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

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.

Finer linkage mapping of a primary hip osteoarthritis susceptibility locus on chromosome 6.

Primary osteoarthritis (OA) is a common late-onset disease that exhibits complex genetic transmittance. A previous genome-wide linkage scan of OA affected sibling pair families (ascertained by total joint replacement surgery) identified a region of suggestive linkage on chromosome 6, with a maximum multipoint-LOD score (MLS) of 2.9 in 194 families containing sibling pairs concordant for total hip replacement (THR-families). However, up to 50 cM of the chromosome had a multipoint-LOD score >2.0, indicating that the susceptibility locus was poorly mapped. We have now genotyped chromosome 6 to a higher density in an expanded cohort of 378 THR-families. We obtained an MLS of 2.8 to an 11.4 cM interval defined by markers D6S452 and 509-8B2, which map between 70.5 to 81.9 cM from the 6p-telomere. Stratification by gender revealed that this linkage was completely accounted for by female THR-families (n=146), with an MLS of 4.0 and with the highest two-point LOD score being 4.6 for marker D6S1573 (75.9 cM). The 11.4 cM interval just encompasses the candidate gene COL9A1 (81.9 cM). We identified and then genotyped twenty common single nucleotide polymorphisms (SNPs) from within COL9A1 in the 146 probands from our female THR-families and in 215 age-matched female controls. No SNP allele, genotype or haplotype demonstrated association to disease. Overall, we have narrowed the chromosome 6 OA susceptibility locus to a point at which linkage disequilibrium/association analysis is feasible, we have demonstrated that this locus is female specific, and found no evidence that COL9A1 encodes for the susceptibility.

Genetic variation including nonsynonymous polymorphisms of a major aggrecanase, ADAMTS-5, in susceptibility to osteoarthritis.

OBJECTIVE: Given the recent characterization of ADAMTS-5 as the main aggrecanase of cartilage destruction in mouse models, we explored whether genetic variation and, in particular, putative damaging polymorphisms in the ADAMTS-5 gene modify susceptibility to osteoarthritis (OA). METHODS: Two likely deleterious nonsynonymous single-nucleotide polymorphisms (SNPs) were identified in ADAMTS-5 by bioinformatics analysis, rs2830585 in exon 5 affecting a thrombospondin 1 motif, and rs226794 in exon 7. Exploration of their role was carried out in 3 steps, discovery, extension, and replication, on samples obtained from 4 European Caucasian collections, comprising a total of 2,715 patients with knee, hip, or hand OA and 1,185 OA-free controls. In addition, 6 tagSNPs were studied to fully evaluate genetic variation in the ADAMTS-5 locus. RESULTS: Initial analyses of 2 sample collections (n = 277 and n = 159) showed a trend toward decreased frequency of the putative deleterious allele of rs226794 among patients with severe knee OA (P = 0.047 versus controls). However, results in patients with knee OA from 2 additional sample collections (n = 360 and n = 265) did not confirm this trend. No association was found with hip OA or hand OA. None of the other SNPs or haplotypes constructed with these SNPs showed a significant association with OA susceptibility. CONCLUSION: Use of several collections of OA samples allowed us to obtain sound evidence against the participation of genetic variation in ADAMTS-5 in OA susceptibility. These results indicate the need to further explore the function of this aggrecanase in human OA to determine whether it is as critical as has been observed in mouse models.

Extreme context specificity in differential allelic expression.

Variability in cis-regulation of gene expression has been implicated in the phenotypic manifestation of complex traits including common, multifactorial diseases. The differential expression of alleles due to polymorphism in cis-regulatory elements is common in the human genome, but there is a paucity of information about the context specificity of these control elements. In this study, we examined the differential allelic expression (DAE) of BMP5 in human mesenchymal tissues obtained from 16 donors undergoing joint replacement for treatment of osteoarthritis. We observed significant differences in BMP5 allelic output, with allelic ratios greater than 4:1 (P < 10(-20)) in the tissues of some donors. We also discovered a significant variability in allelic expression within the different tissues of donors. For 12 of our donors, we examined the allelic expression of BMP5 in two different regions of cartilage: cartilage adjacent to the site of the osteoarthritic lesion and cartilage distal from the lesion. Five of these 12 donors demonstrated highly significant differences (P < or = 10(-8)) in allelic expression between the different regions of their cartilage. Using DAE as a phenotype, we attempted to map tissue-specific cis-regulatory polymorphisms, and we identified a single nucleotide polymorphism located downstream of BMP5, which was significantly associated with DAE in some but not all of the examined tissues. These findings suggest that allelic expression can be highly context specific and that when interrogating the cis-regulatory control of a particular gene, one cannot necessarily assume that allelic expression is conserved across different tissues or even across different regions of the same tissue.

Investigating the aspartic acid (D) repeat of asporin as a risk factor for osteoarthritis in a UK Caucasian population.

OBJECTIVE: A compelling genetic association with osteoarthritis (OA) of 2 functional alleles in the aspartic acid (D) repeat of the asporin gene was recently reported in a Japanese population. Allele D13 of the repeat encoded OA protection, whereas allele D14 encoded OA susceptibility. The 2 alleles mediate differences in the capacity of asporin to inhibit the cartilage growth factor transforming growth factor beta, with the D14 allele being a particularly potent inhibitor. Our objective was to assess whether the D repeat is associated with OA in UK Caucasians. METHODS: The repeat was genotyped in 1,247 patients who had undergone elective joint replacement of the hip or the knee due to end-stage primary OA and in 748 age-matched controls. RESULTS: The D13 allele was more common in controls, and the D14 allele was more common in patients. However, this trend was significant only for men who had undergone hip replacement (P = 0.016, odds ratio 1.48, 95% confidence interval 1.09-2.01). CONCLUSION: Our data suggest that the asporin polymorphism is not a major influence on OA etiology in Caucasians. The results of our study do not question the veracity of the Japanese report. Instead, our study highlights the complex, heterogeneous nature of OA genetic susceptibility.

Association of the interleukin-1 gene cluster on chromosome 2q13 with knee osteoarthritis.

OBJECTIVE: To investigate whether the interleukin-1 (IL-1) ligand gene cluster at 2q13 encodes for genetic susceptibility to primary osteoarthritis (OA). METHODS: Seven single-nucleotide polymorphisms (SNPs) and a variable-number tandem repeat (VNTR) polymorphism from within the IL-1 ligand genes IL1A, IL1B, and IL1RN were genotyped in a cohort of 557 OA cases and 557 age-matched controls. RESULTS: None of the variants demonstrated association in the unstratified data set. However, when cases were stratified according to sex and site of disease (hip or knee), 4 SNPs showed marginal evidence for association (P < 0.1) in knee cases (n = 136) and male knee cases (n = 58). For 2 of these SNPs, evidence for association was enhanced when probands from 60 knee-only affected sibling pair families were genotyped and combined with the original knee cases (P < or = 0.05). Further analysis revealed that the associated alleles at 2 of these SNPs were markers for the same haplotype, the frequency of which was significantly elevated when knee cases and knee probands were combined (P = 0.01, odds ratio [OR] 1.4) and when male knee cases and male knee probands were combined (P = 0.009, OR 1.7). Furthermore, linkage analysis of 2q revealed suggestive evidence for linkage to the IL-1 gene clusters in affected sibling pairs concordant for knee OA but no evidence for linkage in affected sibling pairs concordant for hip OA. CONCLUSION: The IL-1 ligand cluster encodes for susceptibility to knee OA but not to hip OA, highlighting the genetic heterogeneity of this common, complex disease.

Finer linkage mapping of primary hip osteoarthritis susceptibility on chromosome 11q in a cohort of affected female sibling pairs.

OBJECTIVE: To finer linkage-map a primary osteoarthritis (OA) susceptibility locus as a prerequisite to linkage disequilibrium/association analysis. METHODS: A 50-cM interval of chromosome 11q that we had previously identified as harboring susceptibility to hip OA in a female sibling-pair cohort was subjected to finer linkage mapping. Thirty-five microsatellite markers with a mean marker interval of 1.4 cM were genotyped in 146 families containing female sibling pairs who were concordant for hip OA, as ascertained by total hip replacement. RESULTS: Two-point and multipoint linkage analysis revealed 2 distinct regions of linkage within the 50-cM interval. The first locus had a linkage interval of 11.9 cM and was centered at 81.5 cM from the 11p telomere, with a maximum multipoint logarithm of odds (LOD) score of 2.4. The second region had a linkage interval of 6.5 cM and was centered at 93.1 cM from the 11p telomere, with a maximum multipoint LOD score of 1.8. CONCLUSION: Dense linkage mapping has highlighted the presence of 2 loci on chromosome 11q, each conferring susceptibility to hip OA. Both loci are sufficiently narrow for association analysis to be undertaken.

Finer linkage mapping of primary osteoarthritis susceptibility loci on chromosomes 4 and 16 in families with affected women.

OBJECTIVE: To more finely linkage-map primary osteoarthritis (OA) susceptibility loci on chromosomes 4 and 16. METHODS: Two hundred eighteen families, each with 2 or more women concordant for primary OA (ascertained by total hip replacement [THR] or total knee replacement), were genotyped using highly polymorphic microsatellite markers from chromosomes 4 and 16, at an average density of 1 marker every 4 cM. Two-point and multipoint linkage analyses were performed for all 218 families and for the 146 families from the 218 that included women concordant for THR (female-THR families). RESULTS: A single region of linkage was identified on chromosome 4q, with a maximum multipoint logarithm of odds (LOD) score (MLS) of 3.1 in the 146 female-THR families. This locus was centered 79 cM from the 4p telomere and had a 1-LOD support interval of 4 cM. Two regions of linkage were identified on chromosome 16, the first on 16p with an MLS of 1.7 in the female-THR families and the second on 16q with an MLS of 1.9 in all 218 families. The first locus was centered 46 cM and the second 89 cM from the p-telomere. The 1-LOD support intervals were 12 cM and 10 cM, respectively. CONCLUSION: Finer linkage mapping using a high density of microsatellite markers has narrowed female OA susceptibility loci on chromosomes 4 and 16. The regions have been narrowed sufficiently for association analysis.

Microsatellite association mapping of a primary osteoarthritis susceptibility locus on chromosome 6p12.3-q13.

OBJECTIVE: To test a high density of microsatellite markers from within a primary osteoarthritis (OA) locus on chromosome 6 for association with OA as a means of narrowing and focusing our search for the susceptibility gene. METHODS: One hundred forty-six families, each with 2 or more women concordant for primary OA (ascertained by total hip replacement), were genotyped for 36 microsatellite markers from within a narrow interval at 6p12.3-q13 which we had previously shown to be linked to OA. Each marker was tested for linkage and for association, the latter by means of the transmission disequilibrium test and by a case-control analysis. RESULTS: The highest 2-point logarithm of odds (LOD) score was 4.8, with 11 markers having LOD scores > or =2.0. Several markers demonstrated evidence of association, in particular, a cluster of markers positioned within or near the functional candidate gene BMP5. CONCLUSION: Our linkage data reinforce the evidence of a major susceptibility locus on chromosome 6. We had previously failed to detect an association with BMP5 using gene-based single-nucleotide polymorphisms. The association data reported here prompt us to speculate that the chromosome 6 susceptibility may be coded for by cis-acting polymorphism in the regulatory elements of this gene, rather than by variation in its protein coding sequence.

Functional variants within the secreted frizzled-related protein 3 gene are associated with hip osteoarthritis in females.

Osteoarthritis (OA) is a leading cause of disability in Western society with multiple risk factors, including a complex genetic pattern. Identifying loci involved in the heredity of OA might lead to insights into the molecular pathogenesis of this common disorder. A previous genome scan mapped a primary hip OA susceptibility locus to chromosome 2q with a maximum multipoint logarithm of odds score of 1.6 in 378 affected sibling pair families. Here, microsatellite targeting of eight candidate genes in this region from 2q23-2q32 demonstrated significant associations with the tumor necrosis factor alpha-induced protein 6 gene in all probands and the integrin alpha 6 and frizzled motif associated with bone development (FRZB) genes in female probands. However, genotyping showed lack of association for a nonsynonymous single-nucleotide polymorphism in tumor necrosis factor alpha-induced protein 6, whereas a single-nucleotide polymorphism in FRZB resulting in an Arg324Gly substitution at the carboxyl terminus was associated with hip OA in the female probands (P = 0.04). This association was confirmed in an independent cohort of female hip cases (n = 338; P = 0.04). In addition, a haplotype coding for substitutions of two highly conserved arginine residues (Arg200Trp and Arg324Gly) in FRZB was a strong risk factor for primary hip OA, with an odds ratio of 4.1 (P = 0.004). FRZB encodes secreted frizzled-related protein 3, which is a soluble antagonist of wingless (wnt) signaling. Variant secreted frizzled-related protein 3 with the Arg324Gly substitution had diminished ability to antagonize wnt signaling in vitro. Hence, functional polymorphisms within FRZB confer susceptibility for hip OA in females and implicate the wnt signaling pathway in the pathogenesis of this disease.

A novel allelic variant of the human TSG-6 gene encoding an amino acid difference in the CUB module. Chromosomal localization, frequency analysis, modeling, and expression.

Tumor necrosis factor-stimulated gene-6 (TSG-6) encodes a 35-kDa protein, which is comprised of contiguous Link and CUB modules. TSG-6 protein has been detected in the articular joints of osteoarthritis (OA) patients, with little or no constitutive expression in normal adult tissues. It interacts with components of cartilage matrix (e.g. hyaluronan and aggrecan) and thus may be involved in extracellular remodeling during joint disease. In addition, TSG-6 has been found to have anti-inflammatory properties in models of acute and chronic inflammation. Here we have mapped the human TSG-6 gene to 2q23.3, a region of chromosome 2 linked with OA. A single nucleotide polymorphism was identified that involves a non-synonymous G --> A transition at nucleotide 431 of the TSG-6 coding sequence, resulting in an Arg to Gln alteration in the CUB module (at residue 144 in the preprotein). Molecular modeling of the CUB domain indicated that this amino acid change might lead to functional differences. Typing of 400 OA cases and 400 controls revealed that the A(431) variant identified here is the major TSG-6 allele in Caucasians (with over 75% being A(431) homozygotes) but that this polymorphism is not a marker for OA susceptibility in the patients we have studied. Expression of the Arg(144) and Gln(144) allotypes in Drosophila Schneider 2 cells, and functional characterization, showed that there were no significant differences in the ability of these full-length proteins to bind hyaluronan or form a stable complex with inter-alpha-inhibitor.