Allele-frequency determination of BsmI and FokI polymorphisms of the VDR gene by quantitative real-time PCR (QRT-PCR) in pooled genomic DNA samples.
The vdr gene is a candidate for osteoporosis susceptibility, with conflicting results in association studies. We have designed and optimized an individual allele-specific and DNA pooling PCR-based methodology to quantitate BsmI and FokI polymorphisms of the vdr gene and studied single-nucleotide polymorphisms (SNPs) from pooled DNA samples. The allele frequency in DNA pooling experiments has been analyzed by kinetic PCR: quantitative real-time PCR (QRT-PCR). A Spanish cohort of 225 healthy postmenopausal women was studied. Bone mineral density (BMD) was measured by dual-energy X-ray absorptiometry (DEXA) and quantitative ultrasound calcaneous densitometry. Allele-specific PCR amplification of BsmI and FokI genes showed full concordance with the PCR-RFLP approach. The prevalence of the three BsmI VDR genotypes was 19.1, 44.9 and 36.0% for BB, Bb and bb, respectively. In the case of the FokI locus, the prevalence of genotypes was 40.4, 48.0 and 11.6% for FF, Ff and ff, respectively. No positive correlation was found between polymorphism and BMD. The DNA pooling procedure was validated. No differences were found in allele frequencies and T-score data obtained using the high throughput DNA pooling approach, as compared to known individual frequencies. In our hands, this is a very useful approach to study quantitative (thus polygenic) traits like osteoporosis susceptibility.
Quesada
J M
JM
Unidad Metabolismo Mineral Hospital Reina Sofia, Córdoba, Spain. md1qugoj@uco.es
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D015502:Absorptiometry, Photon; D000483:Alleles; D001483:Base Sequence; D015331:Cohort Studies; D004247:DNA / Q000235:genetics*; D017931:DNA Primers; D015252:Deoxyribonucleases, Type II Site-Specific / Q000378:metabolism*; D005787:Gene Frequency; D006801:Humans; D011110:Polymorphism, Genetic; D018167:Receptors, Calcitriol / Q000235:genetics*; D020133:Reverse Transcriptase Polymerase Chain Reaction
D017931:DNA Primers; D018167:Receptors, Calcitriol; D004247:DNA; C053864:endodeoxyribonuclease BsmI; C035042:endodeoxyribonuclease FokI; D015252:Deoxyribonucleases, Type II Site-Specific