Procedimiento de demarcación nacional indígena (comunidad)

To attempt to further elucidate the associations found, a haplotype analysis was performed using the data for the IL4R gene in the Leiden EAC cohort. This resulted in 9 haplotype blocks with a minor haplotype frequency of >0.01 (results available from the correspond- ing author upon request). All haplotypes with a prevalence of >0.1 were tested for an association with joint destruction in the EAC cohort. Because of this cutoff, no haplotypes that included rs119132 were evaluated. Two haplotypes of one haplotype block showed a better association than the independent SNPs (AAA and CGG, consisting of the follow- ing SNPs: rs1805011, rs1805015, and rs1801275, respectively). These haplotypes were analyzed in the replication phase, and no association with the progression of joint damage was observed.

Finally, rs1119132 and rs1805011 were studied in anti–citrullinated protein antibody (ACPA)–positive and ACPA-negative patients separately. This yielded comparable effect sizes in both subgroups (data not shown).

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The severity of RA is reflected by the severity of radiologic joint destruction. It is highly variable between patients, and part of this variance is explained by genetic factors. Several studies of IL-4 at the protein level have suggested that this interleukin is relevant in RA.3-6 _In

addition, several genetic studies of IL4 and IL4R and joint damage have been performed,7-10

though none of the factors identified have been replicated. This prompted us to perform the present multi-cohort candidate gene study. We observed that patients carrying 2 minor alleles of rs1119132 in IL4R had more severe joint damage progression. Although a minor- ity of RA patients may have this genetic variant, individual independent replication was found in some of the replication cohorts as well as in the meta-analysis of the 6 replication cohorts.

In addition to the results for rs1119132, another SNP in IL4R, rs1805011, showed an association with joint destruction. In a conditional analysis including both SNPs, both re- mained significantly associated with the progression of joint damage. These SNPs were in low linkage disequilibrium. Since, after applying the conservative Bonferroni correction for

rs1805011 0 1 2 3 4 5 6 7 0 10 20 30 40 50 AA (n=4) AG (n=111) GG (n=440) P = 0.01 A Time (years) SH S (m ed ia n) rs1119132 0 1 2 3 4 5 6 7 0 10 20 30 40 50 AA (n=14) AG/GG (n=561) P = 0.04 Time (years) SH S (m ed ia n) rs1805011 0.8 1.0 1.2 1.4 Total NARAC Sheffield Lund Groningen 1.06 (1.01-1.11) 0.62 1.00 (0.93-1.08) 0.24 1.03 (0.91-1.16) 0.09 1.12 (0.96-1.30) 0.06 weight beta (95%CI) B P = 0.02 rs1119132 0.5 1.0 1.5 2.0 2.5 Total Sheffield NARAC NDB Lund Groningen Wichita 1.26 (1.11-1.42) 0.48 0.97 (0.85-1.12) 0.35 1.43 (1.10-1.85) 0.10 0.99 (0.66-1.49) 0.04 1.70 (0.95-3.02) 0.02 0.79 (0.34-1.81) 0.01 weight beta (95%CI) P = 0.001

Figure 1 Results of rs1805011 and rs1119132 in the discovery phase (A) and replication phase (B). A. Presented are the median Sharp van der Heijde scores (SHS) over 7-years of follow-up, per genotype in RA-patients of the EAC. The ‘bump’ in the line at year five is caused by missing radiographs of part of the patients with rs1119132 genotype AA.

B. Presented is an inverse variance weighted meta-analysis in four cohorts (rs1805011) and six co- horts (rs1119132). In the NARAC, Wichita and NDB cohort a proxy SNP for rs1119132 was analyzed; rs1859308 (r2_{=0.92). Genotyping data of rs1805011 were not available for the Wichita and NDB cohort.}

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multiple testing only rs1119132 remained significant, we did not draw a definite conclu- sion regarding rs1805011.

SNP I50V (rs1805010) in IL4R was previously found to be associated with joint destruc- tion in RA.8 _{Despite studies of the potential functional relevance of this SNP,}8,20 _{the associa-}

tion of this variant with joint damage was not observed in the study by Marinou et al9 _or

in our study. Another coding variant on IL4R, Q551R (rs1801275), was not associated with joint destruction in prior studies.7-10 _{In the present study, IL4R Q551R was significantly as-}

sociated with joint destruction in the discovery cohort (P = 0.01) but not in the replication phase (P = 0.21).

Despite previous in vitro studies and mouse studies showing that IL-4 plays a role in suppressing arthritis severity, in the present study no association between SNPs in IL4 and joint destruction were observed. An association between IL4 VNTR has been reported previously.7 _{This variant was not included in our study.}

We used the classic candidate gene approach, including immune response factors that had previously been shown to be involved in RA pathogenesis. This method has a larger a priori chance of finding a true association between SNPs and disease severity. However, this approach may also result in false-positive or false-negative findings. We studied 6 replication cohorts in order to reduce the chance of false-positive findings.

In conclusion, we identified and replicated a genetic variant in IL4R predisposing to joint damage progression in RA. Further studies of IL-4R at the protein level are needed to increase insight on the role of this variant in the pathogenesis of RA progression.

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ReFeRenCe list

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