Abstract 2566: Functional studies of HLA SNPs associated with risk of follicular lymphoma.

Abstract

Abstract Background: GWAS suggest an important role of genetic variation in the etiology of follicular lymphoma (FL). We previously identified multiple FL risk loci in the human leukocyte antigen (HLA) Class II region on chromosome 6p21.33-32, including rs10484561 (OR=1.95, P=1.12x10-29) and rs2647012 SNP (OR=0.64, P=2x10-21) (Conde et al., Nat Gen, 2010; Smedby et al. Plos Gen, 2011). HLA typing revealed that the rs10484561 and rs2647012 SNPs are in complete LD (D’=1) with the extended HLA haplotypes DRB1*01:01-DQA1*01:01-DQB1*05:01 and DRB1*15-DQA1*01-DQB1*06, respectively (Skibola et al., Tiss Antigens, 2012). HLA gene variants may alter immune responses to specific antigens and influence tumor development through effects on gene expression or protein structure. By comparing GWAS data with RNA sequencing data, we identified expression quantitative trait loci (eQTL) in LD with rs2647012 that influence gene expression (Conde et al, in press). To gain a further understanding of the mechanisms involved, here we explored the effects of FL-associated HLA SNPs and alleles on expression at both the transcriptional and protein level. Methods: Thirty-eight lymphoblastoid cell lines (Coriell Institute) and monocyte-derived dendritic cells from 28 individuals with HLA typing information were cultured, and RNA and DNA were extracted. Genotype data for rs10484561, rs2647012 and recently identified eQTLs were obtained from HapMap or by Taqman genotyping. mRNA expression levels of HLA Class II genes were measured by RT-qPCR and protein levels were measured by flow cytometry, ELISA and Western blotting. Correlation between genotype and expression data was tested using the Spearman's rank correlation test. Results: The variant (protective) alleles of rs2647012-linked SNPs were significantly associated with higher HLA-DQB1 transcript levels, confirming our previous eQTL analysis. Flow cytometry and ELISA with HLA-specific antibodies also confirmed that these FL-protective alleles were associated with higher HLA-DQB1 (but not HLA-DQA1 or -DRB1) protein levels. No significant changes were observed in HLA-DRB1, -DQB1 or -DQA1 transcript or protein levels when analyzing the correlation of rs10484561-linked SNPs and HLA gene expression. Similar results were found in lymphoblastoid cell lines, and in immature and mature monocyte-derived dendritic cells. Conclusions: eQTL SNPs in LD with the protective rs2647012 variant were associated with higher HLA-DQB1 mRNA and protein expression, suggesting that rs2647012 or a SNP in LD influences FL risk through effects on HLA-DQB1 gene expression. No significant differences were found 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 Citation Format: Christine F. Skibola, Jianqing Zhang, Lucia Conde, Kipp Akers, Jacques Riby, Martyn T. Smith, Fenna CM Sillé. Functional studies of HLA SNPs associated with risk of follicular lymphoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2566. doi:10.1158/1538-7445.AM2013-2566