Genetic Analysis with the Immunochip Platform in Behçet Disease. Identification of Residues Associated in the HLA Class I Region and New Susceptibility Loci

Abstract

Behcet's disease (BD) is an immuno-mediated vasculitis in which knowledge of its etiology and genetic basis is limited. To improve the current knowledge, a genetic analysis performed with the Immunochip platform was carried out in a population from Spain. A discovery cohort comprising 278 BD cases and 1,517 unaffected controls were genotyped using the Immunochip platform. The validation step was performed on an independent replication cohort composed of 130 BD cases and 600 additional controls. The strongest association signals were observed in the HLA class I region, being HLA-B*51 the highest peak (overall P = 6.82E-32, OR = 3.82). A step-wise conditional logistic regression with classical alleles identified HLA-B*57 and HLA-A*03 as additional independent markers. The amino acid model that best explained the association, includes the position 97 of the HLA-B molecule and the position 66 of the HLA-A. Among the non-HLA loci, the most significant in the discovery analysis were: IL23R (rs10889664: P = 3.81E-12, OR = 2.00), the JRKL/CNTN5 region (rs2848479: P = 5.00E-08, OR = 1.68) and IL12A (rs1874886: P = 6.67E-08, OR = 1.72), which were confirmed in the validation phase (JRKL/CNTN5 rs2848479: P = 3.29E-10, OR = 1.66; IL12A rs1874886: P = 1.62E-08, OR = 1.61). Our results confirm HLA-B*51 as a primary-association marker in predisposition to BD and suggest additional independent signals within the class I region, specifically in the genes HLA-A and HLA-B. Regarding the non-HLA genes, in addition to IL-23R, previously reported in our population; IL12A, described in other populations, was found to be a BD susceptibility factor also in Spaniards; finally, a new associated locus was found in the JRKL/CNTN5 region.

Fig 1. Workflow diagram summarising the methodological process of this study.

*The control set was obtained from [17].

Fig 2. Manhattan plot representation of the Immunochip results.

The–log10 of the combined logistic regression test P-values are plotted against its physical chromosomal position. The red line indicate the genome-wide level of significance (P<5x10E-08). The most relevant associations are highlighted. The red dots represent the overall results of IL12A and rs2848479 after the validation phase.

Fig 3. Manhattan plot representation of the Omnibus test on amino acid positions.

A) Unconditioned test. B) Omnibus test conditioning on position 97 of HLA-B. C) Omnibus test conditioning on positions 97 and 66 of HLA-B and HLA-A, respectively. The–log10 of the combined logistic regression test P-values are plotted against the physical chromosomal positions of the centre of codon. The red line represents the threshold for statistical significance (P<3.91E-04).

Fig 4

Ribbon representation of the HLA molecules HLA-B*5101 (A) and HLA-A*0301 (B). The independently associated amino acid positions with Behçet’s disease are highlighted in red. A mesh representation of the HIV immunodominant epitope KM1 and a proteolipid protein peptide are shown in HLA-B*5101 and HLA-A*0301, respectively, according to Protein Data Bank entries 1E27 [46] and 2XPG [37].