Functional Characterization of HLA SNPs Associated with Risk of Follicular Lymphoma

Genome-wide association studies (GWAS) suggest an important role of genetic variation in the etiology of follicular lymphoma (FL). Multiple susceptibility alleles in the human leukocyte antigen (HLA) Class I and II regions on chromosome 6p21.33–32 have been identified for FL, including rs10484561 (OR = 1.95, P = 1.12×10⁻²⁹) and rs2647012 SNP (OR = 0.64, P = 2×10⁻²¹) (Conde et al., Nat Gen, 2010; Smedby et al. Plos Gen, 2011). Moreover, high-resolution HLA typing using next-generation sequencing has identified a number of HLA alleles associated with FL including the extended haplotype DRB1*01:01—DQA1*01:01—DQB1*05:01 that is in complete linkage disequilibrium with rs10484561 (D'=1), and DRB1*15-DQA1*01-DQB1*06, which is highly correlated with rs2647012 (D'=1) (Skibola et al., Tissue Antigens, 2012). We hypothesized that these SNPs or protein variants may alter immune responses to specific antigens and influence tumor development through effects on gene expression or protein structure. To gain a further understanding of the mechanisms involved, here we explored the effects of HLA alleles on expression at the transcriptional and protein level.

HLA-DRB1, HLA-DQB1 and HLA-DQA1 mRNA expression and protein levels were measured by RT-qPCR and flow cytometry, respectively, in 40 lymphoblastoid cell lines (Coriell) where wild type and variant rs10484561 and rs2647012 genotypes were compared.

HLA-DQB1 gene expression changes were significantly associated with rs2647012 genotypes, where the presence of the variant (protective) allele correlated with higher HLA-DQB1 transcript levels. Flow cytometry with HLA-specific antibodies and pan-HLA antibodies (against DR/DQ/DP) also confirmed that the variant rs2647012 allele was associated with higher HLA-DQB1 protein levels. No significant changes were observed in HLA-DRB1, HLA-DQB1 or HLA-DQA1 transcript or protein levels when comparing rs10484561 wild type versus variant alleles.

We did not detect significant differences in transcript or surface protein levels based on rs10484561 genotypes. These findings suggest that the increased risk of FL associated with rs10484561 that is in high LD with the HLA-DRB1*0101—DQA1*0101—DQB1*0501 extended haplotype is not due to transcriptional modulation, but may be due to effects on protein structure. On the other hand, the protective rs2647012 variant was associated with higher HLA-DQB1 mRNA and protein expression, suggesting that rs2647012 or a SNP in LD influences FL risk through effects on gene expression. We are currently testing the influence of additional FL risk alleles in the 6p21.3 region on HLA gene and protein expression, as well as investigating whether FL risk alleles alter HLA expression in other cell types.

No relevant conflicts of interest to declare.