Association of Sequence Variants on Chromosomes 20, 11, and 5 (20q13.33, 11q23.3, and 5p15.33) With Glioma Susceptibility in a Chinese Population

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1 American Journal of Epidemiology ª The Author Published by Oxford University Press on behalf of the Johns Hopkins Bloomberg School of Public Health. All rights reserved. For permissions, please Vol. 173, No. 8 DOI: /aje/kwq457 Advance Access publication: February 24, 2011 Original Contribution Association of Sequence Variants on Chromosomes 20, 11, and 5 (20q13.33, 11q23.3, and 5p15.33) With Glioma Susceptibility in a Chinese Population Hongyan Chen, Yuanyuan Chen, Yao Zhao, Weiwei Fan, Keke Zhou, Yanhong Liu, Liangfu Zhou, Ying Mao, Qingyi Wei, Jianfeng Xu, and Daru Lu* * Correspondence to Dr. Daru Lu, State Key Laboratory of Genetic Engineering, Fudan-VARI Genetic Epidemiology Center and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, 220 Handan Road, Shanghai , China ( drlu@fudan.edu.cn). Initially submitted May 25, 2010; accepted for publication November 29, Two genome-wide association studies of glioma in European populations identified 14 genetic variants strongly associated with risk of glioma, but it is unknown whether these variants are associated with glioma risk in Asian populations. The authors genotyped these 14 variants in 976 glioma patients and 1,057 control subjects to evaluate their associations with risk of glioma, particularly high-grade glioma (glioblastoma; n ¼ 312), in a Chinese population ( ). Overall, the authors identified 3 susceptibility loci for glioma risk at 20q13.33 (RTEL1 rs (P ¼ )), 11q23.3 (PHLDB1 rs (P ¼ )), and 5p15.33 (TERT rs (P ¼ )) in this study population; these loci were also associated with glioblastoma risk (20q13.33: RTEL1 rs (P ¼ ); 11q23.3: PHLDB1 rs (P ¼ ); 5p15.33: TERT rs and TERT rs (P ¼ and P ¼ , respectively)). This study provides further evidence for 3 glioma susceptibility regions at 20q13.33, 11q23.3, and 5p15.33 in Chinese populations. Asian continental ancestry group; association; genetics; genetic variation; genome-wide association study; glioma; polymorphism, single nucleotide Abbreviations: CI, confidence interval; GWAS, genome-wide association study(ies); OR, odds ratio; SNP, single nucleotide polymorphism. Although primary brain tumors are relatively rare (5% 9% of all cancers), approximately 22,070 persons are diagnosed each year in the United States (1). Asian populations generally show lower incidence rates than those in Europe and North America. In China, in 2000, the annual incidence rate of brain tumors was less than 3.9 per 100,000 in men and 2.8 per 100,000 in women (2). However, incidence rates tend to be increased in large cities in China. According to the China s Health Statistics Yearbook 2009, the annual mortality rate in 2008 in China was approximately 3.13 per 100,000 population (3). Glioma derived from glial cells surrounding and supporting neurons (4) is the most common type of primary intracerebral neoplasm in China, as well as in the West, accounting for more than 40% of primary brain tumors in humans (5). Furthermore, fewer than 50% of glioma patients live longer than 5 years after diagnosis (6). The only contributing factor identified currently is ionizing radiation (4, 7), although an increased familial risk has also been recognized. At least 8 susceptibility loci have been found to be associated with glioma risk from a series of case-control studies in at least 2 populations via candidate-gene association studies. These include the DNA repair genes PRKDC (also known as XRCC7) G6721T (8, 9), XRCC1 W399R (9 11), PARP1 A762V (9, 11), MGMT F84L (11, 12), ERCC1 A8092C (13, 14), and ERCC2 Q751K (9, 14); the cell cycle gene EGFþ61 A/G (15, 16); and the inflammation gene IL13 R110G (17, 18). Although these candidate-gene association-study approaches are promising, serious limitations due to small sample sizes (very few studies have had 915

2 916 Chen et al. more than 500 cases and controls) and the characteristic heterogeneity of gliomas have led to failure to confirm these findings in subsequent independent replication studies. With the rapid advances in genotyping technology, it is now possible to characterize inherited variation in very large numbers of genes to comprehensively examine genetic variation in glioma etiology and prognosis. The number of genome-wide association studies (GWAS) has been growing rapidly, leading to the discovery and replication of many new susceptibility loci. Recently, GWAS of glioma in populations of European ancestry were completed in the United States and the United Kingdom (19, 20). One GWAS reported on by Shete et al. (19) implicated 14 single nucleotide polymorphisms (SNPs) in 5 genes meeting the criteria for genome-wide significance (P < ). Among these 14 SNPs, odds ratios ranged from 1.18 to 1.36, with respective P values ranging between and The 5 genes were TERT, CCDC26, CDKN2A/B, PHLDB1, and RTEL1. The other GWAS identified 3 SNPs located in the CDKN2B and RTEL1 genes that were strongly associated with risk of high-grade glioma (glioblastoma) (P < 10 7 ) (20). Among these 3 SNPs, 2 (rs and rs ) were also reported by Shete et al. (19). Because of potential differences in genetic background by ethnicity, it is extremely important to understand the consequences of inheriting these variants in other ethnic populations. Therefore, we comprehensively evaluated all glioma risk variants identified by these 2 GWAS in a Chinese Han population using a hospital-based case-control design. MATERIALS AND METHODS Study subjects Using the same recruitment method described elsewhere (21 23), we recruited 976 patients with histopathologically confirmed glioma and 1,057 healthy controls between October 2004 and July 2009 from the Department of Neurosurgery at Huashan Hospital, Fudan University (Shanghai, China). There were no restrictions on age, sex, or histologic type, but patients with a self-reported history of cancer and patients with previous radiotherapy or chemotherapy for unknown disease conditions were excluded. Additionally, diagnoses of potentially eligible cases were validated by trained abstractors who reviewed pathologic and medical records of all cases to confirm that there were no undiagnosed occult primary tumors at the time of recruitment. All controls, who were frequency-matched to the cases by age (65 years), sex, and residential area (urban area or countryside), were selected from visitors to the trauma outpatient clinic and persons undergoing annual check-ups at the same hospital. These controls had no known central nervous system-related diseases, self-reported history of cancer at any site, or history of radiotherapy/chemotherapy for unknown disease conditions. No evidence of demographic differences was found between the trauma outpatients and the annual check-up subjects. All cases and controls were from Shanghai and the surrounding provinces (Zhejiang, Jiangsu, and Anhui) in eastern China and had a Han Chinese ethnic background. We previously published information on questionnaire design, interview, and DNA sample collection (22). Briefly, a shortened structured questionnaire was adapted from a questionnaire originally created for a brain tumor study in the Department of Epidemiology at the M. D. Anderson Cancer Center (Houston, Texas). The questionnaire was used to elicit information on demographic factors, history of occupational exposure to ionizing radiation, and the health characteristics of study subjects. Prior to enrollment, subjects were informed of the objectives, procedures, and voluntary nature of this study in a cover letter attached to the questionnaire. Among persons who agreed and signed an informed consent form for participation in the study, each subject underwent a face-to-face questionnaire interview and provided a one-time blood sample (3 5 ml) for DNA extraction. A separate signed consent form was obtained for acquiring and reviewing medical records. Participation rates for cases and controls in this study were 85.7% and 79.2%, respectively. Selection of SNPs for genotyping The 14 previously identified SNPs, representing 5 distinct loci, were genotyped in the current study. Of these, 12 SNPs were from the study by Shete et al. (19) and the other 2 (rs and rs ) were excluded because of strong linkage disequilibrium with rs and rs , respectively (D# ¼ 1 and r 2 ¼ 1 in the Chinese population in the HapMap Phase II data). We also included 1 SNP (rs ) implicated in the risk of high-grade glioma (20). In addition, we selected another SNP (rs ) located in the TERT gene that has been reported to be associated with risk of multiple types of cancer (24). We used the white blood cell fractions of the whole blood samples for extraction of genomic DNA using the Qiagen Blood Kit (Qiagen, Chatsworth, California). After extraction, genomic DNA was diluted to a final concentration of ng/ll for the genotyping assays. Polymorphism spanning fragments were amplified by polymerase chain reaction, and genotyping was performed with the Mass- ARRAY iplex platform (Sequenom, San Diego, California) through the use of an allele-specific matrix-assisted laser desorption/ionization time-of-flight mass spectrometry assay (25). Primers for amplification and extension reactions were designed using MassARRAY Assay Design software, version 3.1 (Sequenom), and SNP genotypes were obtained according to the iplex protocol provided by the manufacturer. We examined genotyping quality by means of a detailed quality control procedure that ensured a >95% successful call rate with duplicate calling of genotypes, internal positive control samples, and subsequent Hardy- Weinberg equilibrium testing. Statistical analysis We used Fisher s exact test to test for deviation from Hardy-Weinberg equilibrium among controls for each SNP and the v 2 test to compare the differences in demographic characteristics and frequency distributions of genotypes and alleles between cases and controls. The most common genotype in controls was used as the reference group. We

3 Glioma Susceptibility Genes in Chinese Populations 917 Table 1. Demographic Characteristics of Glioma Cases and Controls in a Chinese Study Population, Cases (n 5 976) Controls (n 5 1,057) P Value From x 2 Test No. % No. % Sex Male Female Missing data Mean age, years 42.3 (16.3) a 42.1 (18.3) Age group Children (18 years) Adults (>18 years) Cigarette smoking 0.34 Never smoker Ever smoker Missing data Family history of cancer (first-degree relatives) No Yes Missing data Histologic type Astrocytic glioma Glioblastoma Other glioma Missing data 8 <1 a Numbers in parentheses, standard deviation. performed unconditional logistic regression analysis, with adjustment for age and sex, to calculate odds ratios and 95% confidence intervals as estimates of the relative risk for each SNP and multiple SNPs. All statistical tests were 2-sided. Akaike s Information Criterion was employed to determine the best-fitting model for each SNP (26). For SNPs in TERT and RTEL1, pairwise linkage disequilibrium was estimated in control subjects using Haploview (27), in which haplotype blocks were inferred using the default option of the solid spine method. The software package Haplo.stats ( was used to perform the haplotype analysis. The statistical significance of the global and haplotype-specific tests (haplo.score) was expressed as a permutation P value (P sim ; minimal simulation: 10,000 with a significance level less than 0.05). Haplotype frequencies and odds ratios (with 95% confidence intervals) were calculated for each haplotype, with adjustment for age and sex, using a generalized linear model (haplo.glm). In stratified analyses, case patients were classified into groups of high-grade tumors (glioblastomas) and low-grade tumors (including astrocytomas, oligodendrogliomas, mixed gliomas, and other low-grade gliomas). We also evaluated the cumulative effects of the risk alleles, which were independently associated with glioma risk, by counting the total number of risk alleles per person from the 3 independently confirmed loci (TERT rs , PHLDB1 rs498872, and RTEL1 rs ) (categories were 0 1, 2, 3, 4, and 5 6). All statistical analyses were performed using SPSS software, version 17.0 (SPSS, Inc., Chicago, Illinois), unless indicated otherwise. Ethics Written informed consent was obtained from each participant, and the study protocol was approved by the School of Life Sciences of Fudan University Ethics Board. RESULTS Sample characteristics Characteristics of the 976 case patients and 1,057 control subjects are presented in Table 1. Because participants were frequency-matched for age, sex, and residential area under our study design, the distributions of age (age at diagnosis for cases and age at recruitment for controls) and sex were comparable between cases and controls. The mean age was 42.3 years for cases and 42.1 years for controls. Approximately 60% of both cases and controls were male. Similar to the previous study (22), cases were slightly more likely to report a family history of cancer (among first-degree relatives) than controls (17.5% vs. 13.1%; P ¼ 0.003).

4 918 Chen et al. Table 2. Association of 14 Previously Identified Single Nucleotide Polymorphisms With the Risks of Glioma and Glioblastoma in a Chinese Population, Single Nucleotide Polymorphism Chromosome Gene Location on Chromosome P Value for HWE Risk Allele Risk Allele Frequency (Proportion) Estimated Risk for All Histologic Types P Value b for Glioblastoma 95% Odds P Controls Cases Ratio a Confidence Value b Interval rs TERT G , e -04* 1.21e -04* rs TERT A , e -03* rs TERT A , e -03* 2.84e -04* rs CCDC C , rs CCDC G , rs CCDC A , rs CCDC T c , rs CCDKN2A/B C , rs CCDKN2A/B G c , rs CCDKN2A/B C , rs CCDKN2A/B G , rs PHLDB T , e -06* 7.24e -03* rs RTEL G c , e -06* 3.57e -07* rs RTEL C , Abbreviation: HWE, Hardy-Weinberg equilibrium. * P < 0.01 a Odds ratio for carriers of the minor allele (adjusted for age and sex). b Adjusted for age and sex. c Ancestral allele. Among cases, 312 (32.2%) had glioblastoma and 656 (67.8%) had low-grade glioma (including 360 (37.2%) astrocytomas and 296 (30.6%) other low-grade gliomas). Analysis of associations with individual SNPs Table 2 shows allele frequencies for the 14 selected SNPs by case/control status. All markers were in Hardy-Weinberg equilibrium in control subjects (P > 0.01). Five of these SNPs were statistically significantly associated with glioma risk, with a significance level of 0.01 (P < 0.01); we assumed that 5 independent tests were performed, and the significance level was adjusted accordingly (rs on 20q13.33 (RTEL1, P ¼ ), rs on 11q23.3 (PHLDB1, P ¼ ), and rs , rs , and rs on 5p15.33 (TERT; P ¼ , P ¼ 0.007, and P ¼ 0.005, respectively)). The remaining 9 SNPs did not reach this predefined statistically significant level. When we evaluated these 14 selected SNPs for associations with glioblastoma risk, rs was identified as having a strong association (P ¼ ; Table 2), and another 2 markers (rs and rs (TERT; P ¼ and P ¼ , respectively) and rs (PHLDB1, P ¼ )) were also significantly associated with giloblastoma risk. Table 3 shows the genotype frequency distributions of the 5 identified risk SNPs in cases and controls. Specifically, significant associations were observed for 3 SNPs (P ¼ for rs , P ¼ for rs498872, and P ¼ for rs ) in a recessive model, for 1 SNP (P ¼ for rs ) in a dominant model, and for 1 SNP (P ¼ for rs ) in a log-additive model, based on the best fit of Akaike s Information Criterion. Analysis of the cumulative effects of multiple risk SNPs We further assessed the cumulative effects of the 3 most significant risk SNPs (TERT rs , PHLDB1 rs498872, and RTEL1 rs ). Overall, glioma risk increased with increasing numbers of risk variant alleles. Persons carrying 5 or 6 risk alleles had a 3-fold increased risk of developing glioma compared with those who carried 0 or 1 risk allele (adjusted odds ratio (OR) ¼ 3.62, 95% confidence interval (CI): 1.79, 7.32; P ¼ (Table 4)). Analysis of haplotype associations Two TERT SNPs (rs and rs ) and 2 RETL1 SNPs (rs and rs ) were in significant linkage disequilibrium (see Web Figure 1, which is posted on the Journal s Web site ( org/)). Haplotype-specific analysis revealed frequency differences between cases and controls for the GA haplotype in TERT (P sim ¼ ) and the GT haplotype in RTEL1 (P sim ¼ 0.00e þ00 ) (Table 5). Further logistic

5 Glioma Susceptibility Genes in Chinese Populations 919 Table 3. Genotype Frequencies for 5 Glioma Susceptibility Single Nucleotide Polymorphisms Among Cases and Controls and Their Association With Glioma Risk in a Chinese Population, Gene, Single Nucleotide Polymorphism, and Haplotype Cases Controls No. % No. % P Value From x 2 Test Adjusted Odds Ratio a Logistic Regression Analysis 95% Confidence Interval P Value a TERT rs TT Reference 8.93e -04 GT , GG , e -04 TT vs. GT/GG , e -04 rs GG e Reference GA , AA , GG/GA vs. AA , e -04 rs GG Reference AG , AA , Log-additive , PHLDB1 rs CC e Reference 7.46e -06 CT , e -05 TT , e -04 CC vs. CT/TT , e -06 RTEL1 rs AA e Reference 7.18e -06 AG , e -04 GG , e -05 AA vs. AG/GG , e -05 a Adjusted for age and sex. regression analyses revealed that haplotypes GG and GA in the TERT block were associated with increased risk of glioma (adjusted OR ¼ 1.22 (95% CI: 1.06, 1.41) and adjusted OR ¼ 1.34 (95% CI: 1.13, 1.60), respectively) and that haplotypes GT and GC in RTEL1 were significantly associated with increased risk of glioma (adjusted OR ¼ 1.46 (95% CI: 1.23, 1.73) and adjusted OR ¼ 1.29 (95% CI: 1.06, 1.56), respectively) compared with the most common haplotypes, TG and AT, respectively. DISCUSSION Performing targeted evaluation of high-risk variants for glioma identified from GWAS in European populations becomes more efficient in other populations, because smaller numbers of SNPs need to be typed and evaluated, thus incurring a lower cost. In this hospital-based case-control validation study of glioma in a Chinese population, we systematically evaluated 14 previously reported glioma risk loci identified through GWAS in European populations. Three glioma susceptibility loci from chromosomes 20, 11, and 5 (20q13.33, 11q23.3, and 5p15.33) were statistically significantly associated with both overall glioma risk (P values ranged from to ) and highgrade glioma (glioblastoma) risk (P values ranged from to ). Consistent with results of single- SNP analysis (allele and genotype), the haplotypes GG and GA in TERT and the haplotypes GT and GC in RTEL1 were significantly associated with an increased risk of glioma. These results suggest that some glioma risk variants identified in European populations are also associated with risk in Chinese Han populations. The strongest signal was located on chromosome 20 at 20q13.33 in RTEL1 (regulator of telomere elongation helicase 1), which is a DNA helicase crucial for regulation of telomere length in mice. The loss of RTEL1 has been associated with shortened telomere length, chromosome breaks, and translocations (28), because it maintains genomic stability directly by suppressing homologous recombination (29). Two SNPs (rs and rs ) in RTEL1 were previously reported to be associated with glioma risk (19, 20). However, only rs was associated with risk in

6 920 Chen et al. Table 4. Association Between the Cumulative Effect of the 3 Most Statistically Significant Single Nucleotide Polymorphisms a and the Risk of Glioma in a Chinese Population, No. of Risk Alleles Cases Controls No. % No. % 95% Odds Ratio b Confidence Interval P Value b Reference , e , e , e , e -04 a Risk alleles included rs (TERT), rs (PHLDB1), and rs (RTEL1). b Adjusted for age and sex in a logistic regression model. our Chinese study population (OR ¼ 1.39, 95% CI: 1.20, 1.59; P ¼ ). Rs is located in intron 12 of RTEL1 and was found to be in strong linkage disequilibrium (D# ¼ 0.933, r 2 ¼ 0.572) with a missense polymorphism (rs , Gln1042His) in exon 32 of RTEL1. This glutamine-to-histidine exchange may cause a conformational change in the RTEL1 protein, thus affecting its activity. Coincidentally, the third-strongest risk locus for glioma was at 5p15.33, where TERT (telomerase reverse transcriptase), also a telomerase-related gene, is located. TERT is the telomerase catalytic subunit that maintains telomeres and cell immortalization, and it has an established role in glioma grade and prognosis (30, 31). In this study population, carriers of the rs G allele and the rs A allele exhibited a statistically significant increased risk of glioma (OR ¼ 1.26 (95% CI: 1.12, 1.43; P ¼ ) and OR ¼ 1.26 (95% CI: 1.07, 1.48; P ¼ ), respectively). Carriers of the GA haplotype containing the G risk allele of rs and the A risk allele of rs had a fold increased risk of developing glioma compared with those carrying the TG haplotype, which was consistent with individual SNP associations. The other SNP, rs , located in exon 2 of TERT, was previously reported to be associated with multiple tumors, including basal cell carcinoma (32), lung cancer (33), bladder cancer (24), and prostate cancer (24). To the best of our knowledge, this is the first report of an association between rs and glioma risk (OR ¼ 1.2, 95% CI: 1.06, 1.38; P ¼ ). Rs498872, which is mapped to the 5#-untranslated region of PHLDB1 within a 101-kilobase linkage disequilibrium block on 11q23.3, was the second most strongly associated marker in our study population (OR ¼ 1.38, 95% CI: 1.20, 1.57; P ¼ ). This SNP was also reported to be associated with rates of change in diastolic blood pressure (34). The 11q23.3 locus is commonly deleted in neuroblastoma (35); however, there is no direct functional evidence for a role of PHLDB1 in initiation of tumors. All of the confirmed variants showed the same direction of association as that seen in the European GWAS. In the previous study, the effect sizes were in the range of odds ratios of (19, 20); in our study, 4 of the 5 risk SNPs had odds ratios of approximately 1.4, and 1 SNP in TERT was associated with an almost 2.5-fold increased risk. To understand the cumulative effects of these variants on glioma risk, we created a variable to combine the effects of risk alleles per person from the 3 independently confirmed loci (rs , rs498872, and rs ). We found that these 3 independent SNPs had a strong cumulative effect on the risk of glioma. For example, persons carrying 5 or 6 risk alleles had a 3-fold increased risk of developing glioma compared with those who carried 0 or 1 risk allele, indicating the importance of the combined effects from independent risk loci in the etiology of glioma; however, this prediction strategy requires further confirmation in Table 5. Frequency Distributions of Haplotypes in the TERT and RTEL1 Genes Among Cases and Controls and Their Associations With Glioma Risk in a Chinese Population, Gene and Haplotype a Total No. of Subjects Cases Controls Logistic Regression Analysis No. % No. % Adjusted Odds Ratio b 95% Confidence Interval P Value b TERT TG 2,217 1, , Reference GG 1, , GA , TA , RTEL1 AT 2,872 1, , Reference GT , e e þ00 GC , AC , NA Abbreviation: NA, not applicable. a The order of polymorphisms was rs and rs in the TERT block and rs and rs in the RETL1 block. b Adjusted for age and sex. c Generated by means of a permutation test with 10,000 simulations. P sim c

7 Glioma Susceptibility Genes in Chinese Populations 921 additional studies before it can be used for future assessment of glioma risk in the general population. Overall, 9 of the previously identified risk SNPs were not associated with glioma in our Chinese Han study population. However, 8 of these 9 SNPs showed the same direction of association as in the previous reports, of which 3 SNPs (2 in CCDKN2A/B (rs and rs ) and 1 in CCDC26 (rs )) reached a nominal significance level of There are several possible reasons for our inability to replicate the previous results for these variants. First, there may have been a lack of statistical power to detect a small effect from very-low-penetrance SNPs; second, the risk allele may have an effect among some populations, but its magnitude may vary in different populations because of genetic heterogeneity, gene-environment interactions, and/or type 1 error; and third, some of the variants may confer risk in one population but not in others. The results from our study strongly suggest the presence of glioma risk variants at 3 genetic loci in Chinese Han populations. However, it is possible that other SNPs in these genes or other genes in or around these regions may be important in Chinese populations as well. Further fine mapping studies of these regions and subsequent functional studies are needed to identify causal variants to understand the etiology of glioma in Chinese populations. ACKNOWLEDGMENTS Author affiliations: State Key Laboratory of Genetic Engineering, Fudan-VARI Genetic Epidemiology Center and MOE Key Laboratory of Contemporary Anthropology, School of Life Sciences, Fudan University, Shanghai, China (Hongyan Chen, Yuanyuan Chen, Weiwei Fan, Daru Lu); Department of Neurosurgery, Huashan Hospital, Fudan University, Shanghai, China (Yao Zhao, KekeZhou, Liangfu Zhou, Ying Mao); Department of Epidemiology, Division of Cancer Prevention and Population Sciences, University of Texas M. D. Anderson Cancer Center, Houston, Texas (Yanhong Liu, Qingyi Wei); and Center for Cancer Genomics and Center for Human Genomics, Wake Forest University School of Medicine, Winston-Salem, North Carolina (Jianfeng Xu). Drs. Hongyan Chen, Yuanyuan Chen, and Yao Zhao contributed equally to this work. Drs. Ying Mao and Daru Lu jointly directed the work. This work was partially supported by the Shanghai Science and Technology Research Program (grants 09JC and ), the Natural Science Foundation of China (grants and ), the Scientific Research Foundation for the Returned Overseas Chinese Scholars (State Education Ministry), the Doctoral Fund of the Ministry of Education of China, and the Shanghai Key Subject Project for Public Health (grant 08GWZX0301). The authors thank Haishi Zhang and Fengping Huang for subject enrollment. They also thank all staff members of the Department of Neurosurgery of Huashan Hospital for their cooperation during data collection. Conflict of interest: none declared. 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