High-Resolution HLA Typing by Next-Generation Sequencing and Risk of Follicular Lymphoma

Abstract 5092

The molecular basis of follicular lymphoma (FL) has been fairly well characterized, although its root causes remain less clear. Recently, in two genome-wide association studies in European-Americans, we identified novel genetic susceptibility loci for FL, narrowed down to two independent regions on chromosome 6p21.3 in the major histocompatibility complex (MHC) (Skibola et al., Nat Gen 41(8):873-5., 2009; Conde et al., Nat Gen 42(8):661-4, 2010). The first susceptibility locus, rs6457327 (combined allelic p-value=4.7 × 10^-11 in 645 patients and 3,377 controls), is part of a 26-kb region of high linkage disequilibrium (LD) in the MHC Class 1 region at 6p21.33 in close proximity to the psoriasis susceptibility region 1 (PSORS1). In a second locus in the MHC Class II region at 6p21.32, upstream of HLA-DQB1, two SNPs in high LD, rs10484561 and rs7755224, were associated with 2-fold increased risks for FL in 1,465 patients and 6,958 controls (combined p-values=1.12×10^-29, 2.00×10^-19). Using tag SNPs as surrogates for HLA allelotypes, the latter loci were found to be part of an extended haplotype that includes HLA-DRB1*0101-HLA-DQA1*0101-HLA-DQB1*0501 and that was associated with increased FL risk [odds ratio (OR) = 2.07, 95% confidence interval (CI) = 1.40–3.06]. To confirm these findings, we determined HLA-DQB1 allelotypes in a subset of 265 FL cases and 757 controls. We corroborated the positive association between FL and the HLA-DQB1*05 allele group (OR=1.56, 95% CI 1.22–1.99; adjusted p-value=7.7×10^-3), and identified an allele group inversely associated with FL risk, HLA-DQB1*06 (OR=0.53, 95% CI 0.40–0.69; adjusted p-value=1.98×10^-5).

To gain further insight into the role of HLA alleles in FL susceptibility, high-resolution HLA typing by next-generation sequencing of the HLA class I (A, B, C) and Class II (DRB1/3/4/5, DQA1, DQB1, DPB1) genes is underway in 222 FL patients and 220 sex- and age-matched controls. HLA typing is being conducted by sequencing the exons that code for the region of the peptide-binding groove of the HLA molecule (exon 2 for the Class II genes, HLA-DQA1, -DQB1, -DRB1, -DRB1/3/4/5 and -DPB1, and exons 2–4 for the Class I genes HLA-A, HLA-B and HLA-C) as described previously (Bentley, G et al. Tissue Antigens. 74:393-403, 2009). Allele and haplotype frequencies for cases and controls will be estimated using the iterative Expectation-Maximization (EM) algorithm implemented in the Pypop software (Lancaster et al. Tissue Antigens. 69 Suppl 1:192-197, 2007). Statistical significance of estimated ORs and 95% CIs, and differences in the distribution of the alleles or haplotypes between cases and controls will be assessed using a two-tailed Fisher's exact test.

To our knowledge, this study will present the highest resolution (up to 8-digit) HLA typing reported in a sample of European-Americans, and is an important next step to determine the role of MHC genetic variation in the pathogenesis of FL.