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Elevated incidence of lymphoma has been observed among carriers of rare high-penetrance mutations in DNA repair genes (e.g., Nijmegen breakage syndrome, Ataxia-telangectasia syndrome, etc.). Common gene variants in DNA repair genes may also influence lymphomagenesis.
Study subjects were pooled from three population-based case-control studies of non-Hodgkin lymphoma (NHL) in the US and Australia. A total of 1,946 cases and 1,808 controls were analyzed. A total of 319 tag single nucleotide polymorphisms (SNPs) in 27 DNA repair gene regions were genotyped. Unconditional logistic regression models were used to estimate the relative risk of NHL and NHL subtypes in relation to SNPs. Tail-strength statistics were used to test for the association between DNA repair pathways and NHL or NHL subtypes. The statistical significance of the smallest P-trend within each gene region was estimated by permutation-based resampling methods.
Overall, DNA repair genetic polymorphisms were associated with NHL (P = 0.005). Tests for the double strand break repair (P = 0.02) and nucleotide excision repair (P = 0.04) pathways were also significant. Four gene regions were significantly associated with NHL or NHL subtypes at the 0.05 level: RAD50, BLM, RAD51/FAM82C, and ERCC3/MAP3K2. Specifically, BLM rs441399 (P trend = 0.004) and FAM82C rs2304583 (P trend = 0.001) were associated with follicular lymphoma, and XRCC4 rs13178127 was associated with NHL overall (P trend = 0.006) significantly. In addition, the ERCC3 rs4150506 was associated with reduced risk for marginal zone lymphoma (P trend = 0.002).
These results support the hypothesis that common genetic polymorphisms in human DNA repair genes may modify the risk of NHL.
DNA repair mechanisms are important in maintaining genomic stability and defects in DNA repair can lead to the development of chromosomal aberrations, a hallmark of lymphoma (Palitti, 2004). Several hereditary syndromes, including Ataxia telangiectasia (OMIM 208900), Bloom syndrome (OMIM 210900), and Nijmegan breakage syndrome (OMIM 251260), are characterized by defective DNA repair and high occurrence of lymphoma, indicating the important role of DNA repair in the pathogenesis and development of NHL.
Recent studies have reported associations between certain DNA repair gene single-nucleotide polymorphisms (SNP) and NHL risk (Shen et al, 2006;Shen et al, 2007;Hill et al, 2006). To investigate this question in greater detail, we conducted a pooled investigation of genetic variation in 27 gene regions involved in human DNA repair based on three case-control studies in the US and Australia.
Study subjects were pooled from three population-based case-control studies of NHL: a study conducted within the Surveillance Epidemiology and End Results (SEER) registry catchment areas of Iowa, Detroit, Los Angeles and Seattle (NCI-SEER study) (Hill et al, 2006), a study conducted among female residents of Connecticut (Shen et al, 2006), and a study conducted among residents of New South Wales (NSW) and the Australian Capital Territory, Australia (Shen et al, 2007).
The final analytic population consisted of 1,946 cases and 1,808 controls (NCI-SEER, 990 / 828; Connecticut, 436 / 515; NSW, 520 / 465) after a few samples were excluded with completion rates <90% (NCI-SEER, N = 17; Connecticut, N = 2; NSW, N = 3).
The four most common NHL subtypes were also evaluated separately: diffuse large B-cell lymphoma (DLBCL), follicular lymphoma (FL), marginal zone lymphoma (MZL), and chronic lymphocytic leukemia / small lymphocytic lymphoma (CLL/SLL).
DNA was extracted from blood clots, buffy coats, or buccal cell samples. Genes were selected based on experimental evidence of functional relevance and findings from previous association studies of lymphoma, related malignancies, and other tumor sites. These genes are involved in different DNA repair pathways (double strand break repair (DSBR), nucleotide excision repair (NER), and base excision repair (BER)).
Tag SNPs for each gene were selected from the designable set of common SNPs (minor allele frequency ≥0.05) genotyped in the HapMap Project (Data Release 20/Phase II, NCBI B35 assembly, dbSNP b125). For each gene, SNPs within the region 20 kb 5′ of the ATG-translation initiation codon and 10kb 3′ of the end of the last exon, which is called gene region, were binned using a binning threshold of r2 > 0.80. Genotyping was performed at the National Cancer Institute Core Genotyping Facility (Gaithersburg, MD) using the Illumina GoldenGate platform. Data for an additional 28 SNPs that had been previously genotyped by TaqMan assay in at least two of the studies and are located within one of the 27 investigated gene regions were also included in the overall analysis. In total, 319 SNPs were analyzed in this report (Supplementary Table 1).
All investigated SNPs had assay completion rates ≥ 98% except BLM rs441399, ERCC2 rs50872 and NBN rs7006322 (97% for each), and genotype concordance rates ≥ 99% except BLM rs16944894 (96%) in quality control duplicates.
The relative risk of NHL and NHL subtypes in relation to SNPs were estimated by calculating odds ratios (OR) and 95% confidence intervals (CI) using polytomous multivariate unconditional logistic regression models, adjusting for age, race/ethnicity, sex and study center. Tests for trend were conducted using an additive model by assigning the ordinal values 0, 1, and 2 to homozygous wild type, heterozygous and homozygous variant genotypes respectively, and by modeling these scores as a continuous variable.
Tail-strength statistics were used to test for the association between pathways and NHL or NHL subtypes. The tail-strength method evaluates the univariate strength of a collection of observed P values (e.g. from a particular pathway) and assesses its statistical significance using a permutation procedure (10,000 permutations) (Taylor & Tibshirani, 2006).
We assessed the statistical significance of the smallest P-trend within each gene region by permutation-based resampling methods (10,000 permutations) that automatically adjust for the number of tag SNPs tested within that gene region and the underlying linkage disequilibrium pattern (Chen et al, 2006). Finally, haplotype analysis was carried out among non-Hispanic Caucasian subjects.
Cases and controls were similar with respect to demographic characteristics including sex, age, race, and ethnicity (Supplementary Table 2). Overall, DNA repair genetic polymorphisms were associated with NHL (P = 0.005). Tests of the DSBR (P = 0.02) and NER (P = 0.04) pathways were also statistically significant (Table 1).
Four gene regions were significantly associated with NHL subtypes at the 0.05 level: DLBCL (RAD50), FL (BLM, RAD51/FAM82C), and MZL (ERCC3/MAP3K2) (Table 2).
Genotype frequencies for cases and controls, and main effects of SNPs are presented in Supplementary Table 3. One or more SNPs in BLM, WRN, RAD50, FAM82C, RAD54L, XRCC4, ERCC3, CCDC16, and IRGQ were associated with NHL or NHL subtypes risk at a significance level of 0.005. Five SNPs (BLM rs441399, RAD50 rs2237060, FAM82C rs2304583, ERCC3 rs4150506, and XRCC4 rs13178127) were particularly noteworthy because their gene regions were significantly associated with NHL or NHL subtypes at the 0.05 level or because of high level of statistical significance and consistent findings across the three studies (Table 3) (Figure 1). Among them, BLM rs441399 (P trend = 0.004) and FAM82C rs2304583 (P trend = 0.001) were associated with follicular lymphoma significantly, and XRCC4 rs13178127 was significantly associated with NHL overall (P trend = 0.006). Most of the statistically significant findings were restricted to DLBCL or FL. However, the ERCC3 rs4150506 was associated with reduced risk for MZL (P trend = 0.002).
Haplotype analysis was conducted to examine different subsets in these gene regions. However, it did not provide additional information for a given gene beyond those significant SNPs found in individual SNP analysis. Subgroup analysis in non-Hispanic Caucasians showed similar results (results not shown).
We studied 319 SNPs in 27 DNA repair genes and found that a number of genetic polymorphisms were significantly associated with altered risk of NHL or NHL subtypes. Among them, genes repairing DNA double strand breaks are particularly notable. These results support the hypothesis that common genetic polymorphisms in DNA repair genes may modify the risk of NHL.
BLM, the Bloom syndrome gene product, is related to the RecQ subset of DNA helicases and has both DNA-stimulated ATPase and ATP-dependent DNA helicase activities. Mutations in the gene causes Bloom syndrome characteristic of high cancer predisposition including lymphoma. BLM plays a role in both homogenous recombination and nonhomologous end joining (NHEJ) manifested by its interaction with RAD51 and LIG4 (So et al, 2004). Two nuclear localization signals (NLS) were found in the C-terminal domain, which is crucial in targeting the BLM protein to the cell nucleus (Kaneko & Kondo, 2004). We found two significant SNPs in this gene and the one in the C-terminal (rs441399) was significantly associated with FL.
The gene region RAD51/FAM82C was associated with NHL overall and especially for follicular lymphoma. FAM82C is a mitochondrial protein with apoptosis-inducing function (Lv et al, 2006). RAD51 is involved in the homologous recombination and repair of DNA. This protein also interacts with BRCA1 and BRCA2, which may be important for the cellular response to DNA damage. RAD51 had been found involved in family breast cancer (Thacker, 2005). The gene FAM82C is located physically close to RAD51. So the association of FAM82C with NHL may be due to functional loci in RAD51.
At the center of the NHEJ, XRCC4 forms a complex with the LIG4 (Sibanda et al, 2001), and further interacts with the DNA-dependent protein kinase for rejoining breaks. The XRCC4/p53 double knockout mice die early from pro-B cell lymphoma (Gao et al, 2000). NHEJ is also critical in the process of V(D)J recombination that generates immunoglobulin and T cell receptor diversity. Errors in the DNA repair genes responsible for ligating the V, D, and J segments are implicated in characteristic chromosomal rearrangements found in some types of NHL. Our findings suggest the XRCC4 and the NHEJ may play a role in the development of NHL.
In summary, we found that variants in genes that play an important role in several DNA repair pathways were associated with risk of NHL. These findings suggest that common genetic polymorphisms in DNA repair genes may be important susceptibility factors for NHL. Identification of the key DNA repair genes that play a role in lymphomagenesis may ultimately help to identify particular exposures or general classes of exposures. Recent genome-wide scan studies of lymphoma identified several loci that were associated with FL and CLL/SLL (Skibola et al, 2009;Di Bernardo et al, 2008). Our findings need to be replicated in other studies.