Association of a functional microsatellite within intron 1 of the BMP5 gene with susceptibility to osteoarthritis

Abstract

Background: In a previous study carried out by our group, the genotyping of 36 microsatellite markers from within a narrow interval of chromosome 6p12.3-q13 generated evidence for linkage and for association to female hip osteoarthritis (OA), with the most compelling association found for a marker within intron 1 of the bone morphogenetic protein 5 gene (BMP5). In this study, we aimed to further categorize the association of variants within intron 1 of BMP5 with OA through an expanded genetic association study of the intron and subsequent functional analysis of associated polymorphisms.

Methods: We genotyped 18 common polymorphisms including 8 microsatellites and 9 single nucleotide polymorphisms (SNPs) and 1 insertion/deletion (INDEL) from within highly conserved regions between human and mouse within intron 1 of BMP5. These markers were then tested for association to OA by a two-stage approach in which the polymorphisms were initially genotyped in a case-control cohort comprising 361 individuals with associated polymorphisms (P < or = 0.05) then genotyped in a second case-control cohort comprising 1185 individuals.

Results: Two BMP5 intron 1 polymorphisms demonstrated association in the combined case-control cohort of 1546 individuals (765 cases and 781 controls): microsatellite D6S1276 (P = 0.018) and SNP rs921126 (P = 0.013). Functional analyses in osteoblastic, chondrocytic, and adipocytic cell lines indicated that allelic variants of D6S1276 have significant effects on the transcriptional activity of the BMP5 promoter in vitro.

Conclusion: Variability in gene expression of BMP5 may be an important contributor to OA genetic susceptibility.

Figure 1

Genomic structure of the BMP5 gene. The exons are represented by boxes with black fields depicting coding regions and white fields representing the untranslated regions. Solid lines represent introns and intergenic regions. The positions of rs9475431 (1), D6S1276 (2), rs9475430 (3), and rs9475429 (4) are shown with flanking sequence within intron 1 of BMP5 along with the two common haplotypes comprised of rs9475431, rs9475430, rs9475429: haplotype I (TTT) and haplotype II (GCA).

Figure 2

Effect of microsatellite D6S1276 polymorphism on BMP5 promoter activity. Luciferase reporter assays of the D6S1276 constructs were performed in MG63 (A), CH8 (B), and SW872 cells (C). Transcriptional activities are given as a percentage of the activity of the Promoter-Luciferase construct for each cell line. Data shown are the mean ± SD of at least 3 experiments done in triplicate. Black bars indicate results for haplotype I (rs9475431 T, rs9475430 T, and rs9475429 T). Checked bars indicate results for haplotype II (rs9475431 G, rs9475430 C, and rs9475429 A). *P < 0.05, ** P < 0.01, *** P < 0.005, **** P < 1.0 × 10^-6, NS is not significant (Student's t-test).

Figure 3

SNP comparison of rs9475431, rs9475430, and rs9475429 on BMP5 promoter activity. Luciferase reporter assays comparing the mean transcriptional activities of haplotypes containing the T allele of rs9475431 and haplotypes containing the G allele, haplotypes containing the T allele of rs9475430 and haplotypes containing the C allele, and haplotypes containing the T allele of rs9475429 and haplotypes containing the A allele were performed in MG63 cells. Data shown are the mean ± SD. * P = 2.4 × 10^-3. NS is not significant.