A Rapid Genotyping Assay for FTO and PPAR-γ Nutrigenetic Variants in Thai Adults with Overweight/Obesity

Nutrigenetic research linking fat mass and obesity-associated (FTO) and peroxisome proliferator-activated receptor gamma (PPAR-γ) variants to dietary response and obesity risk requires accessible genotyping tools, particularly for population studies in low- and middle-income countries. Current genotyping methods are typically limited to single or duplex formats, increasing cost and processing time for multivariant screening.

The aim of this study is to develop, validate, and apply a cost-effective 4-plex high-resolution melting (HRM) assay for simultaneous detection of FTO (rs9939609 and rs1421085) and PPAR-γ (rs1801282 and rs3856806) variants using standard laboratory equipment.

Allele-specific primers with distinct melting temperatures were designed for multiplexed HRM detection of 4 obesity-associated single nucleotide polymorphisms. Analytical validation was performed against Sanger sequencing in 30 samples. Diagnostic accuracy was assessed by sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV). The validated assay was applied to genotype 384 Thai adults (123 normal-weight, 261 with overweight/obesity) from Bangkok, Thailand. Genotype frequencies and Hardy-Weinberg equilibrium (HWE) were evaluated by the χ² test.

The 4-plex HRM assay demonstrated 100% concordance with Sanger sequencing across all 4 variants (sensitivity: 100%; specificity: 100%; PPV: 100%; NPV: 100%). All triplicate measurements produced consistent genotype calls. Total assay time was 70 min. Based on the manufacturer's catalog prices (QIAGEN, 2024), the estimated reagent cost per sample was ∼4- to 5-fold lower than 4 individual TaqMan assays and 6- to 8-fold lower than Sanger sequencing for the same 4 targets. Population screening revealed minor allele frequencies of 5.3%-8.2% for FTO and 0%-5.8% for PPAR-γ variants, with genotype distributions conforming to HWE for 3 of 4 variants.

This validated 4-plex HRM assay provides a rapid, accurate, and cost-effective tool for nutrigenetic screening of obesity-associated variants. The method is suitable for large-scale population studies where budget and infrastructure constraints limit access to high-throughput genotyping platforms.