Evaluation of the association of ERCC2 rs13181 polymorphism with different types of gliomas in patients in Northeast Brazil

Gliomas are the most common primary tumors of the central nervous system with unclear etiology. However, hereditary factors may play an important role in glioma development, with mutations and single nucleotide polymorphisms (SNPs) being prominent among the genetic changes. This study aimed to evaluate the association of the ERCC2 gene rs13181 variant polymorphism between high- and low-grade gliomas in patients from Brazil’s Northeast region. Samples from glioma patients stored in paran blocks were used. DNA extraction was performed using the MagMAX™ FFPE DNA/RNA Ultra kit, and for genotyping, the Taqman assay probe C_3145033_10 corresponding to the SNP rs13181 of the ERCC2 gene was selected. Quantitative and categorical variables were analyzed using the t-test and Fisher’s exact test or Chi-square test (p < 0.05). Patients with low-grade gliomas were younger than those with high-grade gliomas (p = 0.003). Statistically signicant differences were not observed in the expression of the GG, GT, and TT genotypes between low- and high-grade gliomas. The ERCC2 rs13181 genotypes found were TT, GG, and GT; however, no difference in the expression between different glioma types was observed.


Introduction
Tumors of the central nervous system (CNS) account for approximately 2% of all cancers, with a worldwide incidence of 4.2 to 5.4 cases per 100,000 individuals per year, with gliomas being the most common primary brain tumor [1]. In Brazil, more than 10,270 new cases were registered in 2016 [1,2]. These tumors were classi ed into low-grade (grades I and II) and high-grade (grades III and IV) gliomas and this classi cation was important for patient prognosis since low-grade gliomas, such as pilocytic astrocytoma (grade I) and diffuse astrocytoma (grade II), exhibit a better prognosis than high-grade gliomas, such as anaplastic astrocytoma (grade III) and glioblastoma (grade IV) [1].
Environmental and lifestyle factors, such as exposure to ionizing radiation and smoking, have been speculated to be risk factors for the incidence of these neoplasms [3]. However, hereditary factors also play an important role in glioma development, and among the different genetic alterations, mutations and single nucleotide polymorphisms (SNP) are crucial [4][5][6]. Therefore, these tumors are considered to be caused by cumulative DNA damage, which is secondary to the interaction between the environment and genetic predisposition, making the existence of a DNA repair system critically important for cell life, as it guarantees the integrity of the genome by preventing mutagenesis [7][8][9].
The ERCC2 is a gene located on chromosome 19q13.3 and has been found to be responsible for DNA repair via the nucleotide excision repair (NER) pathway. The most frequently identi ed polymorphism in ERCC2 is Lys751Gln (rs13181), characterized by the replacement of thymine (T) by guanine (G) at locus 751, which can alter the enzymatic activity of some encoded proteins and is associated with several types of cancer, including gliomas [10]. However, in literature, ndings on this SNP and its association with gliomas are contradictory. Accordingly, a study conducted by Chen et al. [11] reported increased risk for glioma development, while McKean-Cowdin et al. [12] showed decreased risk associated with gliomas. In addition to the con icting results, the above-mentioned studies are primarily con ned to the Asian population.
To the best of our knowledge, there are no such studies in literature based on the Brazilian population, consisting of a rich racial miscegenation, that evaluate the presence of ERCC2 rs13181 variant polymorphism among patients with gliomas, and this led us to design the current study in patients with different glioma types in Northeast Brazil.

Results
Epidemiological characteristics. Patients with low-grade gliomas had a signi cantly lower mean age than high-grade glioma patients, 20.05 ± 15.9 and 44.47 ± 18.93 years [mean ± standard deviation of the mean (SDM)], respectively (p < 0.001). Patients with low-and high-grade gliomas were considered homogenous with respect to gender, smoking, alcohol use, and cancer familiar history (Table 1).
Genotypic Expression. Statistically signi cant differences were not observed in the expression of the GG, GT, and TT genotypes of the SNP ERCC2 rs13181 between high-and low-grade gliomas ( Table 2).

Discussion
Several studies have demonstrated the existence of DNA repair SNPs responsible for the induction and/or progression of neoplasms [10]. Among these polymorphisms, the rs13181 variant of the ERCC2 gene, also known as ERCC2 Lys751Gly, can alter the enzymatic activity of some encoded proteins, such as helicase, and may be associated with several types of cancer, including gliomas [10,13].
To the best of our knowledge, this is the rst study to evaluate the expression of SNP rs13181 of the ERCC2 gene between high-and low-grade gliomas using DNA samples isolated from neoplastic tissue stored in para n blocks in Brazilian patients. In the current study, the patient groups with high-and lowgrade gliomas were homogeneous regarding sex, smoking, alcohol use, and family cancer history.
However, the mean age of patients with high-grade gliomas was signi cantly higher than those with lowgrade gliomas, while no statistically signi cant difference was observed in the expression of GG, GT, and TT genotypes.
As for the epidemiological characteristics of the patients, some studies reported a higher prevalence of gliomas in males and no association between sex and the histological grade of these tumors [14][15][16], corroborating the results of the present study. There was no difference between family history of cancer and gliomas, perhaps because this association is more common in certain types of syndromes, such as Li Fraumeni, Turcort, neuro bromatosis, among others [17], which did not occur in this study. There was also no association between gliomas and smoking or alcohol consumption; however, some authors reported a higher risk for high-grade gliomas in cases of excessive smoking [18] and alcohol use [19,20]. High-grade gliomas occur in older patients [14][15][16], corroborating the present study results, as cellular aging increases immunological senescence, telomere shortening, chronic in ammation with antigenic stimulation, genomic instability, and mutations forming more aggressive neoplasms such as high-grade gliomas [17].
Some studies have shown an association between the rs13181 variant of the ERCC2 gene and the risk of developing gliomas [21,22,23]. However, most of these studies were conducted only in Asian populations and showed contradictory results regarding the in uence of genotypes of this variant in the development of glial tumors [24][25][26][27][28]. In the present study, the rs13181 SNP of the ERCC2 gene was found in all samples. However, no statistically signi cant differences in the expression of the GG, GT, and TT genotypes between high-and low-grade gliomas were observed, consistent with the ndings of Qian et al. The absence of an association between genotypes and the degree of malignancy of glial tumors in the current study may be due to its small sample size and the lack of a consistent genetic pattern in the Brazilian population. Brazil has a rich mixture of Europeans, Africans, and Indians, presenting different results from those found in other countries, where the populations are predominantly Caucasian, Asian, or African [29].
In conclusion, in the current study, patients with low-grade gliomas had a signi cantly lower mean age than high-grade glioma patients, and the ERCC2 rs13181 genotypes found were TT, GG, and GT; however, no difference in the expression between high-and low-grade gliomas was found in patients in Northeast Brazil. Nevertheless, further studies should be carried out with a larger sample size due to the rich Brazilian racial miscegenation.

Patients
This study was approved by the Internal Review Board of the Federal University of Piauí, Brazil wich was conducted according to the Declaration of Helsink. All patients or legal their legal guardians gave written informed consent. Fifty-one para n-embedded tissue samples from patients with histologically con rmed gliomas were obtained from the pathology department's archives. The selected samples were in storage for less than ve years. The inclusion criteria considered a histological glioma diagnosis without any previous treatment. An informed consent form was not necessary since the material had been previously collected, being part of the disease's surgical treatment.

DNA Extraction
The previously cataloged biopsy specimens stored in para n blocks were cut into 60-µm thick slices using a microtome and placed in sterile 1.5-mL Eppendorf tubes. The samples were treated with xylene and heated at 50 °C overnight to increase DNA yield and then heated again at 90 °C for 1 h to remove the para n. The material was then washed thrice using 100% ethanol and dried using a vacuum centrifuge to completely remove contaminants.
For DNA extraction, MagMAX™ FFPE DNA/RNA Ultra kit supplied piaUIJO Applied Biosystems™ (Foster City, CA, USA) was used, wherein magnetic microbeads were used to enable DNA extraction with high performance for sturdy samples. The protocol diligently followed the manufacturer's recommendations, and the degree of purity and concentration of the extracted DNA was assessed using a Nanodrop 2000 spectrophotometer (Thermo-Fisher Scienti c, Waltham, MA, USA), wherein the ratio between the 260/280 nm wavelength was measured, and the values considered ideal ranged from 1.8 to 2.0. The samples were normalized to a concentration of 30 ng/µL for later analysis through quantitative PCR.
qPCR for SNP genotyping For SNP genotyping, a Taqman assay probe C_3145033_10 corresponding to the SNP rs13181 of the ERCC2 gene was selected ( Table 3). The 20-µL reaction comprised 10 µL of TaqMan™ GTXpress™ Master Mix (Applied Biosystems), 4 µL of DNA, and 6 µL of ultrapure water treated with DEPC.
The reaction was conducted using an Applied Biosystems qPCR 7500 FAST thermocycler in fast mode for 40 cycles. The results were analyzed using the Applied Biosystems TaqMan Genotyper Software ver. 1.6.

Statistical analysis
For comparative analysis between the groups, t-test and Fisher's exact or chi-square test were used for quantitative and categorical variables, respectively. Odds ratios (ORs) with 95% con dence intervals (CIs) were calculated using the adjusted conditional logistic regression for gender and age. The level of signi cance was set at p<0,05. The statistical data were analyzed using the SPSS 20.0 software (SPSS Inc., Chicago, IL, USA).

Declarations
Ethics approval and consent to participate Not applicable Consent for publication Not applicable.

Availability of data and materials
All data generated in this analysis are available from the corresponding author upon request.

Competing interests
The authors declare that they have no competing interests.

Funding
No funding was obtained to conduct this study.

Author Contributions
T CB and dSBB conceived and designed the study; NJEJ, E-DCSM provided study materials and tools; TCB and dSBB were responsible for the collection and assembly of data, data analysis, and interpretation; C-VLC, G-BFCSA, L-CPV, dSAR, PRO was involved in writing the manuscript; TCB, dSBB, and GLH revised the manuscript.