Figures
Abstract
Background
Non-Hodgkin lymphomas are a heterogeneous group of solid tumours that constitute the 5th highest cause of cancer mortality in the United States and Canada. Poor control of cell death in lymphocytes can lead to autoimmune disease or cancer, making genes involved in programmed cell death of lymphocytes logical candidate genes for lymphoma susceptibility.
Materials and Methods
We tested for genetic association with NHL and NHL subtypes, of SNPs in lymphocyte cell death genes using an established population-based study. 17 candidate genes were chosen based on biological function, with 123 SNPs tested. These included tagSNPs from HapMap and novel SNPs discovered by re-sequencing 47 cases in genes for which SNP representation was judged to be low. The main analysis, which estimated odds ratios by fitting data to an additive logistic regression model, used European ancestry samples that passed quality control measures (569 cases and 547 controls). A two-tiered approach for multiple testing correction was used: correction for number of tests within each gene by permutation-based methodology, followed by correction for the number of genes tested using the false discovery rate.
Citation: Schuetz JM, Daley D, Graham J, Berry BR, Gallagher RP, Connors JM, et al. (2012) Genetic Variation in Cell Death Genes and Risk of Non-Hodgkin Lymphoma. PLoS ONE 7(2): e31560. https://doi.org/10.1371/journal.pone.0031560
Editor: Ana Paula Arez, Instituto de Higiene e Medicina Tropical, Portugal
Received: November 7, 2011; Accepted: January 13, 2012; Published: February 7, 2012
Copyright: © 2012 Schuetz et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Canadian Institutes for Health Research (CIHR) and the Canadian Cancer Society. J.M.S. was supported by the Lionel McLeod Award of the Alberta Heritage Foundation for Medical Research and by CIHR. D.D. holds a Tier II Canadian Research Chair (Genetic Epidemiology of Common Complex Diseases). A.B.-W. is a Senior Scholar and D.D. and J.G. are Scholars of the Michael Smith Foundation for Health Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
Non-Hodgkin lymphoma (NHL) is a collection of different subtypes, each with different clinical presentation, preferred treatment regimens and prognosis. These subtypes appear to be derived from progenitor cells at different stages of B- and T-cell development. For example, mantle cell lymphomas are derived from pre-germinal centre B cells while follicular lymphomas arise from germinal centre B-cells [1]. The development of an active and specialized lymphocyte requires many steps [2], and each is controlled by apoptosis. If all requirements to proceed to the next level of development and activation are not met, lymphocytes enter a default program and are selected to die by apoptosis. This commonality interested us in testing the hypothesis that variation in cell death genes would affect risk of NHL.
Programmed cell death, the best characterized form of which is apoptosis, gives cells the ability to auto-destruct following specific triggers. Such triggers can be the presence or absence of a signal, depending on the cell context. Apoptosis has a central role in the development of the immune system, balancing the need for an effective immune response with the need to eliminate auto-reactive cells [2]. The crucial role of apoptosis in lymphoma was established by the discovery that the most common translocation in follicular lymphoma (FL), t(14;18), caused the inappropriate expression of BCL2 by juxtaposing it to IG regulatory elements [3]. BCL2 is now known to be at the centre of a family of proteins with roles in apoptosis.
The number of BCL2-homology domains (or BH domains) determines whether the proteins in this family have anti-apoptotic or pro-apoptotic roles. Members with 4 BH-domains (BCL2, MCL1, and BCL-xL) have anti-apoptotic roles. Both the BH3-only members (including BIM, PUMA/BBC3, NOXA/PMAIP1, BID, BAD, BMF, BIK/BLK/NBK, HRK/DP5), which have only a BH3 domain, and the members with three BH domains (BAX and BAK) have pro-apoptotic functions [4]. Interactions between these diverse family members allow cells to respond to a variety of triggers, such as DNA damage, lack of B-cell receptor signaling from other cells, environmental toxins, or interleukin withdrawal. Tumour cells can acquire mutations that inactivate parts of this signaling network, causing tumourigenesis and drug resistance [5].
Seventeen candidate genes were selected following literature review and were chosen based on their role in apoptosis in lymphocyte development: BAX, BCL2, BCL6 [6], BCL10 [7], BCL2L11, BBC3, BECN1 [8], BMI1 [9], FAS [10], MCL1, MDM2 [11], PMAIP1, RFWD2 [12], SKP2 [13], miR-15a/16 [14], miR-17-92 [15], and miR-155 [16]. The coverage of known SNPs across each candidate gene at the time of initiation of the study was assessed by determining the ratio of the number of known SNPs to tagSNPs in each gene. 11 genes with low coverage were re-sequenced in DNA samples from 47 NHL cases. Targeted re-sequencing provided new, more thorough coverage of genetic information in candidate genes. SNPs were chosen for genotyping based on linkage disequilibrium relationships within the re-sequencing data and based on HapMap data. SNPs selected were genotyped in 797 cases and 790 controls and tested for association with risk of common NHL subtypes.
Materials and Methods
Study population
The study population has been previously described [17], [18], [19] and is summarized in Table S1. Briefly, all NHL cases diagnosed in British Columbia from March 2000 to February 2004, residing in the Greater Vancouver Regional District and Greater Victoria (Capital Regional District), and aged 20 to 79 were invited to participate. HIV-positive cases and cases with prior transplant were excluded. Categorization of ethnicity was based on self-reported ethnicity of each participant's four grandparents. Population controls were identified from the Client Registry of the British Columbia Ministry of Health and were frequency matched to cases by sex, age (within 5-year age groups), and area of residence in an approximate 1∶1 ratio. 828 cases and 848 controls completed at least part of a study questionnaire and 797 cases and 790 controls had DNA (Table S1). The study was approved by the joint University of British Columbia/British Columbia Cancer Agency Research Ethics Board. Written informed consent was obtained from all participants.
From the 1587 samples, DNA was derived from whole blood in 407 samples, lymphocytes isolated from blood in 782 samples, mouthwash in 24 samples and saliva in 48 samples. DNA extraction was done using the PureGene DNA isolation kit (Gentra Systems, Minneapolis, MN) following the manufacturer's instructions. DNA was quantified using PicoGreen (Molecular Probes, Invitrogen, Burlington, ON). Samples with limited amounts of DNA (326/1587 samples) were subjected to whole-genome amplification using the RepliG kit (QIAGEN, Mississauga, ON, Canada); these are hereafter referred to as “WGA” samples.
Sequencing
Forty-seven NHL case samples (Table S2) were selected for SNP discovery via Sanger sequencing by a protocol previously described [20], based on age (preference was given to younger patients to enrich for genetics-based cases) and DNA quantity. Genomic sequences for use in primer design were obtained for each gene using Ensembl, with known SNPs annotated. Primers were designed using Primer 3 [21]. For all 17 candidate genes, 1.5 kb upstream of each gene's transcriptional start site was sequenced to identify genetic variation in promoter sequences near the gene; a subset of the candidate genes were subject to additional re-sequencing of all exons.
Genes were chosen for exon re-sequencing based on the depth of genetic variation already present in HapMap Phase II. For each gene, boundaries were selected by first capturing the gene with an extra 1.5 kb of sequence on either side of the gene within the browser window, and then extending the window boundaries until at least one more SNP in either direction was captured within the genomic region displayed. Genotypes were downloaded into Haploview [22]. Using Tagger [23] within Haploview [22], the set of tagSNPs required to capture the genotypes with r2 = 0.8 was calculated. A gene was selected for exon re-sequencing if the ratio of #SNPs/#tagSNPs was less than 2. A ratio <2 indicates that there is less than 2-fold redundancy of tagSNPs in HapMap data, implying that re-sequencing may reveal as yet untagged variants in that gene. This cut-off was arbitrarily selected, as it can be interpreted to mean that, on average, a tagSNP represents at least one more SNP.
Uniform sequencing conditions were enabled by adding the -21M13F (TGTAAAACGACGGCCAGT) or M13R (CAGGAAACAGCTATGAC) extensions to the 5′ ends of the forward and reverse PCR primers, respectively. Primers and PCR conditions used are listed in Table S3. PCR reactions were followed by product cleanup using the Ampure bead system (Agencourt, Bioscience, Beverly, MA). A 2 uL aliquot of purified PCR product was cycle sequenced using Big Dye Terminator Mix V.3 at 1/24 chemistry in a total volume of 4 uL (Applied Biosystems, Foster City, CA) as described previously [20]. As a quality control measure, both forward and reverse directions were sequenced. Variants had to be present in both read directions to be considered valid. Sequences were assembled and inspected in Mutation Surveyor v3.24 (SoftGenetics, State College, PA). All new sequenced variants were deposited in dbSNP.
TagSNP selection
Sequencing results were exported from Mutation Surveyor and imported into Haploview [22] for tagSNP selection using Tagger [23]. In addition, publicly available data from HapMap phase II was obtained as described above. To obtain tagSNPs representing both in-house re-sequencing data and public data, we used a strategy similar to one previously described [24]. Briefly, for each gene, tagSNPs were selected in four stages. We first selected tagSNPs for the SNPs that were present in both the sequencing results and the HapMap results, within the sequenced samples, using r2 = 1 and a minor allele frequency cut-off (MAF) of 2% (set a). These tagSNPs were then preferentially selected in the next step, selecting tagSNPs with r2 = 0.8 and MAF≥5% in HapMap samples from SNPs that were present in both the sequencing results and the HapMap results (set b). Third, tagSNPs were selected for all the SNPs found by sequencing, using the sequenced samples and force-including set a, with the same criteria as those used for set a (to make set c). Last, tagSNPs were selected from HapMap genotyping results, force-including set b, and using the same criteria as for set b (making up set d). The final genotyped set of SNPs was made up of the combination of sets c+d.
Genotyping
The SNPs selected for this study were part of a larger Golden Gate assay (Illumina, San Diego, CA), which also contained SNPs from candidate genes in other pathways related to other hypotheses. SNPs predicted to fail assay design (most often due to being too close together) were replaced when possible by equivalent tagSNPs. In addition to tagSNPs, 51 ancestry-informative markers (AIMs) selected from Halder et al. [25] were included in the assay.
600 ng of each genomic or WGA DNA sample was arrayed into 18 96-well plates and sent to The Centre for Applied Genomics, the Hospital for Sick Children in Toronto, Canada. The resulting genotypes were assessed using Genome Studio version 2009.1 (Illumina, San Diego, CA). The first steps in genotype quality control (Q/C) were performed in Genome Studio; systems and databases in the laboratory of DD completed the final steps. Genotypes derived from WGA and genomic DNA were subjected to Q/C separately.
Data quality control (SNPs)
For all genotypes, a GenCall Score cutoff of 0.25 was used, following Illumina recommendations for GoldenGate technology. The following SNPs were excluded: SNPs with GenTrain scores <0.4; SNPs with GenTrain scores between 0.4 and 0.7 and poor clustering (unexpected number of clusters, or clusters that were not well defined); SNPs with more than 3 clusters (which potentially indicate copy number variants); mono-allelic SNPs; SNPs with any genotype discrepancies between 53 pairs of duplicate samples; SNPs with call rate <95% in any sample type category (blood, mouthwash or saliva) were excluded for all samples in that category; one X-chromosome SNP that showed heterozygous calls in confirmed male samples; and SNPs out of Hardy-Weinberg equilibrium (p<0.001) in European-ancestry controls.
Of 178 tagSNPs selected for genotyping; 28/178 failed in the design phase, leaving 150 SNPs ordered for genotyping. Twenty-seven SNPs were excluded at the genotype quality control stage (12 SNPs were rejected by the genotyping centre upon initial inspection, 2 for low GenTrain scores, 5 for being potential copy number variants, 3 for being monoallelic, 3 for having a call rate <0.95, and 2 for having 1 error between duplicate genotypes). An additional 16 SNPs failed quality control only in WGA samples (7 for low GenTrain score and 9 for call rate <0.95), and 2 SNPs failed Q/C only in mouthwash or saliva samples. This left 123 SNPs (82%), listed in Table S4, for analysis in all non-WGA samples and 107 SNPs in both blood and WGA samples.
Data quality control (samples)
WGA samples give different genotype intensities than non-WGA samples [26] and so were examined separately. SNPs were excluded from analysis in WGA samples only if they passed the above criteria but showed any discrepancies in genotype between 52 pairs of WGA sample and matched pre-WGA samples.
Samples with sex discrepancies (Qu et al., in press at Frontiers in Statistical Genetics and Methodology) were excluded as they could have been subject to sample swaps during processing, as well as samples with call rate <0.98.
DNA samples from 1587 individuals were genotyped, 326/1587 being WGA samples. 58 non-WGA samples and 115 WGA samples were removed during quality control (23 non-WGA and 1 WGA sample due to gender discrepancies, and 35 non-WGA and 114 WGA samples due to call rate below 98%), leaving 1203 non-WGA samples+211 WGA samples, or 1414 samples in total.
45/51 AIMs passed quality control. Using genomic controls methodology, which has been shown to be appropriate when a modest number of candidate genes are assessed [27], [28], [29], we found no evidence for population stratification. The calculated inflation factor estimate was λ = 1.0.
Self reported ethnicities were confirmed using identity by state allele sharing and Multi-Dimensional Scaling (MDS) plots using HapMap data as the reference. These analyses identified two previously unidentified sets of sib-pairs, and three individuals who self-identified as Asian but clustered close to the HapMap CEU reference cluster. Sib-pair relationships were subsequently confirmed after review of study records; the youngest sibling of each pair (both were non-WGA samples) was excluded from analyses, as excess relatedness within a group of cases is not appropriate for a population-based association study. For the three subjects whose self-reported ethnicity was Asian, a review of study records indicated one subject self-reported being of Chinese ancestry, and two self-reported as being of Iranian descent. The subject of Chinese descent (non-WGA sample) was removed from the analyses due to concerns of a possible sample swap; and for the two subjects of Iranian descent the ethnicity was changed to “other”. This left 1200 non-WGA samples and 211 WGA samples, or 1411 samples in total (Table 1).
After all quality control, 1116/1411 samples (569 cases and 547 controls) of European ancestry were included in statistical analysis.
Statistical analysis
Statistical analysis was performed using SVS Suite 7 (Golden Helix, Bozeman, MT). Logistic regressions under an additive model were fit for diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), mantle cell lymphoma (MCL), all B-cell NHLs and all T-cell NHLs. In all subtype analyses, selected cases were compared to all controls. European-ancestry samples were used for analysis; other ethnicities (Asian, south-east Asian and “other”) were tested only for SNPs that showed association in European-ancestry samples. For each SNP, we calculated p-values for the model including the SNP of interest vs. the basic model (which accounted for 5-year age groups, sex, and region of residence). To test for genetic interaction between SNPs (in this example SNP A and SNP B), we tested a logistic regression model including an interaction term SNPA*SNPB against a model including the effects of SNP A, SNP B, 5-year age groups, sex and region of residence, limiting the analysis to European-ancestry samples.
To correct for multiple testing within each gene, full scan permutation carried out in SVS (10,000 permutations) [30], gave an adjusted p-value (denoted here by pp). For each permutation, the program randomly assigns case status to genotypes and calculates the number of times a more extreme p-value could be found by chance in permutations of all SNPs in the set. This method is similarly conservative to Bonferroni adjustment [30]. Like other lymphoma genetic epidemiology researchers [31], [32], we have used a two-tiered approach to multiple testing correction. The smallest adjusted p-value of SNPs within each gene was taken to represent the gene, and then the Benjamini-Hochberg procedure [33], implemented in R version 2.11.1, was applied to control the false-discovery rate (FDR) across the 17 genes and obtain a corrected p-value (pF). Adjusted p-values<0.05 were considered statistically significant. No multiple-testing correction was implemented for the few genetic interaction tests.
Results
Re-sequencing for SNP discovery
Table 2 lists the candidate genes. Eleven of the 17 genes were selected for re-sequencing in 47 samples because they failed the criterion of #SNPs/#tagSNPs>2. Genes ranged from having a complete lack of genotyped SNPs in HapMap (for example the 10 kb gene BMI1) to densely genotyped genes such as RFWD2 (which had 228 SNPs in HapMap and a #SNPs/#tagSNPs ratio of 7.9) (Table 2). Re-sequencing yielded 155 single nucleotide variants (Table S5), of which 40% was previously reported in dbSNP (build 127). Small insertions/deletions were as common as SNPs (40% vs. 39%, respectively). If one was to look only at relatively common SNPs (i.e. minor allele frequency (MAF) ≥5%), 72% of variants observed were previously reported at the time the sequencing results were analyzed, showing that most of the new information gathered by re-sequencing lay in rare variants. Eighty-eight tagSNPs were needed to capture the in-house sequencing data at r2 = 1, with the exception of singletons which were not included in the next phase of this study. An r2 of 1 was selected as the genetic information gained from sequencing was potentially more relevant because it was derived from lymphoma patient samples. Only 19/88 of these tagSNPs overlapped with tagSNPs used to capture the HapMap data, indicating that the information obtained is non-redundant with that in online databases and that the additional in-house sequencing produced novel genetic information valuable for representing (tagging) genetic variation in some of the candidate genes.
Association results
For a summary of all analysis results see Table 3. One SNP showed an association that was strong enough to survive multiple testing correction both at the individual gene and multi-gene level. rs928883 (close to miR-155) was significantly associated with marginal zone lymphoma (OR: 2.80 [95% CI: 1.63–4.82]; pp = 0.0016; pF = 0.0272). rs2829803, located 4 kb away from rs928883 (r2 = 0.41 between rs2829803 and rs928883), showed a weaker association with this same subtype (OR: 1.85 [95% CI: 1.12–3.08]). These associations were restricted to European ancestry samples, as there was no statistically-significant association in Asian samples or in South-Asian ancestry samples, although the power to detect such association was quite low in these smaller sample sets.
Four other results indicated an association when the permuted p-values for correction within the gene were used (i.e. pp<0.05), but did not have pF<0.05. The SNP rs17041883 in BCL2L11 for marginal zone lymphoma (OR: 0.29 [95% CI: 0.10–0.81]; pp = 0.033; pF = 0.280), rs6676805 in RFWD2 for mantle cell lymphoma (OR: 2.05 [95% CI: 1.22–3.45]; pp = 0.048; pF = 0.823), rs17642969 close to mir17-92 for T-cell lymphoma (OR: 0.409 [95% CI: 0.15–1.14]; pp = 0.047; pF = 0.800), and rs10512488 in BECN1 for diffuse large B-cell lymphoma (OR: 1.36 [95% CI: 1.03–1.80]; pp = 0.034; pF = 0.578).
miR-155 has many known targets, including p53BP1 (the product of TP53BP1), AID (the product of AICDA) and AGTR1 [34]. We had genotypes for 4, 3 and 1 tagSNPs, respectively, for these genes, in this same sample set. These tagSNPs were selected using HapMap Phase II genotypes in the CEU population (r2 = 0.8) for another study in our laboratory. We tested for interaction of each of these eight SNPs with rs928883. None of these eight SNPs were significantly associated with MZL. However, we found evidence for interactions of rs928883 near miR-155 with rs714629 (p = 0.048) and rs9971686 (p = 0.051) in AICDA (interactions odds ratios are tabulated in Table S7). These two SNPs in AICDA are not in linkage disequilibrium with each other (r2 = 0).
Discussion
Association studies are often based on tagSNPs, which are usually selected from HapMap. TagSNPs derived from HapMap have been identified as non-transferable between populations in some cases [35] but transferable in others [36], particularly if tagSNPs are collected from different populations and merged to create a “cosmopolitan” set [36]. Our re-sequencing strategy, in which genes showing low redundancy of SNPs in HapMap were re-sequenced in individuals derived from the population of interest, ensures good representation of genetic variation in our genes of interest in these experiments. We note that between the time of sequencing and publication, continued activity in the sequencing of human genomes further populated databases; 33% of SNPs that were not reported in dbSNP build 127 are now in this public database (build 132).
rs928883, which is associated with MZL in our study, is located 2.3 kb upstream of MIRN155, in the second intron of MIRN155HG, the host gene of this 64 bp microRNA. It is in weak LD (r2 of 0.37) with rs2829803, another SNP weakly associated with MZL. Replication of these findings is needed to validate the results, as our analysis used a relatively small group of 78 MZL samples.
The Eμ-myc/miR-155 double hit mouse [37], which overexpresses both myc and miR-155, has an enlarged spleen, a decrease in white blood cells and high grade malignant lymphomas that are similar to human acute lymphoblastic leukemia or lymphoblastic lymphoma, beginning at 6 months of age. miR-155 targets p53BP1 (p53-binding protein 1) [38], AID (activation-induced cytidines deaminase) [39], AGTR1 [34], and other genes. Known miR-155-targets are important players in lymphocyte development, marking miR-155 as an important connection point between these pathways.
miRNAs are ∼22 nucleotide RNAs that act as negative post-transcriptional regulators of gene expression by binding to imperfectly complementary sites on target mRNAs. 50% of miRNA genes are located in fragile sites or cancer-associated genomic regions [40], as well as being drastically reprogrammed in cancer and leukemia by copy number variation and differential expression [41]. Because miRNAs are short and highly conserved, mutations within miRNAs are expected to be uncommon. Consistent with this expectation, we found no polymorphisms in the transcribed region of the three miRNAs studied. Changes in allele frequencies of the SNPs located in miRNA target sites are potentially associated with human cancers [42]. In the past year, a few studies have explored the role of germline variants in microRNAs (particularly miR-146a, miR-196a and miR-499) in susceptibility to a variety of epithelial cancers, both familial [43] and sporadic – with both positive [44], [45] and negative reports [46], [47]. To our knowledge, however, no reports of tagSNPs or other SNPs near microRNAs affecting susceptibility to lymphoma or subtypes have been published thus far.
Although little is known about the role of miR-155 in MZL, its role has been investigated in other subtypes of NHL. High miR-155 expression is a marker of poor prognosis in HL and DLBCL [48], particularly in activated B-cell type DLBCL [49]. Its roles in modulation of cytokine production and AID regulation allow it to regulate the germinal centre response. Suppression of miR-155 in EBV-infected cells impedes entry into S phase and causes an increase in apoptosis [50]. The functions miR-155 has in lymphocytes and its documented effects in lymphoma certainly make it an interesting candidate gene for further study. As the causal variant may be another SNP in LD with rs928883, testing other variants in LD with this SNP could help to identify a variant that could affect miR-155's ability to repress its target mRNAs.
Morton et al. [31] examined risk of NHL with BCL10, BCL2L11, BCL2, BAX and BCL6 and found associations of BCL2L11 with FL, of BCL6 with DLBCL and BCL2 with MZL. Although we genotyped SNPs that either overlapped or were in high LD with these SNPs (r2>0.9), we did not replicate their findings. Two SNPs in BCL2L11 previously reported to be associated with NHL by Kelly et al. [32], with one SNP associated with all NHL and the other also associated with CLL/SLL and FL in particular, were also genotyped and we were unable to replicate these results. rs4934436 in FAS, previously associated with NHL risk by Wang et al. [51], particularly with the DLBCL and FL subtypes, was not tagged at sufficiently high LD (r2 = 0.6 with the haplotype CGG in the SNPs rs2234978/rs2147420/rs1571013, genotyped in this study) to test for replication. rs1056932 in BCL6 was previously found to be associated with NHL, B-cell lymphomas and T-cell lymphomas [52]. This SNP was genotyped but did not show association with any NHL subtype tested. We examined SNPs in LD with rs2279744 (“SNP 309”) in MDM2, in female cases of DLBCL as suggested by Bond et al. [53] but found no association. Previous reports of no association with NHL risk with variants in BCL2 [31], [54], [55], BCL6 [56], [57], BCL10 [31], [58], BAX [31], [54], FAS [59], and MDM2 [60], [61], [62] are consistent with our negative findings. With the exception of the findings of Morton et al. [31] (which, based on our smaller sample size after quality control measures are applied, we had <80% power to replicate) and the association reported by Wang et al. [51] (for which the SNP was not adequately tagged in our study), our lack of replication of the other reported associations may be due to differences between populations, or could indicate false positive associations in those studies, as our study did have >80% power to replicate the reported associations.
Some studies have found that further classifying DLBCLs according to cell-of-origin subtypes Germinal-Centre B-cell like (GCB) and Activated B-Cell like (ABC) can clarify associations; for example, rs80031434 (G397C) was associated only with the GCB subtype of DLBCL [63]. We do not have comprehensive ABC and GCB classifications for our samples, however, and are unable to address this question. Another limitation of our study is the small number of cases for some NHL subtypes, particularly when analyzing non-European ethnicities. Finally, a large number of comparisons have been conducted and, while our multiple-testing correction methods are conservative, this nevertheless affects the power of this study.
In summary, we report an association for rs928883, located near miR-155, with marginal zone lymphoma. To our knowledge this is the first reported association between germline polymorphisms involving a miRNA gene and lymphoma, though the relevance of microRNAs to cancer in general is well established. We were not able to replicate some previously reported associations of SNPs in other apoptosis genes with subtypes of NHL. Replication of our finding in other studies is needed to confirm its significance.
Supporting Information
Table S1.
Characteristics of the study population. DLBCL = Diffuse Large B-Cell Lymphoma, FL = Follicular Lymphoma, MZ/MALT = Marginal Zone lymphoma/Mucosa-Associated Lymphoma Tissue lymphoma,MCL = Mantle Cell lymphoma, SLL = Small Lymphocytic Lymphoma, LPL = Lymphoplasmacytic Lymphoma, Misc. B-cell = Miscellaneous B-cell lymphoma, MF = Mycosis Fungoides, PTCL = Peripheral T-Cell Lymphoma, Misc. T-cell = Miscellaneous T-cell lymphoma.
https://doi.org/10.1371/journal.pone.0031560.s001
(PDF)
Table S2.
Characteristics of the samples sequenced. DLBCL = Diffuse Large B-Cell Lymphoma, FL = Follicular Lymphoma, MZ/MALT = Marginal Zone lymphoma/Mucosa-Associated Lymphoma Tissue lymphoma,MCL = Mantle Cell lymphoma, SLL = Small Lymphocytic Lymphoma, LPL = Lymphoplasmacytic Lymphoma, Misc. B-cell = Miscellaneous B-cell lymphoma, MF = Mycosis Fungoides, PTCL = Peripheral T-Cell Lymphoma, Misc. T-cell = Miscellaneous T-cell lymphoma.
https://doi.org/10.1371/journal.pone.0031560.s002
(PDF)
Table S3.
PCR primers and reaction conditions.
https://doi.org/10.1371/journal.pone.0031560.s003
(PDF)
Table S4.
Variants available for testing after data cleaning.
https://doi.org/10.1371/journal.pone.0031560.s004
(PDF)
Table S5.
Variants discovered by re-sequencing.
https://doi.org/10.1371/journal.pone.0031560.s005
(XLS)
Table S6.
Summary of results for top-ranked SNP (ranked by pp) in each gene in European-ancestry samples for the major subtypes of non-Hodgkin lymphoma (odds ratio for additive model, p-value* adjusted within gene and p-value* adjusted overall). All analyses are done against 547 European-ancestry controls. p-values<0.05 are in bold. DLBCL = Diffuse Large B-Cell Lymphoma, FL = Follicular Lymphoma, MZ/MALT = Marginal Zone lymphoma/Mucosa-Associated Lymphoma Tissue lymphoma,MCL = Mantle Cell lymphoma.
https://doi.org/10.1371/journal.pone.0031560.s006
(XLS)
Table S7.
Interaction table displaying odds ratios for rs928883 and two AICDA SNPs, rs714629 and rs9971686, derived under an additive model.
https://doi.org/10.1371/journal.pone.0031560.s007
(PDF)
Acknowledgments
The authors thank Stephen Leach for his longstanding lab assistance and Dr. Tara Paton of the Centre for Applied Genomics at The Hospital for Sick Children, Toronto, Canada for assistance with genotype data processing and quality control.
Author Contributions
Conceived and designed the experiments: JMS RPG JJS ABW. Performed the experiments: JMS. Analyzed the data: JMS DD JG JJS. Contributed reagents/materials/analysis tools: DD. Wrote the paper: JMS ABW. Did the pathology review: RDG BRB. Responsible for clinical review for all cases: JMC.
References
- 1. Shaffer AL, Rosenwald A, Staudt LM (2002) Lymphoid malignancies: the dark side of B-cell differentiation. Nat Rev Immunol 2: 920–932.
- 2. Strasser A (2005) The role of BH3-only proteins in the immune system. Nat Rev Immunol 5: 189–200.
- 3. Willis TG, Dyer MJ (2000) The role of immunoglobulin translocations in the pathogenesis of B-cell malignancies. Blood 96: 808–822.
- 4. Danial NN (2007) BCL-2 family proteins: critical checkpoints of apoptotic cell death. Clin Cancer Res 13: 7254–7263.
- 5. Kelly PN, Strasser A (2011) The role of Bcl-2 and its pro-survival relatives in tumourigenesis and cancer therapy. Cell Death Differ 18: 1414–1424.
- 6. Cattoretti G, Shaknovich R, Smith PM, Jack HM, Murty VV, et al. (2006) Stages of germinal center transit are defined by B cell transcription factor coexpression and relative abundance. J Immunol 177: 6930–6939.
- 7. Hu S, Du MQ, Park SM, Alcivar A, Qu L, et al. (2006) cIAP2 is a ubiquitin protein ligase for BCL10 and is dysregulated in mucosa-associated lymphoid tissue lymphomas. J Clin Invest 116: 174–181.
- 8. Yue Z, Jin S, Yang C, Levine AJ, Heintz N (2003) Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor. Proc Natl Acad Sci U S A 100: 15077–15082.
- 9. Lessard J, Sauvageau G (2003) Bmi-1 determines the proliferative capacity of normal and leukaemic stem cells. Nature 423: 255–260.
- 10. Villa-Morales M, Gonzalez-Gugel E, Shahbazi MN, Santos J, Fernandez-Piqueras J (2010) Modulation of the Fas-apoptosis-signalling pathway by functional polymorphisms at Fas, FasL and Fadd and their implication in T-cell lymphoblastic lymphoma susceptibility. Carcinogenesis 31: 2165–2171.
- 11. Eischen CM, Alt JR, Wang P (2004) Loss of one allele of ARF rescues Mdm2 haploinsufficiency effects on apoptosis and lymphoma development. Oncogene 23: 8931–8940.
- 12. Dornan D, Wertz I, Shimizu H, Arnott D, Frantz GD, et al. (2004) The ubiquitin ligase COP1 is a critical negative regulator of p53. Nature 429: 86–92.
- 13. Gstaiger M, Jordan R, Lim M, Catzavelos C, Mestan J, et al. (2001) Skp2 is oncogenic and overexpressed in human cancers. Proc Natl Acad Sci U S A 98: 5043–5048.
- 14. Aqeilan RI, Calin GA, Croce CM (2010) miR-15a and miR-16-1 in cancer: discovery, function and future perspectives. Cell Death Differ 17: 215–220.
- 15. Xiao C, Srinivasan L, Calado DP, Patterson HC, Zhang B, et al. (2008) Lymphoproliferative disease and autoimmunity in mice with increased miR-17-92 expression in lymphocytes. Nat Immunol 9: 405–414.
- 16. Vigorito E, Perks KL, Abreu-Goodger C, Bunting S, Xiang Z, et al. (2007) microRNA-155 regulates the generation of immunoglobulin class-switched plasma cells. Immunity 27: 847–859.
- 17. Sipahimalani P, Spinelli JJ, MacArthur AC, Lai A, Leach SR, et al. (2007) A systematic evaluation of the ataxia telangiectasia mutated gene does not show an association with non-Hodgkin lymphoma. Int J Cancer 121: 1967–1975.
- 18. Spinelli JJ, Ng CH, Weber JP, Connors JM, Gascoyne RD, et al. (2007) Organochlorines and risk of non-Hodgkin lymphoma. Int J Cancer 121: 2767–2775.
- 19. Schuetz JM, MaCarthur AC, Leach S, Lai AS, Gallagher RP, et al. (2009) Genetic variation in the NBS1, MRE11, RAD50 and BLM genes and susceptibility to non-Hodgkin lymphoma. BMC Med Genet 10: 117.
- 20. Brooks-Wilson AR, Kaurah P, Suriano G, Leach S, Senz J, et al. (2004) Germline E-cadherin mutations in hereditary diffuse gastric cancer: assessment of 42 new families and review of genetic screening criteria. J Med Genet 41: 508–517.
- 21. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol 132: 365–386.
- 22. Barrett JC, Fry B, Maller J, Daly MJ (2005) Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 21: 263–265.
- 23. de Bakker PI, Yelensky R, Pe'er I, Gabriel SB, Daly MJ, et al. (2005) Efficiency and power in genetic association studies. Nat Genet 37: 1217–1223.
- 24. Halaschek-Wiener J, Amirabbasi-Beik M, Monfared N, Pieczyk M, Sailer C, et al. (2009) Genetic variation in healthy oldest-old. PLoS One 4: e6641.
- 25. Halder I, Shriver M, Thomas M, Fernandez JR, Frudakis T (2008) A panel of ancestry informative markers for estimating individual biogeographical ancestry and admixture from four continents: utility and applications. Hum Mutat 29: 648–658.
- 26. Mead S, Poulter M, Beck J, Uphill J, Jones C, et al. (2008) Successful amplification of degraded DNA for use with high-throughput SNP genotyping platforms. Hum Mutat 29: 1452–1458.
- 27. Devlin B, Roeder K, Wasserman L (2000) Genomic control for association studies: a semiparametric test to detect excess-haplotype sharing. Biostatistics 1: 369–387.
- 28. Devlin B, Roeder K, Wasserman L (2001) Genomic control, a new approach to genetic-based association studies. Theor Popul Biol 60: 155–166.
- 29. Devlin B, Bacanu SA, Roeder K (2004) Genomic Control to the extreme. Nat Genet 36: 1129–1130; author reply 1131.
- 30. Zhao JH, Curtis D, Sham PC (2000) Model-free analysis and permutation tests for allelic associations. Human Heredity 50: 133–139.
- 31. Morton LM, Purdue MP, Zheng T, Wang SS, Armstrong B, et al. (2009) Risk of non-Hodgkin lymphoma associated with germline variation in genes that regulate the cell cycle, apoptosis, and lymphocyte development. Cancer Epidemiol Biomarkers Prev 18: 1259–1270.
- 32. Kelly JL, Novak AJ, Fredericksen ZS, Liebow M, Ansell SM, et al. (2010) Germline variation in apoptosis pathway genes and risk of non-Hodgkin's lymphoma. Cancer Epidemiol Biomarkers Prev 19: 2847–2858.
- 33. Benjamini Y, Hochberg Y (1995) Controlling the false discovery rate: a practical and powerful approach to multiple testing. J R Stat Soc Ser B 57: 289–300.
- 34. Sethupathy P, Borel C, Gagnebin M, Grant GR, Deutsch S, et al. (2007) Human microRNA-155 on chromosome 21 differentially interacts with its polymorphic target in the AGTR1 3′ untranslated region: a mechanism for functional single-nucleotide polymorphisms related to phenotypes. Am J Hum Genet 81: 405–413.
- 35. Johansson A, Vavruch-Nilsson V, Cox DR, Frazer KA, Gyllensten U (2007) Evaluation of the SNP tagging approach in an independent population sample–array-based SNP discovery in Sami. Hum Genet 122: 141–150.
- 36. de Bakker PI, Burtt NP, Graham RR, Guiducci C, Yelensky R, et al. (2006) Transferability of tag SNPs in genetic association studies in multiple populations. Nat Genet 38: 1298–1303.
- 37. Costinean S, Zanesi N, Pekarsky Y, Tili E, Volinia S, et al. (2006) Pre-B cell proliferation and lymphoblastic leukemia/high-grade lymphoma in E(mu)-miR155 transgenic mice. Proc Natl Acad Sci U S A 103: 7024–7029.
- 38. Gironella M, Seux M, Xie MJ, Cano C, Tomasini R, et al. (2007) Tumor protein 53-induced nuclear protein 1 expression is repressed by miR-155, and its restoration inhibits pancreatic tumor development. Proc Natl Acad Sci U S A 104: 16170–16175.
- 39. Teng G, Hakimpour P, Landgraf P, Rice A, Tuschl T, et al. (2008) MicroRNA-155 is a negative regulator of activation-induced cytidine deaminase. Immunity 28: 621–629.
- 40. Calin GA, Sevignani C, Dumitru CD, Hyslop T, Noch E, et al. (2004) Human microRNA genes are frequently located at fragile sites and genomic regions involved in cancers. Proc Natl Acad Sci U S A 101: 2999–3004.
- 41. Volinia S, Galasso M, Costinean S, Tagliavini L, Gamberoni G, et al. (2010) Reprogramming of miRNA networks in cancer and leukemia. Genome Res 20: 589–599.
- 42. Yu Z, Li Z, Jolicoeur N, Zhang L, Fortin Y, et al. (2007) Aberrant allele frequencies of the SNPs located in microRNA target sites are potentially associated with human cancers. Nucleic Acids Res 35: 4535–4541.
- 43. Shen J, Ambrosone CB, Zhao H (2009) Novel genetic variants in microRNA genes and familial breast cancer. Int J Cancer 124: 1178–1182.
- 44. Jazdzewski K, Murray EL, Franssila K, Jarzab B, Schoenberg DR, et al. (2008) Common SNP in pre-miR-146a decreases mature miR expression and predisposes to papillary thyroid carcinoma. Proc Natl Acad Sci U S A 105: 7269–7274.
- 45. Hoffman AE, Zheng T, Yi C, Leaderer D, Weidhaas J, et al. (2009) microRNA miR-196a-2 and breast cancer: a genetic and epigenetic association study and functional analysis. Cancer Res 69: 5970–5977.
- 46. Yang R, Dick M, Marme F, Schneeweiss A, Langheinz A, et al. (2010) Genetic variants within miR-126 and miR-335 are not associated with breast cancer risk. Breast Cancer Res Treat.
- 47. Catucci I, Yang R, Verderio P, Pizzamiglio S, Heesen L, et al. (2010) Evaluation of SNPs in miR-146a, miR196a2 and miR-499 as low-penetrance alleles in German and Italian familial breast cancer cases. Hum Mutat 31: E1052–1057.
- 48. Eis PS, Tam W, Sun L, Chadburn A, Li Z, et al. (2005) Accumulation of miR-155 and BIC RNA in human B cell lymphomas. Proc Natl Acad Sci U S A 102: 3627–3632.
- 49. Jung I, Aguiar RC (2009) MicroRNA-155 expression and outcome in diffuse large B-cell lymphoma. Br J Haematol 144: 138–140.
- 50. Linnstaedt SD, Gottwein E, Skalsky RL, Luftig MA, Cullen BR (2010) Virally induced cellular microRNA miR-155 plays a key role in B-cell immortalization by Epstein-Barr virus. J Virol 84: 11670–11678.
- 51. Wang SS, Purdue MP, Cerhan JR, Zheng T, Menashe I, et al. (2009) Common gene variants in the tumor necrosis factor (TNF) and TNF receptor superfamilies and NF-kB transcription factors and non-Hodgkin lymphoma risk. PLoS One 4: e5360.
- 52. Zhang Y, Lan Q, Rothman N, Zhu Y, Zahm SH, et al. (2005) A putative exonic splicing polymorphism in the BCL6 gene and the risk of non-Hodgkin lymphoma. J Natl Cancer Inst 97: 1616–1618.
- 53. Bond GL, Hirshfield KM, Kirchhoff T, Alexe G, Bond EE, et al. (2006) MDM2 SNP309 accelerates tumor formation in a gender-specific and hormone-dependent manner. Cancer Res 66: 5104–5110.
- 54. Enjuanes A, Benavente Y, Bosch F, Martin-Guerrero I, Colomer D, et al. (2008) Genetic variants in apoptosis and immunoregulation-related genes are associated with risk of chronic lymphocytic leukemia. Cancer Res 68: 10178–10186.
- 55. Fernberg P, Chang ET, Duvefelt K, Hjalgrim H, Eloranta S, et al. (2010) Genetic variation in chromosomal translocation breakpoint and immune function genes and risk of non-Hodgkin lymphoma. Cancer Causes Control 21: 759–769.
- 56. Au WY, Fung A, Liang R (2005) Molecular epidemiology of follicular lymphoma in Chinese: relationship with bcl-2/IgH translocation and bcl-6 397G/C polymorphism. Ann Hematol 84: 506–509.
- 57. Susova S, Trneny M, Soucek P (2006) Single nucleotide polymorphism in 5′-flanking region of BCL6 is not associated with increased risk of non-Hodgkin's lymphoma. Cancer Lett 238: 142–145.
- 58. Yuille MR, Stone JG, Bradshaw PS, Houlston RS (1999) Bcl10 in chronic lymphocytic leukaemia and T-cell prolymphocytic leukaemia. Br J Haematol 107: 384–385.
- 59. Scott K, Adamson PJ, Willett EV, Worrillow LJ, Allan JM (2008) Genetic variation in genes expressed in the germinal center and risk of B cell lymphoma among Caucasians. Haematologica 93: 1597–1600.
- 60. Zenz T, Habe S, Benner A, Kienle D, Dohner H, et al. (2008) The MDM2 −309 T/G promoter single nucleotide polymorphism does not alter disease characteristics in chronic lymphocytic leukemia. Haematologica 93: 1111–1113.
- 61. Wrench D, Waters R, Carlotti E, Iqbal S, Matthews J, et al. (2009) Clinical relevance of MDM2 SNP 309 and TP53 Arg72Pro in follicular lymphoma. Haematologica 94: 148–150.
- 62. Do TN, Ucisik-Akkaya E, Davis CF, Morrison BA, Dorak MT (2009) TP53 R72P and MDM2 SNP309 polymorphisms in modification of childhood acute lymphoblastic leukemia susceptibility. Cancer Genet Cytogenet 195: 31–36.
- 63. Berglund M, Amini RM, Book M, Rosenquist R, Roos G, et al. (2006) Subtype preference of the BCL6397G/C polymorphism in germinal-center and non-germinal-center subtypes of diffuse large B-cell lymphoma. Blood 108: 3623–3624.