Rapid and Simultaneous Detection of Vitamin D Receptor Gene Polymorphisms by a Single ARMS-PCR Assay

Abstract

Vitamin D has various roles in many biological actions such as calcium homeostasis, cell proliferation, and cell differentiation to many target tissues. These effects are mediated by the active form of vitamin D, 1,25(OH)2D3, which binds to a cytoplasmic protein called vitamin D receptor (VDR). VDR gene has four common single nucleotide polymorphisms (SNPs) that are defined by the presence of restriction sites for FokI (F/f), TaqI (T/t), BsmI (B/b), and ApaI (A/a). The association of VDR gene polymorphisms with several diseases has been investigated. In most studies, VDR genotyping was performed by polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) assays, which are cumbersome and time consuming, and their results are sometimes difficult to interpret. We modified previously reported primers for VDR genotyping and set up a single amplification-refractory mutation system (ARMS)-PCR method for simultaneous genotyping of four common VDR polymorphisms. In this study, 218 DNA samples were analyzed for VDR genetic variants by this ARMS-PCR technique; 136 of them were re-genotyped by PCR-RFLP assays to compare genotyping results. We obtained allelic frequencies of 69 vs. 31% for F/f, 34 vs. 66% for B/b, 70 vs. 30% for T/t, and 52 vs. 48% for A/a in this sample of the Iranian population. In addition, comparisons of the results of these two methods showed good uniformity in VDR genotypes; although, in some samples, ambiguity in restriction patterns was present. As ARMS-PCR is more rapid, economic, and user friendly than PCR-RFLP, its substitution would be welcomed in disease association and pharmacogenetic studies of VDR variants.