ANKHvariants associated with ankylosing spondylitis: gender differences

Ankylosing spondylitis (AS) is a disorder that results in chronic joint and entheseal inflammation, and ankylosis of axial and peripheral joints. It affects approximately 0.1–0.8% of Caucasians [1]. The disease usually begins in young adulthood and can be associated with chronic pain and significant disability. AS is strongly associated with HLA-B27 [2], but analyses of recurrence risk among family members [3] suggest that at least three other genetic loci in addition to HLA-B27 are required to confer full susceptibility to AS. However, genome-wide linkage studies have detected very few strongly linked non-major histocompatibility complex (MHC) loci [4‐6], implying that non-MHC susceptibility loci have small effects and/or that heterogeneous sets of loci combine with HLA-B27 to confer susceptibility to AS. This complexity highlights the strategic advantage of testing predetermined candidate genes. In addition, although several chromosomal regions showed potential linkage in several genome-wide linkage studies conducted in AS families [5, 6], the identities of the predisposing genes in these regions remain largely unknown.

Normal osteogenesis depends critically on maintaining the physiological level of inorganic pyrophosphate (PPi). Abnormal PPi levels can be associated with aberrant bone formation. PPi export from the cell is regulated by the ANK protein [7], and mutant mice (ank/ank), which have a premature stop codon in the 3' end of the ank gene, develop severe ankylosis. As a first step in testing the hypothesis that specific polymorphisms in the ANKH gene might contribute to AS susceptibility, we previously reported the identification of two novel polymorphic sites, one in the 5' noncoding region (ANKH-OR) and the other in the promoter region (ANKH-TR), of ANKH [8]. These two marker alleles are in complete linkage disequilibrium (LD). Our results from a linkage analysis of 124 North American AS families [8] indicated that AS is genetically linked to ANKH, and the locus-specific sibling recurrence risk of ANKH to AS susceptibility (λ_S) is 1.9 (λ_S for HLA-B27 is 5.2). Our family-based association analysis on the same families [8] showed that AS is modestly associated with ANKH-OR allele 1 (additive model: P = 0.03). Because of insufficient numbers of informative families, our results did not allow us to distinguish between different modes of inheritance. In addition, our analyses were focused on the 5' end of the gene, using only two markers. For these reasons, we have now carried out fine mapping of the complete ANKH region, including not only the AS families used in the previous study but also an additional 77 multiplex AS families (a total of 201 multiplex AS families).

The prevalence of AS is 2.5 times higher in men than in women [9]. Extensive fusion of the spine is a phenotype of the mouse model ank. There has been a clinical impression that radiographic severity (e.g. the bamboo spine) may be relatively less common in affected women than in men [10‐13]. It has also been observed that long-term outcome in AS is worse in men than in women [14, 15], but the basis for this difference in severity of clinical expression remains unclear. It is unlikely that the major genetic factors that account for these differences are X-linked because there is no linkage of AS susceptibility with X-chromosome markers [16]. Gender also has a significant impact on heritability in AS. AS has a higher prevalence in the offspring of women than men with AS, and sons of men with AS are 2.5 times more likely than daughters to inherit the disease [17, 18]. It remains unclear whether there is gender heterogeneity in non-MHC loci that confer susceptibility to AS. In the present study, we asked whether there is any gender difference in the association of ANKH with AS in multiplex families.

In this study of the association of ANKH genetic markers with AS, including 201 AS multiplex families, we found that ANKH variants located in two different regions of the ANKH gene were associated with AS. A more striking finding was that the genetic association for men and women with AS differed. In men, AS was associated with genetic markers at the 3' end of the ANKH gene, whereas in women AS appeared to be associated with genetic markers at the 5' end of the ANKH gene. As expected, when the genders of AS patients were analyzed separately, we observed more than one SNP in each region (within the same haplotype block) showing significant association with AS. Haplotype analyses appeared to confirm the results of the single-marker tests (FBAT analyses), indicating that the predisposing polymorphism(s) for men with AS probably lies at the 3' end of the ANKH gene, whereas those for affected women are probably at the 5' end of the gene. After Bonferroni correction for the number of haplotypes and models, the haplotype combination of rs26307 [C] and rs27356 [C] was significantly associated with men with AS; and the haplotype combination of rs28006 [C] and rs25957 [C] was significantly associated with women with AS. However, in both cases, the significance level was modest. We attribute this to the fact that we have not identified the aetiological variants in the men/women with AS. Despite the modest P values (which are a function of sample size), the calculated 'odds ratios' for increased risk (which provide estimates of the magnitude of the effect) were close to 2 for the transmission of rs26307 [C] rs27356 [C] to affected men, and close to 3 for the transmission of rs28006 [C] rs25957 [C] to affected women in the subset of families with affected individuals of both genders.

The rationale for studying multiplex AS families is to enhance the chances of identifying the genes involved. There are very few studies that directly compare familial versus sporadic AS. In one study [26], familial versus sporadic Dutch AS patients exhibited no difference in age at disease onset, age at diagnosis, or prevalence of peripheral arthritis and acute anterior uveitis. In another study, familial AS disease was significantly milder than sporadic disease, as assessed by spinal mobility score, Arthritis Impact Measurement Scales (AIMS) overall impact score, AIMS physical activity score, AIMS social function score and AIMS pain score [27]. Thus, findings from multiplex families might not be directly applicable to individuals affected with sporadic AS. Most studies assessing the impact gender has on age at AS onset or diagnosis have been conducted without addressing whether the individuals had familial or sporadic disease [28‐30]; these studies showed that the age at disease onset is similar between genders. However, in our cohort of AS multiplex families, men had a significantly earlier age at diagnosis compared with that for women (for men 28 ± 11 years [n = 213]; and for women 30 ± 11 years [n = 149]). Because these are AS multiplex families, it is unlikely that there is a bias leading physicians to delay diagnosis in affected women. The misconception that AS is exclusively a male disease may yet be a confounding factor. In the subset of families with affected individuals of both genders, men have an even earlier age at diagnosis (27.8 ± 11 years [n = 94]) compared with that for women (32.6 ± 11 years [n = 101]), whereas both men and women have similar ages of diagnosis in the subsets of families with affected individuals of only one gender (for men 28.6 ± 12 years [n = 130]; for women 29.9 ± 12 years [n = 53]). This finding suggests that there is heterogeneity even in multiplex AS families.

The ANKH variants that were significantly associated with AS are located in introns 1, 8 and 12. It is likely that the predisposing polymorphisms affect gender-specific regulation of ANKH expression. Very little is known regarding the molecular mechanisms that underlie the regulation of ANKH expression. One study [31] reported that ANKH is a growth factor responsive gene. Three recent reports [22‐24] showed that ANKH is an androgen responsive gene. In androgen-treated prostate cancer cell lines, the abundance of ANKH transcripts was sixfold higher than in the untreated cells. In the ANKH promoter, there is a sequence at position -1015 (AGAACAcacTtTcCT) with 83% match to an androgen response element (ARE) consensus sequence [22]. It remains unclear whether this ARE-like motif is functional. In view of the locations of the ANKH variants associated with AS, it remains unclear whether this ARE-like motif at the promoter region can directly contribute to the regulation of ANKH expression by the predisposing polymorphisms. It is also unknown whether there is a different mode of ANKH regulation in women.

A report recently concluded that ANKH did not significantly contribute to susceptibility or specific disease expression in AS patients from the UK [32]. In that report, a case–control study was conducted using five ANKH SNPs within the coding region and flanking splice sites and three known promoter variants. There was no association between these polymorphisms and AS or the clinical pattern of the disease. In addition, using 185 affected sib-pair AS families, no linkage between ANKH and AS was observed. However, the exact linkage results were not shown. Using multipoint exclusion mapping of the ANKH region, the presence of a gene contributing more than 10% of the recurrence risk to AS (λ_S = 1.4) was excluded. Using λ_S of 1.4 as the cutoff may exclude genes with modest effects. In that report, the LD between markers was not shown. In situations where the aetiological variant is not typed, haplotype-based analysis may be a more powerful analytical method when there is significant LD.

The basis for the discrepancy between the UK results [32] and ours is not entirely clear, but there are several possible explanations. First, the UK group focused on analyzing exonic variants, variants near splice junctions and in the promoter region. Their analysis did not include any ANKH variants in the 3' region, where we detected association with AS in men. Second, although the UK group included a gender breakdown of their patients (63.5% men and 36.5% women), the analysis did not include a breakdown of AS patients by gender, and variants with modest gender-specific effects might have been missed. Third, it is possible that there are some intrinsic differences between the two populations (UK versus North American). Genome-wide linkage scans performed in the two groups revealed some similar susceptibility regions, such as on chromosomes 6p (the MHC), 5q and 10q [5, 6]. However, the linkage identified on chromosome 11q23 in the North American Spondylitis Consortium families was not seen in the UK study. In addition, the linkage identified on chromosome 2q in the UK study was not seen in the North American Spondylitis Consortium study. The intrinsic differences could reflect clinical differences in the patient population recruited, or they could be due to population-specific mechanisms of genetic susceptibility. Finally, because both groups analyzed about 200 AS families, there might not be sufficient power to detect genes with 'small effects' consistently, leading to discrepancies between results.

In our cohort of North American multiplex AS families, the age at diagnosis was significantly younger in men than in women. However, FBAT analyses using the offset option (-o; an option which works for both quantitative and qualitative traits) did not show any significant association of age at diagnosis in the men or women with AS, even in subsets of families, using the ANKH markers (data not shown), suggesting that ANKH variants are responsible for disease susceptibility. Our finding of gender-specific polymorphisms in the ANKH gene conferring differential susceptibility to AS might shed light on the biological basis of these clinical observations.

In view of the difficulty in locating susceptibility loci with modest effects in recent genome-wide linkage studies conducted in AS families, it will be of interest to assess whether gender subsetting in the analyses of genome-wide linkage studies might yield further insights into the genetic basis of rheumatic diseases, many of which have a strong gender predilection.

Taken together, our findings showed that, after Bonferroni correction, two intronic markers at the 3' end of the ANKH gene were significantly associated with AS only in affected men, and two intronic markers at the 5' end of the ANKH gene were significantly associated with AS only in affected women. This may partly account for the gender difference in the prevalence of AS.