Impact of GTF2H1 and RAD54L2 gene polymorphisms on the non-small cell lung cancer with Platinum chemotherapy in high altitude areas CURRENT STATUS: POSTED

Background Repair pathway genes are closely related to the sensitivity of tumor patients to drugs. It is particularly important to study the correlation between non-small cell lung cancer (NSCLC) and DNA repair gene ( GTF2H1 and RAD54L2 ) in high altitude areas.Materials and methods Four SNPs on RAD54L2 and five SNPs on GTF2H1 were genotyped via the Agena MassARRAY in 506 age-and gender-matched patients with NSCLC and 510 healthy controls. The influence of GTF2H1 and RAD54L2 on lung cancer was assessed by logistic regression analysis. The effects of polymorphisms of these two genes on the efficacy and side effects of NSCLC patients with platinum-based chemotherapy were further evaluated.Results RAD54L2 rs9864693 GC heterozygote genotype was increased the risk of NSCLC (OR = 1.33, 95%CI: 1.01 – 1.77, p = 0.045). Stratified analysis found that GTF2H1 rs4150667 promoted the risk of lung cancer in individuals younger than 59 years group (OR = 1.32, 95%CI: 1.00 – 1.75, p = 0.048). However, the individuals with GTF2H1 rs3802967 CT-TT reduced the risk of the lung squamous cell carcinoma by 32% (OR = 0.68, 95%CI: 0.46 – 0.99, p = 0.045). The patients with GTF2H1 rs3802967 TT genotypes showed a significantly higher curative effect (OR = 2.65, 95%CI: 1.14 - 6.15, p = 0.023); patients with GTF2H1 rs3802967 TT genotypes showed a significantly lower risk of side effects (OR = 0.32, 95%CI: 0.10 - 0.98, p = 0.047).Conclusion Our study indicated that RAD54L2 and GTF2H1 gene was associated with NSCLC susceptibility.


Abstract
Background Repair pathway genes are closely related to the sensitivity of tumor patients to drugs. It is particularly important to study the correlation between non-small cell lung cancer (NSCLC) and DNA repair gene ( GTF2H1 and RAD54L2 ) in high altitude areas.Materials and methods Four SNPs on RAD54L2 and five SNPs on GTF2H1 were genotyped via the Agena MassARRAY in 506 age-and gender-matched patients with NSCLC and 510 healthy controls. The influence of GTF2H1 and RAD54L2 on lung cancer was assessed by logistic regression analysis. The effects of polymorphisms of these two genes on the efficacy and side effects of NSCLC patients with platinum-based chemotherapy were further evaluated.Results RAD54L2 rs9864693 GC heterozygote genotype was increased the risk of NSCLC (OR = 1.33, 95%CI: 1.01 -1.77, p = 0.045). Stratified analysis found that GTF2H1 rs4150667 promoted the risk of lung cancer in individuals younger than 59 years group (OR = 1.32, 95%CI: 1.00 -1.75, p = 0.048). However, the individuals with GTF2H1 rs3802967 CT-TT reduced the risk of the lung squamous cell carcinoma by 32% (OR = 0.68, 95%CI: 0.46 -0.99, p = 0.045). The patients with GTF2H1 rs3802967 TT genotypes showed a significantly higher curative effect (OR = 2.65, 95%CI: 1.14 -6.15, p = 0.023); patients with GTF2H1 rs3802967 TT genotypes showed a significantly lower risk of side effects (OR = 0.32, 95%CI: 0.10 -0.98, p = 0.047).Conclusion Our study indicated that RAD54L2 and GTF2H1 gene was associated with NSCLC susceptibility.

Background
Lung cancer is called primary bronchogenic carcinoma and is a malignant tumor derived from the trachea, bronchial mucosa or glands, and is currently the most common malignant tumor in the world [1]. According to statistics, in 2015, the incidence of lung cancer in Chinese men and women was 50.9 per 100,000 person-years and 22.4 per 100,000 person-years, respectively, which is the most important cause of death in cancer patients [2]. At present, surgery is still the treatment of choice for early stage lung cancer, most lung cancer patients have been diagnosed at an advanced stage, lost the best treatment opportunity, and its 5-year survival rate is only 19.7% [1,3] GTF2H1 (general transcription factor IIH subunit 1, GTF2H1, p62) protein plays an important role in the nucleotide excision repair (NER) pathway, participates in the early damage recognition of XPC-HR23B protein, and recruits endonuclease XPG to the injury site to complete the enzymatic cleavage process [10][11][12]. In addition, the GTF2H1 protein is involved in transcription and regulates transcriptional activation of multiple genes [13].
One research found that the variants of rs3802967 and rs4150606 on GTF2H1 gene increased the risk of lung cancer, and rs4150667 on GTF2H1 gene variant reduced the risk of lung cancer [14]. So, the genetic polymorphism of GTF2H1 gene may be involved in the pathogenesis of lung cancer.
RAD54L2, also known as ARIP4 (Androgen Receptor-Interacting Protein 4), is a protein-coding gene, which belongs to the RAD54 subfamily of SNF2-type chromatin remodeling factor superfamily [15], and has the double-stranded DNA-dependent ATPase activity. In fact, Rad54 interacts with Rad51 and thereby enhances its ability to form cruciform and D-loops. RAD51 catalyzes DNA repair by homologous recombination (HR) to ensure the stability of cell genome. In a study of the effects of RAD51 G135C polymorphism on NSCLC patients treated with platinum-paclitaxel / gemcitabine Wrst line chemotherapy, we found that the G135C allele was associated with a higher survival time and had a better prognosis [16]. However, the effect of RAD54L2 gene on the occurrence and development of lung cancer is unknown.
The occurrence and development of tumors are closely related to genetic susceptibility and mutation. The genotypes have racial differences. Gene mutation is related to regional environmental factors, perennial hypobaric hypoxia and strong ultraviolet radiation in high altitude areas. The occurrence and development of non-small cell lung cancer at altitude is not known. Lung cancer is not only the most common malignant tumor in China, but also one of the most common malignant tumors in our high altitude areas. Therefore, it is particularly important to study the correlation between lung cancer and DNA repair gene polymorphism in high altitude areas. Therefore, we chose two genes GTF2H1 and RAD54L2 related to DNA repair to explore their impact on the risk of non-small cell lung cancer in Chinese Han population of living high altitude areas and the efficacy and side effects of platinum chemotherapy.

Study participants
In high altitude areas, 506 age-and gender-matched patients with lung cancer and 510 healthy controls were selected from the Qinghai Province Cancer Hospital. The inclusion criteria were lung cancer confirmed by histopathology and no history of malignant tumors in other organs. Record whether patients received platinum chemotherapy, and the treatment effect (complete response, CR; partial response PR; stable disease SD; progressive disease, PD), toxicity, lymph node metastasis, clinical stage, smoking and drinking, Body Mass Index (BMI). The inclusion criteria for the control group included medical or family history without cancer or any lung disease. At the time of recruitment, each subject was interviewed by trained personnel using a structured questionnaire to obtain information about demographic characteristics.
This study was conducted under the approval of the Institutional Review Boards of Qinghai Province Cancer Hospital. All participants were aware of the content of the study and signed an informed consent.

SNP selection and genotyping
We selected the GoldMag-Mini Whole Blood Genomic DNA Purification Kit (GoldMag Co. Ltd. Xi'an City, China) to extract the DNA from the 5 ml peripheral venous blood; and Nanodrop 2000 (Gene Company Limited) was used to detect the concentration and purity of samples, DNA to ensure that the samples could be used for subsequent experiments.
rs11720298, rs4687721, rs4687592, and rs9864693 four SNPs on RAD54L2 gene and rs4150530, rs3802967, rs4150606, rs4150658, and rs4150667 on GTF2H1 were selected in our study based on minor allele frequency data more than 0.05 in the global population.

Statistical analysis
Demographic characteristics were counted. The Hardy-Weinberg equilibrium (HWE) was calculated by χ2 test [22]. Five genetic models were used to evaluate the association between gene polymorphisms and lung cancer risk. Odds ratios (ORs) and its corresponding 95%CI were estimated using an logistic regression model with adjustments for age and gender through the PLINK software [23]. Further analysis to assess the impact of polymorphism on lung cancer based on age, gender, smoking, drinking, lymph node metastasis, clinical stages, toxic and side effects, and curative effect. The threshold of p was set to 0.05.

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The information of the astrocytoma patients and healthy participants was shown in table The relationship between gene polymorphism and the efficacy and side effects of platinum chemotherapy in patients with NSCLC was further analyzed (Table 5). The results found that patients with GTF2H1 rs3802967 TT genotypes showed a significantly higher curative effect, when compared with the CC-CT genotype (OR = 2.65, 95%CI: 1.14 -6.15, p = 0.023); patients with GTF2H1 rs3802967 TT genotypes showed a significantly lower risk of side effects, when compared with the CC-CT genotype (OR = 0.32, 95%CI: 0.10 -0.98, p = 0.047).

Discussion
In our research, we found that RAD54L2 rs9864693 and GTF2H1 rs4150667 increased the risk of NSCLC. GTF2H1 rs3802967 reduced the risk of the lung squamous cell carcinoma, GTF2H1 rs3802967 "T" allele showed a significantly higher curative effect and lower risk of side effects.
GTF2H1 interacts with the C-terminus and N-terminus of XPC protein, participates in the recruitment of other protein subunits in TFIIH, and initiates the NER repair process. In our study, it was found that GTF2H1 rs3802967 CT-TT reduced the risk of the lung squamous cell carcinoma. In the previous study, the variant of GTF2H1 rs3802967 was also significantly associated with the risk of lung cancer [14], which is consistent with our findings. In addition, in the evaluation of efficacy and side effects after platinum-based chemotherapy, GTF2H1 rs3802967 "T" allele showed a significant higher curative effect and lower risk of side effects. It is indicated that GTF2H1 rs3802967 may play a protective role in the development of NSCLC. The study found that the expression of GTF2H1 was down-regulated in lung cancer tissues [24]. The luciferase activity of the vector carrying the T allele was enhanced by lung adenocarcinoma experiments [14], suggesting that the mutation of rs3802967 may affect the binding of GTF2H1 gene to transcription factor AP-1.

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In turn, it affects the expression level of GTF2H1 gene in lung cancer, and further protects the development of lung cancer.
Stratified analysis found that GTF2H1 rs4150667 promoted the risk of NSCLC in individuals younger than 59 years group. While one research found that rs4150667 on GTF2H1 gene variant reduced the risk of lung cancer [14]. This is contradictory to our findings. In the research of Gong et al., SNP rs4150667 from GTF2H1 also decreased the risk of ovarian cancer (OR = 0.64) [25]. Whether the variant at this site increases or decreases the risk of the disease requires further confirmation. Besides, rs4150606 on GTF2H1 gene increased the risk of lung cancer [14]. However, we found no correlation between the variant of rs4150606 on GTF2H1 gene and the risk of NSCLC. This may be due to the false negative results of our small sample size, and whether these two sites are associated with the risk of lung cancer need to be further expanded. The effect of RAD54L2 gene on lung cancer is not clear. Rad54 interacts with Rad51, which functions during DNA repair. It has been proved that Rad51 G135C allele was associated with a higher survival time and had a better prognosis in NSCLC patients treated with platinum-paclitaxel / gemcitabine Wrst line chemotherapy [16]. The role of RAD54L2 gene in the occurrence and development of lung cancer needs to be further clarified.

Conclusions 9
The result indicates that RAD54L2 rs9864693 and GTF2H1 rs3802967 and rs4150667 play an important role in the susceptibility of NSCLC. GTF2H1 rs3802967 "T" allele showed a significantly higher curative effect and lower risk of side effects. In the next step, we will focus on exploring the molecular mechanism of mutations in these two genes in the occurrence and development of lung cancer.

Ethics approval and consent to participate
This study was conducted under the approval of the Institutional Review Boards of Qinghai Province Cancer Hospital. All participants were aware of the content of the study and signed an informed consent.

Competing interests
The authors have declared that they have no conflict of any interests.   Tables Table 1 The