RESUMO
Osteoporosis is a multifactorial disease in which genetic factors and epigenetic modifications play a major role. DNA methylation is known for gene silencing and its effect on BMP2 promoter has been studied here to understand its regulatory activity in osteoporosis pathogenicity. CpG methylation in the BMP2 promoter was analyzed by performing bisulfite specific PCR on the gDNA samples extracted from whole blood of osteoporotic (n = 24) and healthy (n = 24) individuals. Disproportionate allele frequency of CpG sites was calculated statistically. Differential BMP2 expression was estimated using quantitative RT-PCR technique. Luciferase reporter assay was performed to determine and confirm differential transcriptional activity of BMP2 promoter due to methylation. Total of 14 CpG sites were reporter in the BMP2 promoter of which, CpG site at - 267th position upstream to TSS was found to have disproportionate allele frequency among osteoporotic and healthy individuals and was found to be significantly associated with osteoporosis condition. Functional and gene expression analysis of this methylated site using luciferase reporter vector and Real Time PCR approach, suggested reduced transcriptional activity of BMP2 promoter as well as decreased gene expression in disease condition. BMP2 is being a central signaling molecule, aberrant methylation in the promoter region may result into down regulation of osteoblast markers involved in bone formation.
Assuntos
Proteína Morfogenética Óssea 2/genética , Osteoporose/genética , Adulto , Idoso , Alelos , Proteína Morfogenética Óssea 2/fisiologia , Linhagem Celular Tumoral , Ilhas de CpG/genética , Metilação de DNA/genética , Regulação para Baixo , Epigênese Genética/genética , Feminino , Regulação Neoplásica da Expressão Gênica/genética , Frequência do Gene/genética , Inativação Gênica , Humanos , Índia , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas/genética , Processamento de Proteína Pós-Traducional/genéticaRESUMO
The large amount of positive genetic association data in a number of bone diseases suggests functional consequences of Vitamin D receptor (VDR) gene polymorphism. In the present study, four microsatellite markers viz., D12S1633, D12S1635, D12S347, and D12S96, that lie in the vicinity of the VDR gene on chromosome 12 were selected to assess the allele distribution pattern and diversity among three groups of individuals - normal, osteopenia and osteoporosis. Genetic association study was performed using allele frequency data. Total genomic DNA was isolated from the whole blood of 226 individuals, after recording their bone mineral density (BMD) using Dual X-ray absorptiometry (DXA). All DNA samples were subjected to multiplex Polymerase Chain Reaction (PCR) - genotyping. Allele frequencies and genetic diversity parameters like - number of alleles, average variance and average heterozygosity across all the four markers among three groups were computed. Effect of population stratification was excluded by investigating population structure. A trend of decreasing genetic diversity across four loci from normal to pre- and post-disease condition has been observed. Lesser recombination rate (θ) indicates linkage between studied microsatellite markers and VDR gene. Statistically significant linkage disequilibrium was detected for the allele - 22 of locus D12S96 with osteoporosis. A positive association of allele - 22 suggests susceptibility to disease whereas predominance of allele - 27 among non - diseased group implicates its association with normal bone health.