University of Oxford, Institute of Molecular Medicine, Oxford,
1 Nuffield Orthopaedic Centre, Oxford,
2 Musgrave Park Hospital, Belfast, UK and
3 Departments of Biomathematics and Biostatistics, University of California, Los Angeles, USA
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Abstract |
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Objective. To test chromosome 2q for linkage to idiopathic osteoarthritis.
Methods. Using a cohort of 481 OA families that each contained at least one affected sibling pair with severe end-stage disease (ascertained by hip or knee joint replacement surgery), we conducted a linkage analysis of chromosome 2q using 16 polymorphic microsatellite markers at an average spacing of one marker every 8.5 cM.
Results. Our results provide suggestive evidence for a locus at 2q31 with a maximum multipoint logarithm of the odds score (MLS) of 1.22 which increased to 2.19 in those families concordant for hip-only disease (n = 311). This suggestive linkage was greater in male-hip families (MLS = 1.57, n = 71) than in female-hip families (MLS = 0.71, n = 132).
Conclusions. Chromosome 2q is likely to contain at least one susceptibility locus for OA.
KEY WORDS: Osteoarthritis, Linkage, Affected sibling pairs, Chromosome 2q, Susceptibility loci
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Introduction |
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Patients and methods |
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Markers and genotyping
The microsatellite markers used to provide coverage of chromosome 2q were obtained from the Genome Data Base (http://www.hgmp.mrc.ac.uk/gdb/gdbtop.html) or from the ABI Prism Linkage Mapping Set (Version 2, PE Applied Biosystems, http://www.perkin-elmer. com/ab). The markers were amplified with either the forward or the reverse primer in a polymerase chain reaction (PCR) pair fluorescently labelled. The amplification products were electrophoresed through 6% acrylamide using an Applied Biosystems 377 Automated DNA Sequencer [9]. Alleles were sized using PE Applied Biosystems Genescan version 2.0.2 and Genotyper version 1.1 software. All markers were monitored for genotyping errors with independent typing by a second individual.
Linkage and stratification analysis
Error checking of the data, single-point linkage analysis (using the ANALYZE package), multipoint linkage analysis (using the ASPEX program) and stratification [by sex, by joint replaced (hip or knee) and by sex combined with joint replaced] were performed as described previously [8].
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Results |
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Finer mapping of 2q
The evidence of suggestive linkage for two adjacent markers at P < 0.01, combined with the previous reports of linkage of OA to 2q, prompted us to genotype the stage 2 families for seven other chromosome 2q markers from our original marker set that flanked D2S202 and D2S72. In addition we genotyped seven new chromosome 2q markers for all 481 families. Multipoint analysis gave a maximum multipoint LOD score (MLS) of 1.22 near to marker D2S72 (Fig. 1).
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Overall, our single-point and multipoint analyses provide suggestive evidence for linkage of OA to chromosome 2q. This is the third report indicating that 2q may harbour an OA susceptibility gene.
Stratification
Epidemiological, segregation and twin pair studies have suggested that the genetic contribution to OA differs between males and females [1, 4, 11]. Furthermore, differences in the heritability values between joint groups have been reported [3, 12, 13]. Overall, these studies have contributed to the hypothesis that there may be genetic heterogeneity of OA between the sexes and between different joint groups. We therefore stratified our results for chromosome 2q by six strata: those families that were affected females-only (196 families), affected males-only (102 families), hips-only (male and/or female) (311 families), knees-only (male and/or female) (54 families), affected females-only who had undergone hip replacement but not knee replacement (female-hip pairs) (132 families) and affected males-only who had undergone hip replacement but not knee replacement (male-hip pairs) (71 families). We did not stratify for female-knee or male-knee as the number of families was too low (21 and eight, respectively) to allow reliable inference of linkage.
The following MLS values were obtained for the six strata tested: 0.55 for females-only between D2S2330 and D2S326, and 0.96 for males-only between D2S117 and D2S202 (Fig. 2A and Table 1
), 2.19 for hips-only between D2S117 and D2S202, and <0.50 for knees-only near to D2S364 (Fig. 2B
), 0.71 for female-hips between D2S325 and D2S157, and 1.57 for male-hips between D2S117 and D2S202 (Fig. 2C
). These results suggest that hips-only pairs are the major contributing strata to the suggestive linkage on 2q. There were much greater numbers of hips-only pairs than knees-only pairs (311 vs 54) and this could account for our inability to detect linkage in the knees-only strata. However, when the unstratified multipoint plot and the hips-only multipoint plots are compared (Figs 1
and 2B
), the hips-only stratum is more significant across the chromosome. This suggests that the linkage in the unstratified families is being obscured, presumably due to the presence of knees-only pairs, or hip and knee pairs. Of our 481 families, 65 pairs were of mixed joint replacement status (one sibling had undergone hip replacement only, the second sibling knee replacement only). These 65 mixed joint replacement pairs were not one of the original six strata tested. The highest MLS in these 65 pairs was 0.41 between D2S335 and D2S326; there was no peak between D2S117 and D2S202 (data not shown). These results suggest that the linkage on 2q is principally accounted for by hip disease.
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In conclusion, this stratification analysis indicates that the suggestive linkage to 2q is principally accounted for by hip-only disease and that this hip-only linkage may be more pronounced in males than females.
Nodal OA
The families used by Wright et al. [6] and Leppavuori et al. [7] were ascertained by OA of the hand. Of our 481 families, 46 affected pairs (from 46 families) were concordant for the presence of three or more Heberden's nodes. When these families were analysed, none of the 16 chromosome 2 markers were significant at P 0.05, with the lowest P-value being 0.13 for D2S72. The MLS was 0.19 between D2S139 and D2S160 (data not shown). Our results do not therefore support a restriction of the suggestive 2q locus to hand OA.
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Discussion |
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The factor that two adjacent markers supported moderate linkage in both stages suggests that our result for 2q is robust: increasing the number of families genotyped increased the evidence for linkage at these markers.
For genes that have only moderate effects, independent confirmation is essential to confirm true as opposed to false positive susceptibility status. When our data are considered in the context of the reports of Wright et al. [6] and Leppavuori et al. [7], there are reasonable grounds for assuming that 2q may contain an OA susceptibility locus.
Since there is evidence to suggest that the heritability of OA varies between the sexes and between different joint groups, these strata merited independent analysis. Stratification of our 2q data highlighted a substantial difference between families that have hip OA and those that have knee OA. These results, if confirmed in additional studies, could provide biological clues as to the nature of the suggestive susceptibility on 2q which might assist in the choosing of candidate genes.
The average density of the 2q markers used is one marker every 8.5 cM but these markers are not equally distributed. The largest gap is 23 cM, between D2S139 and D2S160. A finer linkage map of this chromosome is therefore important to quantify the nature of the susceptibility on this chromosome more accurately and to determine whether the troughs that are clearly evident in the multipoint analyses are real or simply reflect low marker density, a lack of power to detect linkage in these regions, or both. If real, these may indicate that this chromosome harbours more than one OA susceptibility locus.
As pointed out by Leppavuori et al. [7], 2q12-q22 contains several interleukin and interleukin-associated genes. The expression of certain interleukin genes, including IL-1ß, is altered in OA joint tissue [15]. Furthermore, interleukins regulate a number of enzymes that degrade the cartilage extracellular matrix, including metalloproteinases [16]. The interleukin cluster is therefore a logical candidate for OA susceptibility.
Although OA is primarily characterized by the degeneration of articular cartilage, one finding that is commonly observed is an increase in the density and mass of the subchondral bone below the articulating cartilage [17]. This has led to the suggestion that increased bone mass precedes any gross changes and that cartilage loss is secondary to this change. Under this assumption, genes that are determinants of bone integrity or bone mass can be considered candidates for OA susceptibility loci. Type V collagen is a minor constituent of bone where it forms heterotypic fibrils with the major bone collagen, type I. The type V collagen gene COL5A2 maps within 1 cM of D2S152. Although this section of 2q is part of a trough between two of our linkage regions, COL5A2 cannot be excluded as a susceptibility locus until finer mapping of this region has been completed. The parathyroid hormone receptor 2 (PTHR2) gene is located approximately 6 cM distal to D2S202/D2S72 at 2q33 [18]. PTHR2 is predominantly expressed in the brain where it is thought to bind parathyroid hormone [19]. Parathyroid hormone is a regulator of calcium and phosphate homeostasis and is therefore important for bone integrity. PTHR2 should therefore also be considered as a candidate.
Overall, the results presented here, together with those reported previously, indicate that an OA susceptibility locus may reside on chromosome 2q. With an MLS of 2.19 in our hip pairs, this linkage should be described as suggestive at this stage. More detailed linkage analysis with additional markers and additional families, combined with association analysis, will define this locus further.
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Acknowledgments |
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Notes |
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References |
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