Genome-wide association study in an admixed case series reveals IL12A as a new candidate in Behçet disease

The etiology of Behçet's disease (BD) is unknown, but widely considered an excessive T-cell mediated inflammatory response in a genetically susceptible host. Recent genome-wide association studies (GWAS) have shown limited number of novel loci-associations. The rarity and unequal distribution of the disease prevalence amongst different ethnic backgrounds have hampered the use of GWAS in cohorts of mixed ethnicity and sufficient sample size. However, novel statistical approaches have now enabled GWAS in admixed cohorts.We ran a GWAS on 336 BD cases and 5,843 controls. The cases consisted of Western Europeans, Middle Eastern and Turkish individuals. Participants from the Generation R study, a multiethnic birth cohort in Rotterdam, The Netherlands were used as controls. All samples were genotyped and data was combined. Linear regression models were corrected for population stratification using Genomic Principal Components and Linear Mixed Modelling. Meta-analysis was performed on selected results previously published.We identified SNPs associated at genome-wide significant level mapping to the 6p21.33 (HLA) region. In addition to this known signal two potential novel associations on chromosomes 6 and 18 were identified, yet with low minor allele frequencies. Extended meta-analysis reveal a GWS association with the IL12A variant rs17810546 on chromosome 3.We demonstrate that new statistical techniques enable GWAS analyses in a limited sized cohort of mixed ethnicity. After implementation, we confirmed the central role of the HLA region in the disease and identified new regions of interest. Moreover, we validated the association of a variant in the IL2A gene by meta-analysis with previous work. These findings enhance our knowledge of genetic associations and BD, and provide further justification for pursuing collective initiatives in genetic studies given the low prevalence of this and other rare diseases

Genome-wide association study in an admixed case series reveals IL12A as a new candidate in Behçet disease

INTRODUCTION: The etiology of Behçet's disease (BD) is unknown, but widely considered an excessive T-cell mediated inflammatory response in a genetically susceptible host. Recent genome-wide association studies (GWAS) have shown limited number of novel loci-associations. The rarity and unequal distribution of the disease prevalence amongst different ethnic backgrounds have hampered the use of GWAS in cohorts of mixed ethnicity and sufficient sample size. However, novel statistical approaches have now enabled GWAS in admixed cohorts.METHODS: We ran a GWAS on 336 BD cases and 5,843 controls. The cases consisted of Western Europeans, Middle Eastern and Turkish individuals. Participants from the Generation R study, a multiethnic birth cohort in Rotterdam, The Netherlands were used as controls. All samples were genotyped and data was combined. Linear regression models were corrected for population stratification using Genomic Principal Components and Linear Mixed Modelling. Meta-analysis was performed on selected results previously published.RESULTS: We identified SNPs associated at genome-wide significant level mapping to the 6p21.33 (HLA) region. In addition to this known signal two potential novel associations on chromosomes 6 and 18 were identified, yet with low minor allele frequencies. Extended meta-analysis reveal a GWS association with the IL12A variant rs17810546 on chromosome 3.DISCUSSION: We demonstrate that new statistical techniques enable GWAS analyses in a limited sized cohort of mixed ethnicity. After implementation, we confirmed the central role of the HLA region in the disease and identified new regions of interest. Moreover, we validated the association of a variant in the IL2A gene by meta-analysis with previous work. These findings enhance our knowledge of genetic associations and BD, and provide further justification for pursuing collective initiatives in genetic studies given the low prevalence of this and other rare diseases.</p

SNP association plot for Behçet’s susceptibility-associated region of Chromosome 18q22.3.

<p>Dots represent GWAS P-values (EMMAX approach) and positions of SNPs found within the 18q22.3 locus. The top SNP, i.e. rs17087141, is denoted by a diamond. Different colours indicate varying degrees of pair-wise linkage disequilibrium (1000Genomes CEU) between the top SNP and all other genotyped SNPs. Genetic coordinates are per 1000 Genomes Nov 2010-CEU. Bottom, LD heat map based on D’ values from the combined population under study including all SNPs in the 500Kb region.</p

Behcet GWAS results using Linear Mixed Models Genomic approach.

<p>Each dot represents an SNP in the dataset. QQ-plot (left). Associated SNPs deviating from the null hypothesis of no association (identity line). Manhattan plot (right). SNPs showing association with the disease map to two different signals in chromosome 6 and a singleton in chromosome 18.</p

SNP association plot for Behçet’s susceptibility-associated region of chromosome 6q25.3.

<p>Dots represent GWAS P-values (EMMAX approach) and positions of SNPs found within the 6q25.3 locus. The top SNP, i.e. rs8187722, is denoted by a diamond. Different colours indicate varying degrees of pair-wise linkage disequilibrium (1000 Genomes Nov 2010 CEU) between the top SNP and all other genotyped SNPs. Genetic coordinates are per 1000 Genomes Nov 2010-CEU. Bottom, LD heat map based on D’ values from the combined population under study including all SNPs in the 500Kb region.</p

Meta-analysis of leading SNP in IL12A.

<p>Association results for rs17810456 (G-allele) in two Turkish cohorts (14) and the current study.</p><p>* From <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0119085#pone.0119085.t001" target="_blank">Table 1</a> Kirino et al. 2013</p><p>Meta-analysis of leading SNP in IL12A.</p

Cases before and after QC.

<p>Collection at [1] Erasmus MC at Rotterdam, The Netherlands, [2] University Hospital of Cukurova, Turkey, [3] St John’s Ophthalmic Hospital, Jerusalem, Israel, [4] St Thomas’ Hospital, London, UK, [5] University Hospital, Damascus, Syria, Ethnicity of cases after QC.</p><p>Cases before and after QC.</p

GWS SNPs associated with Behçet’s disease susceptibility using GPC and EMMAX approaches.

<p>Results for the association analysis when correcting for 20 PCs to adjust for stratification. In italic font the SNPs which show association only by one of the two approaches.</p><p>* Odd ratios cannot be calculated by EMMAX approach.</p><p>GWS SNPs associated with Behçet’s disease susceptibility using GPC and EMMAX approaches.</p

Genetic substructure of the Combined GWAS dataset.

<p>Two-dimensional scatterplots from multidimensional scaling analyses of the Generation R Study and Behçet collected data together with the three initial Panels form the HapMap Project. Each dot represents an individual in the dataset. Color codes: Grey = Generation R, Black = Behcet Cases, Yellow = Jordan controls. Blue = CEU, Red = YRB, Green = JPT.</p

Behçet GWAS results using Genomic Principal Components (GPC) adjustment.

<p>Each dot represents an SNP in the dataset. QQ-plot (left). Associated SNPs deviating from the null hypothesis of no association (identity line). Manhattan plot (right). SNPs showing association with the disease map to chromosome 6 and a singleton in chromosome 18.</p