Development and validation of a tetra-primer ARMS-PCR assay for genotyping the MTHFR (rs1801131) c.1286 A > C (p.Glu429Ala) polymorphism: a comparative study with KASP

BACKGROUND: The tetra-primer amplification refractory mutation system (T-ARMS) PCR assay is a cost-effective and rapid method for SNP genotyping. The methylenetetrahydrofolate reductase (MTHFR) c.1286 A > C (rs1801131) polymorphism can influence MTHFR enzyme activity and homocysteine levels. While its independent clinical impact remains a subject of debate, it is frequently screened in thrombophilia panels, particularly in the context of compound heterozygosity. METHODS AND RESULTS: This cross-sectional validation study aims to develop and validate a T-ARMS-PCR assay on the MTHFR c.1286 A > C mutation. The results were validated using the Kompetitive Allele-Specific PCR (KASP) as the reference method across 30 clinical DNA samples from a thrombophilia-susceptible cohort. The T-ARMS-PCR assay successfully distinguished genotypes with a clear electrophoretic separation and without non-specific amplification. The results exhibited 100% concordance with the KASP assay (95% CI: 88.4-100%; κ: 1.0). The total turnaround time including the reaction and electrophoretic separation was 107.5 min. CONCLUSIONS: T-ARMS-PCR offers a reliable, rapid, and cost-effective alternative for MTHFR c.1286 A > C genotyping. This method is particularly suitable for resource-limited clinical settings that require accurate genotyping without the need for high-end sequencing or specialized instrumentation.