Primary biliary cirrhosis associated with HLA, IL12A, and IL12RB2 variants

Abstract

Background: Primary biliary cirrhosis is a chronic granulomatous cholangitis, characteristically associated with antimitochondrial antibodies. Twin and family aggregation data suggest that there is a significant genetic predisposition to primary biliary cirrhosis, but the susceptibility loci are unknown.

Methods: To identify genetic loci conferring a risk for primary biliary cirrhosis, we carried out a genomewide association analysis in which DNA samples from 2072 Canadian and U.S. subjects (536 patients with primary biliary cirrhosis and 1536 controls) were genotyped for more than 300,000 single-nucleotide polymorphisms (SNPs). Sixteen of the SNPs most strongly associated with primary biliary cirrhosis were genotyped in two independent replication sets. We carried out fine-mapping studies across three loci associated with primary biliary cirrhosis.

Results: We found significant associations between primary biliary cirrhosis and 13 loci across the HLA class II region; the HLA-DQB1 locus (encoding the major histocompatibility complex class II, DQ beta chain 1) had the strongest association (P=1.78x10(-19); odds ratio for patients vs. controls, 1.75). Primary biliary cirrhosis was also significantly and reproducibly associated with two SNPs at the IL12A locus (encoding interleukin-12alpha), rs6441286 (P=2.42x10(-14); odds ratio, 1.54) and rs574808 (P=1.88x10(-13); odds ratio, 1.54), and one SNP at the IL12RB2 locus (encoding interleukin-12 receptor beta2), rs3790567 (P=2.76x10(-11); odds ratio, 1.51). Fine-mapping analysis showed that a five-allele haplotype in the 3' flank of IL12A was significantly associated with primary biliary cirrhosis (P=1.15x10(-34)). We found a modest genomewide association (P<5.0x10(-5)) with the risk of disease for SNPs at the STAT4 locus (encoding signal transducer and activator of transcription 4) and the CTLA4 locus (encoding cytotoxic T-lymphocyte-associated protein 4) and 10 other loci.

Conclusions: Our data show significant associations between primary biliary cirrhosis and common genetic variants at the HLA class II, IL12A, and IL12RB2 loci and suggest that the interleukin-12 immunoregulatory signaling axis is relevant to the pathophysiology of primary biliary cirrhosis. (ClinicalTrials.gov number, NCT00242125.)

Figure 1. Quality Control and Study Design

Panel A shows the outcomes of genotyping quality control for single-nucleotide polymorphisms (SNPs) and genomic DNA from individual subjects. The requirements for SNPs to meet quality-control standards were call rates of greater than 95%, P>0.0001 for the test for Hardy–Weinberg equilibrium, and P>0.01 for the test for minor allele frequency. Panel B shows the stages of analysis in the study: from stage 1, the genomewide association screening, through stage 2, the replication analysis, to fine-mapping and haplotype analyses. IL12A denotes the gene encoding interleukin-12α, IL12RB2 the gene encoding interleukin-12 receptor β2, IL23R the gene encoding interleukin-23 receptor, and STAT4 the gene encoding signal transducer and activator of transcription 4.

Figure 1. Quality Control and Study Design

Panel A shows the outcomes of genotyping quality control for single-nucleotide polymorphisms (SNPs) and genomic DNA from individual subjects. The requirements for SNPs to meet quality-control standards were call rates of greater than 95%, P>0.0001 for the test for Hardy–Weinberg equilibrium, and P>0.01 for the test for minor allele frequency. Panel B shows the stages of analysis in the study: from stage 1, the genomewide association screening, through stage 2, the replication analysis, to fine-mapping and haplotype analyses. IL12A denotes the gene encoding interleukin-12α, IL12RB2 the gene encoding interleukin-12 receptor β2, IL23R the gene encoding interleukin-23 receptor, and STAT4 the gene encoding signal transducer and activator of transcription 4.

Figure 2. Results of the Genomewide Association Analysis

The y axis represents the level of significance (from the EIGENSTRAT method) for each single-nucleotide polymorphism on each chromosome along the x axis. The dotted line indicates the threshold for genomewide association significance. The P values were adjusted in EIGENSTRAT for the 10 eigenvectors having the highest eigenvalues. HLA-DQB1 denotes the gene encoding major histocompatibility complex class II DQ β chain 1, IL12A the gene encoding interleukin-12α, IL12RB2 the gene encoding interleukin-12 receptor β2, and STAT4 the gene encoding signal transducer and activator of transcription 4.

Figure 3. Results of Association Analyses and Linkage-Disequilibrium Blocks for the IL12A and IL12RB2 Loci

Data are shown for the IL12A (encoding interleukin-12α) locus (Panel A) and the IL12RB2 (encoding interleukin-12 receptor β2) locus (Panel B). The results of chi-square analyses are presented in the top plots. Underneath the plots, the organization of the target genes and surrounding loci in humans is shown (not to exact scale). At the bottom, the haplotype block structure is depicted for 25 genotyped SNPs in the IL12A locus (Panel A) and 30 genotyped SNPs in the IL12RB2 locus (Panel B). The block structure is based on criteria established by Gabriel et al., with the use of pairwise estimates of standardized Lewontin's disequilibrium coefficient (D′), whereas the linkage disequilibrium among pairs of SNPs was characterized on the basis of the square of the correlation coefficient (r²). Regions with high r² values are dark gray, and regions with lower r² values are lighter gray (i.e., the shade lightens with decreasing r² values).

Figure 3. Results of Association Analyses and Linkage-Disequilibrium Blocks for the IL12A and IL12RB2 Loci

Data are shown for the IL12A (encoding interleukin-12α) locus (Panel A) and the IL12RB2 (encoding interleukin-12 receptor β2) locus (Panel B). The results of chi-square analyses are presented in the top plots. Underneath the plots, the organization of the target genes and surrounding loci in humans is shown (not to exact scale). At the bottom, the haplotype block structure is depicted for 25 genotyped SNPs in the IL12A locus (Panel A) and 30 genotyped SNPs in the IL12RB2 locus (Panel B). The block structure is based on criteria established by Gabriel et al., with the use of pairwise estimates of standardized Lewontin's disequilibrium coefficient (D′), whereas the linkage disequilibrium among pairs of SNPs was characterized on the basis of the square of the correlation coefficient (r²). Regions with high r² values are dark gray, and regions with lower r² values are lighter gray (i.e., the shade lightens with decreasing r² values).