RTEL1 tagging SNPs and haplotypes were associated with glioma development

  • Gang Li1Email author,

    Affiliated with

    • Tianbo Jin2Email author,

      Affiliated with

      • Hongjuan Liang1,

        Affiliated with

        • Zhiguo Zhang1,

          Affiliated with

          • Shiming He1,

            Affiliated with

            • Yanyang Tu3,

              Affiliated with

              • Haixia Yang1,

                Affiliated with

                • Tingting Geng4,

                  Affiliated with

                  • Guangbin Cui5,

                    Affiliated with

                    • Chao Chen2Email author and

                      Affiliated with

                      • Guodong Gao1Email author

                        Affiliated with

                        Diagnostic Pathology20138:83

                        DOI: 10.1186/1746-1596-8-83

                        Received: 6 April 2013

                        Accepted: 12 May 2013

                        Published: 17 May 2013

                        Abstract

                        As glioma ranks as the first most prevalent solid tumors in primary central nervous system, certain single-nucleotide polymorphisms (SNPs) may be related to increased glioma risk, and have implications in carcinogenesis. The present case–control study was carried out to elucidate how common variants contribute to glioma susceptibility. Ten candidate tagging SNPs (tSNPs) were selected from seven genes whose polymorphisms have been proven by classical literatures and reliable databases to be tended to relate with gliomas, and with the minor allele frequency (MAF) > 5% in the HapMap Asian population. The selected tSNPs were genotyped in 629 glioma patients and 645 controls from a Han Chinese population using the multiplexed SNP MassEXTEND assay calibrated. Two significant tSNPs in RTEL1 gene were observed to be associated with glioma risk (rs6010620, P = 0.0016, OR: 1.32, 95% CI: 1.11-1.56; rs2297440, P = 0.001, OR: 1.33, 95% CI: 1.12-1.58) by χ 2 test. It was identified the genotype “GG” of rs6010620 acted as the protective genotype for glioma (OR, 0.46; 95% CI, 0.31-0.7; P = 0.0002), while the genotype “CC” of rs2297440 as the protective genotype in glioma (OR, 0.47; 95% CI, 0.31-0.71; P = 0.0003). Furthermore, haplotype “GCT” in RTEL1 gene was found to be associated with risk of glioma (OR, 0.7; 95% CI, 0.57-0.86; Fisher’s P = 0.0005; Pearson’s P = 0.0005), and haplotype “ATT” was detected to be associated with risk of glioma (OR, 1.32; 95% CI, 1.12-1.57; Fisher’s P = 0.0013; Pearson’s P = 0.0013). Two single variants, the genotypes of “GG” of rs6010620 and “CC” of rs2297440 (rs6010620 and rs2297440) in the RTEL1 gene, together with two haplotypes of GCT and ATT, were identified to be associated with glioma development. And it might be used to evaluate the glioma development risks to screen the above RTEL1 tagging SNPs and haplotypes.

                        Virtual slides

                        The virtual slides for this article can be found here: http://​www.​diagnosticpathol​ogy.​diagnomx.​eu/​vs/​1993021136961998​

                        Keywords

                        Tagging single nucleotide polymorphism (tSNP) Glioma RTEL1 Haplotype Case - control studies

                        Introduction

                        The overall incidence of brain tumors for benign and malignant tumors combined is 18.71 per 100,000 person-years; 11.52 per 100,000 person-years for benign tumors and 7.19 per 100,000 person-years for malignant tumors [1]. Though the age- standardized incidence recently reported varied greatly than ever, non-malignant tumours still accounted for 66% of all newly diagnosed primary brain tumours with the age-standardized incidence rate of 3.57 per 100,000 person-years, while malignant tumours incidence rate was 1.82 per 100,000 person-years (crude incidence rates were 3.69 and 1.92 per 100,000 respectively) [2]. Gliomas, most aggressive malignant brain tumours (astrocytic, oligodendroglial, oligoastrocytic and ependymal origin), represent 20.8% of all brain tumours [2], and account for almost 80% of primary malignant brain tumors, usually resulting in poor survival compared to other types of brain tumors [3].

                        Current evidence suggests that inherited risks play a significant role in glioma susceptibility, as with other cancers, and a majority of the inherited risk is due to the co-inheritance of multiple low-risk variants. These variants are commonly seen gene variants and hence can be identified through association studies [4]. The epidemiology of glioma has focused on identifying factors that can be modified to prevent this disease [57]. Recently studies of genetic risk factors for brain tumors have expanded to genome-wide association studies, and have focused on identifying germline polymorphisms associated with the risk of glioma as well as using molecular markers to classify glial tumors in more homogenous groups [6, 7].

                        A research group from the University of Texas M.D. Anderson Cancer Center conducted a meta-analysis of two genome-wide association studies (GWAS) by genotyping 550 K tagging single nucleotide polymorphisms (tSNPs) in a total of 1,878 cases and 3,670 controls, with validation in three additional independent series totaling 2,545 cases and 2,953 controls. They identified five risk loci for glioma including rs6010620 intronic to RTEL1 gene (P = 2.52 × 10-12) to be associated with glioma risk [6]. Another study in Chinese also identified rs60106203 for glioma risk (P = 2.793 × 10-6), and the locus also associated with glioblastoma risk (P = 3.573 × 10-7) [8]. The subsequent study found that rs6010620 was statistically significantly associated with glioma risk in US female population [9]. Recently, a new independent GWAS of glioma using 1,856 cases and 4, 955 controls has found evidence of strong replication for three of the seven previously reported associations at 20q13.33 (RTEL), 5p15.33 (TERT), and 9p21.3 (CDKN2BAS), and consistent association signals for the remaining four at 7p11.2 (EGFR both loci), 8q24.21 (CCDC26) and 11q23.3 (PHLDB1) [7]. These data tend to show that common susceptibility alleles contribute to the risk of developing glioma and provide insight into disease causation of this primary brain tumor.

                        As the Chinese Han population is by far the population with the largest number in the world, we comprehensively analysized in this study the associations between RTEL1 genotypes and haplotypes with glioma risk, to uncover how germ-line genetic variants of the RTEL1 gene play a complex role in the development of glioma, to offer important insights into the etiology of glioma in the certain Chinese Han population.

                        Patients and methods

                        Study population

                        A total of 629 patients with glioma, includes well-differentiated pilocytic astrocytoma [World Health Organization (WHO) grade I], low grade ependymomas [WHO grade II], low grade astrocytomas [WHO grade II], low grade oligodendrogliomas [WHO grade II], anaplastic astrocytomas [WHO grade III] and glioblastoma multiforme (GBM) [WHO grade IV] [10], between November 2008 and December 2012 were recruited into an ongoing molecular epidemiological study at the Department of Neurosurgery of the Tangdu Hospital affiliated with The Fourth Military Medical University (FMMU) in Xi’an city, China. All glioma cases had no previous history of other cancers, or prior chemotherapy or radiotherapy. There were no age, sex, or disease stage restrictions for case recruitment. There were no age, sex, or disease stage restrictions for case recruitment. All the slides of glioma tissues were re-evaluated according to WHO classifications [10] by two pathologists, with differences resolved by careful discussion. The median age was 43 years (age range, 1–81). The clinicopathological features and the treatment strategies of all the patients were indicated in Table 1.
                        Table 1

                        Clinicopathological features of 629 glioma patients

                        Clinicopathological features

                        WHO I

                        WHO II

                        WHO III

                        WHO IV

                         

                        Pilocytic astrocytoma

                        Astrocytoma

                        Oligodendroglioma

                        Ependymoma

                        Anaplastic astrocytoma

                        Glioblastoma multiforme

                        Case Number

                         

                        Total

                        20

                        433

                        24

                        34

                        81

                        37

                        Male

                        11

                        241

                        13

                        17

                        39

                        21

                        Female

                        9

                        192

                        11

                        17

                        42

                        16

                        Mean age (Age range) (ys)

                         

                        Total

                        12(2-51)

                        42(1-81)

                        46(17-76)

                        29(1-71)

                        50(2-81)

                        47(6-70)

                        Male

                        13(3-35)

                        41(1-81)

                        53(35-76)

                        34(5-60)

                        50(2-73)

                        48(17-70)

                        Female

                        9(2-51)

                        42(2-79)

                        41(17-55)

                        20(1-71)

                        52(10-81)

                        44(6-67)

                        KPS

                         

                        ≥70

                        19

                        423

                        24

                        33

                        79

                        35

                        <70

                        1

                        10

                        0

                        1

                        2

                        2

                        Surgery

                         

                        Gross total resection

                        20

                        412

                        22

                        32

                        76

                        36

                        Partial resection

                        0

                        10

                        2

                        1

                        3

                        1

                        Biopsy

                        0

                        11

                        0

                        1

                        2

                        0

                        Adjuvant treatment

                         

                        Radiotherapy

                        0

                        280

                        18

                        17

                        4

                        0

                        Chemotherapy

                        0

                        55

                        1

                        1

                        0

                        0

                        Radiotherapy and Chemotherapy combination

                        0

                        98

                        5

                        16

                        77

                        37

                        KPS Karnofsky performance score.

                        A total of 645 healthy unrelated individuals as the controls between June 2010 and August 2012 were recruited from the medical examination center at Tangdu Hospital, for genetic association research of human complex diseases, such as lung cancer, stomach cancer, and glioma. The median age was 45 years (age range 4–83). The detailed recruitment and exclusion criteria were used. Generally, subjects with chronic diseases and conditions involving vital organs (heart, lung, liver, kidney, and brain) and severe endocrinological, metabolic, and nutritional diseases were excluded from this study. The purpose of the above exclusion procedures was to minimize the known environmental and therapeutic factors that influence the variation of human complex diseases.

                        In our study population, all analyses were restricted to the Han Chinese living in Xi’an city and its surrounding areas. A written informed consent was obtained from all the subjects or their custodians, and we collected all the blood samples from the controls and the patients before chemotherapy or radiotherapy. All specimens were handled and made anonymous according to the ethical and legal standards. The protocol was approved by the Medical Ethics Committee of the Fourth Military Medical University.

                        Demographic and clinical data

                        Demographic and personal data were collected through an in-person interview using a standardized epidemiological questionnaire, including age, sex, ethnicity, residential region, smoking status, alcohol use, education status, and family history of cancer. For patients, detailed clinical information was collected through a medical chart review or consultation with treating physicians. Plasma carcinoembryonic antigen and alpha-fetoprotein were tested in control subjects to make sure they did not have any cancers.

                        Blood samples collection, DNA extraction and SNP selection and genotyping

                        Peripheral blood was taken from the 629 glioma patients and 645 apparently healthy individuals, and from the elbow vein or the head superficial vein, and treated immediately with an anticoagulant containing sodium citrate (22 g/L) and sodium chloride (8.5 g/L). The blood samples were then stored at -70˚C before use. Genomic DNA was isolated from the samples by using an extraction kit (GoldMag, China). DNA concentration and purity were determined by an ultraviolet spectrophotometer (Eppendorf, Hamburg, Germany).

                        Candidate tSNPs in the seven genes were selected from previously published polymorphisms associated with glioma. Validated tSNPs were selected with a minor allele frequency (MAF) > 5% in the HapMap Asian population. A total of 10 tSNPs were selected for further genotyping. Genomic DNA was extracted from whole blood using the phenol-chloroform extraction method. DNA concentration was measured by spectrometry (DU530 UV/VIS spectrophotometer, Beckman Instruments, Fullerton, CA, USA). A multiplexed SNP MassEXTEND assay was designed with the Sequenom MassARRAY Assay Design 3.0 Software [11]. SNP genotyping was performed using the Sequenom MassARRAY RS1000 with standard protocol recommended by the manufacturer [11]. Data management and analyses were performed using Sequenom Typer 4.0 software as previously described [11, 12].

                        Statistical analysis

                        Statistical analyses were performed using Microsoft Excel and SPSS 16.0 statistical packages (SPSS, Chicago, IL). All P values in this study were two-sided. A P ≤ 0.05 was considered the threshold of statistical significance. Genotypic frequencies in control subjects for each tSNP were tested for departure from Hardy–Weinberg equilibrium (HWE) using an exact test. Genotype frequencies and allele frequencies of glioma patients and control subjects were compared using the χ 2 test [13]. Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated by unconditional logistic regression analysis with adjustment for age and sex [14]. We did not divide subjects into subgroups because of limited sample size. The possibility of sex differences as a source of population sub-structure was evaluated by a genotype test for each tSNP in male and female controls, and the number of significant results at the 5% level was compared with the number expected by the χ 2 test. We did not detect population stratification because all participants’ ethnicity was Han Chinese. The four genetic models (dominant, recessive, additive and genotypic) were applied by PLINK software (http://​pngu.​mgh.​harvard.​edu/​purcell/​plink/​) to assess the association of single tSNP with the risk of glioma. ORs and 95% CIs were calculated by unconditional logistic regression analysis adjusted for age and sex [14, 15].

                        We used the Haploview software package (version 4.2) and SHEsis software platform (http://​www.​nhgg.​org/​analysis/​) for analyses of linkage disequilibrium, haplotype construction, and genetic association at polymorphism loci [16, 17] ORs and 95% CIs were calculated by unconditional logistic regression analysis with adjustment for age and gender [14]. Additionally, the likelihood ratio test was performed to determine the genotype frequencies among various grade groups. The χ2 test was also used for comparison of categorical variables. A P value of <0.05 (two-tailed) was considered statistically significant.

                        Results

                        A total of 629 cases (310 male, 319 female; median age at diagnosis 41 ± 18 yrs) and 645 controls (329 male, 316 female; median age at 45 ± 12 yrs) were included in the current study. Basic characteristics of the cases are listed in Table 1 including gender, age, and pathology. As listed in Table 2, a multiplexed SNP MassEXTEND assay was designed with Sequenom MassARRAY Assay Design 3.0 Software. Ten SNPs of seven candidate genes were genotyped in glioma patients and the control group, the average tSNPs call rate was 99.6% in cases and controls, and all of the tested tSNPs are in Hardy–Weinberg equilibrium (HWE) in the control population of this study (Table 3). Two significant tSNPs in the RTEL1 gene were observed to be associated with glioma risk at a 5% level (rs6010620, P = 0.0016, OR: 1.32, 95% CI: 1.11-1.56; rs2297440, P = 0.001, OR: 1.33, 95% CI: 1.12-1.58) by χ 2 test.
                        Table 2

                        Primers used in the study

                        SNP_ID

                        1st-PCR primer sequences

                        2nd-PCR primer sequences

                        UEP sequences

                        rs2992

                        G/A

                        ACGTTGGATGTCAAGTATCTGCTCTGTGGG

                        ACGTTGGATGACTGGGTGCATCCTGAGAG

                        cgatAGCAGGGGTGACGTATGTAGAA

                        rs12022378

                        C/T

                        ACGTTGGATGAGATGCCTGGACCAGCTCT

                        ACGTTGGATGCAATTACAGCCCACCTCTTG

                        cctaaAGCCCACCTCTTGCATCGT

                        rs12917

                        T/C

                        ACGTTGGATGCGAGGCTATCGAAGAGTTCC

                        ACGTTGGATGAGATGGCTTAGTTACCGACC

                        gctaGAAAACGGGATGGTGAA

                        rs12645561

                        T/C

                        ACGTTGGATGTTACAGTTCTCTTTCACAG

                        ACGTTGGATGGCAGAGCCTAGTTTCATGAC

                        TTGCTCATTACTGTAAGAAATAATAC

                        rs7003908

                        C/A

                        ACGTTGGATGGGGGAGAAAATATTCCTGTT

                        ACGTTGGATGTCCTACCTCACGAACTCAGC

                        AGCAATTGCCTAAGAGTC

                        rs6010620

                        G/A

                        ACGTTGGATGGCCTGTTTTCCCTTTTTGAG

                        ACGTTGGATGCCTCTCAACATCTCAGCAAC

                        tGATCATGCAAAGCAGG

                        rs2297440

                        T/C

                        ACGTTGGATGACGAGGTCTGGTGGCACAT

                        ACGTTGGATGCACTGTCCTTTGCGTCCTC

                        gtTCCTCCCTCACCAGC

                        rs4809324

                        C/T

                        ACGTTGGATGGAGAAGTCAAGTGACATCAG

                        ACGTTGGATGAGCCGGTGCACAGATTCCAA

                        gagggCAAGGGCCTGGAATCTGT

                        rs3770502

                        A/G

                        ACGTTGGATGCTATATGGGTGCAGATGCAG

                        ACGTTGGATGACAGGCGTGAACCACTGTA

                        ACCCGGCCCCTCCAC

                        rs9288516

                        A/T

                        ACGTTGGATGACAGGCCAAGGGCAATAATC

                        ACGTTGGATGGCTTCCTAAGATTTCCTATTC

                        CATTTCAAAAGAAATGGAGAAT

                        UEP Unextended mini-sequencing primer.

                        Table 3

                        Tagging SNPs information that were examined

                        SNP No.

                        Gene

                        chr

                        Position

                        MA

                        MAF (CHB)

                        MAF

                        HWE P

                        P

                        OR (95%CI)

                        P adj.

                        Genotype rate

                              

                        Case

                        Control

                             

                        rs12022378

                        AP4B1

                        chr1

                        114448389

                        C

                        0.411

                        0.360

                        0.393

                        0.4943

                        0.0889

                        1.15(0.98-1.35)

                        0.889

                        99.34

                        rs12917

                        MGMT

                        chr10

                        131506283

                        T

                        0.31

                        0.105

                        0.082

                        0.2082

                        0.0497

                        0.76(0.58-1)

                        0.497

                        99.67

                        rs12645561

                        NEIL3

                        chr4

                        178260872

                        T

                        0.114

                        0.275

                        0.268

                        0.1388

                        0.6903

                        0.97(0.81-1.15)

                        6.903

                        99.34

                        rs7003908

                        PRKDC

                        chr8

                        48770702

                        C

                        0.283

                        0.236

                        0.209

                        0.1246

                        0.1074

                        0.86(0.71-1.03)

                        1.074

                        99.83

                        rs6010620

                        RTEL1

                        chr20

                        62309839

                        G

                        0.393

                        0.269

                        0.327

                        0.194

                        0.0016

                        1.32(1.11-1.56)

                        0.016

                        99.83

                        rs2297440

                        RTEL1

                        chr20

                        62312299

                        C

                        0.226

                        0.266

                        0.325

                        0.3127

                        0.001

                        1.33(1.12-1.58)

                        0.01

                        100

                        rs4809324

                        RTEL1

                        chr20

                        62318220

                        C

                        0.12

                        0.114

                        0.120

                        0.6994

                        0.6328

                        1.06(0.83-1.35)

                        6.328

                        99.67

                        rs2992

                        UBXN6

                        chr19

                        4443046

                        A

                        0.295

                        0.436

                        0.433

                        0.4229

                        0.8701

                        0.99(0.84-1.16)

                        8.701

                        99.67

                        rs3770502

                        XRCC5

                        chr2

                        217045059

                        A

                        0.208

                        0.148

                        0.160

                        0.7558

                        0.4221

                        1.09(0.88-1.36)

                        4.221

                        99.83

                        rs9288516

                        XRCC5

                        chr2

                        217053264

                        A

                        0.489

                        0.480

                        0.450

                        0.4774

                        0.1288

                        0.89(0.76-1.04)

                        1.288

                        99.67

                        Association results between tSNP genotypes and the risk of glioma were listed in Table 4. We found that the genotype “GG” of rs6010620 as the protective genotype for glioma (OR, 0.46; 95% CI, 0.31-0.7; P = 0.0002), and the genotype “CC” of rs2297440 as the protective genotype in glioma (OR, 0.47; 95% CI, 0.31-0.71; P = 0.0003).
                        Table 4

                        Association between tSNP genotypes and the risk of glioma

                        SNP ID

                        Genotype

                        No. (frequency)

                        Logistic regression

                        Case

                        Control

                        OR (95% CI)

                        P value

                        rs12022378

                        CC

                        114(18.2)

                        87(13.6)

                        0.71(0.51-0.99)

                        0.0418

                        rs12022378

                        CT

                        265(42.2)

                        288(44.9)

                        1.01(0.8-1.29)

                        0.9127

                        rs12022378

                        TT

                        249(39.6)

                        267(41.6)

                        1(referent)

                         

                        rs12917

                        TT

                        6(1)

                        10(1.6)

                        1.7(0.61-4.72)

                        0.3011

                        rs12917

                        TC

                        91(14.5)

                        115(17.9)

                        1.29(0.96-1.74)

                        0.0956

                        rs12917

                        CC

                        530(84.5)

                        519(80.6)

                        1(referent)

                         

                        rs12645561

                        TT

                        43(6.8)

                        56(8.7)

                        1.26(0.83-1.93)

                        0.2784

                        rs12645561

                        TC

                        251(40)

                        241(37.6)

                        0.93(0.74-1.18)

                        0.5537

                        rs12645561

                        CC

                        334(53.2)

                        344(53.7)

                        1(referent)

                         

                        rs7003908

                        CC

                        31(4.9)

                        43(6.7)

                        1.44(0.89-2.33)

                        0.1387

                        rs7003908

                        CA

                        201(32)

                        217(33.7)

                        1.12(0.88-1.42)

                        0.3536

                        rs7003908

                        AA

                        397(63.1)

                        383(59.6)

                        1(referent)

                         

                        rs6010620

                        GG

                        75(11.9)

                        40(6.2)

                        0.46(0.31-0.7)

                        0.0002

                        rs6010620

                        GA

                        261(41.5)

                        267(41.5)

                        0.89(0.71-1.12)

                        0.3212

                        rs6010620

                        AA

                        293(46.6)

                        337(52.3)

                        1(referent)

                         

                        rs2297440

                        CC

                        73(11.6)

                        40(6.2)

                        0.47(0.31-0.71)

                        0.0003

                        rs2297440

                        CT

                        263(41.8)

                        263(40.8)

                        0.86(0.68-1.08)

                        0.1902

                        rs2297440

                        TT

                        293(46.6)

                        342(53)

                        1(referent)

                         

                        rs4809324

                        CC

                        11(1.8)

                        7(1.1)

                        0.62(0.24-1.6)

                        0.3162

                        rs4809324

                        CT

                        129(20.5)

                        133(20.7)

                        1(0.76-1.31)

                        0.9901

                        rs4809324

                        TT

                        488(77.7)

                        504(78.3)

                        1(referent)

                         

                        rs2992

                        AA

                        126(20.1)

                        117(18.2)

                        0.98(0.71-1.35)

                        0.9003

                        rs2992

                        AG

                        292(46.5)

                        328(50.9)

                        1.19(0.92-1.52)

                        0.1822

                        rs2992

                        GG

                        210(33.4)

                        199(30.9)

                        1(referent)

                         

                        rs3770502

                        AA

                        12(1.9)

                        15(2.3)

                        1.18(0.54-2.54)

                        0.6809

                        rs3770502

                        AG

                        177(28.1)

                        161(25)

                        0.86(0.67-1.1)

                        0.2198

                        rs3770502

                        GG

                        440(70)

                        468(72.7)

                        1(referent)

                         

                        rs9288516

                        AA

                        128(20.4)

                        153(23.8)

                        1.28(0.93-1.74)

                        0.1249

                        rs9288516

                        AT

                        308(49.1)

                        312(48.4)

                        1.08(0.84-1.4)

                        0.5539

                        rs9288516

                        TT

                        191(30.5)

                        179(27.8)

                        1(referent)

                         

                        OR odd ratio, CI confidence interval.

                        We assumed that the minor allele of each tSNP was a risk allele compared to the wild type allele. Four tSNPs were detected to be associated with glioma by model association analyses including rs6010620 and rs2297440 in the RTEL1 gene, rs12022378 in the DCLRE1B gene, and rs12917 in the MGMT gene (Table 5). We observed two tSNPs in RTEL1 gene to be associated with the risk of glioma by recessive model (rs6010620, OR, 2.09; 95% CI, 1.39-3.13; P = 0.0004, and rs2297440, OR, 2.02; 95% CI, 1.35-3.04; P = 0.0007). Rs12022378 in the DCLRE1B gene was also found by recessive model associated with glioma risk (OR, 1.42; 95% CI, 1.05-1.93; P = 0.0246). Rs6010620 and rs2297440 were also detected by Dominant Model with increased risk of glioma (rs6010620, OR, 1.26; 95% CI, 1.01-1.57; P = 0.041, and rs2297440, OR, 1.3; 95% CI, 1.04-1.62; P = 0.022). Another SNP, rs12917 in the MGMT gene, was associated with decreased glioma risk by recessive model analysis (OR, 0.73; 95% CI, 0.54-0.98; P = 0.036). Rs6010620, rs2297440 and rs12917 were also found to be associated with glioma risk by additive model analyses (rs6010620, OR, 1.32; 95% CI, 1.11-1.57; P = 0.0015, rs2297440, OR, 1.34; 95% CI, 1.12-1.59; P = 0.001 and rs12917, OR, 0.75; 95% CI, 0.58-0.99; P = 0.038). Genotypic model analyses results shown that three tSNPs were significant to be associated with glioma risk (rs6010620, OR, 1.48; 95% CI, 1. 2-1.83; P = 0.0002, rs2297440, OR, 1.47; 95% CI, 1.19-1.82; P = 0.000, and rs12022378, OR, 1.19; 95% CI, 1.0-1.4; P = 0.043).
                        Table 5

                        Association between tSNPs and the risk of glioma and their heterozygote and homozygote odds ratios, per allele odds ratios and confidence intervals

                        SNP No.

                        Dominant model

                        Recessive model

                        Additive model

                        Genotypic model

                        OR

                        95% CI

                        P-value

                        OR

                        95% CI

                        P-value

                        OR

                        95% CI

                        P value

                        OR

                        95% CI

                        P value

                        rs12022378

                        1.08

                        0.86

                        1.36

                        0.4966

                        1.42

                        1.05

                        1.93

                        0.0246

                        1.14

                        0.97

                        1.33

                        0.1031

                        1.19

                        1.01

                        1.40

                        0.0426

                        rs2992

                        0.90

                        0.71

                        1.14

                        0.3963

                        1.14

                        0.86

                        1.51

                        0.3714

                        0.99

                        0.85

                        1.16

                        0.9496

                        1.02

                        0.87

                        1.19

                        0.8340

                        rs12917

                        0.73

                        0.54

                        0.98

                        0.0359

                        0.70

                        0.25

                        1.94

                        0.4882

                        0.75

                        0.58

                        0.98

                        0.0383

                        0.81

                        0.49

                        1.36

                        0.4362

                        rs12645561

                        1.02

                        0.82

                        1.28

                        0.8315

                        0.74

                        0.49

                        1.12

                        0.1499

                        0.96

                        0.81

                        1.15

                        0.6584

                        0.87

                        0.70

                        1.08

                        0.2142

                        rs7003908

                        0.87

                        0.69

                        1.10

                        0.2372

                        0.74

                        0.46

                        1.19

                        0.2102

                        0.87

                        0.73

                        1.05

                        0.1492

                        0.84

                        0.66

                        1.08

                        0.1684

                        rs6010620

                        1.26

                        1.01

                        1.57

                        0.0410

                        2.09

                        1.39

                        3.13

                        0.0004

                        1.32

                        1.11

                        1.57

                        0.0015

                        1.48

                        1.20

                        1.83

                        0.0002

                        rs2297440

                        1.30

                        1.04

                        1.62

                        0.0221

                        2.02

                        1.35

                        3.04

                        0.0007

                        1.34

                        1.12

                        1.59

                        0.0010

                        1.47

                        1.19

                        1.82

                        0.0003

                        rs4809324

                        1.03

                        0.79

                        1.34

                        0.8386

                        1.76

                        0.67

                        4.60

                        0.2490

                        1.06

                        0.83

                        1.36

                        0.6257

                        1.33

                        0.82

                        2.14

                        0.2513

                        rs3770502

                        1.16

                        0.91

                        1.48

                        0.2314

                        0.79

                        0.37

                        1.72

                        0.5557

                        1.11

                        0.89

                        1.38

                        0.3668

                        0.91

                        0.62

                        1.34

                        0.6410

                        rs9288516

                        0.90

                        0.70

                        1.14

                        0.3803

                        0.84

                        0.64

                        1.10

                        0.1972

                        0.90

                        0.77

                        1.05

                        0.1896

                        0.90

                        0.77

                        1.05

                        0.1789

                        OR odd ratio, CI confidence interval.

                        Only one block was detected in RTEL1 gene by haplotype analysis. Global result for the block was: total case = 1286, total control =1256, global χ 2 = 13.0855 while df = 2, Fisher’s P value = 0.0015, and Pearson’s P value = 0.0014. The results of the association between the RTEL1 gene haplotypes and the risk of glioma are listed in Table 6. Haplotype “GCT” in RTEL1 gene was found to be associated with risk of glioma (OR, 0.7; 95% CI, 0.57-0.86; Fisher’s P = 0.0005; Pearson’s P = 0.0005). Haplotype “ATT” was found to be associated with risk of glioma (OR, 1.32; 95% CI, 1.12-1.57; Fisher’s P = 0.0013; Pearson’s P = 0.0013).
                        Table 6

                        Haplotype frequencies of RTEL1 gene and association with risk of glioma in cases and controls

                        Haplotype

                        Freq(case)

                        Freq(ctrl)

                        Chi2

                        Fisher’s P

                        Pearson’s P

                        Odds ratio

                        [95% CI]

                        A T T

                        0.7294

                        0.6728

                        10.3722

                        0.0013

                        0.0013

                        1.32

                        [1.12,1.57]

                        G C C

                        0.1135

                        0.1194

                        0.1959

                        0.6581

                        0.6581

                        0.95

                        [0.74,1.21]

                        G C T

                        0.1524

                        0.2054

                        11.9905

                        0.0005

                        0.0005

                        0.70

                        [0.57,0.86]

                        Note: Loci chosen for hap-analysis: rs6010620, rs2297440 and rs4809324 (RTEL1); OR odd ratio, CI confidence interval.

                        Furthermore, the associations between different clinicopatholiogical features and genotype frequency of GG in rs6010620, together with CC in rs2297440 in glioma patients (n = 75, 73, respectively) were analyzed. GG frequency in various grade goups were determined, being 16.0% (12/75), 46.7% (35/75), 16.0% (12/75), and 21.3% (16/75), respectively, in grade I,II,III, and IV group (P > 0.05), and CC frequency in various grade goups were determined as 14.7% (11/73), 56.2% (35/73), 17.8% (13/73), and 19.2% (14/73), respectively, in grade I, II, III, and IV group (P >0.05) (Table 7). No significant association was found between genotype frequency of GG or CC, and other parameters including WHO grading, gender, age at diagnosis, or Karnofsky performance score (KPS).
                        Table 7

                        Associations between different clinicopatholiogical features and genotype frequency of GG in rs6010620, and CC in rs2297440 of RTEL1 gene in glioma patients ( n = 75, 73, respectively)

                        Clinicopatholiogical features

                        GG frequency

                        P value

                        CC frequency

                        P value

                         

                        n (%)

                         

                        n (%)

                         

                        WHO grade

                         

                        I

                        12 (16.0)

                        >0.05

                        11 (14.7)

                        >0.05

                        II

                        35 (46.7)

                         

                        35 (56.2)

                         

                        III

                        12 (16.0)

                         

                        13 (17.8)

                         

                        IV

                        16 (21.3)

                         

                        14 (19.2)

                         

                        Age

                         

                        ≥40

                        36 (48.0)

                        NS

                        40 (54.8)

                        NS

                        <40

                        39 (52.0)

                         

                        33 (45.2)

                         

                        Gender

                         

                        Male

                        30 (40.0)

                        NS

                        31 (42.5)

                        NS

                        Female

                        45 (60.0)

                         

                        42 (57.5)

                         

                        KPS

                         

                        ≥70

                        72 (96.0)

                        >0.05

                        70 (95.9)

                        >0.05

                        <70

                        3 (4.0)

                         

                        3 (4.1)

                         

                        NS not significant, KPS Karnofsky performance score.

                        Discussion

                        As known, biomarker detection and screening is an emerging field for oncology [1823]. Especially for gliomas considerable progresses have been made in identifying, characterizing, and attempting to apply molecular markers, e.g. in the previous study, we initially found the increased expression of miR-372 in glioma tissues was significantly correlated with advanced tumor progression and aggressive clinicopathological features [24]. And subsequently a series of have determined the associations between lots of SNPs in ABCB 1, NR 1/2, VEGFR 3, etc. and therapy outcome [2527].

                        In this case–control study in Han Chinese population, we found two susceptibility tSNPs in RTEL1 gene that were associated with increased risk of glioma (rs6010620 and rs2297440). The genotype “GG” of rs6010620 was the protective genotype for glioma, and the genotype “CC” of rs2297440 was the protective genotype in glioma. We also observed in the RTEL1 gene a haplotype “GCT” that was associated with a decreased the risk and a haplotype of “ATT” with an increased risk of developing glioma.

                        As described initially, however, we failed to determine the associations between genotype frequency of GG or CC, and other parameters including WHO grading, gender, age at diagnosis, or KPS status. Additionally, we also tried to elucidate the relationship of genotype frequency of GG or CC, with overall surviaval (OS) in the correlated patients. During the follow-up period, only 11 of the patients with genotype of GG or CC had died [6 (8%) from the 75 patients with genotype GG, and 5 (6.8%) from the 73 patients with genotype CC], and most of the patients are alive and are being traced continuously.

                        Albeit the correlated survival data in the present study are still accumulating, our findings in this study have provided new evidence for the association between common SNPs (or haplotypes) and the risk of glioma in the Chinese Han population, suggesting an important determinant of glioma development by RTEL1 gene. RTEL1 gene locates in 20q13.3 with the length of 40.889 kb, including 40 exons. Known functions of RTEL1 include nucleic acid binding, ATP-dependent DNA helicase activity, DNA repair, apoptosis and anti-apoptosis, and so on. Previous study proposed that RTEL1 maintains genomic stability by suppressing homologous recombination [28, 29], and implements the second level of meiotic crossover control by promoting non-crossovers [30, 31]. A recent review point out that RTEL1 was an essential helicase for telomere maintenance and the regulation of homologous recombination [32, 33]. RTEL1 didn’t involve any KEGG pathway (http://​www.​genome.​jp/​kegg/​) so far. The frequencies of rs6010620 risk genotypes were highly correlated with high-grade disease (P < 0.001), indicating that genetic variations at the locus has subtype-specific effects on the risk of developing glioma [34]. RTEL1 gene was over expressed in human gastrointestinal tract tumors [35]. Polymorphism in the RTEL1 gene was associated with glioblastoma survival [36].

                        Some limitations are inherent in this case–control study and must be noted here. Glioma patients were not sub-grouped by age or gender, and gender-specific significant variants were not tested. We selected tSNPs with frequencies higher than 5% in HapMap Asian populations to affirm the statistical power was large enough for analyzing data. We also designed a haplotype-based study to ensure sufficiently high power to detect the risk of glioma associated with candidate tSNPs. Another potential concern was population admixture, which is a known confounding factor for association analysis and may also result in inflated type-I error (false positive). In this study, glioma patients and controls were used in the same hospital to avoid the possibility that one may have a more pronounced selection bias. However, this bias is unlikely to be of significance because they did not differ in the distributions of demographic variable and genotype frequencies. We limited all subjects’ ethnicity to Han Chinese, and a living area to Xi’an City and its surrounding area, thus there is no substantial population admixture in our study populations.

                        In the upcoming studies, our team will go on to follow up the subjects recruited into the present study, and carry out additional research with larger subject numbers and grade types to further characterize the relationship among grades within the individuals, clinical features and the mentioned RTEL1 tagging SNPs & haplotypes. Furthermore, to elucidate the role of the RTEL1 gene in gliomagenesis, serum RTEL1 expression levels between different mutations or haplotype groups will be compared. And, we will also investigate the association between germline RTEL1 variants and somatic RTEL1 mutations, and the relationship between serum RTEL1 expression and somatic RTEL1 expression in the same glioma subjects.

                        Conclusion

                        In conclusion, our comprehensive analysis of tSNPs suggests that the genotypes of “GG” of rs6010620 and “CC” of rs2297440 (rs6010620 and rs2297440) in the RTEL1 gene, together with two haplotypes of GCT and ATT, were identified to be associated with glioma development. And it might be used to evaluate the glioma development risks to screen the above RTEL1 tagging SNPs and haplotypes.

                        Declarations

                        Acknowledgments

                        This work is supported by grants from the National Natural Science Foundation of China (No. 81272776), China Postdoctoral Science Foundation funded projects (No. 20100471628 and No. 201104634), Wu Jieping Medical Foundation funded Project (320.6750.12161), Shaanxi Province Programs for Science and Technology Development (No. 2012 K 13-01-13 and 2011 K12-47) and the Talents Program 2010, Tangdu hospital, the Fourth Military Medical University. We are grateful to all the patients and individuals for their participation. We would also like to thank the clinicians and other hospital staff who contributed to the blood sample and data collection for this study.

                        Authors’ Affiliations

                        (1)
                        Department of Neurosurgery, Tangdu hospital, the Fourth Military Medical University
                        (2)
                        National Engineering Research Center for Miniaturized Detection Systems, School of Life Sciences, Northwest University
                        (3)
                        Department of Clinical Experimental Surgery, Tangdu hospital, the Fourth Military Medical University
                        (4)
                        Shaanxi Lifegen Co. Ltd
                        (5)
                        Department of Radiology, Tangdu hospital, the Fourth Military Medical University

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                        © Li et al.; licensee BioMed Central Ltd. 2013

                        This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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