Mechanisms of Osteoporosis Associated With Common Allelic Variants of the COL1A1 Gene

Abstract

INTRODUCTION: Osteoporosis is a common disease with a strong genetic component and the collagen type I alpha I gene (COL1A1) is an important candidate for susceptibility. Three single nucleotide polymorphisms (SNP) have been identified in the 5' flank of the COLIA1 gene (-1997G/T; -1663IndelT and +1245G/T) which have been associated with osteoporosis in various populations. The mechanisms by which these SNP predispose to osteoporosis are unclear however.METHODS: I analyzed the effects of the individual SNPs and associated haplotypes on bone mineral density (BMD) and risk of osteoporotic fracture in women in association studies. I also studied associations between the SNPs, haplotypes and bone strength ex-vivo by biomechanical testing of bone cores obtained from femoral heads of patients undergoing surgery for hip fracture. The effects of the SNPs and haplotypes on gene expression were studied using promoter-reporter assays and gel shift assays were performed to investigate whether the SNPs were situated at transcription factor binding sites and whether the polymorphic variants affected DNA protein binding. In order to investigate the possible effects of the polymorphisms in vivo, gene targeting constructs were prepared containing two common haplotypes and these were transfected into ES cells.RESULTS: The individual polymorphisms were all associated with BMD, but the haplotypes defined by all three SNPs showed a stronger association with BMD, biomechanical strength of bone and hip fracture. Two haplotypes increased in frequency with age suggesting an effect on survival. However these haplotypes were particularly enriched in hip fracture patients. Biomechanical testing showed that all three SNPs were strongly associated with reduced bone strength, independently of BMD. Gel shift assays showed that the region surrounding the -1663insdelT polymorphism recognized the nuclear binding proteins Spl, Nmp4 and Osterix and the -1663delT allele had greater binding affinities for Nmp4 and Osterix than -1663insT allele. The region surrounding the -1997 G/T polymorphism also recognized DNA binding proteins but the polymorphism did not affect DNA protein binding significantly. Reporter assays showed significant differences between the ability of different haplotypes to drive gene expression and constructs containing haplotype2 (G-delT-T) had the highest transcriptional activity (pO.OOl). Transgenic constructs containing different 5' COL1A1 haplotypes were completed and transformed into mouse embryonic stem cells by electroporation in an attempt to generate a disease model of osteoporosis associated with common variants in the COLIA1 geneCONCLUSION: The studies suggest that haplotypes, rather than individual polymorphisms in the 5' flank of COL1A1 predispose to osteoporosis, by affecting DNA protein interactions and gene expression.