Development and Validation of PCR-RFLP Assays for Detection of MYBPC3 Variants Associated with Hypertrophic Cardiomyopathy in Maine Coon and Ragdoll Cats.

PCR-RFLP is a feasible low-resource method for MYBPC3 genotyping, but larger studies are needed for clinical risk prediction.

This study developed and validated two PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) assays for detecting MYBPC3 variants associated with hypertrophic cardiomyopathy (HCM) in Maine Coon (c.91G>C; A31P) and Ragdoll (c.2453C>T; R820W) cats. The A31P assay used HaeIII digestion of a 128-bp amplicon, while the R820W assay used BccI digestion of a 319-bp amplicon. Both assays demonstrated concordance with in silico predictions and Sanger sequencing validation. Applied to a Vietnamese cohort of 25 Maine Coon and 34 Ragdoll client-owned cats with concurrent echocardiographic phenotyping, heterozygous carriers were identified in 3/25 Maine Coons (12.0%; allele frequency 0.06) and 3/34 Ragdolls (8.8%; allele frequency 0.044). No homozygous mutant cats were detected. Clinical HCM was confirmed in one heterozygous cat per breed; however, Fisher's exact tests found no statistically significant genotype-HCM associations, likely due to insufficient sample size. Heterozygous cats demonstrated exploratory differences in selected echocardiographic variables. The study confirms that PCR-RFLP is technically feasible for MYBPC3 genotyping in resource-limited laboratory settings. However, the small cohort size limits conclusions regarding variant penetrance, clinical risk stratification, or population-level allele frequency estimation. Larger, prospective studies are needed before these genetic findings can be reliably integrated into clinical breeding or screening programs.

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