Objective: Genome-wide studies have identified the chromosomal region 16p13 in the susceptibility to type 1 diabetes (T1D) and multiple sclerosis (MS). This region includes the CLEC16A/KIAA0350 gene and an adjacent gene, MHC2TA (MHC class II transactivator), previously associated with susceptibility to MS and rheumatoid arthritis (RA). The role of CLEC16A polymorphisms in the pathogenesis of T1D, MS and RA and its relationship with the association reported with a MHC2TA haplotype were investigated.
Methods: CLEC16A (rs2903692/rs6498169/rs11074956) polymorphisms were analysed in 435 patients with MS, 316 with T1D and 600 with RA and in 550 ethnically matched controls. The MHC2TA rs3087456G/rs4774C risk haplotype was studied in an independent RA cohort.
Results: rs2903692 conferred a protective effect on patients with T1D, MS and RA. The described association of rs6498169 with MS was replicated in MS and RA cohorts. The effect of the MHC2TA rs3087456G/rs4774C haplotype on RA susceptibility was confirmed, and the haplotype was found to be in negative linkage disequilibrium with the CLEC16A rs2903692A/rs6498169A haplotype.
Conclusions: Associations of CLEC16A polymorphisms with T1D and MS were successfully replicated in a Spanish population. A novel association of rs6498169 with a predisposition to RA was described which is consistent with previous MHC2TA results. These data provide evidence for the influence of variants within this chromosomal region on the development of complex diseases.
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Genome-wide association (GWA) studies have offered exciting findings in the biology of complex disorders. These “hypothesis-free” studies have proved useful in identifying previously unsuspected genes involved in predisposition to disease. Replication of these findings is essential to validate the initial association in independent populations. One GWA study detected a susceptibility locus for type 1 diabetes (T1D) within a 233 kb linkage disequilibrium block on chromosome 16p13 where the CLEC16A/KIAA0350 gene maps.1 This gene encodes a protein of unknown function but of predicted C-type lectin domain structure and, therefore, potentially related to the immune response.2 C-type lectins function both as adhesion and as pathogen recognition receptors.3 Another GWA scan in British patients with T1D showed an association with six chromosomal regions, one of which was 16p13.4 Moreover, another polymorphism in the CLEC16A gene was shown to modulate the predisposition to multiple sclerosis (MS).5 We aimed to investigate whether polymorphisms within this chromosomal region act as genetic predisposition factors for T1D and MS and also for other autoimmune diseases such as rheumatoid arthritis (RA) in our population.
Interestingly, the MHC2TA gene—which maps close to the CLEC16A gene—was recently found to be associated with RA and MS.6 7 MHC2TA encodes the master regulator of MHC class II expression.8 A promoter polymorphism at –168A/G (rs3087456) was originally found which modified the expression and was associated with susceptibility to RA and MS; however, later studies in independent populations were not able to replicate the effect of this SNP as a risk factor for RA.9 Nonetheless, some evidence of an association between MHC2TA and autoimmune diseases has been reported,7 10 11 12 underlining the need for a more complete study of the region.
Two polymorphisms within intron 22 of CLEC16A which were previously found to be associated with either T1D (rs2903692) or MS (rs6498169) were selected for a case-control study. Homozygosity for the first one was found in an NK cell line which presented a higher expression of CLEC16A.1 Two MHC2TA polymorphisms (rs3087456 and rs4774, a missense Ala500Gly at exon 11) delineated a RA risk haplotype in the Spanish population and we attempted to replicate this association with RA in an independent cohort. Moreover, imputation of the available data from the Wellcome Trust Case-Control Consortium13 revealed several polymorphisms within the common 3′-region shared by CLEC16A and SOCS1 genes which increased susceptibility to RA, T1D and Crohn’s disease. We analysed one SNP (rs11074956) in this intergenic region in our cohorts.
The study group consisted of 435 unrelated patients with MS (60% women), 316 with T1D (51% women) and 600 with RA (74% women) consecutively recruited from one centre, either Hospital Clínico San Carlos or Hospital Ramón y Cajal (Madrid).
The diagnosis of MS was made according to the Poser criteria14 and 36% of the patients with MS carried HLA DRB1*1501. Most patients (79%) had a relapsing remitting clinical course, 11% were secondary progressive and 9% were primary progressive patients.
The median age at onset of T1D was 15 years (range 1–55). Patients were diagnosed according to the criteria of the American Diabetes Association and were insulin-dependent at the time of the study. More than 90% of our patients had at least one copy of the class II HLA-DR3 or DR4 allele compared with 45% found in the general population.
The diagnosis of RA was based on the American College of Rheumatology criteria15 and blood samples were previously genotyped for HLA-DRB1. Mean (SD) age at onset was 51 (16) years; 57% of the patients carried the shared epitope; 67% and 50% of the patients were positive for rheumatoid factor and for anticyclic citrullinated peptide, respectively.
A group of 550 healthy unrelated subjects from Madrid (mainly hospital employees and blood donors) were selected as controls. Cases and controls were all white Spanish individuals and were included in the study after giving written informed consent.
To assess new evidence of the association between the MHC2TA susceptibility haplotype and RA disease, we chose to divide the RA patient group into samples already analysed (reported by Martinez et al7) and new patients (n = 230). To add a new control group we made use of non-transmitted alleles from healthy parents of patients with coeliac disease (n = 454). No differences were observed, as expected, when this sample was compared with healthy controls.
The CLEC16A polymorphisms (rs2903692, rs6498169 and rs11074956) were genotyped using pre-designed TaqMan assays in an ABI 7900HT following the manufacturer’s instructions (Applied Biosystems, Foster City, California, USA). The call rate success was over 95%. The MHC2TA polymorphisms were genotyped as described elsewhere.7
Allele and genotype frequencies in patients and controls were compared by the χ2 test using a standard package (Epi Info V.6.02, CDC, Atlanta, USA).
CLEC16A polymorphisms were found to be in Hardy-Weinberg equilibrium in the control subjects (see linkage disequilibrium data in fig 1).
Table 1 shows the frequencies of these polymorphisms in our Spanish cohorts. In agreement with the previously described association with T1D,1 the effect of rs2903692 was replicated in T1D and MS, and there was a trend for an association with RA. For rs6498169, which was originally reported to be associated with MS,5 this association was replicated in MS but no association was seen in patients with T1D. Moreover, there was evidence of an association of rs6498169 with susceptibility to RA. No differences were observed after stratification by the presence of either shared epitope (SE+ vs SE−, rs2903692: 124:142:39 vs 81:116:234 and rs6498169: 51:132:109 vs 32:91:99) or anticyclic citrullinated peptides (anti-CCP+ vs anti-CCP−, rs2903692: 97:126:32 vs 89:111:42 and rs6498169: 36:109:101 vs 35:97:98).
The rs11074956 polymorphism located in the 3′ region common to CLEC16A and SOCS1 genes was analysed and no association with MS, T1D and RA was detected in our cohorts (table 1).
We previously described the association of a haplotype (rs3087456G/rs4774C) along the adjacent MHC2TA gene with RA7 and aimed to replicate this finding in a new population of patients with RA and controls. This independent replication (OR 1.93 (95% CI 1.1 to 3.41), p = 0.014) confirmed the previous findings with MHC2TA (see table 1 in online supplement). Finally, we found the CLEC16A rs2903692A/rs6498169A haplotype to be in negative linkage disequilibrium with the MHC2TA haplotype associated with RA susceptibility in our population (MHC2TA GC susceptibility haplotype extended with either the protective CLEC16A AA haplotype or with the other remaining CLEC16A haplotypes: 2% (8/409) vs 7% (39/567); p = 0.0004).
Recently performed genome-wide searches for susceptibility loci have provided new insights into disease pathogenesis. Lectins modulate innate and adaptive immune responses under physiological or pathological conditions, and the CLEC16A gene contains a lectin domain. In agreement with the GWA study conducted in patients with MS,5 rs6498169 was strongly associated with MS in our population; moreover, the same size effect was also detected for the other polymorphism originally reported to affect T1D susceptibility.1 Our work therefore validates these CLEC16A polymorphisms (or others in strong linkage disequilibrium with them) as modulators of MS risk. Recently, two independent studies found an association between several CLEC16A polymorphisms and T1D,1 4 which was also seen in our cohort for rs2903692. Certainly, the studied polymorphisms are not genetically equivalent (fig 1), so the observed lack of association between T1D and rs6498169 could be due to a lesser degree of linkage disequilibrium with the aetiological variant. We report here the identification of the rs6498169 polymorphism within the CLEC16A gene as a modifier of RA risk.
The transactivator of MHC class II genes, MHC2TA, maps close to the CLEC16A gene. Both genes are putative good candidates to be involved in the predisposition to autoimmune diseases. The role of a MHC2TA promoter polymorphism in RA and MS susceptibility was previously reported,6 although replication studies in independent populations yielded inconsistent results which might be explained by the possible distinct linkage disequilibrium with the aetiological variant in different populations. In our cohorts, the study of haplotypes within the MHC2TA gene showed an association indicative of the importance of this region for MS and RA susceptibility,7 which has now been corroborated in an independent RA cohort. We are not able to elucidate whether any of the studied polymorphisms is the primary signal for the association found with this region, or whether several polymorphisms participate independently to increase the risk.
Moreover, the intergenic region between CLEC16A and SOCS1 showed a common signal for association with RA, T1D and also with Crohn’s disease in the Wellcome Trust genome-wide study.13 However, no similar effect could be found in our population for rs11074956. Multiethnic studies will contribute to a better definition of the variants involved in the observed effect of this region, as they have previously helped to clarify which variants are truly causal for other common diseases.
Replication of GWA findings is essential to distinguish an artefact from a true positive result. Our work lends support to the consideration of this 16p13 chromosomal region as carrying genetic susceptibility factors for autoimmune diseases. Interestingly, the genome-wide study performed by the Wellcome Trust Consortium13 showed a strong signal for this chromosomal region in T1D, but seemingly a weaker effect in RA. In our Spanish population, maybe due to the lower statistical power of the diabetic cohort or to population heterogeneity, the difference was not so evident.
Even though modest effects are found, a complete picture is required to gain insight into the disease pathogenesis. Targets for the pursuit of therapies will hopefully be provided by confirmation in multiple studies of the contribution to the risk of disease of genome-wide discoveries.
The authors thank Carmen Martínez for her technical assistance.
▸ Additional data are published in table 1 in the online supplement at http://ard.bmj.com/content/vol69/issue1
Funding This work was supported by grants from Fundación Mutua Madrileña, FIS PI07/0369, FIS PI07/0353, FIS 04/1691. AM and JLS hold research FIS contracts (CP04/00175 and CM05/00216, respectively). NP holds a grant from Ministerio de Educación y Ciencia, JV is a fellow and EU works for the Fundación para la Investigación Biomédica-Hospital Clínico San Carlos.
Competing interests None.
Ethics approval Cases and controls gave written informed consent and the ethics committee of Hospital Clínico (Madrid) approved the study.
Provenance and Peer review Not commissioned; externally peer reviewed.