Development of an ARMS-PCR protocol for FTO rs9939609 genotyping in the Kinh Vietnamese population

Overweight and obesity are polygenic diseases, in which the rs9939609 single nucleotide polymorphism (SNP) of the FTO gene is an established genetic marker closely linked to obesity risk and several metabolic disorders. Existing genotyping methods, such as Sanger sequencing, RFLP-PCR, and TaqMan real-time PCR, are limited by cost, turnaround time, or the need for specialized equipment, and are not well suited to large-scale screening at testing facilities in Vietnam. To develop an ARMS-PCR (Amplification Refractory Mutation System - PCR) protocol for genotyping FTO rs9939609 in the Vietnamese population. A set of four primers (two outer primers and two allele-specific primers) was designed with Primer-BLAST based on the NCBI reference sequence. The protocol's operating parameters were evaluated on 3 control DNA samples of known genotype; it was then tested on 105 DNA samples from the Kinh Vietnamese population, with genotyping results cross-validated against RFLP-PCR and Sanger sequencing. ARMS-PCR showed concordant results with RFLP-PCR in 102 of 105 samples, with a sensitivity of 94.29%, a specificity of 98.57%, and a Cohen’s kappa of κ≈0.935 for A-allele carrier status. Sanger sequencing of three selected samples, representing the TT, AT, and AA genotypes, confirmed the corresponding ARMS-PCR genotype calls. The ARMS-PCR protocol developed here is highly accurate, simple to perform, and low-cost, with potential for use in population studies and genetic screening for FTO rs9939609 in Vietnam