Meta-analysis of genome-wide association studies discovers multiple loci for chronic lymphocytic leukemia

Chronic lymphocytic leukemia (CLL) is a common lymphoid malignancy with strong heritability. To further understand the genetic susceptibility for CLL and identify common loci associated with risk, we conducted a meta-analysis of four genome-wide association studies (GWAS) composed of 3,100 cases and 7,667 controls with follow-up replication in 1,958 cases and 5,530 controls. Here we report three new loci at 3p24.1 (rs9880772, EOMES, P=2.55 × 10−11), 6p25.2 (rs73718779, SERPINB6, P=1.97 × 10−8) and 3q28 (rs9815073, LPP, P=3.62 × 10−8), as well as a new independent SNP at the known 2q13 locus (rs9308731, BCL2L11, P=1.00 × 10−11) in the combined analysis. We find suggestive evidence (P<5 × 10−7) for two additional new loci at 4q24 (rs10028805, BANK1, P=7.19 × 10−8) and 3p22.2 (rs1274963, CSRNP1, P=2.12 × 10−7). Pathway analyses of new and known CLL loci consistently show a strong role for apoptosis, providing further evidence for the importance of this biological pathway in CLL susceptibility.

C hronic lymphocytic leukemia (CLL) is the most common leukemia among adults in western countries 1 . Although advances in treatment options have been made, CLL remains an incurable malignancy. Genome-wide association studies (GWAS) have identified multiple susceptibility loci for CLL [2][3][4][5][6][7] with at least three loci having more than one independent signal 5,8 . However, these discovered loci only account for about a third of the estimated heritability attributed to common variants 5 . In a combined analysis of four GWAS and follow-up replication, including 3,888 cases and 12,539 controls of European ancestry, we recently discovered 11 independent single-nucleotide polymorphisms (SNPs) in nine novel loci associated with CLL risk 5 . To discover additional loci associated with susceptibility to CLL, we more than doubled our replication sample size in the present study, slightly increasing our statistical power, and investigated the association with 14 other promising SNPs identified from our GWAS meta-analysis.
Here, we identify four new independent SNPs in three novel loci as well as two promising new loci associated with the risk of CLL. Pathway analyses with these new loci as well as the previously identified loci suggest a strong role for the apoptosis in susceptibility to CLL, further enhancing our understanding.

Results
Discovery meta-analysis. We conducted a meta-analysis of four genome-wide association studies 4,5,9 comprising 3,100 unrelated cases and 7,667 controls of European ancestry (see 'Methods' section, Supplementary Tables 1-3). As these studies used different commercial SNP microarrays, we imputed the B8.5 million common SNPs present in the 1000 Genomes Phase 1 integrated data (version 3) 10 for each study using IMPUTE2 (ref. 11; Supplementary Table 2) and tested for associations with CLL risk assuming a log-additive genetic model. After quality control exclusions, B8.5 million SNPs with minor allele frequency 41% were meta-analysed in the discovery stage using a fixed effects model.
A quantile-quantile plot of the meta-analysis results in the discovery stage showed an enrichment of small P values from the fixed-effects model compared with the null distribution, which persisted even after removal of the known loci ( Supplementary  Fig. 1). There was little evidence for inflation due to population stratification (lambda ¼ 1.028). Under a log-additive genetic model, a total of 16 unique loci (defined as separated by at least 1 Mb) reached genome-wide significance (Po5 Â 10 À 8 ; Supplementary Fig. 2), all of which had been previously reported 2,3,5,8 . For each previously reported locus, we identified the SNP with the strongest P value within 1 Mb of the published index SNP. Of the 29 published loci, 21 were at least suggestively associated with CLL under a log-additive model in our discovery meta-analysis with Po5 Â 10 À 7 (Supplementary Table 4). As the original reported SNPs at two loci (4q26 and 6q25.2) failed to show nominal significance (Po0.05) in our study, we metaanalysed our results with the published results for known loci from two other GWAS 6,7 . In this larger meta-analysis, 25 of the published loci were at least suggestively associated with CLL risk (Po5 Â 10 À 7 ) based on a fixed-effects model; however, both rs6858698 at 4q26 and rs11631963 at 15q25.2 showed attenuated odds ratios and weak P values even with this increased sample size (P ¼ 0.002 and P ¼ 0.0003, respectively; Supplementary Table 5), questioning the certainty of these loci.

Discussion
All the three novel loci are located in or near genes implicated in apoptosis and/or immune function. The novel 3p24.1 SNP (rs9880772) resides 13 kb 5 0 of eomesodermin (EOMES), a member of the T-box gene family and a key regulator in cellmediated immunity and CD8 þ T-cell differentiation 12 . EOMES is critical for lymphoproliferation due to Fas-deficiency 13 , which has been observed in inherited lymphoproliferative disorders associated with autoimmunity 14,15 . Overexpression of EOMES has been observed among extranodal natural killer/T (NK/T)-cell and peripheral T-cell lymphomas 16 . Interestingly, highly correlated SNPs within the same 15 kb region 5 0 of EOMES have also been associated with two autoimmune diseases, rheumatoid arthritis 17 (rs3806624, r 2 ¼ 0.96) and multiple sclerosis 18 (rs11129295, r 2 ¼ 0.72), as well as Hodgkin's lymphoma 19 (rs3806624, r 2 ¼ 0.96), underscoring the importance of this genetic region for susceptibility to both lymphoma and autoimmune disease. Regions locally centromeric and telomeric of rs9880772 show strong regulation and promoter signatures by histone marks, DNaseI hypersensitivity and transcription factor binding sites, and the correlated SNP, rs3806624, is located within a poised promoter in the lymphoblastoid cell line, GM12878 (Supplementary Table 8).
The novel 6p25.2 SNP (rs73718779) is located within an intron of SERPINB6, which encodes a member of the serine protease inhibitor (serpin) superfamily. Although the physiological role of SERPINB6 is not well understood, it inhibits cathepsin G 20 , which activates the pro-apoptotic proteinase caspase 7 (ref. 21). In eQTL and methylation QTL analyses, we found that the T allele for rs6939693, an SNP completely correlated with rs73718779 (r 2 ¼ 1), was associated with significantly reduced SERPINB6 expression in blood in a weighted z-score metaanalysis (P ¼ 1.40 Â 10 À 52 , Supplementary Table 9) and increased DNA methylation levels based on a linear mixed model (P ¼ 1.70 Â 10 À 11 , Supplementary Table 10), suggesting strong potential functional relevance.
The suggestive 4q24 SNP (rs10028805) is located within an intron of B-cell scaffold protein with ankyrin repeats 1 (BANK1), which encodes a protein adaptor that is predominantly expressed in B-cells. BANK1 is a putative tumour suppressor gene in B-cell lymphomagenesis 26 , and BANK1-deficient cells show enhanced CD40-mediated proliferation and survival with Akt activation 27 . Rs10028805 is moderately correlated with rs10516487 (r 2 ¼ 0.70), a non-synonymous SNP in exon 2 that has been associated with systemic lupus erythematosus 28 and shown to alter mRNA splicing and the quantity of the BANK1 protein 29 . Consistent with this, we observed rs10028805 to be associated with BANK1 expression in lymphoblastoid cells (P ¼ 6.89 Â 10 À 13 , Supplementary Table 11).
The 3p22.2 SNP (rs1274963) is an intronic variant in the gene CSRNP1 (cysteine-serine-rich nuclear protein 1), which is induced by AXIN1, a scaffold protein that is a negative regulator of the Wnt/signalling pathway 30 . A putative tumour suppressor with potential apoptosis activity 31 , CSRNP1 plays an important role in the development of haematopoiesis progenitors in zebrafish 32 and has been shown to be expressed in many tissues, with leukocytes being among those with the highest abundance 30 . The SNP resides in an area with strong regulatory potential based on histone marks, DNaseI hypersensitivity and transcription factor binding sites (Supplementary Table 8) and is located within a strong enhancer in the lymphoblastoid cell line, GM12878 ( Supplementary Fig. 4). Of potential functional relevance, in lymphocytes and blood, the rs1274963A risk allele was associated with reduced WDR48 expression (Supplementary  Tables 9 and 11), a gene shown to induce apoptosis and suppress tumour cell proliferation 33 .
We constructed a polygenetic risk score that included the four new SNPs from this study as well as 30 previously identified SNPs at known loci (Supplementary Table 5) to evaluate the possibility of risk stratification for CLL (see 'Methods' section). Those in the top 20% of the risk distribution had a 1.9-fold increased risk (95% confidence interval: 1.70-2.21) compared with those in the middle quintile of the distribution. The newly discovered SNPs explain B1% of the familial risk. Together with the previously identified loci, we estimate that the identified loci for CLL thus far explain B16.5% of the familial risk, which is similar to previous estimates 5, 6 .
In conclusion, our meta-analysis of GWAS identified four new independent SNPs and two additional promising loci for CLL, furthering our knowledge of the underpinnings of genetic susceptibility to CLL. Pathway analyses of known and new CLL  35,36 . All the studies obtained informed consent from their participants and approval from their respective Institutional Review Boards for this study 5 .
To maximize our statistic power, all cases with sufficient DNA and a subset of available controls were genotyped for this study. Subjects in these studies were genotyped using the Illumina OmniExpress, Omni2.5, HumanHap610K, HumanCNV360-Duo or Affymetrix 6.0. For the NCI GWAS, the majority of subjects were genotyped with the Illumina OmniExpress; however, a subset of controls (N ¼ 3,536) and one case were genotyped using the Omni2.5, so to prevent potential platform artifacts, extensive quality control metrics were used, including the removal of assays with low completion rates or monomorphic calls from either platform, before combining the data 5 . For all four GWAS, rigorous quality control metrics were applied to each study to ensure high quality results. Samples with poor call rates, gender discordance, abnormal heterozygosity or of non-European ancestry were excluded, and SNPs with a call rate o95% or Hardy-Weinberg equilibrium P value o1 Â 10 À 6 were removed from the analysis (Supplementary Table 2).
Each GWAS was imputed separately using IMPUTE2 (ref. 11). In contrast to the previous study 5 where a hybrid reference panel was used for imputation, all the studies in this analysis were imputed using the 1000 Genomes Project version 3 (March 2012 release) as the reference panel. Poorly imputed SNPs (INFO score o0.3) and SNPs with minor allele frequency o1% were excluded from each study, leaving roughly B8.5 million SNPs for analysis. After quality control filters, a total of 3,100 cases and 7,667 controls across the four studies remained for analysis (Supplementary Table 3). For each study, principal component analyses were conducted separately. Association testing was conducted for each study separately using SNPTEST version 2, adjusting for age, sex and significant principal components (Po0.05 in null model with age and sex). Meta-analyses were performed using the fixed-effects inverse variance method based on the beta estimates and standard errors from each study.
Replication and technical validation. Replication of potential novel SNPs was undertaken in 1,958 additional cases and 5,530 controls from six different studies ( Supplementary Tables 1 and 3). Fourteen promising SNPs that reached a significance threshold of Po5 Â 10 À 6 in the discovery meta-analysis were taken forward for replication, including 10 SNPs in novel regions (defined as at least 1 Mb from a known CLL locus) and four SNPs in known regions that appeared to be possible secondary signals (r 2 o0.1 with the reported SNPs and Po5 Â 10 À 7 in the discovery meta-analysis). To conduct conditional analyses with the potential secondary signals, the previously reported index SNP(s) in each of these four   regions were also genotyped. TaqMan custom genotyping assays (Applied Biosystems) were designed and optimized for the 14 promising SNPs as well as five previously reported index SNPs. Taqman or Sequenom genotyping was conducted separately for each replication study at their own centre. Each study included duplicates for quality control, and HapMap samples genotyped across the centres yielded excellent concordance (100%). Association testing was conducted separately for each study, adjusting for age, sex and for MSKCC, Ashkenazi ancestry. The replication studies were then meta-analysed together and with the discovery GWAS using an inverse variance fixed effects model. All the SNPs reaching genome-wide or suggestive significance in the joint meta-analysis were either directly genotyped or well imputed (INFO40.78 for all SNPs with average INFO ¼ 0.95) in the GWAS. Technical validation comparing genotype calls or imputed data from the NCI GWAS with Taqman assays for 639 samples revealed moderate concordance for rs9815073 (r 2 ¼ 0.67), but high concordance (r 2 40.97) for the other SNPs. Although the concordance was lower than expected and further confirmation is needed, an analysis of the Taqman validation data for rs9815073 showed an odds ratio ¼ 1.30, which is similar to the odds ratio observed in the full discovery data set.
Polygenic risk score analysis. To evaluate possible stratification for CLL risk based on the 34 independent SNPs from the 30 loci, we performed a polygenic risk score analysis using the discovery sample data. Polygenic risk scores were derived for each person by taking the weighted sum of the risk alleles (0, 1 or 2) for each of the 34 SNPs. The weights for each SNP were the per-allele log odds ratios estimated from our meta-analysis of the discovery data. We then computed the quintiles of the polygenic risk scores and used logistic regression models to estimate the odds ratio for CLL risk for each quintile with the middle quintile as the reference.
Departures from a multiplicative model were assessed by testing for all pair-wise SNP interactions. No evidence of significant interactions was observed.
Heritability analysis. To estimate the familial risk explained by both the novel and previously established loci for CLL, we estimated the contribution of each independent SNP to the heritability using the equation is the log-odds ratio per copy of the risk allele from the replication stage analyses and f is the allele frequency, and summed the contributions of all novel and established SNPs 37 . We then estimated the total heritability from the sibling relative risk (relative risk ¼ 8.5 from Goldin et al. 38 ), using the equation derived by Pharoah et al. 39 We then calculated the proportion of familial risk explained by dividing the summed contributions of the novel and established SNPs by the total heritability.
Expression quantitative trait loci and other related analyses. To explore the potential functional relevance of the CLL-associated SNPs, we conducted expression quantitative trait loci (eQTL) and methylation quantitative trait loci (meQTL) analyses using three independent data sets: (1) a childhood asthma study of gene expression in lymphoblastoid cell lines 40 , (2) a meta-analysis of eQTL associations from whole blood 41 , and (3) meQTL in CD4 þ lymphocytes from the GOLDN study 42 . In the childhood asthma study 40 , RNA was extracted from lymphoblastoid cell lines from 830 parents and offspring from 206 families of European ancestry. Gene expression was assessed with the Affymetrix HG-U133 Plus 2.0 chip, and subjects were genotyped using the Illumina Human-1 and HumanHap300K beadchips with subsequent imputation using data from the 1000 Genomes Project. The four new and two suggestive SNPs were tested for cis associations (defined as gene transcripts within 1 Mb), adjusting for non-genetic effects in the gene expression value and relatedness using MERLIN 43 . To gain insight into the relative importance of associations with our SNPs compared with other SNPs in the region, conditional analyses were also conducted, in which both the CLL SNP and the most significant SNP for the particular gene transcript (that is, the peak SNP) were included in the same model. The meta-analysis of eQTL associations from whole blood 41 included eQTL data generated using Illumina gene expression arrays from seven studies consisting of a total of 5,311 unrelated Europeans. Gene expression arrays were harmonized by matching probe sequences, and all the studies were imputed using the HapMap European reference panel. SNPs that were strongly correlated (r 2 40.8) with the newly discovered and suggestive CLL SNPs were examined for possible cis associations. In the GOLDN study 42 , over 450,000 CpG methylation sites were genotyped in CD4 þ T-cells  from 593 participants. Subjects were genotyped with the Affymetrix Human SNP Array 6.0, and the 2.5 million SNPs available in the HapMap2 release were imputed. We updated the analysis by including more participants (n ¼ 717) and expanded the scope of cis-meQTL to SNPs and CpG sites within 50 kb of each other. The association between the CLL-associated SNPs (as well as strongly correlated SNPs, r 2 40.8) and methylation beta values was tested using the linear mixed models, adjusting for family structure and other covariates including age, sex, recruitment centres and principal components. Finally, we also utilized HaploReg 44 , a tool for exploring noncoding functional annotation using ENCODE data, to evaluate the genome surrounding our SNPs.
Pathway analyses. To explore potential biological pathways underlying known CLL loci to date, we conducted analyses using GRAIL 34 , Webgestalt 45 and GeneMania 46 . GRAIL 34 is a text-based mining tool that is used to evaluate the relationship between genes at different disease loci. Genes within 250 kb of known loci were included, and the 2006 text database was used to avoid overweighting the previously published loci. Webgestalt 45 is a web-based pathway analysis server offering hypergeometric tests for Gene Ontology (GO) term enrichments and visualization of enriched GO terms in a graph depicting the GO hierarchy. GeneMania 46 is a network-based analysis server that finds an expanded set of genes including the query genes and additional genes closely linked with the query genes via protein and genetic interactions, pathways, co-expression, co-localization and protein domain similarity. For both Webgestalt and GeneMania, the nearest gene for each locus was included. For all pathways analyses, only newly discovered loci and the previously identified loci that reached at least Po1 Â 10 À 5 in the combined meta-analysis with the published results from two other GWAS 6,7 (Supplementary Table 5) were included.
Chromatin state dynamics analysis. To assess chromatin state dynamics, we used Chromos 47 , which utilizes Chip-Seq data from ENCODE 48 on nine cell types: