‐dependent, L type, alpha 1S subunit (CACNA1S), and L‐type voltage‐gated calcium channel alpha 2/delta‐subunit (CACNL2A). Twenty‐nine different RYR1 mutations were discovered in 40 families. Three other MH genes were tested in negative cases. Fourteen RYR1 amino acid changes were novel, of which 12 were located outside the mutational ‘hot spots'. In two families, the known mutation p.R3903Q was also observed in malignant hyperthermia‐nonsusceptible (MHN) individuals. Unexpectedly, four changes were also found in the same family and two in another. Our study confirms that MH is genetically heterogeneous and that a consistent number of cases are not due to RYR1 mutations. The discordance between in vitro contracture test status and the presence of a proven causative RYR1 mutation suggests that the penetrance may vary due to as yet unknown factors.