Validation and functional characterization of GWAS-identified variants for chronic lymphocytic leukemia: a CRuCIAL study

During the past years, considerable efforts have been made to uncover the genetic component of chronic lymphocytic leukemia (CLL) susceptibility. To date, several genome-wide association studies (GWAS) and their meta-analysis have identified not only single-nucleotide polymorphisms (SNPs) associated with CLL risk [ 1 ] but also patient survival [ 2 ]. However, despite these noticeable results, it becomes evident that both validation and functional characterization of the genetic variations identified are still required before they can be used in a clinical setting. Hence, we decided to validate the association of 41 GWAS-identified hits for CLL in 1158 CLL cases and 1947 controls ascertained through the Consortium for Research in Chronic lymphocytIc Leukemia (CRuCIAL) and to investigate their impact on modulating host immune responses and their utility to predict disease onset. Study participants were of European ancestry and gave their written informed consent to participate in the study, which was approved by the ethical review committee of participant institutions. CLL patients had often Binet stage A and Rai stage I (67.00% and 79.83%) and, compared to controls, had a higher mean age (66.19 ± 12.66 vs. 55.60 ± 11.50) and an increased male/female ratio (1.54 vs. 0.91). SNPs selection was based on published GWAS, functionality according to HaploReg data, and linkage disequilibrium between the SNPs. Genotyping of genetic variants was performed using KASP TM and Taqman® assays. Hardy–Weinberg equilibrium was assessed in the controls ( P > 0.001) and the association between CLL and SNPs was tested using a multivariate unconditional logistic regression analysis adjusted for age, sex, and country of origin. A meta-analysis of the CRuCIAL results with those from previous GWAS was conducted to validate genetic associations and the I [ 2 ] statistic was used to assess statistical heterogeneity between the studies ( P Het > 0.01). The pooled OR was computed using the fixed-effect model and the significance threshold for the meta-analysis was set to 5.0 × 10 −8 . Mechanistically, we evaluated the correlation of the GWAS-identified SNPs with a production of nine cytokines after in vitro stimulation of whole blood, peripheral mononuclear cells, and monocyte-derived macrophages from 408 healthy subjects of the Human Functional Genomic Project (HFGP) with LPS, PHA, Pam3Cys, CpG and Borrelia burgdorferi and Escherichia coli . In parallel, we also tested the correlation between selected SNPs and circulating concentrations of 103 serum and plasmatic inflammatory proteins, 7 plasma steroid hormones, and absolute numbers of 91 blood-derived immune cell populations. The HFGP study was approved by the Arnhem-Nijmegen Ethical Committee (42561.091.12) and biological specimens were collected after informed consent was obtained. A detailed description of the study population and participating centers, selected SNPs and protocols and reagents used in the functional experiments are included in the Supplementary Material available on the Blood Cancer Journal website. In order to account for multiple comparisons, we used a significance threshold of 2.3 × 10 −5 , 1.2 × 10 −5 , 1.34 × 10 −5 , and 1.74 × 10 −4 for the cytokine quantitative trait loci, proteomic, blood cell counts, and steroid hormone analyses, respectively.