Studies on genetic susceptibility to osteoporosis : analysis of cis-acting sequences in the collagen type I alpha 1 gene
In osteogenesis imperfecta, an osteoporotic phenotype results from mutations in the protein coding regions of the collagen type I genes - COLIA1 and COLIA2. Genetic factors are known to play an important role in determining bone mass. For this reason, it was considered that more subtle mutations in the transcriptional control regions of the collagen type I genes might be associated with osteoporosis. The polymerase chain reaction was used, followed by single-stranded conformation polymorphism detection (PCR/SSCP) to screen for polymorphisms in the transcriptional control regions (the promoter and first intron) of the type I 1 collagen gene. Three polymorphisms were found; two were rare (allelic frequency 3% and 4%) but the third was more common (allelic frequency 23%) and comprised a guanosine to thymidine substitution at the first base of a consensus sequence for an Sp1 binding motif - an important regulator of transcription in this collagen gene and many other genes. Since osteoporosis is a common disease and the polymorphism was at a functional site, this was investigated further. Electrophoresis mobility shift and supershift assays revealed that the G to T polymorphism was indeed an Sp1 binding motif. This technique also revealed that the T variant (designated "s") had twice the affinity for Sp1 than the G variant (designated "S"). Differential allelic expression analysis showed an increased rate in transcription associated with "s". On analysis of bone mineral density (BMD) in a cohort of 156 women, those with the "Ss/ss" genotype had significantly reduced BMD at the lumbar spine (p<0.03) when compared with "SS" homozygotes. A significant over-representation of the "s" containing genotypes was found in osteoporotic individuals with vertebral fracture (2=6.76;p<0,01). In combination with VDR genotypes, an 'at risk' genotype of "bb/s" was identified.