Individual single tube genotyping and DNA pooling by allele-specific PCR to uncover associations of polymorphisms with complex diseases.

BACKGROUND: The genotyping for the study of the SNPs in different complex diseases require a great number of patients. In this sense, the determination of allele frequencies and genotypes requires a rapid and economical procedure. METHODS: The genotype has been carried out by allele-specific PCR in single tube with the discrimination of the products of PCR by its T(m). For this purpose a GC tail was added to 5' extreme of the specific primer. The allele frequencies were also calculated by DNA pooling and QRT-PCR using allele-specific primers. RESULTS: The use of the genotyping in single tube through allele-specific PCR and melting curves has led us to the accurate genotype of three polymorphisms of vdr (cdx-2), osteoprotegerin (A-163G) and ppar-gamma (C-681G) genes in 225 postmenopausal women to be associated to osteoporosis. Only the cdx-2 polymorphism was associated with a reduced bone mineral density (BMD). These data were similar to those obtained when the allele frequencies were calculated using QRT-PCR in DNA pools. CONCLUSIONS: Individual genotyping with allele-specific PCR in single tube and melting curve analysis is a fast, trustworthy and economic method to study any SNP. We propose the following approach to determine the possible association of SNPs with complex and multifactorial diseases like osteoporosis, in which hundreds of individuals should be analyzed: construct control and problem groups, make DNA pools, and calculate pooled allelic frequencies. Genotyping each individual further permits to determine the genotypic distribution when differences in allelic frequencies are observed, thus allowing more complex statistical analyses (including other variables like age, weight, etc.).

Vitamin D status and the Cdx-2 polymorphism of the vitamin D receptor gene are determining factors of bone mineral density in young healthy postmenopausal women.

UNLABELLED: Bone mineral density (BMD) is a main determinant of osteoporotic fractures. A cross-sectional study was conducted in 229 young, healthy postmenopausal women (PMW) to evaluate the contribution of the vitamin D endocrine system and other clinical, biochemical and genetic parameters. Clinical risk factors for osteoporosis were obtained by a questionnaire. Serum concentrations of 25OHD, 1,25(OH)2D, PTH, and bone turnover markers were measured. The BsmI, FokI and Cdx-2 polymorphisms of the vitamin D receptor (VDR) gene were determined. DXA and the WHO criteria were applied for the diagnosis of osteoporosis. Univariate logistic and multivariate logistic regression analyses were carried out. RESULTS: The prevalence of vitamin D deficiency (<50nmol/l) was 50%. Age increased osteoporosis risk; whereas body mass index (BMI), number of reproductive years, 25OHD level and the Cdx-2 polymorphism in the VDR gene (when allele A is present) were found to be protective. Therefore, both serum 25OHD and VDR polymorphism should be taken into account in the evaluation and implementation of therapeutic strategies concerning PMW, especially as the prevalence of vitamin D deficiency is still alarmingly high even at Southern latitudes. This article is part of a Special Issue entitled 'Vitamin D Workshop'.