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Carcinogenesis Advance Access originally published online on October 27, 2006
Carcinogenesis 2007 28(4):823-827; doi:10.1093/carcin/bgl196
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Published by Oxford University Press 2006

Genetic variants in caspase genes and susceptibility to non-Hodgkin lymphoma

Qing Lan1,*, Tongzhang Zheng2, Stephen Chanock1,3, Yawei Zhang2, Min Shen1, Sophia S. Wang1, Sonja I. Berndt1, Shelia H. Zahm1, Theodore R. Holford2, Brian Leaderer2, Meredith Yeager1, Robert Welch1, Dean Hosgood1, Peter Boyle4 and Nathaniel Rothman1

1 Division of Cancer Epidemiology and Genetics, National Cancer Institute NIH, DHHS, Bethesda, MD, USA
2 Department of Epidemiology and Public Health, Yale School of Medicine New Haven, USA
3 Pediatric Oncology Branch, Center for Cancer Research NCI, NIH, DHHS, Bethesda, MD, USA
4 International Agency for Research on Cancer Lyon, France

*To whom correspondence should be addressed at: Occupational and Environmental Epidemiology Branch, Division of Cancer Epidemiology and Genetics, National Cancer Institute, NIH, DHHS, MSC 7240, 6120 Executive Boulevard, EPS 8109, Bethesda, MD 20892-7240, USA. Tel: +1 301 435 4706; Fax: +1 301 402 1819 Email: qingl{at}mail.nih.gov


   Abstract
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 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
The caspase proteins are essential for the regulation of normal B cell development and regulation of apoptosis. We investigated five single nucleotide polymorphisms in four key caspase genes, CASP3 [Ex8-280C>A (rs6948) and Ex8+567T>C (rs1049216)], CASP8 Ex14-271A>T (rs13113), CASP9 Ex5+32G>A (rs1052576) and CASP10 Ex3-171A>G (rs3900115) to determine whether they alter risk for non-Hodgkin lymphoma (NHL) in a population-based case–control study of women in Connecticut (461 cases and 535 controls). Variants in CASP3 and CASP9 were significantly associated with a decreased risk for NHL, particularly follicular lymphoma [e.g. CASP3 Ex8+567T>C odds ratio (OR)CC+TC = 0.4, 95% confidence interval (CI) = 0.3–0.7; and CASP9 Ex5+32G>A ORAA+AG = 0.6, 95% CI = 0.4–1.0]. Further, variants in CASP3, CASP8 and CASP10 were associated with a decreased risk of marginal zone lymphoma and variants in CASP3 and CASP10 were associated with a lower risk of chronic lymphocytic leukemia and related subtypes. The striking protective associations observed for polymorphisms in all four genes for NHL and/or one or more subtypes suggest that genetic variation in CASP genes may play an important role in the etiology of NHL.

Abbreviations: HWE, Hardy–Weinberg equilibrium; LD, linkage disequilibrium; NHL, non-Hodgkin lymphoma; SNP, single nucleotide polymorphisms


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Caspases are highly conserved intracellular cysteine proteases that are fundamental components of apoptosis as well as regulators of B cell development and homeostasis (1). Expression array analyses of non-Hodgkin lymphoma (NHL) tumors have shown that caspase genes may be differentially expressed in different subtypes of NHL (2,3). Further, somatic mutations in CASP genes have been reported in NHL, multiple myeloma and other human cancers (4–8). These observations provide evidence that genetic variation in CASP genes could play a role in lymphomagenesis. We therefore examined the association between single nucleotide polymorphisms (SNPs) in four key CASP genes (CASP3, CASP8, CASP9 and CASP10) in a population-based case–control study of NHL among women in Connecticut. SNPs analyzed in this study were chosen in part based on prior observations in association studies (9–12). We genotyped either the same SNP or a SNP in linkage disequilibrium (LD) and the priority was on SNPs with expected functional consequences, i.e. an amino acid change or within the 3'-untranslated region (3'-UTR), which contains regulatory sequences and binding sites that could alter the stability of the mRNA transcript (13).


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
The study population has been described previously in detail (14–16). Briefly, from 1996 to 2000 all histologically confirmed incident NHL cases without a previous diagnosis of cancer except for non-melanoma skin cancer, age 21–84 years old, among women in Connecticut were identified. The cases were restricted to those alive at the time of interview. Of 832 eligible cases, 601 (72%) completed in-person interviews. Population-based controls, identified through random digit dialing and the Centers for Medicare and Medicaid files, were frequency matched with cases based on age (±5 years) and sex. The participation rate was 69% for those contacted by random digit dialing and 47% for those contacted through health care records. In total, 461 interviewed cases and 535 interviewed controls provided a blood sample, and 57 cases and 62 controls provided a buccal cell sample. DNA from only blood samples was genotyped for this report as DNA available from buccal cells was either not analyzable or present in insufficient quantities. Genotyping was carried out by real-time PCR on an ABI 7900HT sequence detection system as described on the SNP500 website (http://snp500cancer.nci.nih.gov) (17). Duplicate samples from 100 study subjects and 40 replicate samples from each of two blood donors were interspersed throughout the plates used for genotype analysis. The concordance rates for QC samples were 100% for all assays.

The chi-square test was used to test for departures from Hardy–Weinberg equilibrium (HWE) among non-Hispanic Caucasian controls. A SNP was considered to be in HWE if the P-value of the chi-square test was >0.05. We analyzed five SNPs in four key caspase genes CASP3 [Ex8-280C>A (rs6948), Ex8+567T>C (rs1049216)], CASP8 Ex14-271A>T (rs13113), CASP9 Ex5+32G>A (rs1052576) and CASP10 Ex3-171A>G (rs3900115), all of which were in HWE among controls. Odds ratios were estimated using unconditional logistic regression, adjusted for age (<50, 50–70 and >70) and race (Caucasian, African-American and other). Further analyses adjusting for family history or limited to non-Hispanic Caucasians (representing 93.2 and 91.6% of all cases and controls, respectively) resulted in similar results. Gene dosage effects were conducted by assigning the ordinal values 1, 2 or 3 to a genotype trend variable in terms of the subject's number of variant alleles (zero, one and two variant alleles, respectively). To maximize statistical power, risks for NHL subtypes were carried out using all controls as the comparison group.

Haplotype frequencies and analyses for CASP3 were conducted within non-Hispanic Caucasians. The HaploView program (http://www.broad.mit.edu/personal/jcbarret/haploview/) was used to evaluate the haplotype block structure (18), the Expectation–Maximization algorithm (19) was used to estimate the haplotype frequencies, and the omnibus test that was implemented in SAS Genetics was used to test for overall differences in haplotype frequencies between non-Hispanic Caucasian cases and controls while adjusting for age. An unconditional logistic regression model was used to estimate the effect of individual haplotypes while assuming the most likely haplotype pairs for each person. Unless otherwise specified, all analyses were conducted using SAS 8.2 (SAS Institute Inc., Cary, NC).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Demographic characteristics of cases and controls have been previously described and were comparable with regard to age and ethnicity (16). Two variants in CASP3 (Ex8-280A and Ex8+567C) and one SNP in CASP9 (Ex5+32A) were significantly associated with a decreased risk for NHL. In subtype analyses, protective associations were significant for only B cell lymphomas (Table I), although power to detect associations for T cell lymphomas was low. Among B cell lymphomas, the risks were particularly pronounced for follicular lymphoma, but not for diffuse large B cell lymphoma (DLBCL) (Table I). The two CASP3 polymorphisms were in moderate LD (r2 = 0.38). Interestingly, when a haplotype analysis was conducted with the two CASP3 SNPs, only the AC haplotype was significantly associated with a decreased risk for NHL (Table II). The AT haplotype was neither associated with risk of NHL overall nor with most subtypes suggesting that the CASP3 Ex8+567C variant, additional SNPs in strong LD, or the combination of both the A and C alleles are associated with risk of NHL. The CASP3 Ex8-280A, CASP8 Ex14-271A and CASP10 Ex3-171G variants were significantly associated with a decreased risk for marginal zone B cell lymphoma and variants CASP3 Ex8+567C and CASP10 Ex3-171G were associated with decreased risk of B cell chronic lymphocytic leukemia/prolymphocytic leukemia/small lymphocytic lymphoma (Table I). Most histology-specific analyses showed a protective effect for these polymorphisms. There was no evidence of gene–gene interactions between CASP SNPs, although the power was low to detect such effects.


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  		Table I Association between CASP polymorphisms and non-Hodgkin lymphoma (NHL) and its major subtypes*

 


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  		Table II Odds ratios and 95% confidence intervals for the association between common CASP3 haplotypes in exon 8 and all non-Hodgkin lymphoma and major histologic subtypes among non-Hispanic Caucasians

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
Overall, we observed that common genetic variants in CASP genes were associated with a decreased risk for NHL with some evidence that the effects were specific to lymphoma subtypes. Caspase-3, a negative regulator of B cell cycling, is essential in the regulation of B cell homeostasis (8). Knockout CASP3 mice studies have shown that casepase-3 plays an essential role in DNA degradation, plasma membrane blebbing and nuclear condensation. Caspase-3-deficient mice (CASP3–/–) showed increased numbers of splenic B cells with increased proliferation in vivo and hyperproliferation in vitro after mitogenic stimulation. Further, CASP3–/– mice had a substantial reduction (50%) in B lineage cells, and in pro-B, pre-B and immature B populations. The two CASP3 variants are located in the 3'-UTR and could alter the stability of the mRNA transcript through ribosome binding, initiation or elongation (13). Additional work is needed to determine their functionality. Alternatively, these polymorphisms could be in LD with another variant in the region truly associated with disease risk that we did not study.

CASP9 knockout mice die perinatally due to defects in the brain (20). The CASP9 (Ex5+32G>A) polymorphism encodes for a glutamine to arginine amino acid change at codon 221 of the gene and thus may have functional significance. CASP8 plays a broad role in regulating lymphocyte homeostasis, including affected T cell activation and proliferation, natural killer cell activation and immunoglobulin production. Mutations in caspase-8 result in altered autoimmunity (21). Mutations in the CASP10 gene result in abnormal lymphocyte proliferation (22). Analysis of NHL tumor tissues found about 15% of the NHL had CASP10 mutations (4). Apoptosis activation occurs in part through specific functions by members of the caspase family in both mitochondrial and death receptor pathways (23). It is plausible that SNPs in multiple genes in these pathways could affect risk for a given tumor, as caspase-8, -9 and -10 initiate a cascade of caspase activation by cleaving downstream effector molecules such as caspase-3, which in turn causes cell death (24,25). Interestingly, recent reports have shown a protective effect for SNPs in both CASP8 and CASP10 and risk of breast cancer (9,10,12). Further, a study found that CASP10 Ex3-171A>G SNP was associated with idiopathic talipes equinovarus (11).

Our study is a population-based, case–control study with incident cases that are histologically confirmed, and accurate genotype data. We note that study participation rates were moderate and DNA for genotyping was available only for subjects who provided a blood sample, which could potentially result in selection bias. However, we observed no significant differences in age, race or family history of NHL between subjects who provided a blood sample and other subjects enrolled in the study. Also, a recent report observed that there are minimal differences in genotype frequencies for genes in several pathways by subject participation status (26). In addition, the overall sample size of our study is modest and the number of cases in some histologic subgroups was small. These limitations resulted in low power for testing some of the associations, which could result in both false negative and false positive (27) findings.

In summary, our study provides the first evidence that common genetic variants in four CASP genes (CASP3, CASP8, CASP9 and CASP10) are associated with decreased risk of NHL. Our results warrant replication in larger studies and ultimately in pooled analyses. Further, a comprehensive strategy of analyzing common genetic variants across each of the four genes in order to eventually identify all potentially functional SNPs should be pursued. If replicated, our findings could provide insight into the pathogenesis of one or more subtypes of NHL and lead to further understanding of the role of caspase-related pathways in this and other diseases.


   Acknowledgments
 
This study was supported in part by the Intramural Research Program of the NIH, National Cancer Institute and NIH grant CA62006 from the National Cancer Institute. We gratefully acknowledge the assistance of Peter Hui (Information Management Services, Inc., Silver Spring, MD) for programming support.

Conflict of Interest Statement. None declared.


   References
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 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received May 18, 2006; revised October 5, 2006; accepted October 8, 2006.


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