Each variant should be fully characterised at the genetic level, including:
- A full description at the DNA and protein level, considering aspects of evolutionary conservation and change in charge, polarity or structure introduced by the amino acid replacement,
- Co-segregation of the variant with the disease in the family or families affected,
- Assessment of the prevalence of the variant in a relevant population by means of database searches, e.g., dbSNP, 1000 Genomes, exome variant server. It is anticipated that pathogenic variants will have a minor allele frequency, MAF <1%. The estimate of MAF should be based on a sample size of > 150 subjects.
The effect of each variant on RYR1 function should be assayed by one or more of the following test systems:
Recombinant in vitro expression on a defined genetic background.
- The standard system, introduced by D.H. MacLennan’s group, uses the expression of a rabbit RYR1 cDNA construct (with appropriate mutations) in HEK293 cells. Calcium release is measured fluorimetrically in response to trigger agents    . Although this is a non-muscle cell type, the advantage of the system is the defined cDNA and the standardised genetic background of the recipient cell line. This allows for direct comparison between mutations and eliminates the potential influence of mutations in other genes which could modify RYR1 function in cells taken from patients.
- Alternatively, myotubes of the dyspedic mouse (RYR1-knock out) have been used as recipients for the expression of cDNA constructs. Again, cDNA construct and genetic background are well defined and standardised. The genetic expression profile of myotubes may be closer to mature muscle. For this reason, results may not be directly comparable to the HEK system.
- Assays of RYR1 function in ex vivo tissues.
- Calcium measurements and ligand binding studies have been performed on tissues from MHS patients with characterised RYR1 variants:
- In myotubes , in microsomal sarcoplasmic reticulum preparations from muscle biopsies, and in lymphoblasts . Interpretation of altered RYR1 function was based on Ca2+ flux and resting [Ca2+] or ryanodine binding to sarcoplasmic reticulum RYR1 preparations. Myotubes and lymphoblasts were derived from individual patients and, therefore, the potential influence of other individual genetic factors cannot be excluded. For the sarcoplasmic reticulum preparations, muscle biopsies of several patients were pooled thus eliminating individual variation.
- In order to avoid the interference of genetic factors other than RYR1, it is recommended that all assays which are based on cells taken from patients should be performed on samples from at least two independent patients with the same mutation.
Criteria for inclusion on EMHG list of diagnostic variants
Genetic and functional characterization both must be consistent with a pathological role in MH. For variants that have been functionally characterized using any of the previously described methods (section 2 above) data can be submitted directly to the EMHG through its website. Functional data acquired using novel methods will require validation through publication in a peer reviewed journal.
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