Detection of the T1640C RYR1 mutation indicating malignant hyperthermia in dogs

Abstract

Malignant hyperthermia (MH) is a clinical syndrome exhibiting elevation of expired carbon dioxide, hyperthermia, muscle rigidity, rhabdomyolysis, acidosis and hyperkalaemia, as well as cardiac dysrhythmia and renal failure. The syndrome manifests itself as a response to anaesthetic agents, such as e.g., halothane, desflurane, and succinylcholine. Depending on the animal species, MH is characterised by autosomal dominant or recessive inheritance, and so far two genes have been identified whose mutations can be linked to MH: RYR1 and CACNA1S. In different species, various mutations of the RYR1 gene have been described which may underlie MH. One of these mutations in dogs is T1640C, which results in the substitution of alanine for valine of the amino acid 547 (V547A) in the RYR1 protein. In our work, we aimed to investigate MH at the DNA level by identifying the T1640C mutation in a group of 50 dogs. For this purpose we used the PCR-RFLP technique, and in six dogs also direct sequencing of PCR products and subsequent comparison of their sequences with the RYR1 gene sequence in an online database. The results of our study show that none of the dogs analysed had any mutant allele of the RYR1 gene, indicating that none should be affected by MH.

Keywords: allele; anaesthesia; disease; gene; sequencing.

Figure 1. Detection of the T1640C RYR1 mutation in DNA of dogs coming from breeders in the Slovak Republic

(A) Electrophoretic analysis of PCR-RFLP profiles. Line 1: molecular weight marker (GeneRuler 50 bp DNA ladder; Thermo Fisher Scientific Baltics UAB, Vilnius, Lithuania). Line 2: plasmid pEX-A128-RYR1 WT digested with MlsI. Line 3: plasmid pEX-A128-RYR1 MUT digested with MlsI. Line 4–9: dog PCR products digested with MlsI (line 4 – dog 1; line 5 – dog 6; line 6 – dog 14; line 7 – dog 15; line 8 – dog 16; line 9 – dog 17). Arrow indicates the 487 bp band. (B) Alignment of the dog 15 PCR product sequence (bottom line) with the RYR1 gene sequence using Clustal Omega software; arrow indicates tymin within the RYR1 sequence which has not undergone the T1640C mutation, and stars indicate the consensual nucleotides