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RYR1 Sequence Variants in Myopathies: Expression and Functional Studies in Two Families.

ABSTRACT: The skeletal muscle ryanodine receptor (RyR1), i.e., the Ca2+ channel of the sarco/endoplasmic reticulum (S/ER), and the voltage-dependent calcium channel Cav1.1 are the principal channels involved in excitation-contraction coupling in skeletal muscle. RYR1 gene variants are linked to distinct skeletal muscle disorders, including malignant hyperthermia susceptibility and central core disease (CCD), mainly with autosomal dominant inheritance, and autosomal recessive myopathies with a broad phenotypic and histopathological spectrum. The age at onset of RYR1-related myopathies varies from infancy to adulthood. We report the identification of four RYR1 variants in two Italian families: one with myopathy and variants c.4003C>T (p.R1335C) and c.7035C>A (p.S2345R), and another with CCD and variants c.9293G>T (p.S3098I) and c.14771_14772insTAGACAGGGTGTTGCTCTGTTGCCCTTCTT (p.F4924_V4925insRQGVALLPFF). We demonstrate that, in patient-specific lymphoblastoid cells, the c.4003C>T (p.R1335C) variant is not expressed and the in-frame 30-nucleotide insertion variant is expressed at a low level. Moreover, Ca2+ release in response to the RyR1 agonist 4-chloro-m-cresol and to thapsigargin showed that the c.7035C>A (p.S2345R) variant causes depletion of S/ER Ca2+ stores and that the compound heterozygosity for variant c.9293G>T (p.S3098I) and the 30-nucleotide insertion increases RyR1-dependent Ca2+ release without affecting ER Ca2+ stores. In conclusion, we detected and functionally characterized disease-causing variants of the RyR1 channel in patient-specific lymphoblastoid cells. This paper is dedicated to the memory and contribution of Luigi Del Vecchio.

SUBMITTER: Zullo A

PROVIDER: S-EPMC6500691 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

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RYR1 Sequence Variants in Myopathies: Expression and Functional Studies in Two Families.

Zullo Alberto A Perrotta Giuseppa G D'Angelo Rossana R Ruggiero Lucia L Gravino Elvira E Del Vecchio Luigi L Santoro Lucio L Salvatore Francesco F Carsana Antonella A

BioMed research international 20190421

The skeletal muscle ryanodine receptor (RyR1), i.e., the Ca2+ channel of the sarco/endoplasmic reticulum (S/ER), and the voltage-dependent calcium channel Cav1.1 are the principal channels involved in excitation-contraction coupling in skeletal muscle. RYR1 gene variants are linked to distinct skeletal muscle disorders, including malignant hyperthermia susceptibility and central core disease (CCD), mainly with autosomal dominant inheritance, and autosomal recessive myopathies w ...[more]

PMID: 31165076

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Project description:RYR1 mutations are typically associated with core myopathies and are the most common overall cause of congenital myopathy. Dominant mutations are most often associated with central core disease and malignant hyperthermia, and genotype-phenotype patterns have emerged from the study of these mutations that have contributed to the understanding of disease pathogenesis. The recent availability of genetic testing for the entire RYR1 coding sequence has led to a dramatic expansion in the identification of recessive mutations in core myopathies and other congenital myopathies. To date, no clear patterns have been identified in these recessive mutations, though no systematic examination has yet been performed.In this study, we investigated genotype-phenotype correlations in a large combined cohort of unpublished (n?=?14) and previously reported (n?=?92) recessive RYR1 cases.Overall examination of this cohort revealed nearly 50% of cases to be non-core myopathy related. Our most significant finding was that hypomorphic mutations (mutations expected to diminish RyR1 expression) were enriched in patients with severe clinical phenotypes. We also determined that hypomorphic mutations were more likely to be encountered in non-central core myopathies. With analysis of the location of non-hypomorphic mutations, we found that missense mutations were generally enriched in the MH/CCD hotspots and specifically enriched in the selectivity filter of the channel pore.These results support a hypothesis that loss of protein function is a key predictive disease parameter. In addition, they suggest that decreased RyR1 expression may dictate non-core related pathology though, data on protein expression was limited and should be confirmed in a larger cohort. Lastly, the results implicate abnormal ion conductance through the channel pore in the pathogenesis in recessive core myopathies. Overall, our findings represent a comprehensive analysis of genotype-phenotype associations in recessive RYR1-myopathies.

Project description:Epigenetic regulation of gene expression is a source of genetic variation, which can mimic recessive mutations by creating transcriptional haploinsufficiency. Germline epimutations and genomic imprinting are typical examples, although their existence can be difficult to reveal. Genomic imprinting can be tissue specific, with biallelic expression in some tissues and monoallelic expression in others or with polymorphic expression in the general population. Mutations in the skeletal-muscle ryanodine-receptor gene (RYR1) are associated with malignant hyperthermia susceptibility and the congenital myopathies central core disease and multiminicore disease. RYR1 has never been thought to be affected by epigenetic regulation. However, during the RYR1-mutation analysis of a cohort of patients with recessive core myopathies, we discovered that 6 (55%) of 11 patients had monoallelic RYR1 transcription in skeletal muscle, despite being heterozygous at the genomic level. In families for which parental DNA was available, segregation studies showed that the nonexpressed allele was maternally inherited. Transcription analysis in patients' fibroblasts and lymphoblastoid cell lines indicated biallelic expression, which suggests tissue-specific silencing. Transcription analysis of normal human fetal tissues showed that RYR1 was monoallelically expressed in skeletal and smooth muscles, brain, and eye in 10% of cases. In contrast, 25 normal adult human skeletal-muscle samples displayed only biallelic expression. Finally, the administration of the DNA methyltransferase inhibitor 5-aza-deoxycytidine to cultured patient skeletal-muscle myoblasts reactivated the transcription of the silenced allele, which suggests hypermethylation as a mechanism for RYR1 silencing. Our data indicate that RYR1 undergoes polymorphic, tissue-specific, and developmentally regulated allele silencing and that this unveils recessive mutations in patients with core myopathies. Furthermore, our data suggest that imprinting is a likely mechanism for this phenomenon and that similar mechanisms could play a role in human phenotypic heterogeneity.

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RYR1 Sequence Variants in Myopathies: Expression and Functional Studies in Two Families.

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<i>RYR1</i> Sequence Variants in Myopathies: Expression and Functional Studies in Two Families.

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