Updated variant curation expert panel criteria and pathogenicity classifications for 251 variants for RYR1-related malignant hyperthermia susceptibility.

The ClinGen malignant hyperthermia susceptibility (MHS) variant curation expert panel specified the American College of Medical Genetics and Genomics/Association of Molecular Pathologists (ACMG/AMP) criteria for RYR1-related MHS and a pilot analysis of 84 variants was published. We have now classified an additional 251 variants for RYR1-related MHS according to current ClinGen standards and updated the criteria where necessary. Criterion PS4 was modified such that individuals with multiple RYR1 variants classified as pathogenic (P), likely pathogenic (LP), or variant of uncertain significance (VUS) were not considered as providing evidence for pathogenicity. Criteria PS1 and PM5 were revised to consider LP variants at the same amino-acid residue as providing evidence for pathogenicity at reduced strength. Finally, PM1 was revised such that if PS1 or PM5 are used PM1, if applicable, should be downgraded to supporting. Of the 251 RYR1 variants, 42 were classified as P/LP, 16 as B/LB, and 193 as VUS. The primary driver of 175 VUS classifications was insufficient evidence supporting pathogenicity, rather than evidence against pathogenicity. Functional data supporting PS3/BS3 was identified for only 13 variants. Based on the posterior probabilities of pathogenicity and variant frequencies in gnomAD, we estimated the prevalence of individuals with RYR1-related MHS pathogenic variants to be between 1/300 and 1/1075, considerably higher than current estimates. We have updated ACMG/AMP criteria for RYR1-related MHS and classified 251 variants. We suggest that prioritization of functional studies is needed to resolve the large number of VUS classifications and allow for appropriate risk assessment. RYR1-related MHS pathogenic variants are likely to be more common than currently appreciated.

Increased Sensitivity of Diagnostic Mutation Detection by Re-analysis Incorporating Local Reassembly of Sequence Reads.

BACKGROUND: Diagnostic genetic testing programmes based on next-generation DNA sequencing have resulted in the accrual of large datasets of targeted raw sequence data. Most diagnostic laboratories process these data through an automated variant-calling pipeline. Validation of the chosen analytical methods typically depends on confirming the detection of known sequence variants. Despite improvements in short-read alignment methods, current pipelines are known to be comparatively poor at detecting large insertion/deletion mutations. METHODS: We performed clinical validation of a local reassembly tool, ABRA (assembly-based realigner), through retrospective reanalysis of a cohort of more than 2000 hereditary cancer cases. RESULTS: ABRA enabled detection of a 96-bp deletion, 4-bp insertion mutation in PMS2 that had been initially identified using a comparative read-depth approach. We applied an updated pipeline incorporating ABRA to the entire cohort of 2000 cases and identified one previously undetected pathogenic variant, a 23-bp duplication in PTEN. We demonstrate the effect of read length on the ability to detect insertion/deletion variants by comparing HiSeq2500 (2 × 101-bp) and NextSeq500 (2 × 151-bp) sequence data for a range of variants and thereby show that the limitations of shorter read lengths can be mitigated using appropriate informatics tools. CONCLUSIONS: This work highlights the need for ongoing development of diagnostic pipelines to maximize test sensitivity. We also draw attention to the large differences in computational infrastructure required to perform day-to-day versus large-scale reprocessing tasks.

The role of CACNA1S in predisposition to malignant hyperthermia.

BACKGROUND: Malignant hyperthermia (MH) is an inherited pharmacogenetic disorder of skeletal muscle, characterised by an elevated calcium release from the skeletal muscle sarcoplasmic reticulum. The dihydropyridine receptor (DHPR) plays an essential role in excitation-contraction coupling and calcium homeostasis in skeletal muscle. This study focuses on the gene CACNA1S which encodes the alpha1 subunit of the DHPR, in order to establish whether CACNA1S plays a major role in MH susceptibility in the UK. METHODS: We investigate the CACNA1S locus in detail in 50 independent MH patients, the largest study to date, to identify novel variants that may predispose to disease and also to characterise the haplotype structure across CACNA1S. RESULTS: We present CACNA1S cDNA sequencing data from 50 MH patients in whom RYR1 mutations have been excluded, and subsequent mutation screening analysis. Furthermore we present haplotype analysis of unphased CACNA1S SNPs to (1) assess CACNA1S haplotype frequency differences between susceptible MH cases and a European control group and (2) analyse population-based association via clustering of CACNA1S haplotypes based on disease risk. CONCLUSION: The study identified a single potentially pathogenic change in CACNA1S (p.Arg174Trp), and highlights that the haplotype structure across CACNA1S is diverse, with a high degree of variability.

A RYR1 mutation associated with recessive congenital myopathy and dominant malignant hyperthermia in Asian families.

In this study we present 3 families with malignant hyperthermia (MH), all of Indian subcontinent descent. One individual from each of these families was fully sequenced for RYR1 and presented with the non-synonymous change c.11315G>A/p.R3772Q. When present in the homozygous state c.11315*A is associated with myopathic symptoms.

Mutation analysis of two patients with hypokalemic periodic paralysis and suspected malignant hyperthermia.

Hypokalemic periodic paralysis (HypoPP) and malignant hyperthermia (MH) are autosomal-dominant genetically heterogeneous ion channelopathies. MH has been described in patients with HypoPP, suggesting a potential link between these disorders. However, a common genetic determinant has not been described. With the aim of corroborating this association, four candidate genes were screened in two independent HypoPP patients, one of whom was also diagnosed as MH-susceptible and the other as MH-normal by the in vitro contracture test (IVCT). An A>G change at nucleotide 7025 was detected in the RYR1 gene in the HypoPP/MH-susceptible patient. Detection of the same mutation in three independent MH families suggested that 7025A>G represents a novel MH-susceptibility allele and that MH and HypoPP occurred independently in the case presented. Conclusive evidence in support of the hypothesis that MH and HypoPP are allelic was therefore not obtained.

RYR1 mutations causing central core disease are associated with more severe malignant hyperthermia in vitro contracture test phenotypes.

Malignant hyperthermia (MH) and central core disease (CCD) are autosomal dominant disorders of skeletal muscle. Susceptibility to MH is only apparent after exposure to volatile anesthetics and/or depolarizing muscle relaxants. CCD patients present with diffuse muscular weakness but are also at risk of MH. Mutations in RYR1 (19q13.1), encoding a skeletal muscle calcium release channel (ryanodine receptor), account for the majority of MH and CCD cases. Fifteen RYR1 N-terminal mutations are considered causative of MH susceptibility, five of which are also associated with CCD. In the first extensive UK population survey, eight of 15 mutations were detected in 85 out of 297 (29%) unrelated MH susceptible cases, with G2434R detected in 53 cases (18%). Mutation type was shown to affect significantly MH phenotypes (in vitro contracture test (IVCT) response to caffeine, halothane, and ryanodine). RYR1 mutations associated with both CCD and MH (R163C, R2163H, R2435H) had more severe caffeine and halothane response phenotypes than those associated with MH alone. Mutations near the amino terminal (R163C, G341R) had a relatively greater effect on responses to caffeine than halothane, with a significantly increased caffeine:halothane tension ratio compared to G2434R of the central domain. All phenotypes were more severe in males than females, and were also affected by muscle specimen size and viability. Discordance between RYR1 genotype and IVCT phenotype was observed in seven families (nine individuals), with five false-positives and four false-negatives. This represents the most extensive study of MH patient clinical and genetic data to date and demonstrates that RYR1 mutations involved in CCD are those associated with one end of the spectrum of MH IVCT phenotypes.