Non-Hodgkin Lymphoma Risk and Variants in Genes Controlling Lymphocyte Development.
ABSTRACT Non-Hodgkin lymphomas (NHL) are a heterogeneous group of solid tumours of lymphoid cell origin. Three important aspects of lymphocyte development include immunity and inflammation, DNA repair, and programmed cell death. We have used a previously established case-control study of NHL to ask whether genetic variation in genes involved in these three important processes influences risk of this cancer. 118 genes in these three categories were tagged with single nucleotide polymorphisms (SNPs), which were tested for association with NHL and its subtypes. The main analysis used logistic regression (additive model) to estimate odds ratios in European-ancestry cases and controls. 599 SNPs and 1116 samples (569 cases and 547 controls) passed quality control measures and were included in analyses. Following multiple-testing correction, one SNP in MSH3, a mismatch repair gene, showed an association with diffuse large B-cell lymphoma (OR: 1.91; 95% CI: 1.41-2.59; uncorrected p = 0.00003; corrected p = 0.010). This association was not replicated in an independent European-ancestry sample set of 251 diffuse large B-cell lymphoma cases and 737 controls, indicating this result was likely a false positive. It is likely that moderate sample size, inter-subtype and other genetic heterogeneity, and small true effect sizes account for the lack of replicable findings.
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ABSTRACT: Abstract PURPOSE: Evaluation diagnostic usefulness of immunoglobulin light chains (Igκ, Igλ) and incomplete IGH D-J clonal gene rearrangements in formalin-fixed, paraffin-embedded (FFPE) tissue of patients with B-cell non-Hodgkin lymphomas (B-NHL). MATERIALS AND METHODS: This study was performed on samples from 70 patients with B-NHL, including two cases of follicular lymphoma (FL), 20 cases of diffuse large B-cell lymphoma (DLBCL), one case of mantle cell lymphoma (MCL), and 47 cases of B-cell neoplasm (non-classified), which had been previously assessed for complete IGH clonality, and failure to clarify gene rearrangements. We used a gold standard multiplex PCR protocol provided by European Biomedicine and Health (BIOMED-2) Concerted Action Project BMH4-CT98-3936 for improvement of diagnosis and analysis of clonality gene rearrangement in lymphoma malignancies. RESULTS: Our results revealed a total positive monoclonality of 89 % (62/70) in Igκ, Igλ, and 11.4 % (8/70) polyclonality in gene rearrangements assay. The samples with positive clonality consisting (Igκ: 45 %, Igλ: 55 %) in DLBCL, (Igκ: 100 %) in FL, (Igλ: 100 %) in MCL, and (Igκ: 47 %, Igλ: 36 %) in B-cell neoplasm non-classified. None of the incomplete IGH D-J immunoglobulin gene families (0 %) showed monoclonality, and all samples demonstrated polyclonality pattern. CONCLUSIONS: Our findings on FFPE tissue revealed that immunoglobulin light chains clonality gene rearrangements assays using BIOMED-2 protocol, could be considered a valuable and reliable method for clonality detection, particularly in cases of failure of complete IGH gene rearrangements analysis. Clonal Ig gene rearrangements assay is applicable for routine diagnostic testing of lymphoproliferative disorders and as a reliable method for differentiating between malignant and benign lymphoma disorders.Clinical and Translational Oncology 05/2014; · 1.60 Impact Factor
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ABSTRACT: The expression and biologic function of SGTA in Non-Hodgkin's Lymphomas (NHL) was investigated in this study. Clinically, by immunohistochemistry analysis we detected SGTA expression in both reactive lymphoid tissues and NHL tissues. In addition, we also correlated high expression of SGTA with poor prognosis. Functionally, SGTA expression was positively related with cell proliferation and negative related with cell adhesion. Finally, SGTA knockdown induced adhesion-mediated drug resistance. Our finding supports a role of SGTA in NHL cell proliferation, adhesion and drug resistance, and it may pave the way for a novel therapeutic approach for CAM-DR in NHL.Leukemia Research 08/2014; · 2.69 Impact Factor
Non-Hodgkin Lymphoma Risk and Variants in Genes
Controlling Lymphocyte Development
Johanna M. Schuetz1, Denise Daley2, Stephen Leach1, Lucia Conde3, Brian R. Berry4,
Richard P. Gallagher5, Joseph M. Connors6, Randy D. Gascoyne7, Paige M. Bracci8, Christine F. Skibola3,
John J. Spinelli5,9, Angela R Brooks-Wilson1,10*
1Canada’s Michael Smith Genome Sciences Centre, BC Cancer Agency, Vancouver, British Columbia, Canada, 2Faculty of Medicine, University of British Columbia,
Vancouver, British Columbia, Canada, 3Department of Epidemiology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, United
States of America, 4Department of Pathology, Royal Jubilee Hospital, Victoria, British Columbia, Canada, 5Cancer Control Research, BC Cancer Agency, Vancouver, British
Columbia, Canada, 6Division of Medical Oncology and Centre for Lymphoid Cancer, BC Cancer Agency, Vancouver, British Columbia, Canada, 7Department of Pathology
and Centre for Lymphoid Cancer, BC Cancer Agency, Vancouver, British Columbia, Canada, 8Department of Epidemiology and Biostatistics, University of California San
Francisco, San Francisco, California, United States of America, 9School of Population and Public Health, University of British Columbia, Vancouver, British Columbia,
Canada, 10Department of Biomedical Physiology and Kinesiology, Simon Fraser University, Burnaby, British Columbia, Canada
Non-Hodgkin lymphomas (NHL) are a heterogeneous group of solid tumours of lymphoid cell origin. Three important
aspects of lymphocyte development include immunity and inflammation, DNA repair, and programmed cell death. We have
used a previously established case-control study of NHL to ask whether genetic variation in genes involved in these three
important processes influences risk of this cancer. 118 genes in these three categories were tagged with single nucleotide
polymorphisms (SNPs), which were tested for association with NHL and its subtypes. The main analysis used logistic
regression (additive model) to estimate odds ratios in European-ancestry cases and controls. 599 SNPs and 1116 samples
(569 cases and 547 controls) passed quality control measures and were included in analyses. Following multiple-testing
correction, one SNP in MSH3, a mismatch repair gene, showed an association with diffuse large B-cell lymphoma (OR: 1.91;
95% CI: 1.41–2.59; uncorrected p=0.00003; corrected p=0.010). This association was not replicated in an independent
European-ancestry sample set of 251 diffuse large B-cell lymphoma cases and 737 controls, indicating this result was likely a
false positive. It is likely that moderate sample size, inter-subtype and other genetic heterogeneity, and small true effect
sizes account for the lack of replicable findings.
Citation: Schuetz JM, Daley D, Leach S, Conde L, Berry BR, et al. (2013) Non-Hodgkin Lymphoma Risk and Variants in Genes Controlling Lymphocyte
Development. PLoS ONE 8(9): e75170. doi:10.1371/journal.pone.0075170
Editor: Kristy L. Richards, University of North Carolina at Chapel Hill, United States of America
Received March 5, 2013; Accepted August 13, 2013; Published September 30, 2013
Copyright: ? 2013 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. DD holds a Tier II Canada
Research Chair (Genetic Epidemiology of Common Complex Diseases). AB-W is a Senior Scholar, and DD and JG are Scholars of the Michael Smith Foundation for
Health Research. For the UCSF study, CFS and PMB have support from the National Institutes of Health, National Cancer Institute (R01CA87014, R03CA143947,
R03CA150037, R01CA104682, RO1CA122663, RO1CA154643). 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.
* E-mail: firstname.lastname@example.org
Non-Hodgkin lymphoma (NHL) is a collection of malignancies
of lymphocyte origin. In Western countries, 85% of NHLs have a
B-cell origin. NHL subtypes vary in prognosis, treatment options
and outcome. Diffuse large B-cell lymphoma (DLBCL) patients
with different molecular or genetic abnormalities can have diverse
presentation and outcomes. Risk of developing NHL can be
influenced by both environmental and genetic factors that affect
the survival of lymphocytes.
Lymphocyte development is a complex process, with check-
points in place to ensure that the cells whose function is to quickly
and effectively protect the host from a variety of offences, will also
withhold such an assault on host cells. Cell growth and cell death
need to be regulated so that the number of lymphocytes is con-
trolled in such a way that they are sufficient to fight infections, but
not so numerous that they are a burden to maintain. Three im-
portant aspects of this control are: 1) immunity and inflammation
to respond to stimuli that cause their activation and rapid cell cycle
division; 2) DNA repair to counteract errors from cell division or
lymphocyte receptor gene rearrangement; and 3) cell death to
remove lymphocytes that are not able to meet cell cycle
checkpoints and/or reduce autoimmunity.
Previous work by several research groups has identified genetic
variants associated with NHL in genes related to B-cell survival
[1,2], DNA repair  and immunity and inflammation[4–8].
Collectively, genetic variants in these types of genes are likely to
play a role in susceptibility to NHL. To survey for genetic factors
associated with NHL in genes involved in immunity and
inflammation, DNA repair or cell death, we selected 118 genes
(listed in Table S1 in File S1) related to these biological
processes, tagged them with SNPs and tested them for association
with NHL in 569 cases and 547 controls. In addition, we selected
39 SNPs that had previously been associated with NHL in the
literature, and tested them for replication in our study. After
correction for multiple testing, we found evidence that a SNP in
PLOS ONE | www.plosone.org1 September 2013 | Volume 8 | Issue 9 | e75170
MSH3, a gene that has never before been implicated in NHL, may
affect susceptibility to DLBCL; however, this association did not
replicate in an independent NHL population.
Materials and Methods
The samples and genes tested in this study were part of a 1536-
SNP Illumina GoldenGate panel that included SNPs from
candidate genes related to other pathways and hypotheses .
Details of the population, samples and methodology have been
previously described .
Study Subjects and Samples
All new NHL cases in the Greater Vancouver Regional District
and Greater Victoria (Capital Regional District), British Colum-
bia, from March 2000 to February 2004 were invited to
participate. Cases aged 20 to 79 were included. Patients with
prior transplant or HIV-positivity were excluded. Population
controls were frequency matched by age (within 5-year groups),
sex and area of residence. Family history of cancer was based on
subject-reported data. Of 821 cases and 848 controls were
available for this study, 797 cases and 790 controls had sufficient
DNA for genotyping. The study was approved by the joint
University of British Columbia/British Columbia Cancer Agency
Research Ethics Board; all participants gave written informed
DNA was extracted from whole blood (407 samples), lympho-
cytes isolated from blood (782 samples), mouthwash (24 samples),
or saliva (48 samples) as previously described . 326/1587
samples, referred to as ‘WGA samples’, had low DNA yields; their
DNA was amplified by whole genome amplification using the
RepliG kit (QIAGEN, Mississauga, ON, Canada) .
The 118 genes selected for this study (Table 1) were based on a
review of the biological literature. For each gene, publicly
available data from HapMap phase II was imported into
Haploview  for tagSNP selection using Tagger at r2=0.8.
TagSNP selection was restricted to SNPs with minor allele
frequency (MAF) .5%. In addition, 39 specific SNPs previously
reported as associated with NHL, autoimmune disease or cancer
were included to test for replication of these associations in our
study. These ‘replication’ SNPs are listed in Table S2 in File S1.
51 ancestry-informative markers (AIMs) selected from Halder et al.
 were also included in the assay. Genotyping was done using
the Golden Gate system (Illumina, San Diego, CA), at The Centre
for Applied Genomics, the Hospital for Sick Children in Toronto,
Canada; as described previously .
Quality control (Q/C) was conducted using Genome Studio
version 2009.1 (Illumina, San Diego, CA) and systems and
databases developed in the laboratory of DD . Genotypes
derived from WGA DNA and genomic DNA were subjected to
Q/C separately. 1411 samples (717 cases and 694 controls) passed
Q/C (Table 2); 1116/1411 samples (569 cases and 547 controls)
were of European ancestry and subsequently included in statistical
analysis . AIMS analysis in this study has been previously
described , and supported analysis of the European-ancestry
samples as one group.
Of 708 SNPs selected for genotyping of variants in genes related
to lymphocyte development, 109 were excluded at the genotype
Q/C stage (32 SNPs were rejected by the genotyping centre upon
initial inspection, 14 for low GenTrain scores, 26 for being
potential copy number variants, 12 for being monoallelic, 8 for
having a call rate ,0.95, 15 for having any error between
duplicate genotypes, and 2 for deviating significantly from Hardy-
Weinberg equilibrium [HWE]). An additional 160 SNPs failed Q/
C only in WGA samples (8 upon initial inspection by the
genotyping centre, 49 for low GenTrain score, 64 for call rate
,0.95, 38 SNPs that had discrepant genotypes between WGA
samples and pre-WGA matched DNA, and 1 SNP for being out of
HWE), and 4 SNPs failed Q/C only in mouthwash or saliva
samples. This left 599 SNPs (85%), listed in Table S3 in File S1,
for analysis in all non-WGA samples and 439 SNPs in both blood
and WGA samples.
Statistical analyses were conducted in SVS Suite 7 (Golden
Helix, Bozeman, MT). Logistic regression (additive model) was fit
for diffuse large B-cell lymphoma (DLBCL), follicular lymphoma
(FL), marginal zone lymphoma (MZL), all B-cell NHLs and all T-
cell NHLs. Other NHL subtypes were not individually tested, as
sample numbers were insufficient. In all subtype analyses, selected
cases were compared to all controls. The analysis was restricted to
European-ancestry samples, with other ethnicities (Asian, south-
east Asian and ‘‘other’’) only tested when SNPs showed association
in European-ancestry samples, corresponding to 148 DLBCL, 165
FL, 55 MZL, 523 B-cell NHL, 45 T-cell NHL and 547 control
samples. This corresponded to a minimum detectable odds ratio of
1.54 for DLBCL, 1.51 for FL, 1.88 for MZL, 1.33 for B-cell NHL
and 1.99 for T-cell NHL. For each SNP, p-values were calculated
for the model with the SNP of interest vs. the basic model (which
accounted for 5-year age groups, sex, and region). For only the
SNPs that showed a statistically significant association, to find the
model with the best fit we then tested dominant and recessive
models in genotypic tests using the chi-squared test, as well as a
recessive model by logistic regression with the adjustments listed
above (i.e. age groups, sex and region). SNPs that showed an
association were also tested for interaction with sex by comparing
a model including the SNP, age group, sex and region to a model
that also included the SNP*sex interaction. In genes that
Table 1. Genes and categories.
Immunity and Inflammation AICDA, BRD2, CCL5, CD69, CD74, CD81, CTLA4, HFE, IFNAR2, IFNB1, IFNG, IL10RA, IL1RN, IL4, IL6, IL7, IL7R, IRF4, IRF5, ITGAM,
JAK1, JAK3, KIAA1542, LTA, PKX, PRDM1, PRMT5, SPIB, SPP1, STAT3
Cell deathAGTR1, APAF1, BAD, BAK1, BCL11A, BCL2L1, BCL2L2, BID, BIK, BIRC3, BMF, CASP1, CASP10, CASP3, CASP4, CASP8, CD40, CDH22,
CFLAR, FASLG, IGFBP3, IL2, IL8, IL8RB, ITCH, MDM4, MYC, NEDD4, NFKB1, NFKB2, PARP1, PAX5, RASSF1, REL, RELA, RELB, TLR2,
TNFSF10, TP73, ZFX
DNA repairAPEX1, ATR, BIN3, C11orf30, CCND1, CDK7, CDKN2A, CHEK1, CHEK2, E2F1, E2F2, E2F3, ERCC2, ERCC5, EXO1, H2AFZ, HIC1, HINT1,
LIG1, LIG3, LIG4, MGMT, MSH2, MSH3, MSH6, MTHFR, MTR, OGG1, PLK1, PMS2, POLB, POLD1, PTEN, RAD51, RAD52, RAD54B,
RAG1, RB1, RPA1, TP53BP1, TYMS, UNG, WRN, XRCC1, XRCC3, XRCC4, XRCC5, YY1
NHL Risk and Lymphocyte Development Genes
PLOS ONE | www.plosone.org2 September 2013 | Volume 8 | Issue 9 | e75170
contained multiple SNPs with an association, the SNPs that
showed an association were tested for interaction by comparing a
model including that included the two SNPs, age group, sex and
region vs. a model with the addition of the SNP*SNP interaction.
In addition, for genes with an association, haplotype analysis was
conducted in SVS Suite 7.
To correct for multiple testing, we have used a two-tiered
approach, as previously described . The Benjamini-Hochberg
procedure , implemented in R version 2.11.1, was applied to
control the false-discovery rate (FDR) for SNPs within each gene,
giving a corrected p-value denoted as pG. The smallest adjusted p-
value for each gene was taken to represent the gene, and FDR was
applied again across the genes in each of the three hypotheses (i.e.
gene categories) tested (cell death, DNA repair and immunity and
inflammation). This second corrected p-value was denoted pH.
Adjusted p-values ,0.05 were considered statistically significant.
No multiple-testing correction was applied for the few interaction
or haplotype tests.
Since genes involved in mismatch repair pathways have been
shown to be important for colorectal cancer risk, we tested
whether rs33003 in MSH3 were associated with a family history of
colorectal cancer. Colorectal cancer in one or more first-degree
relatives of the NHL cases and controls was coded as a true/false
‘‘family history of colorectal cancer’’ variable, and was used in
logistic regression analysis in European-ancestry samples, adjusting
for sex, region and 5-year age groups. 28/569 cases and 33/547
controls of European-ancestry had a family history of colorectal
The association of rs33003 with DLBCL was tested in a
previously described independent population from the San
Francisco Bay Area . Briefly, cases were identified through
the Northern California Cancer Center between 2001 and 2005.
All were residents of the San Francisco Bay Area, 20–84 years old,
and provided informed consent. For this analysis, we used
genotypes imputed by BEAGLE v.3.3  for 737 controls and
251 DLBCL cases that self-reported as ‘‘non-Hispanic white’’ and
also clustered with Caucasian samples by principal component
analysis. The imputation yielded 391 samples of GG genotype,
417 samples of GA genotype, 103 samples of AA genotype and 77
samples with unknown genotype. A logistic regression model
under the additive model, with correction for age and sex was used
to estimate odds ratios.
Table S4 in File S1 lists all SNPs with p,0.05 (before any
multiple testing correction). Table 3 lists the 59 SNPs with
pG,0.05. Of note, none of the 39 SNPs selected to replicate
previously reported associations were associated with lymphoma in
our population. Only one SNP showed an association that was
significant after multiple testing correction both at the individual
gene and multi-gene (hypothesis) level. rs33003, located in MSH3,
was significantly associated with DLBCL (OR per allele: 1.91
[95% CI: 1.41–2.59]; pG=0.0002; pH=0.0103). It is a common
SNP, with MAF 0.32. We found the recessive model best fits the
inheritance mode of rs33003 (Table S5 in File S1). Many SNPs
in the same region had low p-values in the analysis with DLBCL
(Figure 1). The second most strongly associated SNP in MSH3,
rs181747, is in moderate linkage disequilibrium with rs33003, with
r2=0.55 in HapMap data and r2=0.65 in our data set. There is
evidence for an interaction between these two SNPs (p=0.0014).
However, no haplotype of SNPs in this region was more strongly
associated with DLBCL than either of these two SNPs alone.
There was no statistically-significant association of rs33003 or
rs181747 with DLBCL in 21 cases and 69 controls of Asian
descent (OR: 0.68 [95% CI: 0.28–1.64], pG=1.00; and OR: 0.94
[95% CI: 0.45–1.93], pG=1.00, respectively) or 6 cases and 31
controls of South-Asian ancestry descent (OR: 1.86 [95% CI:
0.45–7.61], pG=0.5483; and OR: 2.43 [95% CI: 0.62–9.50],
pG=0.4849, respectively); the number of samples in these groups
is too small to make a statement about associations in these groups.
There was also no evidence for interaction between rs33003 and
rs181747 in Asian ancestry samples (p=0.1957) or South-Asian
ancestry samples (p=0.9873).
Testing rs33003 for association with increased risk of family
history of colorectal cancer showed an association under the
recessive model (OR: 0.20 [95% CI: 0.03–1.43], p=0.034) but
not under the additive or dominant models. The 95%
confidence interval overlaps 1, however, indicating this result
could be a chance finding. Furthermore, adjusting the DLBCL
Table 2. Samples that passed Q/C.
Controls (%)Cases (%)
DLBCL– 189 (26%)
FL– 205 (29%)
MZL/MALT– 78 (11%)
MCL– 43 (6%)
SLL/CLL– 39 (5%)
LPL– 40 (6%)
MISC BCL– 54 (8%)
MF– 38 (5%)
PTCL– 24 (3%)
MISC TCL– 7 (1%)
Caucasian547 (79%)569 (79%)
Asian 69 (10%)66 (9%)
South Asian31 (4%)26 (4%)
Mixed/Other29 (4%)33 (5%)
Refused/Unknown18 (2%)23 (3%)
Male 360 (52%)416 (58%)
Female 334 (48%)301 (42%)
Age group (years)
20–49172 (25%) 131 (18%)
50–59153 (22%)173 (24%)
60–69 185 (27%)196 (27%)
184 (27%) 217 (30%)
Total 694 (100%) 717 (100%)
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.
NHL Risk and Lymphocyte Development Genes
PLOS ONE | www.plosone.org3September 2013 | Volume 8 | Issue 9 | e75170
Table 3. Logistic regression analysis results for SNPs with pG,0.05.
Subtype CategoryGeneSNP Coordinates* Alleles Odds ratio 95% CIp-valuepG
BCL Cell death TP73 rs3765703 1:3592436T/G 0.720.61–0.860.00032 0.00443 0.17282
TP73rs3765702 1:3592187C/T 0.710.58–0.860.000470.00443–
TP73rs18858591:3583692 C/G 0.760.64–0.91 0.00249 0.01577–
NEDD4 rs1163078015:56128445A/C 1.29 1.08–1.530.003900.03120 0.40560
CASP10rs12693932 2:202093395T/C 1.231.04–1.460.01461 0.01461 0.28494
DNA repair MSH3 rs1817475:80016874T/C1.36 1.13–1.640.00128 0.012770.19655
E2F2 rs3218203 1:23837560C/G1.41 1.13–1.760.002360.014190.19655
APEX1rs113040914:20925154G/T0.76 0.64–0.910.00282 0.002820.13525
E2F3rs109463846:20495546C/T 1.40 1.12–1.740.002830.04528 0.40187
MSH3 rs33003 5:80171134G/A 1.36 1.11–1.670.00287 0.01433–
MSH3 rs2453975:80101773 C/T1.38 1.11–1.73 0.004300.01433–
RB1rs4151510 13:48945175 G/A 0.680.52–0.89 0.005460.016380.19655
MSH3rs16507375:80001785A/G 1.301.07–1.58 0.00801 0.01771–
MSH3rs6151627 5:79965536A/G0.77 0.63–0.940.008860.01771–
DLBCLCell death TP73rs37657031:3592436 T/G0.660.51–0.87 0.00272 0.030230.83626
TP73 rs37657021:3592187 C/T 0.640.48–0.87 0.003180.03023 –
LTArs2844484 6:31536224G/A0.650.49–0.86 0.001830.01466 0.43980
IL7Rrs1494571 5:35880087 G/C 0.660.48–0.90 0.007000.035020.52530
DNA repairMSH3 rs33003 5:80171134 G/A1.91 1.41–2.590.00003 0.00022
MSH3 rs1817475:80016874 T/C1.791.35–2.360.00004 0.00022–
E2F3rs2328524 6:20488234 G/A 1.551.18–2.020.001350.008570.20573
MSH3rs245397 5:80101773 C/T1.691.23–2.320.001400.00349–
E2F3 rs10946384 6:20495546C/T1.701.23–2.36 0.001570.00857–
E2F3rs4134945 6:20483603C/T1.851.27–2.70 0.001610.00857–
E2F3rs23284886:20418329C/T1.491.12–1.99 0.00726 0.02902–
E2F3rs911361 6:20415053G/A0.710.54–0.940.01432 0.04581–
ERCC5rs1765513:103528002 G/C0.670.48–0.95 0.020040.020040.32065
LIG3rs1003918 17:33332177A/G1.371.05–1.79 0.021290.03194–
FLDNA repairE2F2rs3218203 1:23837560 C/G1.661.23–2.25 0.001170.007000.16809
APEX1 rs1130409 14:20925154G/T 0.680.52–0.88 0.003610.003610.16809
C11orf30rs1939469 11:76236220A/G0.570.37–0.870.00714 0.035710.57140
MZL Cell deathRELB rs12609547 19:45532009G/T2.031.34–3.07 0.000730.001450.05666
TP73rs11818681:3651126 T/G1.991.31–3.01 0.001340.025540.24900
RELBrs1560725 19:45543787 T/C1.861.22–2.82 0.003380.00338–
CASP10rs12693932 2:202093395T/C1.751.16–2.64 0.006310.006310.12305
IL8RBrs1126579 2:219000734 T/C0.590.37–0.93 0.021480.021480.24900
LTArs915654 6:31538497 T/A0.420.24–0.74 0.001370.010970.32901
DNA repairCHEK2rs5762746 22:29088123C/T1.871.25–2.78 0.001980.01595 0.38291
CHEK2rs1033667 22:29130300C/T1.831.22–2.73 0.003550.01595–
YY1rs490594114:100725438 A/G0.510.31–0.86 0.007610.015230.38291
CHEK2rs5762763 22:29132389G/C0.550.34–0.91 0.015180.04553–
MCL Cell deathCASP8rs1035142 2:202153078 G/T2.25 1.41–3.600.000560.004500.17541
BAK1rs17627049 6:33537802C/A0.400.19–0.830.00640 0.044780.58215
CDH22rs391573720:44871381A/C 0.490.26–0.94 0.021470.021470.41874
NHL Risk and Lymphocyte Development Genes
PLOS ONE | www.plosone.org4September 2013 | Volume 8 | Issue 9 | e75170
susceptibility analysis by family history of colorectal cancer (in
addition to 5-year age group, sex, and region) did not change
the OR or p-values of the association of rs33003 with DLBCL
susceptibility. We find no evidence that family history of
colorectal cancer influences the association between rs33003 and
susceptibility to DLBCL.
The association of rs33003 with DLBCL did not replicate in the
San Francisco sample set (OR 1.03 [95% CI: 0.83–1.29],
p=0.774). The minor allele frequencies of rs33003 are similar in
the original population (MAF=0.32) and the San Francisco set
(MAF=0.34). Furthermore, the r2value between rs33003 and
rs181747 is similar in the two populations (r2=0.65 in the original
population and r2=0.67 in the San Francisco population). This
indicates that the failure to replicate is unlikely to be due to
population-specific differences in minor allele frequencies or LD
structure in that area of the genome.
One other SNP, rs12609547, in RELB, was mildly associated
with marginal zone lymphoma (OR: 2.03 [95% CI: 1.34–3.07],
pG=0.0015). This association was not significant, however, after
multiple testing correction at the hypothesis level (pH=0.0570).
After multiple testing correction within genes, there was
evidence for associations of NHL subtypes with SNPs in two
genes: RELB with MZL and MSH3 with DLBCL. Only the MSH3
association, however, was significant after the additional correc-
tion for multiple testing between genes. This association, however,
did not replicate in another North American population ,
indicating that it was likely a type I error.
MSH3 is involved in DNA mismatch repair (MMR), which
corrects mismatched or unmatched bases and small insertion/
deletion loops that result from DNA replication before cell division
or from DNA repair processes . The MMR pathway is an
important repair mechanism in normal lymphocyte development
as evidenced by mouse models and human patients deficient in this
pathway . Studies of MMR deficiency and MMR gene
deregulation in lymphomas have also illustrated the potential role
of this pathway in NHL[19–23].
Because of the MMR pathway’s established role in hereditary
non-polyposis colorectal cancer (HNPCC), we tested whether
Figure 1. Association results and linkage disequilibrium in
MSH3. r2values for our genotyped samples are shown in the top
section (‘‘r2values in NHL data’’) and r2from the CEU population of
HapMap are shown in the bottom section (‘‘r2values in HapMap CEU
data’’). The gene model of MSH3 is shown on top, 59 to 39 from left to
right, with vertical lines marking exons. p-values (before correction for
multiple testing) are from the analysis in DLBCL samples of European
Table 3. Cont.
SubtypeCategoryGeneSNP Coordinates*AllelesOdds ratio 95% CIp-valuepG
PRDM1rs6924807 6:106531266 A/G0.440.25–0.76 0.00185 0.014780.28303
JAK3 rs10419991 19:17938891A/G0.460.27–0.78 0.00270 0.01887 0.28303
JAK3rs3212760 19:17947546A/G0.510.30–0.88 0.01140 0.03989–
DNA repairLIG4rs115140213:108858030 C/T0.400.23–0.69 0.00042 0.002500.12007
LIG4 rs1242816213:108871915 G/C2.421.44–4.05 0.000840.00253–
LIG4 rs1805386 13:108861913A/G 2.461.43–4.24 0.00156 0.00312–
TCL Cell deathCASP4rs1944900 11:104838471 C/T0.340.15–0.750.00199 0.01196 0.46639
ITCHrs4911154 20:32996101G/A 2.061.21–3.52 0.010270.030820.47519
RASSF1rs22369473:50371432 C/A1.781.10–2.88 0.018280.036550.47519
IFNB1rs10519229:21077716 G/A 1.751.14–2.68 0.01126 0.01126 0.33787
IL6 rs2069840 7:22768572C/G 1.861.13–3.050.014210.04263 0.57505
*Coordinates obtained from Ensembl 64.
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rs33003 was associated with a family history of colorectal cancer in
first degree relatives. Adjusting the DLBCL susceptibility analysis
by family history of colorectal cancer in addition to 5-year age
group, sex, and region did not change the analysis results,
indicating that family history of colorectal cancer is not a
confounder for susceptibility to DLBCL. Furthermore, we did
not find that rs33003 was associated with a family history of
colorectal cancer. This is not entirely surprising, as colorectal
cancer is not associated with lymphoma , although mismatch
repair cancer syndrome is characterized in part by a combination
of colorectal polyposis,  and early-onset hematologic cancers
DLBCL can be subdivided into at least three subgroups using
molecular signatures . It is therefore possible that the MSH3
association is confined to patients with tumours belonging to
specific DLBCL subgroups. We do not, however, have molecular
signature data for the tumours of the DLBCL patients included in
this study. It is also possible that there are true associations with
NHL susceptibility that we are not able to detect in this study. This
could be due to low sample sizes for some subtypes of NHL in our
study, or perhaps population-specific effects. This could explain
our inability to replicate candidate gene (Table 1) associations of
SNPs in IRF4 with FL , or our observation of weak associations
(i.e. a SNP with p,0.05 but that does not pass multiple testing
correction) of SNPs in BID, APAF1 and CASP10 with NHL . We
were also unable to replicate other associations for SNPs in the
‘‘replication’’ category, listed in Table S2 in File S1. Further-
more, HapMap coverage may not have been adequately deep to
represent causal variants present in some genes we assayed,
making our tagSNP approach vulnerable to false negative results.
As in most other lymphoma studies[1–8], multiple testing
correction was not done for the number of subtypes tested as
the subtypes are considered separate disease entities, with different
presentation, possible etiology and hypotheses. Finally, any
association reported here could be an association with survival
as opposed to susceptibility, as patients who have less aggressive
disease are more likely to have time to participate in the study and
provide a DNA sample. This is not likely, however, given the low
percentage of cases who died prior to contact (10.5% in the British
Columbia study  and 14.2% in the San Francisco set ).
In summary, we found no replicated associations in the genes
studied related to immunity and inflammation, DNA repair and
programmed cell death.
interest. Table S2. SNPs tested for replication. Table S3. SNPs
that passed quality control. Table S4. Logistic regression analysis
results for SNPs with pG,0.05 before multiple testing correction.
Table S5. The best model for rs33003 in European-ancestry
DLBCL vs. controls is the recessive model.
Table S1. Candidate genes chosen based on biological
For their help with previous work on genotype data processing and quality
control, the authors would like to thank Dr. Tara Paton of the Centre for
Applied Genomics at The Hospital for Sick Children, Toronto, Canada;
and Dr. Jinko Graham and Conghui Qu of Simon Fraser University. We
also thank the study staff - Agnes Lai, Carmen Ng, Kuldip Bagga, Agnes
Bauzon, Betty Hall, Lina Hsu, Pat Ostrow, Lynne Tse and Anthony Tung,
the computer support of Dr. Tim Lee and Zenaida Abanto, and the
technical assistance of Rozmin Janoo-Gilani. We also would like to thank
the continued support of the members of the BC Cancer Agency
Lymphoma Tumour Group. Finally, we thank all the participants of the
study for making this research possible.
Conceived and designed the experiments: RPG CFS JJS AB-W. Performed
the experiments: JMS SL LC BRB JMC RDG. Analyzed the data: JMS
DD PMB. Contributed reagents/materials/analysis tools: DD SL BRB
JMC JJS AB-W. Wrote the paper: JMS AB-W.
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