In this case-control study, we demonstrate gene- and SNP-level association of BCL2L11
(BIM), BCLAF1, BAG5,
with NHL risk that remained noteworthy after accounting for multiple testing. While the pFDR q-values estimated for BCL2L11
SNPs do indicate that there is a moderate chance that any of these associations individually may be false positives, the tail strength estimates indicated that the distribution of p-values for the group of SNPs from these genes is more extreme than we would have expected by chance alone. Moreover, the significant gene-level associations for BCL2L11
were consistent with previously published data from a pooled analysis of three studies (NCI-SEER, Connecticut, and New South Wales) that included 1946 cases and 1808 controls (32
). While there was minimal overlap in the individual tagSNPs genotyped across our study and the pooled study, we were able to impute genotypes for the SNPs in our study that were not directly observed, and we found associations that were largely consistent in magnitude and direction across all four significant BCL2L11
SNPs and 2 of 3 CASP9
SNPs from the pooled study, making this the first independent replication of these results. The associations from our study with BCLAF1
have not been tested in an independent dataset and thus require replication.
Strengths of our study include a carefully designed case-control study, central pathology review, and high quality genotyping. Although this study was not population-based, both case and control participation was restricted to those residing in the region surrounding Mayo Clinic (Minnesota, Iowa, and Wisconsin), thus minimizing the effect of referral bias, and increasing the internal validity of using frequency-matched general medicine controls from the same region. Common HapMap SNPs were used to tag genes of interest, and through other genotyping projects we have ruled out the presence of significant population stratification (27
). The major limitations are the use of an exclusively Caucasian population, which limits generalizability, and the relatively small sample size, which in particular precludes robust estimation of NHL subtype associations. There are several apoptosis genes evaluated in this study for which the genotyped tagSNPs provided <70% gene coverage, and thus an association between these genes and NHL risk cannot yet be ruled out. In addition, there are greater than 100 human genes more broadly identified with apoptosis pathway involvement in which germline variants may play a role in NHL risk, including genes with caspase-recruitment domains, death domains, and death effector domains (43
). The genes in the current analysis represent only the core of a larger set of genes involved in apoptosis-related pathways.
The two genes that replicated have strong biologic plausibility in NHL pathogenesis. BCL2L11
balances the anti-apoptotic influence of BCL2
and coordinates pro-apoptotic signaling through the intrinsic apoptosis pathway (4
). In addition, BCL2L11
is required for negative selection of autoreactive lymphocytes (44
). Functional silencing of BCL2L11
through methylation has been observed in Burkitt lymphoma cell lines and primary tumor biopsies, and reduced BCL2L11
mRNA and protein expression has also been documented in other tumors including renal cell carcinoma, melanoma, and colon cancer (45
). Of note, we did not identify any association between BCL2
and NHL risk at the gene level, and only one of the 53 genotyped SNPs was significant at the SNP-level. While these 53 SNPs comprised only 74% gene coverage, it does suggest that BCL2
germline variation may not play a role in NHL risk. This would be consistent with the hypothesis that most of the BCL2
variation in lymphoma tumors is a result of hypermutation following the t(14;18) translocation event (4
). This hypothesis should be further explored by comparing somatic and germline mutation among patients stratified by the t(14;18) translocation.
, the other gene to be replicated, is a pro-apoptotic protease integral to the intrinsic apoptotic pathway, and is responsible for effector caspase activation and apoptosis execution following activation by Apaf-1 bound to cytochrome c
released from mitochondria (46
). Hyperphosphorylation of caspase-9 may lead to aberrant apoptosis inhibition, and the relevance of this process has been demonstrated in a number of other cancer types (46
). Also of note, upon mitochondrial release of cytochrome c
via both the intrinsic and extrinsic apoptosis pathways, the cytoplasmic protein Apaf-1 binds caspase-9 to form the apoptosome, in turn activating the caspase cascade (4
did not reach gene-level statistical significance in our population, although 6 of the 13 genotyped tagSNPs in this gene were significant at p<0.05. Given the gene-level significance of CASP9
, there may be some clinical relevance of the individual tagSNPs significance in APAF1
, and further follow-up on this gene is warranted.
To our knowledge, this is the first report of an association between germline variation in BAG5
with regard to lymphoma risk. BCLAF1
are both Bcl-2 family members that suppress BAX
(pro-apoptotic) gene expression, in turn suppressing the APAF1
gene and inhibiting apoptosis (4
). These associations should be confirmed in follow-up with an independent study population.
While underpowered to assess NHL subtypes, our data provide some evidence that there may be subtype-specific associations in the apoptosis pathway, particularly BCL2L11 and CASP9
. Evidence of differential association of germline variants in the BCL2
families was observed in the pooled analysis of 3 case-control studies (32
), although the BCL2L11
association was largely limited to follicular lymphoma (33
), a pattern we observed for both follicular and CLL/SLL in our study.
In conclusion, our results support an association of four genes from the apoptosis pathway, with NHL risk, and these associations may vary by NHL subtype. In light of the importance of the apoptosis pathway to human lymphomagenesis, further characterization of the key players within this pathway is warranted.