Common variation at 12q24.13 (OAS3) influences chronic lymphocytic leukemia risk

Chronic lymphocytic leukemia (CLL) is the most common form of lymphoid malignancy in Western countries1. Recent multi-stage genome-wide association studies (GWAS) have shown that part of the eight-fold increased risk of CLL seen in first-degree relatives of patients can be ascribed to the co-inheritance of multiple low-risk variants.2, 3, 4, 5, 6

The discovery phase comprised two previously described GWAS conducted in the United Kingdom 2,5 (see Supplementary Methods). UK-GWAS-1; 517 CLL cases (155 enriched for genetic susceptibility by virtue of family history) genotyped using Illumina HumanCNV370-Duo BeadChips 5 and 2698 controls from the Wellcome Trust Case Control Consortium 2 (WTCCC2) 1958 Birth cohort, typed using Hap1.2M-Duo Custom array. 7 UK-GWAS-2; 1271 CLL cases genotyped using the Illumina Omni Express BeadChip and 2501 UK Blood Service Donor controls typed using Hap1.2M-Duo Custom arrays. 2 To harmonise GWAS data sets we recovered untyped genotypes by imputation using IMPUTEv2 with 1000genomes as a reference (phase 1 integrated variant set (b37) from March 2012) (Supplementary Methods). Genomic control lambda values for UK-GWAS1 and UK-GWAS2 were 1.04 and 1.05, respectively, thereby excluding significant differential genotyping or cryptic population substructure. 2 Post quality control the two GWAS provided data on 1739 cases and 5199 controls. In a meta-analysis we identified 156 common SNPs (minor allele frequency40.01), typed in either UK GWAS-1 or 2, that showed good evidence of an association (ie P o1.5 × 10 − 4 ) and did not map to any of the 30 loci that have previously been associated with CLL risk. 2 Seven SNPs chosen on the basis of strength of association and/or biological plausibility of the annotated gene (that is, a role in B-cell or cancer biology) were genotyped in the UK replication series ( Two SNPs, rs10735079 and rs17512800, provided further evidence for an association with CLL risk (ie Po 0.05) and these two SNPs were taken forward for genotyping in a further replication series from Sweden, which comprised 347 CLL cases and 342 controls (Supplementary Table 1). This case control analysis provided additional evidence for an association between rs10735079 and CLL risk. Subsequently we genotyped rs10735079 in three further case-control series, Poland-1 (105 cases, 101 controls), Poland-2 (176 cases, 209 controls) and Italy (186 cases, 155 controls) (Supplementary Methods). In the combined analysis of all series the association between rs10735079 and CLL attained genome-wide significance (combined OR per allele = 1.18, 95% CI:1.12-1.26, P = 2.34 × 10 − 8 ) (Figure 1). The association was not restricted to IGHV mutation and showed no relationship with either sex or age (Supplementary Table 2). rs10735079 maps to intron 2 of the 2′-5′-oligoadenylate synthetase 3 (OAS3) gene, one of three OAS genes clustering at 12q24.13 (Figure 2), and is in LD (r 2 = 0.87) with the splice acceptor variant of OAS1, rs10774671, which mediates alternative splicing of OAS1 transcription and affects enzymatic activity. 9  Although attractive as the basis of the 12q24.13 association the association with CLL is stronger for rs10735079 than rs10774671 (P = 1.16 × 10 − 5 and 1.74 × 10 − 4 , respectively; Supplementary Table 3).
The significant dose relationship between rs10735079 genotype and OAS3 expression in blood, with the risk allele being associated with reduced levels of mRNA (P = 5.4 × 10 − 29 ; Supplementary  Table 4), supports a role for rs10735079 genotype mediating its effect on CLL through differential OAS3 expression rather than impacting on OAS1.
Although rs10735079 is not predicted to lie in an active promoter or strong enhancer element, the correlated SNP rs6489879 (r 2 = 0.99) that maps to intron 1 of OAS3 resides in a region predicted to be a strong enhancer in lymphoblastoid GM12878 cells and to be involved in binding of a number of transcription factors including IRF4 (interferon regulatory factor-4), a lymphocyte-specific transcription factor (Figure 2; Supplementary Table 3).
OAS is induced by interferon in response to viral infection activating 2-5A-dependent RNase L degradation of viral RNA 10 and variation in OAS genes has been reported to be a determinant of viral susceptibility. 9,11-13 Given the possible role of viral response in the pathogenesis of CLL, although speculative, it is therefore possible that genetic variation in OAS3 influences risk of developing CLL through differing response to antigenic challenge. Moreover, OAS3 is a B-cell receptor (BCR) signature gene. 14 Intriguingly as variation in the BCR genes IRF4 (ref. 5), BCL2 (ref. 3) and HLA-DQA1 (ref. 15) has previously been implicated by GWAS as determinants of CLL risk this suggests a common aetiological pathway through differential BCR-activation.
Although further functional studies are required to fully elucidate the biological basis of the 12q24.13 association, our finding brings the total number of risk loci identified for CLL thus far to 31 and provides additional support for the role of inherited genetic factors in the aetiology of CLL.
URLS The study made use of genotyping data on the 1958 Birth Cohort; a full list of the investigators who contributed to the generation of these data is available at http:// www.wtccc.org.uk/. We thank L. Padyukov (Karolinska Institutet) and the Epidemiological Investigation of Rheumatoid Arthritis (EIRA) group for providing control samples from the Swedish population for the Swedish replication study. This study makes use of data generated by the Wellcome Trust Case-Control Consortium 1 and 2. A full list of the investigators who contributed to the generation of the data is available at http://www.wtccc.org.uk/. We are grateful to all investigators and all the patients and individuals for their participation. We also thank the clinicians, other hospital staff and study staff that contributed to the blood sample and data collection for this study.

AUTHOR CONTRIBUTIONS
RSH obtained financial support, designed the project and provided overall project management; GPS and RSH drafted the manuscript; GPS and HES performed project management and supervised genotyping; JMA obtained financial support and supervised genotyping; GPS and MCDB performed bioinformatic and statistical analyses; GPS and AH performed genotyping; YW and MCDB performed imputation analysis;  MJSD, NJS, HM, LM, SD, LK, IF, DW, KJ, GJ, KES, SJ, AM, CD, AGH, TM, GHJ, GS, RJH, ARP,  DJA, JRB, GP, CP, CF, RR, DC and JMA acquired samples for the replication cohorts.