A Single Nucleotide Polymorphism Under the Reverse Primer Binding Site May Lead to BsmI Mis‐Genotyping in the Vitamin D Receptor Gene

The BsmI polymorphism in the VDR gene has been extensively investigated by PCR and restriction digestion in bone genetics. A SNP within the corresponding region for the previously published reverse primer was observed and confirmed by DNA sequencing. BsmI mis‐genotyping caused by this SNP could confound genetic findings. Introduction: By analyzing the FokI, BsmI, ApaI, and TaqI polymorphisms in the vitamin D receptor (VDR) gene, we observed a significantly different genotype distribution in the BsmI polymorphic locus with a deviation from Hardy‐Weinberg equilibrium. One of the reasons for polymerase chain reaction (PCR) non‐amplification may be a mismatched base at the primer binding region. Therefore, the aim of this study was to analyse whether a single nucleotide polymorphism (SNP), which has been recently described as TruI, is responsible for the discrepancy between expected and observed genotype frequencies. Materials and Methods: The VDR genotypes were identified in a cohort of 165 peri‐ and postmenopausal women of white origin. PCR amplification was carried out using the originally published primers and followed by restriction cleavage. The BsmI genotypes were further verified with a reverse primer external to the original binding site. The presence of the TruI polymorphism under the previously published reverse primer was confirmed by a restriction digestion and DNA sequencing. In Bb subjects, the colocalization of b allele with the TruI restriction site on the same chromosome was confirmed by a simultaneous digestion of the PCR product with both BsmI and TruI restriction enzymes. Results: The BsmI reanalysis with an external primer provided a higher number of heterozygous subjects with a proportionally smaller number of BB subjects, and the changed genotype distribution was under Hardy‐Weinberg equilibrium (BB, 31; Bb, 80; bb, 54; r = 0.0203; p = 0.90). In our primary analysis, the presence of the TruI polymorphism led to a drop out of b allele during PCR amplification and thus to the false prevalence of BB genotypes (BB, 50; Bb, 61; bb, 54; r = 11.17; p = 0.01). Conclusion: The SNP in the region corresponding to the reverse primer may lead to BsmI mis‐genotyping, which may have confounded some previous genetic studies.