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.


BACKGROUND
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.


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).


CONCLUSIONS
The SNP in the region corresponding to the reverse primer may lead to BsmI mis-genotyping, which may have confounded some previous genetic studies.
Institute of Endocrinology, Prague, Czech Republic. katka2222@volny.cz
K
K
Zajícková
A
A
Krepelová
I
I
Zofková
D000483:Alleles; D001665:Binding Sites; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism; D005838:Genotype; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D008957:Models, Genetic; D011110:Polymorphism, Genetic; D020641:Polymorphism, Single Nucleotide; D011485:Protein Binding; D018167:Receptors, Calcitriol / Q000235:genetics*; D017422:Sequence Analysis, DNA
D018167:Receptors, Calcitriol; C053864:endodeoxyribonuclease BsmI; D015252:Deoxyribonucleases, Type II Site-Specific