RESEARCH ARTICLE

Association of RECQL5 gene polymorphismsand osteosarcoma in a Chinese Han population

Li-Qiang Zhi & Wei Ma & Hong Zhang & Si-Xiang Zeng &

Bo Chen

Received: 22 October 2013 /Accepted: 13 November 2013# International Society of Oncology and BioMarkers (ISOBM) 2013

Abstract Despite the knowledge on many genetic variantspresent in osteosarcoma, the complexity of this diseaseprecludes placing its biology into a simple conceptualframework. RECQL is a DNA helicase involved in DNAmismatch repair and has been reported to be associatedwith many human cancers. We aimed to investigate theassociation of RECQL genetic polymorphism with osteo-sarcoma in a Chinese population. We selected three poly-morphisms of the RECQL5 gene (rs820196, rs820200,and rs4789223) in the present study. TaqMan methodwas utilized for genotyping these three SNPs in 212 pa-tients with osteosarcoma and 240 age- and sex-matchednoncancer controls. In our study, we found that CC geno-type in rs820196 (17.5 vs 8.3 %, P =0.005) and AAgenotype in rs4789223 (21.7 vs 14.2, P <0.001) weremore frequent in osteosarcoma group compared to thecontrol group, respectively. We also found that the C alleleof rs820196 (OR=1.492, 95 % CI 1.138∼1.951; P =0.004)and A allele of rs4789223 (OR=1.767, 95 % CI: 1.354∼2.301; P <0.001) were common in the osteosarcoma pa-tients than those in the control subjects, respectively. Hap-lotype analysis showed that TTA (OR=3.469, 95 % CI1.798∼6.695; P <0.001) was associated with increased

risk for osteosarcoma. However, the TTG (OR=0.578,95 % CI 0.442∼0.756) was associated with decreased riskfor osteosarcoma. Our results suggested that RECQL5genetic polymorphisms were associated with osteosarco-ma in a Chinese population.

Keywords RECQL5 . Osteosarcoma . Polymorphism .

Haplotype

Introduction

Osteosarcoma (OS) is the most common pediatric bonemalignancy in the world [1–3]. The exact mechanism ofthe development of OS carcinogenesis still remainspoorly understood. Patients with localized OS at presen-tation have a 60–80 % rate of long-term survival, whilemetastatic disease carries a poorer prognosis [4, 5].Cumulative evidences suggest that OS is a complexdisease resulting from the interaction between environ-mental factors and genetics [6–10]. However, severalpublished literature suggested that the genetic factorscould play key roles in the pathogenesis of OS [11–15].

It is suggested that DNA damage may lead to genomicinstability, cancer transformation, or cell death [16–18].The RECQ family is a highly conserved group of DNAhelicases required for the maintenance of genome stabilityand integrity. They play important roles in DNA replica-tion and repair pathways including mismatch repair, nu-cleotide excision repair, and direct repair [19, 20]. There-fore, the RECQL5 is considered a candidate gene forcancer. Several studies suggested that RECQL familygene mutations were associated with human cancers [19,21–23].

RECQL5 is one of the members of RECQL family. Therelation between the polymorphism and osteosarcoma is not

Wei Ma and Hong Zhangcontributed equally to this work.Si-Xiang Zeng and Bo Chen contributed equally to this work.

L.<Q. Zhi :W. Ma (*) : S.<X. ZengDepartment of Orthopedics, The First Affiliated Hospital of Xi’anJiaotong University, No. 227, YanTa West Rd, Xi’an 710061, Chinae-mail: zhiliqiang2011@yeah.net

H. ZhangMedical Research Center of Shaanxi University of ChineseMedicine, Xi’an, China

B. ChenDepartment of Orthopedics, Xijing Hospital, The Fourth MilitaryMedical University, Xi’an, China

Tumor Biol.DOI 10.1007/s13277-013-1425-4

understood. In the present study, we aimed to investigate theassociation of RECQL5 gene polymorphisms with OS in aChinese population.

Material and methods

Study population

A total of 212 diagnosed OS patients (aged 8–58 yearsold) and 240 healthy subjects (aged 12–60 years old) whohad no history of OS and other cancer as controls wererecruited from the First Affiliated Hospital of Xi anJiaotong University, the Second Affiliated Hospital of Xian Jiaotong University, the First Affiliated Hospital ofShaanxi University of Chinese Medicine, and the ForthMilitary Medical University between March 2008 andMarch 2013. All subjects were unrelated Chinese withHan ethnicities. The cancer-free controls were matched

with OS patients with regards to gender, age, and resi-dence area. The general characteristics of the participantswere shown in Table 1. The written informed consent wasobtained from each participant. The protocol of this studywas approved by the Ethics Committee of the First Affil-iated Hospital of Xi’an Jiaotong University.

Genotyping

Using the Haploview 4.2 software and the HapMap phases Iand II database, we obtained three tagging SNPs (rs820196,rs820200, and rs4789223) for Han Chinese with the minorallele frequency (MAF) ≥0.15 and r2≥0.8. Genomic DNAwas extracted from the peripheral blood leukocytes by using aDNA Extraction Kit (Beijing Biotech Co. Ltd). Genotypingwas confirmed by TaqMan method.

Statistical analyses

The chi-squared (χ2) test was utilized to assess the Hardy–Weinberg equilibrium (HWE) in allele and genotype dis-tribution, and general characteristics between OS patientsand cancer-free controls. The odds ratios (ORs) and 95 %confidence intervals (95 % CIs) were calculated by uncon-ditional logistic regression and utilized to evaluate thepotential associations between RECQL5 genetic variantsand OS risk. All statistical analyses were analyzed by theStatistical Package for Social Sciences software (SPSS,Windows version release 15.0; SPSS Inc., Chicago, IL,USA). Based on the genotype data of the genetic varia-tions, we performed linkage disequilibrium (LD) analysisand haplotype-based case–control analysis, using theSHEsis software (http://analysis2.bio-x.cn/myAnalysis.php)

Table 1 The clinical characteristics of OS and controls

Characteristics Cases Controls P value

Number 212 240

Gender (male) (n , %) 121 (82.0) 130 (81.3) 0.765

Age (years) 42.3±12.4 43.1±13.1 0.446

Tumor location (n , %)

Long tubular bones 161 (75.9) –

Axial skeleton 61 (24.1) –

Metastasis (n , %)

Yes 84 (39.6) –

No 128 (60.4) –

Table 2 Genotype distribution of RECQL5 SNPs between cases and controls

SNPs Genotype and allele Osteosarcoma (n =212) Control (n =240) OR (95 % CI) P value

rs820196 CC 37 (0.175) 20 (0.083) 0.005CT 109 (0.514) 122 (0.508)

TT 66 (0.311) 98 (0.408)

C 183 (0.432) 162 (0.338) 1.492 (1.138∼1.951) 0.004T 241 (0.568) 162 (0.338)

rs820200 GG 42 (0.198) 39 (0.163) 0.591GT 114 (0.538) 132 (0.550)

TT 56 (0.264) 69 (0.287)

G 198 (0.467) 210 (0.438) 1.126 (0.866∼1.464) 0.374T 226 (0.533) 270 (0.562)

rs4789223 AA 46 (0.217) 34 (0.142) <0.001AG 128 (0.604) 114 (0.475)

GG 38 (0.179) 92 (0.383)

A 220 (0.519) 182 (0.379) 1.767 (1.354∼2.301) <0.001G 204 (0.481) 298 (0.621)

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[24, 25]. In the haplotype-based case–control analysis,haplotypes with a frequency of <0.02 were excluded.Statistical significance was established at P <0.05.

Results

General characteristics of subjects

In this study, we enrolled 212 OS patients and 240 healthycontrol subjects. The general characteristics of OS patientsand cancer-free controls were shown in Table 1. There are nosignificant differences between OS patients and cancer-freecontrols with regard to gender, age, tumor location, andmetastasis.

The genotype distribution of each SNP did not show sig-nificant differences from the Hardy–Weinberg equilibriumvalues (data not shown). As shown in Table 2, CC genotypein rs820196 (17.5 vs 8.3 %, P=0.005) and AA genotype inrs4789223 (21.7 vs 14.2, P <0.001) were more frequent in theOS group compared to the control group, respectively. Wealso found that the C allele of rs820196 (OR=1.492, 95 % CI1.138∼1.951; P=0.004) and A allele of rs4789223 (OR=1.767, 95 % CI 1.354∼2.301; P <0.001) were common inthe OS patients than that in the control subjects, respectively.

Figure 1 shows the patterns of LD in the RECQL5 gene,with their |D ′| and r2 values. All these three SNPs are locatedin one haplotype block. In the haplotype-based case–controlanalysis, haplotypes were established using these three SNPs.As shown in Table 3, haplotype analysis showed that TTA

Fig. 1 Linkage disequilibrium (LD) blocks across the locus. LD value is shown: a |D ′|×100; |D ′| color scheme: |D ′|=0: white; 0<|D ′|<1: shades of pink; |D ′|=1: red; b r2×100

Table 3 Distribution ofhaplotypes Haplotype Laryngeal cancer Control P value OR [95 % CI]

CGA 140.75 (0.332) 135.11 (0.281) 0.099 1.268 [0.955∼1.684]CTA 30.75 (0.073) 26.89 (0.056) 0.310 1.318 [0.772∼2.249]TGA 12.50 (0.029) 7.50 (0.016) 0.157 1.914 [0.767∼4.779]TGG 44.75 (0.106) 67.39 (0.140) 0.112 0.722 [0.483∼1.081]TTA 36.00 (0.085) 12.50 (0.026) <0.001 3.469 [1.798∼6.695]TTG 147.75 (0.348) 230.60 (0.480) <0.001 0.578 [0.442∼0.756]

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(OR=3.469, 95 % CI 1.798∼6.695; P <0.001) was associatedwith increased risk for OS. However, the TTG (OR=0.578,95 % CI 0.442∼0.756) was associated with decreased risk forOS.

Discussion

OS is a primary bone tumor and caused by complex interac-tions between environmental factors and genetic factors. Inthis study, we found that RECQL5 gene polymorphisms wereassociated with OS risk in a Chinese population. This is thefirst study to study on the relation between RECQL5 geneticpolymorphisms and OS.

RECQ helicase family members possess intrinsicDNA-dependent ATPase and ATP-dependent DNA-unwinding activity, with variable preferences for specificDNA structures. Human cells express five RECQ ho-mologues—RECQL1, BLM, WRN, RECQL4, andRECQL5. According to the previous studies, RECQL5plays a role in DNA replication, DNA repair, homolo-gous recombination, and RNA polymerase II (RNAPII)-mediated transcription [21, 22]. Recently, RECQL5polymorphism was reported to be associated with Telo-mere length and colon cancer [26]. However, the rela-tionship between RECQL5 polymorphism and OS hasnot been revealed in the past decades.

We performed a case–control study and genotypedthree SNPs in RECQL5 gene in a Chinese population.We found that the genotype distribution for rs820196and rs4789223 was significantly different between OSpatients and control subjects, which suggested that thesusceptibility of OS is increased in participants with themutant allele of rs820196 or rs4789223. In addition, wesuccessfully established haplotypes for the RECQL5gene from the different combinations of these threeSNPs. The frequency of the TTA was associated withincreased risk, but the TTG was associated with de-creased risk for OS, respectively.

There were several limitations in our study. Firstly,the sample size is relatively small, which may affect thepower in statistical analysis; secondly, we performedthis case–control study in one ethnic population anddid not replicate the results in another ethnic group;finally, we have not carried out a functional study tofurther reveal the mechanism of how the genetic poly-morphisms in RECQL5 affect the OS risk.

In conclusion, our findings indicate that OS is asso-ciated with the RECQL5 gene polymorphisms. The TTAhaplotype may be a useful genetic marker, and the TTGhaplotype might be a protective factor of OS in ChineseHan population.

References

1. Dorfman HD, Czerniak B. Bone cancers. Cancer. 1995;75:203–10.2. Mirabello L, Troisi RJ, Savage SA. Osteosarcoma incidence and

survival rates from 1973 to 2004: data from the Surveillance,Epidemiology, and End Results Program. Cancer. 2009;115:1531–43.

3. Hameed M, Dorfman H. Primary malignant bone tumors—recentdevelopments. Semin Diagn Pathol. 2011;28:86–101.

4. Wittig JC, Bickels J, Priebat D, Jelinek J, Kellar-Graney K,Shmookler B, et al. Osteosarcoma: a multidisciplinary approach todiagnosis and treatment. Am Fam Physician. 2002;65:1123–32.

5. Kager L, Zoubek A, Potschger U, et al. Primary metastatic osteosar-coma: presentation and outcome of patients treated on neoadjuvantCooperative Osteosarcoma Study Group protocols. J Clin Oncol.2003;21:2011–8.

6. Zhou Y, Liu B,WangM, Ni J. Endothelin-1 gene polymorphisms andrisk of chemoresistant pediatric osteosarcoma. Pediatr Blood Cancer.2013. doi:10.1002/pbc.24790.

7. Wang L, Liu Z, Jing P, Shao L, Chen L, He X, Gong W. Effects ofmurine double minute 2 polymorphisms on the risk and survival ofosteosarcoma: a systemic review and meta-analysis. Tumour Biol.2013. doi:10.1007/s13277-013-1227-8

8. He M, Wang Z, Zhao J, Chen Y, Wu Y. COL1A1 polymorphism isassociated with risks of osteosarcoma susceptibility and death.Tumour Biol. 2013. doi:10.1007/s13277-013-1172-6.

9. Wang J, Nong L, Wei Y, Qin S, Zhou Y, Tang Y. Association ofinterleukin-12 polymorphisms and serum IL-12p40 levels with oste-osarcoma risk. DNA Cell Biol. 2013;32:605–10. doi:10.1089/dna.2013.2098.

10. He J, Wang J, Wang D, Dai S, Yv T, Chen P, Ma R, Diao C, Lv G(2013) Association analysis between genetic variants ofMDM2 geneand osteosarcoma susceptibility in Chinese. Endocr J. (in press).

11. Salinas-Souza C, Petrilli AS, de Toledo SR. Glutathione S-transferasepolymorphisms in osteosarcoma patients. Pharmacogenet Genomics.2010;20:507–15.

12. Wang W, Wang J, Song H, Liu J, Song B, Cao X. Cytotoxic T-lymphocyte antigen-4 +49G/A polymorphism is associated withincreased risk of osteosarcoma. Genet Test Mol Biomarkers.2011;15:503–6.

13. Liu Y, He Z, Feng D, Shi G, Gao R, Wu X, et al. Cytotoxic T-lymphocyte antigen-4 polymorphisms and susceptibility to osteosar-coma. DNA Cell Biol. 2011;30:1051–5.

14. Liu Y, Lv B, He Z, Zhou Y, Han C, Shi G, et al. Lysyl oxidasepolymorphisms and susceptibility to osteosarcoma. PLoS One.2012;7:e41610.

15. Caronia D, Patino-Garcia A, Perez-Martinez A, et al. Effect ofABCB1 and ABCC3 polymorphisms on osteosarcoma survivalafter chemotherapy: a pharmacogenetic study. PLoS One.2011;6:e26091.

16. Futreal PA, Liu Q, Shattuck-Eidens D, et al. BRCA1 mutations inprimary breast and ovarian carcinomas. Science. 1994;266:120–2.

17. Khanna KK, Jackson SP. DNA double-strand breaks: signaling,repair and the cancer connection. Nat Genet. 2001;27:247–54.

18. O’Driscoll M, Jeggo PA. The role of double-strand break repair-insights from human genetics. Nat Rev Genet. 2006;7:45–54.

19. Sharma S, Doherty KM, Brosh RM. Mechanisms of RecQ helicasesin pathways of DNA metabolism and maintenance of genomic sta-bility. Biochem J. 2006;398:319–37.

20. Bohr VA. Rising from the RecQ-age: the role of human RecQhelicases in genome maintenance. Trends Biochem Sci. 2008;33:609–20.

21. Li D, Suzuki H, Liu B, Morris J, Liu J, Okazaki T, et al. DNA repairgene polymorphisms and risk of pancreatic cancer. Clin Cancer Res.2009;15:740–6. doi:10.1158/1078-0432.CCR-08-1607.

Tumor Biol.

22. Hu Y, Raynard S, Sehorn MG, Lu X, Bussen W, Zheng L,et al. RECQL5/Recql5 helicase regulates homologous recom-bination and suppresses tumor formation via disruption ofRad51 presynaptic filaments. Genes Dev. 2007;21:3073–84.

23. Raynard S, Bussen W, Sung P. A double Holliday junctiondissolvasome comprising BLM, topoisomerase IIIalpha, andBLAP75. J Biol Chem. 2006;281:13861–4.

24. Shi YY, He L. SHEsis, a powerful software platform foranalyses of linkage disequilibrium, haplotype construction,

and genetic association at polymorphism loci. Cell Res.2005;15(2):97–8.

25. Li Z, Zhang Z, He Z, et al. A partition-ligation-combination-subdivisionEM algorithm for haplotype inference with multiallelic markers: updateof the SHEsis. Cell Res. 2009;19:519–23 (http://analysis.bio-x.cn).

26. Pellatt AJ, Wolff RK, Lundgreen A, Cawthon R, Slattery ML.Genetic and lifestyle influence on telomere length and subsequentrisk of colon cancer in a case control study. Int J Mol EpidemiolGenet. 2012;3:184–94.

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