Analysis of the stable levels of messenger RNA derived from different polymorphic alleles in the vitamin D receptor gene

Abstract:

The association between polymorphisms in the vitamin D receptor (VDR) gene and bone mineral density (BMD) has been studied by many investigators. However, the question of how polymorphisms in the gene modulate the function of the VDR remains to be answered. To address this issue, we examined the mRNA levels of the VDR in relation to polymorphisms. First, we compared the levels of mRNA between the allele with the polymorphic TaqI-digestive site (t) and nondigestive site (T) located at exon 9 of the VDR gene determined by reverse transcription-polymerase chain reaction (RT-PCR). Total RNA was extracted from peripheral mononuclear cells in volunteers whose genotype is Tt. After the amplification of cDNA by PCR, the amplified fragments were digested by TaqI. The digested (t) and undigested (T) fragments were visualized by ethidium bromide and semiquantified by an image analyzer. In 24 subjects, the mRNA levels of allele t were significantly higher than those of allele T (1.35 fold, P < 0.001). Second, the VDR mRNA levels were estimated by competitive RT-PCR in 60 healthy subjects (35 TT, 24 Tt, 1 tt). The competitive template was 47 bases shorter than the product of the wild-type gene. After RT-PCR, the mRNA level was determined by a comparison with the competitive fragments. No significant difference in the mRNA level was observed between two groups (1.75 ± 0.84 and 1.65 ± 0.99 10⁻¹³ mol/g total RNA in TT and Tt, respectively). In addition, no significant relationship was observed between the VDR mRNA levels and BMD in the 23 subjects whose BMD data were available. In conclusion, higher mRNA levels of allele t than T were detected, but the difference did not result in higher levels of VDR mRNA in subjects with the Tt genotype compared to those with the TT genotype.