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Published on: August 8, 2022
Development and validation of PCR-RFLP assays for detection of MYBPC3 variants associated with hypertrophic
Thao Phuong Vu1, Thong Quang Le2, Truc Thi Thanh Truong2
1Animal Genetics Group, Faculty of Biotechnology, Ho Chi Minh city Open University, Ho Chi Minh city, Vietnam.
Insights
This study developed PCR-RFLP assays to detect MYBPC3 gene variants linked to hypertrophic cardiomyopathy (HCM) in Maine Coon and Ragdoll cats. The assays are technically feasible for genetic testing in cats, but the study cohort was too small for definitive clinical associations.
Area of Science:
- Genetics
- Veterinary Medicine
- Molecular Biology
Background:
- Hypertrophic cardiomyopathy (HCM) is a prevalent myocardial disease in cats.
- Specific MYBPC3 gene variants are associated with HCM in Maine Coon (A31P) and Ragdoll (R820W) breeds.
- Genetic testing is crucial for understanding HCM heritability and management.
Purpose of the Study:
- To develop and validate Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) assays for detecting MYBPC3 variants (A31P and R820W) in cats.
- To assess the technical feasibility of these assays in a resource-limited setting.
- To explore potential genotype-phenotype correlations in a small cohort of cats.
Main Methods:
- Development of two PCR-RFLP assays targeting MYBPC3 variants: c.91 G>C (A31P) in Maine Coons and c.2453 C>T (R820W) in Ragdolls.
- Analytical validation of assays using in silico predictions and Sanger sequencing.
- Application of assays to a cohort of 25 Maine Coons and 34 Ragdolls with echocardiographic phenotyping.
Main Results:
- Successful development of PCR-RFLP assays yielding specific amplicon sizes and digestion patterns.
- Detection of heterozygous carriers for the A31P variant in 12.0% of Maine Coons and R820W variant in 8.8% of Ragdolls.
- No significant association found between genotypes and clinical HCM, though exploratory differences in echocardiographic variables were noted in heterozygous cats.
Conclusions:
- PCR-RFLP assays are technically feasible for targeted MYBPC3 genotyping in cats, suitable for resource-limited laboratories.
- The current study cohort is underpowered to estimate penetrance, predict clinical risk, or infer population allele frequencies.
- Further research with larger cohorts is needed to establish definitive genotype-HCM associations and clinical implications.
Abstract:
Hypertrophic cardiomyopathy (HCM) is the most common myocardial disease in domestic cats and breed-associated variants in MYBPC3 have been reported in Maine Coon and Ragdoll cats. This study developed and analytically evaluated two polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assays for detecting the MYBPC3 c.91 G>C (A31P) variant in Maine Coon cats and the c.2453 C>T (R820W) variant in Ragdoll cats, and applied the assays to a small Vietnamese cohort with concurrent echocardiographic phenotyping. A total of 25 Maine Coon and 34 Ragdoll client-owned cats were examined and classified using echocardiographic criteria based primarily on left ventricular wall thickness after exclusion of secondary causes of hypertrophy. The A31P assay generated a 128-bp amplicon digested with HaeIII, whereas the R820W assay generated a 319-bp amplicon digested with BccI. Restriction profiles were concordant with in silico predictions and representative Sanger sequencing results. Heterozygous carriers were detected in 3/25 Maine Coons (12.0%; mutant allele frequency 0.06) and 3/34 Ragdolls (8.8%; mutant allele frequency 0.044); no homozygous mutant cats were identified. Clinical HCM was observed in one heterozygous cat of each breed, but Fisher's exact tests did not provide evidence of genotype-HCM associations. Heterozygous cats showed exploratory differences in selected echocardiographic variables. These findings support the technical feasibility of PCR-RFLP for targeted MYBPC3 genotyping in resource-limited laboratory settings, but this cohort is underpowered for penetrance estimation, clinical risk prediction, or population-level allele-frequency inference.
