Construction and Expression of Ryanodine Receptor Mutants Relevant to Malignant Hyperthermia Patients in Japan

Malignant hyperthermia (MH) is a potentially fatal pharmacogenetic disorder triggered by exposure to commonly used volatile anesthetics. Pharmacological and genetic analyses implicated the type 1 of ryanodine receptor (RyR1) /Ca2+ release channel as the main candidate gene for causing MH. Genetic diagnosis of MH was proposed to replace conventional methods using biopsied muscle samples that are painful for patients and require skillful diagnosticians to interpret. However, more than 250 RyR1 gene variants have now been reported in MH-susceptible patients, although most have yet to be associated with functional abnormalities using exogenous constructs of these mutants expressed in living cells. To directly compare the pharmacological characteristics of some of the MH-related RyR1 mutants, we have established doxycycline -inducible cell lines expressing two of the unconfirmed rabbit RyR1 mutants, Q156K or R534H (corresponding to the Q155K or R533H mutations in human RyR1 reported in MH patients in Japan) and a confirmed mutant, R164C RyR1 (corresponding to the R163C mutation in human). The caffeine sensitivity of Q156K-expressing cells was remarkably enhanced compared to wild-type RyR1 and similarly to previously reported levels for R164C-expressing cells, while that of the R534H mutants was not different from wild-type cells. The resting cytosolic Ca2+ concentrations of cell lines expressing Q156K or R164C were much higher than those expressing R534H or wild-type RyR1. These results indicated that the RyR1 gene mutation causing the Q156K phenotype (Q155K in human) is potentially susceptible to MH, and that screening for this mutation could be useful for the noninvasive genetic diagnosis of MH in humans

Congenital chloride diarrhea needs to be distinguished from Bartter and Gitelman syndrome

Pseudo-Bartter/Gitelman syndrome (p-BS/GS) encompasses a clinically heterogeneous group of inherited or acquired disorders similar to Bartter syndrome (BS) or Gitelman syndrome (GS), both renal salt-losing tubulopathies. Phenotypic overlap frequently occurs between p-BS/GS and BS/GS, which are difficult to diagnose based on their clinical presentation and require genetic tests for accurate diagnosis. In addition, p-BS/GS can occur as a result of other inherited diseases such as cystic fibrosis, autosomal dominant hypocalcemia, Dent disease, or congenital chloride diarrhea (CCD). However, the detection of the variants in genes other than known BS/GS-causing genes by conventional Sanger sequencing requires substantial time and resources. We studied 27 cases clinically diagnosed with BS/GS, but with negative genetic tests for known BS/GS genes. We conducted targeted sequencing for 22 genes including genes responsible for tubulopathies and other inherited diseases manifesting with p-BS/GS symptoms. We detected the SLC26A3 gene variants responsible for CCD in two patients. In Patient 1, we found the SLC26A3 compound heterozygous variants: c.354delC and c.1008insT. In Patient 2, we identified the compound heterozygous variants: c.877G > A, p.(Glu293Lys), and c.1008insT. Our results suggest that a comprehensive genetic screening system using targeted sequencing is useful for the diagnosis of patients with p-BS/GS with alternative genetic origins