Document 3J1JbRd61479rqnZ2rwBKkEM0

Carcinogenesis vol.27 no.10 pp.20832089, 2006 doi:10.1093/carcin/bgl061 Advance Access publication May 25, 2006 Polymorphisms in genes involved in DNA double-strand break repair pathway and susceptibility to benzene-induced hematotoxicity Downloaded from http://carcin.oxfordjournals.org by on March 30, 2010 Min Shen1,, Qing Lan1, Luoping Zhang2, Stephen Chanock1,3, Guilan Li4, Roel Vermeulen1, Stephen M.Rappaport5, Weihong Guo2, Richard B.Hayes1, Martha Linet1, Songnian Yin4, Meredith Yeager1,3, Robert Welch1,3, Matthew S.Forrest2, Nathaniel Rothman1 and Martyn T.Smith2 1Division of Cancer Epidemiology and Genetics, NCI, NIH, DHHS, Bethesda, MD 20892, USA, 2School of Public Health, University of California, Berkeley, CA 94720, USA, 3Center for Cancer Research, NCI, NIH, DHHS, Bethesda, MD 20892, USA, 4Institute of Occupational Health and Poison Control, Chinese Center for Disease Control and Prevention, Beijing, China and 5School of Public Health, University of North Carolina, Chapel Hill, NC 27599, USA 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, Bethesda, MD 20892-7240, USA. Tel: 301 451 8791; Fax: 301 402 1819; Email: shenmi@mail.nih.gov Benzene is a recognized hematotoxicant and carcinogen that produces genotoxic damage. DNA double-strand breaks (DSB) are one of the most severe DNA lesions caused directly and indirectly by benzene metabolites. DSB may lead to chromosome aberrations, apoptosis and hematopoietic progenitor cell suppression. We hypothesized that genetic polymorphisms in genes involved in DNA DSB repair may modify benzene-induced hematotoxicity. We analyzed one or more single nucleotide polymorphisms (SNPs) in each of seven candidate genes (WRN, TP53, NBS1, BRCA1, BRCA2, XRCC3 and XRCC4) in a study of 250 workers exposed to benzene and 140 controls in China. Four SNPs in WRN (Ex4 16 G > A, Ex6 +9 C > T, Ex20 88 G > T and Ex26 12 T > G), one SNP in TP53 (Ex4 +119 C > G) and one SNP in BRCA2 (Ex11 +1487 A > G) were associated with a statistically significant decrease in total white blood cell (WBC) counts among exposed workers. The SNPs in WRN and TP53 remained significant after accounting for multiple comparisons. One or more SNPs in WRN had broad effects on WBC subtypes, with significantly decreased granulocyte, total lymphocyte, CD4+-T cell, CD8+-T cell and monocyte counts. Haplotypes of WRN were associated with decreased WBC counts among benzene-exposed subjects. Likewise, subjects with TP53 Ex4 +119 C > G variant had reduced granulocyte, CD4+-T cell and B cell counts. The effect of BRCA2 Ex11 +1487 A > G polymorphism was limited to granulocytes. These results suggest that genetic polymorphisms in WRN, TP53 and BRCA2 that maintain genomic stability impact benzene-induced hematotoxicity. Abbreviations: BMI, body mass index; DSB, double-strand break; LD, linkage disequilibrium; SNP, single nucleotide polymorphism; WBC, white blood cell. Introduction Benzene is an important industrial chemical and is a component of cigarette smoke, gasoline, crude oil and automobile emissions. Several million workers worldwide are exposed to benzene in the oil industry, shipping, automobile repair, shoe manufacture, and so on. (1). Epidemiological studies have shown that exposure to benzene results in an increased risk of aplastic anemia, myelodysplastic syndromes, leukemia and other blood disorders (1). Benzene must be metabolized in order to elicit toxicity to the blood and bone marrow (2). These benzene metabolites, that is, reactive quinones, are capable of binding to and damaging macromolecules including DNA, glutathione, tubulin, histones, topoisomerase II and other DNA-related proteins. Additionally, benzene metabolites may give rise to reactive oxygen species (ROS) (3). Direct attack by ROS and benzene metabolites or replication of unrepaired DNA damage can result in DNA double-strand breaks (DSB) (4). DSBs are repaired in vivo by nonhomologous end joining or, after replication when a second identical DNA copy is present, homologous recombination (5). DSB is especially genotoxic because (i) it affects both DNA strands and no intact template is available for repair; (ii) the repair is intrinsically more difficult than other types of DNA repair mechanisms because erroneous rejoining of broken DNA may occur. Therefore, a DNA DSB is potentially highly cytotoxic and can induce chromosomal aberrations (CA) and disrupt the genomic integrity of a cell. It was observed that chromosomal instability can be induced by the benzene metabolite, hydroquinone, and may contribute to the development of acute myeloid leukemia by increasing the number of genetic lesions in hematopoietic cells (6). Therefore, the prompt and efficient repair of DSBs is fundamental for genomic stability and cancer prevention in the presence of benzene (7). Stem cells in bone marrow undergo active cell proliferation and differentiation throughout life and are sensitive to DNAdamaging factors. Hematopoietic progenitor cells have been found to be suppressed after benzene exposure (8,9). In addition, DNA DSB repair genes may be implicated in hematopoiesis (10). In a study by Bender et al. (11) mice with defects in DSB repair genes demonstrated progressive hematopoietic stem cell failure. Genetic polymorphism in genes involved in the repair of DSB may modify the function of DNA DSB repair and confer genetic susceptibility to hematotoxicity caused by benzene. We, therefore, examined the association between a number of single nucleotide polymorphisms (SNPs)/haplotypes in seven genes, which are either DSB repair genes (NBS1, BRCA1, BRCA2, XRCC3, and XRCC4) or genes that play an important role in this pathway (WRN and TP53), and benzene-induced hematotoxicity in a cross-sectional study in China. # The Author 2006. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org 2083 M.Shen et al. Downloaded from http://carcin.oxfordjournals.org by on March 30, 2010 Materials and methods Study population and exposure assessment The details of this cross-sectional study have been described elsewhere (8). Briefly, the study population included 250 workers who were exposed to benzene in two shoe manufacturing factories, and 140 unexposed controls from comparable populations who worked in three clothing-manufacturing factories in the same region of China. Data were obtained from 28 benzeneexposed workers in both study years (2000 and 2001) and are treated as independent observations in the study. Controls were frequency-matched by sex and age to exposed workers. Blood samples were collected from all workers. Individual exposure to benzene and toluene, as well as other organic solvents, was monitored repeatedly up to 16 months before phlebotomy by wearing an organic vapor passive monitor badge, and post-shift urine samples were collected from each subject (8). Subjects were administered a questionnaire for information on lifetime occupational history, hobbies, environmental exposures, medical history and current medications, and past and current tobacco and alcohol use. Individuals carrying out exposure assessment activities were blinded with regard to hematologic data on study subjects. Hematology All subjects provided a 29 ml peripheral blood sample and a buccal cell mouth rinse sample, and underwent a physical exam. Blood samples were delivered to the lab within 6 h of being collected; the complete blood cells (CBC) and differentials were analyzed using a T540 blood counter, and the major lymphocyte subsets were analyzed by an FACS Calibur flow cytometer (Software: SimulSET v3.1). Genotyping DNA was extracted from blood samples using phenolchloroform extraction (12) and genotyped by TaqMan-based real-time PCR at Core Genotyping Facility of NCI (http://snp500cancer.nci.nih.gov) (13). SNPs were selected for study on the basis of a minimum allele frequency of 0.05, and evidence of association in previous epidemiology studies, evidence of function, or to extend genomic coverage for a given gene. Individuals carrying out genotyping were blinded with regard to hematologic data on study subjects. DNA was successfully extracted from all samples, and >95% of the DNA samples were successfully genotyped for all candidate SNPs except for three SNPs in WRN [Ex6 +9 C > T (91%), Ex20 88 G > T (90%) and IVS33 95 C > T (81%)] and four SNPs in BRCA2 [Ex11 +1487 A > G (88%), Ex11 +1898 T > C (90%), Ex14 194 A > G (90%) and Ex2 +14 A > G (88%)]. Blind replicate samples were randomly interspersed, and the concordance rates were 99100% for all assays. Statistical analysis The HardyWeinberg equilibrium for each SNP was tested with a Pearson c2 or exact test. Measure of pairwise linkage disequilibrium (LD) and the test for LD at one gene were carried out using the HaploView program (http:// www.broad.mit.edu/personal/jcbarret/haploview/). Genotype data were analyzed with the homozygotes of the common allele as the reference group. Generalized Estimating Equations (GEE) were used to model the relationship between SNPs and blood cell counts, adjusting for age, sex, body mass index (BMI), any recent smoking and alcohol consumption for controls, plus occupational exposure to benzene and toluene on a log scale 1 month before phlebotomy for exposed subjects. Interaction was estimated by adding a multiplicative term between SNP (variant carriers versus Wild-type carriers) and benzene exposure (yes/no) into a model with all subjects. Haplotype block structure was examined for SNPs within the same gene using HaploView. Overall association between quantitative blood cell counts and haplotypes was assessed separately for unexposed and exposed subjects adjusting for the potential confounders using the HaploStats program (14) in R (Version 2.0.1) (15). Individual haplotypes were estimated using SAS/Genetics, and the effects of each haplotype were estimated using the best haplotype pairs in a GEE model with the most common haplotype as the reference. We controlled for multiple hypothesis testing by calculating the false discovery rate, using the BenjaminiHochberg method (16), which was defined as the expected ratio of erroneous rejections of the null hypothesis to the total number of rejected hypotheses. An FDR of 0.05 was used as a critical value to assess if the obtained P-values are still significant. Those SNPs demonstrating significant gene-dosage effects (i.e. trend test) among workers exposed to benzene were further explored for their influence on specific white blood cell (WBC) subtypes. All P-values presented are two-sided and data were analyzed using the Statistical Analysis Software, version 8.02 (SAS Institute Inc, 1996) if not specified elsewhere. 2084 Results Demographic characteristics were essentially the same between controls and benzene-exposed workers. The majority of the study subjects were females (65%) and relatively young (30 8). The subjects in the benzene-exposed and nonexposed groups were comparable in alcohol use, recent infection, smoking status and BMI (8). The benzene-exposed workers had been employed an average of 6.1 2.9 years. The average benzene air exposure in the month before phlebotomy was 5.4 (SD: 12.1) p.p.m. in the exposed group (Table I). WBCs and most WBC subtypes as well as the platelet counts were significantly reduced in benzene-exposed workers compared with controls (Table I). Natural killer (NK) cell counts and hemoglobin levels were significantly decreased only among the most highly exposed workers (8), and CD8+-T cells did not vary with benzene-exposure levels. Twenty-four SNPs in seven genes involved in the DNA DSB repair pathway were genotyped, covering substitutions in the coding and non-coding regions (Table II). Only those SNPs that are associated with total WBC counts in benzeneexposed group are shown in Table III. Those SNPs that were not associated with total WBC counts in benzene-exposed group are shown in a Supplementary Table. In the exposed group, homozygous variants in four SNPs in WRN (Ex4 16 G > A, Ex6 +9 C > T, Ex20 88 G > T and Ex26 12 T > G) had significantly reduced WBCs in the range of 818%. For the TP53 Ex4 +119 C > G polymorphism, the magnitude of reduction in WBC counts was $12%, and subjects Table I. Hematological characteristics of study population by benzene-exposure statusa Controls (n 140) Exposed (n 250) P Benzene-exposure measurements Benzene air level (p.p.m.)b Benzene 0.4 1.2 urine (mg/l)c Peripheral blood cell countsd Total WBCs 6484 1712 Granulocytes 4111 1410 Lymphocytes CD4+-T cells CD8+-T cells CD4+: CD8+ ratio 2129 577 742 262 553 208 1.5 0.6 B cells 218 93.8 NK cells 586 318 Monocytes Platelets 241 92.1 230 59.7 103 Hgb (g/dl) 14.5 1.6 5.4 12.1 158 536 5488 1350 3334 1053 1939 521 622 183 553 213 1.2 0.4 173 88.5 542 277 215 93.2 (202 52.0) 103 14.5 1.6 <0.0001 <0.0001 0.0014 <0.0001 0.88 <0.0001 <0.0001 0.30 0.002 <0.0001 0.83 aThere are up to 418 observations on 390 unique subjects (140 controls and 250 benzene-exposed workers). Data were obtained from 28 exposed subjects in both years (2000 and 2001) and are treated as independent observations in summary data shown. bBenzene air level is the arithmetic mean (standard deviation) of an average of two measurements per subject collected during the month before phlebotomy. cUrinary benzene (mean, standard deviation) and mean individual air levels of benzene were strongly correlated (Spearman r 0.88, P < 0.0001). dUnadjusted mean (standard deviation) cells per microliter of blood. Linear regression was used for statistical analyses adjusted for repeated measures by GEEs. Models were adjusted for age, sex, current smoking, current alcohol drinking, BMI and recent infections and, where appropriate, ln benzene and ln toluene air level. DNA repair SNPs and benzene-induced hematotoxicity Downloaded from http://carcin.oxfordjournals.org by on March 30, 2010 Table II. Genes and SNPs assessed in this study Gene Chromosome location SNP Region (dbSNP ID)a WRN 8p12-p11.2 TP53 NBS1 BRCA1 17p13.1 8q21-q24 17q21 BRCA2 13q12.3 XRCC3 14q32.3 XRCC4 5q13-q14 Ex4 16 G > A (rs4987236) Val114Ile; Ex6 +9 T > C (rs2725349) Cys171Cys; Ex20 88 T > G (rs1800392) Leu787Leu; Ex26 12 T > G (rs2725362) Phe1074Leu; IVS33 95 C > T (rs4987036); Ex34 93 T > C (rs1346044) Cys1367Arg Ex4 +119 G > C (rs1042522) Arg72Pro Ex5 32 G > C (rs1805794) Glu185Gln Ex12 +1641 T > C (rs16940) Leu730Leu; Ex12 1485 C > T (rs799917) Pro830Leu; Ex12 984 A > G (rs16941) Glu997Gly; Ex12 549 A > G (rs16942) Lys1142Arg; Ex17 150 A > G (rs1799966) Ser1613Gly Ex2 +14 A > G (rs1799943) (50 UTR); Ex10 +72 A > C (rs766173) Asn289His; Ex10 +321 A > C (rs144848) Asn372His; Ex11 +1062 A > G (rs1799944) Asn991Asp; Ex11 +1487 A > G (rs1801406) Lys1132Lys; Ex11 +1898 T > C (rs543304) Val1269Val; Ex14 194 A > G (rs1799955) Ser2414Ser Ex8 53 C > T (rs861539) Thr241Met Ex6 7 G > T (rs3734091) Ala247Ser; IVS7 1 G > A (rs1805377); Ex8 +34 T > G (rs1056503) Ser307Ser aIncluding amino acid change if applicable. homozygous for the uncommon allele of BRCA2 Ex11 +1487 A > G had a 7% greater reduction in WBCs. There was a linear trend of reduced WBC counts associated with the increase of carried variant for WRN Ex6 +9 C > T, Ex20 88 G > T, Ex26 12 T > G, TP53 Ex4 +119 C > G and BRCA2 Ex11 +1487 A > G. Although most SNPs located within the same gene were in LD and as such do not constitute true independent tests, we assessed all 24 SNP associations for possible FDR and found that the five SNPs in WRN and TP53 remained significantly associated with total WBC counts. There was a statistically significant interaction (P 0.021) between benzene exposure and the WRN Ex26 12 T > G polymorphism. All these SNPs were not found to influence WBC counts among unexposed subjects except that the polymorphism of BRCA2 Ex11 +1487 A > G was associated with increased WBCs with borderline significance. SNPs in other genes were not associated with reduced WBC counts in exposed subjects. We further evaluated the influence on several major WBC subtypes of the SNPs that had significant gene-dosage effects on total WBC counts among exposed workers (Table IV). One or more SNPs in WRN had broad effects on WBC subtypes, with significant decreases of granulocyte, total lymphocyte, CD4+-T cell, CD8+-T cell and monocytes counts. Likewise, TP53 Ex4 +119 C > G was associated with reduced granulocyte, CD4+-T cell and B cell counts, while the effect of BRCA2 Ex11 +1487 A > G polymorphism was limited to granulocytes. Haplotype analysis of genes in which two or more SNPs in LD were available (i.e. WRN, BRCA1, BRCA2 and XRCC4) was carried out. Six SNPs in WRN constituted one haplotype block. There was an overall association between WBC counts and haplotypes of WRN (P 0.007) in the benzene-exposed group (Table V). A haplotype with substitutions in three SNPs (Ex4 16 G > A, Ex20 88 G > T and Ex26 12 T > G) was Table III. Effect on total WBC counts of SNPs in WRN, TP53 and BRCA2 by benzene-exposure statusa Genotype Controls WBCb P Exposed WBCb P WRN Ex4 -16 G > A (rs4987236) Val114Ile GG 133 6430 1658 GA 7 7514 2473 Ex6 +9 T > C (rs2725349) Cys171Cys TT 96 6516 1615 TC 30 6600 2046 CC 1 6500 Trend Ex20 -88 T > G (rs1800392) Leu787Leu TT 46 6517 1642 TG 59 6592 1744 GG 20 6550 1789 Trend Ex26 -12 T > G (rs2725362)c Phe1074Leu TT 53 6392 1726 TG 68 6479 1719 GG 18 6756 1757 Trend TP53 Ex4 +119 G > C (rs1042522) Arg72Pro GG 41 6534 1763 GC 67 6621 1758 CC 31 6190 1532 Trend BRCA2 Ex11 +1487 A > G (rs1801406) Lys1132Lys AA 54 6280 1502 AG 61 6674 1843 GG 9 7489 1883 Trend Ref. 0.489 Ref. 0.988 0.983 Ref. 0.895 0.831 0.821 Ref. 0.791 0.350 0.393 Ref. 0.684 0.531 0.530 Ref. 0.091 0.092 0.035 267 9 179 62 10 90 104 56 105 111 61 103 109 63 96 121 27 5524 1356 Ref. 4511 752 0.0003 5463 1340 Ref. 5398 1342 0.575 4710 1090 0.0008 0.022 5764 1391 Ref. 5295 1270 0.010 5159 1367 0.002 0.001 5866 1409 Ref. 5320 1232 0.002 5144 1316 0.0006 0.0003 5764 1393 Ref. 5489 1367 0.149 5067 1150 0.0009 0.001 5611 1546 Ref. 5326 1197 0.127 5193 1059 0.073 0.045 aModels were adjusted for age, sex, current smoking, current alcohol drinking, BMI, recent infections and, in exposed workers, ln air benzene exposure and ln air toluene exposure in the month before phlebotomy. There are two controls without BMI data and they are excluded from the statistical analysis. bUnadjusted total WBC count (/ml) as mean standard deviation. cThere is a significant interaction (P 0.021) between benzene exposure (yes/no) and this polymorphism [(TG + GG)/TT]. associated with a 20% reduction in WBCs following benzene exposure. Discussion We studied the effect of 24 SNPs in 7 genes that survey the genome and participate in DNA DSB repair on peripheral WBC counts among 250 benzene-exposed workers and 140 unexposed controls. Of these genes, our results demonstrate that four SNPs in WRN (Ex4 16 G > A, Ex6 +9 C > T, Ex20 88 G > T and Ex26 12 T > G), one SNP in TP53 (Ex4 +119 C > G) and one SNP in BRCA2 (Ex11 +1487 A > G) are associated with decreased WBCs and particular WBC subtypes in benzene-exposed workers. The effect of all SNPs on WBC counts in controls was not statistically significant after adjustment for multiple comparisons using the FDR method. In addition, the two unadjusted borderline associations between WRN (IVS33 95 C > T) and XRCC4 2085 Downloaded from http://carcin.oxfordjournals.org by on March 30, 2010 M.Shen et al. 2086 Table IV. Effect on WBC subsets of SNPs in WRN, TP53 and BRCA2 among benzene-exposed subjectsa Genotype Exposed Granulocytesb P Lymphocytesb P CD4+ -T cellsb P CD8+ -T cellsb P B Cellsb P NK cellsb P Monocytesb WRN Ex4 16 G > A (rs4987236) Val114Ile GG 267 3353 1060 GA 9 2800 742 Ex6 +9 T > C (rs2725349) Cys171Cys TT 179 3324 1067 TC 62 3305 998 CC 10 2620 736 Trend Ex20 88 T > G (rs1800392) Leu787Leu TT 90 3532 1142 TG 104 3215 909 GG 56 3102 1129 Trend Ex26 12 G > T (rs2725362) Phe1074Leu TT 105 3593 1159 TG 111 3226 881 GG 61 3085 1072 Trend TP53 Ex4 +119 G > C (rs1042522) Arg72Pro GG 103 3515 1151 GC 109 3367 1040 CC 63 3000 830 Trend BRCA2 Ex11 +1487 A > G (rs1801406) Lys1132Lys AA 96 3493 1235 AG 121 3222 915 GG 27 2981 791 Trend Ref. 0.011 1954 519 1556 448 Ref. 0.830 0.0001 0.027 1925 505 1874 532 1850 584 Ref. 0.042 0.004 0.003 2004 502 1866 546 1848 466 Ref. 0.020 0.002 0.001 2048 498 1882 547 1854 488 Ref. 0.518 0.003 0.005 2021 557 1914 525 1860 440 Ref. 0.067 0.005 0.005 1909 544 1887 467 1974 522 Ref. 626 184 0.038 513 128 Ref. 0.307 0.178 0.111 626 193 598 154 556 176 Ref. 0.045 0.048 0.031 654 197 599 174 589 174 Ref. 0.008 0.015 0.007 667 196 593 170 595 171 Ref. 0.124 0.124 0.097 656 195 605 179 599 166 Ref. 0.927 0.601 0.721 615 202 621 176 604 140 Ref. 557 216 0.130 440 105 Ref. 0.394 0.167 0.140 558 212 520 226 464 158 Ref. 0.041 0.025 0.018 583 212 532 211 515 222 Ref. 0.008 0.026 0.014 595 219 537 201 509 215 Ref. 0.045 0.123 0.078 565 224 552 219 536 189 Ref. 0.395 0.773 0.841 532 230 542 187 593 216 Ref. 174 89 0.016 158 87 Ref. 547 277 Ref. 217 93 0.939 405 270 0.207 156 73 Ref. 0.131 0.037 0.022 168 86 171 98 166 55 Ref. 0.436 0.953 0.605 521 267 550 295 621 284 Ref. 0.894 0.752 0.783 214 85 219 114 240 84 Ref. 0.057 0.026 0.019 169 85 164 70 180 118 Ref. 0.968 0.741 0.755 550 274 521 284 537 279 Ref. 0.864 0.956 0.981 228 85 213 102 209 92 Ref. 0.029 0.006 0.004 176 87 167 73 178 114 Ref. 0.697 0.909 0.967 556 264 529 289 541 281 Ref. 0.546 0.810 0.745 225 86 212 101 205 90 Ref. 0.736 0.745 0.723 184 102 173 79 155 79 Ref. 0.534 0.040 0.047 555 262 533 275 543 308 Ref. 0.186 0.496 0.393 228 98 208 84 206 100 Ref. 0.287 0.071 0.077 160 78 174 84 182 136 Ref. 0.170 0.759 0.398 543 298 509 259 573 245 Ref. 0.306 0.297 0.811 209 91 217 86 237 131 aModels were adjusted for age, sex, current smoking, current alcohol drinking, BMI, recent infections and, in exposed workers, ln air benzene exposure and ln air toluene exposure in the month before phlebotomy. There are two controls without BMI data and they are excluded from the statistical analysis. bUnadjusted cell counts (/ml) as mean standard deviation. P Ref. 0.016 Ref. 0.669 0.793 0.845 Ref. 0.274 0.326 0.280 Ref. 0.224 0.224 0.183 Ref. 0.196 0.148 0.123 Ref. 0.257 0.405 0.264 DNA repair SNPs and benzene-induced hematotoxicity Downloaded from http://carcin.oxfordjournals.org by on March 30, 2010 Table V. Haplotype analysis of WRN on total WBC counts by benzene-exposure status Haplotypea Controls WBCb Pc Exposed WBCb Pc G-T-T-T-C-T 173 A-C-G-G-C-C A-T-G-G-C-T 7 G-C-G-G-C-C 26 G-C-G-G-T-T 6 G-C-T-T-C-T G-T-G-G-C-C 3 G-T-G-G-C-T 1 G-T-G-G-T-T 61 G-T-G-T-C-T 2 G-T-G-T-T-T G-T-T-G-C-T G-T-T-T-T-T 1 Omnibus testc 6453 1707 Ref. 311 1 7514 2473 0.484 8 6315 1951 0.712 74 7800 1761 0.057 15 1 4867 569 0.0009 6 6300 6546 1477 0.456 128 4700 848 6 1 1 6100 2 0.844 5658 1355 Ref. 4000 4575 778 0.0002 5385 1369 0.013 5107 1263 0.297 5900 5500 657 0.601 5198 1262 0.008 6600 2117 0.272 5300 4000 6000 1980 0.007 aSix SNPs in WRN are included in the analysis: Ex4 16 G > A, Ex6 +9 T > C, Ex20 88 T > G, Ex26 12 T > G, IVS33 95 C > T and Ex34 93 T > C. bUnadjusted cell counts (/ml) as mean standard deviation. cModels were adjusted for age, sex, current smoking, current alcohol drinking, BMI, recent infections and, in exposed workers, ln air benzene exposure and ln air toluene exposure in the month before phlebotomy. (Ex6 7 G > T) and lower WBC count are based on small numbers of observations. The gene WRN encodes a member of the RecQ subfamily and the DEAH (Asp-Glu-Ala-His) subfamily of DNA and RNA helicases. It possesses an intrinsic 3050 DNA helicase activity, and is also a 3050 exonuclease. WRN plays an important role in monitoring genome integrity and controlling the cell's response to genotoxic stress (17). Initially, together with other related factors, WRN helps recruit the proper DNA repair factors to the site of the lesion. However, the WRN complex may direct the activation of the apoptotic pathway if the damage is excessive. Lack of WRN may result in deregulation of DNA damage monitoring and anomalous activation of DNA repair or apoptosis in response to certain types of DNA damage (18). Specifically, WRN has been found to play a key role in DSB repair (19). Mutations in this gene produce truncated proteins and result in Werner syndrome, an autosomal recessive disorder characterized by premature aging and genomic instability. Four significant SNPs are located in the functional domains of WRN, with the Ex4 16 G > A and Ex6 +9 C > T being in the exonuclease domain, Ex20 88 G > T in the helicase domain and Ex26 12 T > G located in the vicinity of the RecQ C-terminal domain (17,20). These polymorphisms may modify the function of these functional domains, particularly for Ex26 12 T > G, which is located in a region that interacts with multiple proteins (21). Even though the four SNPs are in LD, there may be an accumulation of effects based on the haplotype analysis, indicating that the effects may not be attributable to one particular SNP. However, available reports for the impact of these SNPs on longevity and aging-related diseases demonstrated only weak and inconsistent associations (22,23), and, moreover, the WRN Ex26 12 T > G polymorphism displayed little change in helicase/exonuclease activities relative to wild-type WRN in an in vitro assay (24). This region in WRN (Ex4Ex26) warrants further investigation for the accountable polymorphic position(s) and their precise role in susceptibility to benzene hematotoxicity. Normal function of TP53 is essential in DNA damage response, cell cycle control and cell apoptosis, and mutations in TP53 are the most common genetic alterations in human tumors. It is activated when genetic material is altered, initiating a range of biological defense pathways. In response to DNA damage, TP53-mediated cell cycle is arrested to allow sufficient time for DNA repair before DNA replication or mitosis. It also directly participates in DNA repair, particularly in homologous recombination and non-homologous end joining (25,26). It has been found that bone marrow cells in p53-deficient mice expressed significantly reduced levels of many key genes involved in the p53-regulated DNA damage response pathways after chronic exposure to benzene (27). In cDNA microarray analyses, Yoon et al. (28) demonstrated that p53 appears to play a key role in benzene-induced hematotoxicity. The TP53 codon 72 polymorphism is functional. The Ex4 +119 C > G non-conservative substitution is located in a proline-rich region (residues 6492) of the p53 protein, where the 72Pro amino acid constitutes one of five PXXP (where P represents Pro and X any amino acid) motifs resembling an Src homology 3 (SH3) binding domain (29). The polymorphic variant of wild-type p53 has been shown to have different biochemical properties and biological functions (3032). Significantly higher levels of p53 expression in lymphocytes were detected in subjects bearing the Arg/Arg allele than in subjects with Pro/Pro and Arg/Pro alleles among healthy humans exposed to low doses of ionizing radiation (33). These findings provide support for a model in which loss of p53 function due to Ex4 +119 C > G polymorphism is associated with benzene-induced hematotoxicity. BRCA2 is a tumor-suppressor protein directly implicated in familial breast cancer. It maintains genome stability by participating in multiple biological pathways including DNA transcription, recombination and cell cycle control (34,35). A particular spectrum of bi-allelic mutations in BRCA2 is connected to a rare hematological disorder, Fanconi anemia, characterized by aplastic anemia and hypoplasia of the bone marrow (36). BRCA2 has an important role in DNA recombination reactions mediated by RAD51 in DNA DSB repair (34). Two structural features of BRCA2 were revealed with eight BRC repeats interacting with RAD51 and three oligonucleotide/oligosaccharide-binding (OB) fold domains binding single-strand DNA (37). The BRCA2 Ex11 +1487 A > G polymorphism is located in exon 11, which is the largest exon and encodes the eight BRC motifs (38). The exon 11 and BRC motifs are essential for BRCA2's function and homologous recombination (38,39). The eight BRC repeats are highly conserved and the four BRC repeats in 50 are the most conserved sequences (38). The BRCA2 Ex11 +1487 A > G is located between BRC1 and BRC2 in 50 of Exon 11. As it leads to a synonymous substitution, the observed hematotoxic effect may be attributable to a linked functional polymorphism in that region that can modify the function of BRC repeats and impact DNA DSB repair. In summary, we report that common genetic polymorphisms in WRN, TP53 and BRCA2 may confer susceptibility to hematotoxicity in workers exposed to benzene. All these genetic variants appear to influence granulocytes, while variants in WRN also altered cells of the lymphoid lineage, suggesting that effects may trace back to earlier progenitor and possibly 2087 M.Shen et al. Downloaded from http://carcin.oxfordjournals.org by on March 30, 2010 stem cells. The three gene products play an important role in multiple mechanisms including DNA damage recognition, replication, recombination, repair and cell cycle regulation, all of which are critical to maintain genomic integrity. In addition, BRCA2, TP53 and WRN perhaps act synergistically to prevent accumulation of genomic lesions (40,41). Decreased function of WRN, TP53 or BRCA2 due to genetic polymorphisms may result in genomic instability and increase predisposition to cancer in the presence of a carcinogen such as benzene. Although this is the largest cross-sectional study of DNA repair SNPs and benzene-induced hematotoxicity in the literature, to the best of our knowledge, and our key findings were highly statistically significant, it is possible that some associations are false-positives, particularly those based on small numbers of subjects carrying at-risk alleles. As such, replication of key findings in other benzene-exposed populations is critical. Supplementary material Supplementary material is available online at http://www. carcin.oupjournals.org/. Acknowledgements This study is supported by the Intramural Research Program of the National Institutes of Health (NIH), National Cancer Institute and NIH grants R01ES06721, P42ES04705, P30ES01896 (to M.T.S.), P42ES05948 and P30ES10126 (to S.M.R.). We thank Dr Bingshu Eric Chen for the statistical consultation for the study. Conflict of Interest Statement: None declared. References 1. Goldstein,B.D. (1988) Benzene toxicity. Occup. Med., 3, 541554. 2. 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