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A Single Nucleotide Polymorphism Under the Reverse Primer Binding Site May Lead to Bsm I Mis‐Genotyping in the Vitamin D Receptor Gene
Author(s) -
Zajíčková K,
Křepelová A,
Žofková I
Publication year - 2003
Publication title -
journal of bone and mineral research
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.882
H-Index - 241
eISSN - 1523-4681
pISSN - 0884-0431
DOI - 10.1359/jbmr.2003.18.10.1754
Subject(s) - genotyping , genotype , biology , single nucleotide polymorphism , molecular inversion probe , genetics , restriction site , primer (cosmetics) , microbiology and biotechnology , polymerase chain reaction , restriction enzyme , snp genotyping , locus (genetics) , calcitriol receptor , primer binding site , gene , chemistry , reverse transcriptase , organic chemistry
The Bsm I 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. Bsm I mis‐genotyping caused by this SNP could confound genetic findings. Introduction: By analyzing the Fok I, Bsm I, Apa I, and Taq I polymorphisms in the vitamin D receptor ( VDR ) gene, we observed a significantly different genotype distribution in the Bsm I 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 Tru I, 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 Bsm I genotypes were further verified with a reverse primer external to the original binding site. The presence of the Tru I 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 Tru I restriction site on the same chromosome was confirmed by a simultaneous digestion of the PCR product with both Bsm I and Tru I restriction enzymes. Results: The Bsm I 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 Tru I 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 Bsm I mis‐genotyping, which may have confounded some previous genetic studies.