Effects of the ANXA6 polymorphisms on glioma risk and patients prognosis

BACKGROUND Glioma is the most frequent malignant primary brain tumor, and the outcomes for patients with glioma remain poor. The purpose of this study is to explore the association between ANXA6 polymorphisms and glioma risk as well as the prognosis of glioma patients in the Chinese Han population. METHODS We selected nine single-nucleotide polymorphisms (SNPs) in ANXA6 which were genotyped by Agena MassARRAY from 593 glioma patients and 589 healthy controls. The odds ratio (OR) and 95% condence interval (CI) were calculated by logistic regression analysis to evaluate the association SNPs with glioma risk. The association between polymorphisms and survival of glioma patient were evaluated using the log-rank test, Kaplan-Meier and Cox regression analysis. RESULTS: Overall analysis found that rs3762993 was signicantly associated with an increased glioma risk. Stratication analysis found that rs11960458 was strongly associated with an increased risk of glioma in age >41; rs3762993 was also found to be associated an increased with glioma risk in age >41, ≤ 41, male and low-grade glioma; but rs4958892 was associated with a decreased risk of glioma in age>41 and male. Interestingly, rs11960458 and rs888988 were correlated with poor prognosis of glioma patient. Furthermore, age, extent of resection and chemotherapy were found to be key prognostic factors in survival of glioma patients. CONCLUSIONS In conclusion, our results indicated that ANXA6 polymorphisms were associated with glioma susceptibility and prognosis. Further studies are required to conrm the results and elucidate the mechanisms of the ANXA6 polymorphisms affect the glioma risk and prognosis. for WHO classication, WHO surgical methods, chemotherapy, radiotherapy and survival The case-control study recruited 593 glioma patients (324 males and 269 females) with mean age 40.56 years old and 589 healthy controls (325 males and 264 females) with mean age 40.98 years old. The statistical analysis results showed that no statistically signicant differences was found between glioma patients and controls with regarded to gender and age distribution (p > 0.05).

measured from the date of diagnosis to death or the last follow-up. Progress-free survival (PFS) was calculated from the date of the pathologically con rmed to the progression of the disease, death without progression, or last clinical follow-up.

Data collection
The demographic and clinical information were collected and regularly updated for the patients with glioma through medical records, questionnaires and follow-up. These data included gender, age, date of diagnosis, histologic type, WHO grade, surgical methods, extent of resection, treatment with radiotherapy and/or chemotherapy, date of last follow-up, status of patients (living/deceased).

DNA extraction
The peripheral blood samples were collected from all subjects before radiotherapy and chemotherapy therapy or surgery into the ethylene diamine tetraacetic acid (EDTA)-containing vacutainers, and stored at −80°C until further DNA extraction. Genomic DNA of the samples was extracted using the GoldMag-Mini Whole Blood Genomic DNA Puri cation Kit (GoldMag. Co. Ltd., Xi'an, China). The purity and concentration of the extracted DNA were determined using a spectrophotometer by absorbance measurements at 260nm and 280nm (NanoDrop 2000; Thermo Fisher Scienti c, Waltham, MA, USA). The extracted DNA was stored at −20°C until further SNPs genotype.

SNPs selection and genotyping
Nine SNPs (rs11960458, rs4958892, rs78243462, rs4346760, rs4958897, rs3762993, rs9324677, rs13185706, and rs888988) in the ANXA6 gene were selected with minor allele frequency (MAF) >5% in the global population from the HapMap database. We used the Agena Bioscience Assay Design Suite V2.0 software (https://agenacx.com/online-tools/) to design PCR ampli cation and extension primers of the nine SNPs. Genotyping of the nine ANXA6 polymorphisms were performed using the Agena MassARRAY platform with iPLEX gold chemistry (Agena Bioscience, San Diego, CA, USA) according to the protocol described. The Agena Bioscience TYPER software (version 4.0) was used to manage and analyze data.

Statistical analysis
We used the Pearson's χ 2 test to analyze the differences in distribution of gender between the glioma patients and controls. The Student's t-test was used to evaluate differences in age between the case and control groups. We examined whether the genotype frequencies of SNPs in the control group was consistent with Hardy-Weinberg equilibrium (HWE) using χ 2 test. The association between ANXA6 polymorphisms and glioma risk was assessed under the codominant, dominant, recessive and additive genetic models and haplotype analysis by PLINK software (version 1.07). Odds ratios (ORs) and 95% con dence intervals (CIs) were calculated to estimate the association between ANXA6 polymorphisms and glioma risk with adjustment of gender and age using logistic regression analysis. We used the PLINK software and Haploview software (version 4.2) to assess the linkage disequilibrium (LD) between the each SNP. Survival distributions were estimated by using the Kaplan-Meier method and differences in the survival were tested using the log-rank test. The hazard ratio (HR) and 95% CI were calculated from the univariate Cox proportional hazards model to estimate the association between SNPs of ANXA6 polymorphism with PFS and OS in glioma. Multivariate Cox models were performed to compute HR and 95% CI, after adjustment potential risk factors that could affect glioma outcome such as age, gender, and extent of resection. All P values were 2 sided and P< 0.05 was considered statistically signi cant. Statistical analysis was conducted using SPSS 20.0 statistical package (SPSS, Chicago, IL).

Subject characteristics
We computed basic descriptive statistics for age, gender, WHO classi cation, WHO grade, surgical methods, chemotherapy, radiotherapy and survival condition ( Table 1). The case-control study recruited 593 glioma patients (324 males and 269 females) with mean age 40.56 years old and 589 healthy controls (325 males and 264 females) with mean age 40.98 years old. The statistical analysis results showed that no statistically signi cant differences was found between glioma patients and controls with regarded to gender and age distribution (p > 0.05).

Details of the selected SNPs
The nine SNPs in the ANXA6 gene were successfully genotyped (call rate >95%).The details of the selected SNPs including chromosome id, position, role, allele and potential function predicted by the RegulomeDB software and HaploReg software about these variants are listed in Table 2. The results showed that rs4958892 and rs3762993 are likely to affect expression quantitative trait loci (eQTL), TF binding, and DNase peak. The SNPs rs4346760 and rs9324677 are likely to affect TF binding, any motif and DNase peak. More detailed results are described in Table 2. The distributions of the nine ANXA6 polymorphisms genotypes frequencies among the controls were in agreement with the HWE (p > 0.05), as shown in Table 2. ANXA6 polymorphisms and glioma risk The allele A frequency of rs3762993 in the patients with glioma was signi cantly different from those among the control subjects (p = 0.007) ( Table 2).
And the results found that individuals carrying the allele A frequency of rs3762993 was associated with signi cantly a 1.26-fold increased risk of glioma (OR = 1.26, 95% CI: 1.07-1.49). However, no signi cant association was found between the other eight polymorphisms ANXA6 of and glioma risk in the allele model.
In order to explore the association between the nine polymorphisms of ANXA6 and glioma risk, genetic models (codominant, dominant, recessive, and additive) by logistic regression analysis with adjustment for age and gender were applied ( Table 3). The results showed that individuals with the AG (adjusted OR = 1.32, 95% CI: 1.03-1.69, p = 0.029) and AA genotypes of rs3762993 (adjusted OR = 1.52, 95% CI: 1.07-2.18, p = 0.021) were associated with increased risk of glioma compared with those with the GG genotype, respectively. Moreover, we also found that rs3762993 was associated with increased risk of glioma under the dominant model and the additive model, respectively (AA-AG vs. GG: adjusted OR = 1.36, 95% CI: 1.08-1.72, p = 0.009; adjusted OR = 1.26, 95% CI: 1.06-1.48, p = 0.007). No association was found between other eight SNPs of ANXA6 and glioma risk (Table 3).
We used the PLINK software and Haploview software to assess the LD between the each SNP. The results found that a small haplotype block with strong LD between the two SNPs (rs78243462 and rs4346760), as shown in Figure 1. However, the haplotype analysis results showed that no signi cant association was found between these haplotypes and glioma risk (Table 4).
Moreover, rs3762993 was found to be associated signi cantly associated an increased risk of glioma in the age more than 41 years old (AA-AG vs. GG:  Table 5.

Impact of clinical factors on glioma survival
In this study, we investigated the impact clinical factors, including gender, age, WHO grade, extent of resection, radiotherapy and chemotherapy, on the OS and PFS of glioma patients using the univariate and Cox regression analysis (Table 6, Figure 2). The results showed that the younger patients (age<40) had a longer OS than the older (age≥40 years) (log-rank p = 0.032), and the age≥40 was a hazardous factor with a 1.19-fold increased risk of death on OS in glioma patients, compared with the age<40 (HR = 1.19, 95% CI = 1.00-1.41, p = 0.049). The gross-total resection (GTR) of glioma patients had a longer OS (log-rank p < 0.001) and PS (log-rank p < 0.001) than the near-total resection (NTR) and sub-total resection (STR) of glioma patients, and had a better prognosis of glioma patients (OS: HR = 0.63, 95% CI: 0.52-0.76, p < 0.001, and PFS: HR = 0.59, 95% CI: 0.91-0.71, p < 0.001). Furthermore, we also found that the glioma patients with the chemotherapy treatment was also associated with a reduced risk of death on OS (log-rank p < 0.001, HR = 0.66, 95% CI: 0.55-0.78, p < 0.001) and PFS (log-rank p = 0.007, HR = 0.81, 95% CI: 0.67-0.96, p = 0.017), compared with the no chemotherapy treatment. However, no signi cant associations were found between the gender, WHO grade, radiotherapy and the prognosis of glioma patients as measured by OS and PFS.

Impact of SNPs on glioma survival
We also evaluated the effect of the nine ANXA6 polymorphisms on the glioma patients with OS and PFS using the log-rank tests and Cox regression analysis. Interestingly, we found the CC genotype of rs888988 was signi cantly correlated with poor prognosis in glioma patients on OS (log-rank p = 0.026, HR = 1.79, 95% CI: 1.05-3.05, p = 0.033) and PFS (log-rank p = 0.018, HR = 1.97, 95% CI: 1.14-3.41, p = 0.016) compared to the GG genotype (Table 7, Figure 3). The CG genotype of rs888988 was also found to have a negative effect on OS (HR = 1.80, 95% CI: 1.05-3.07, p = 0.031) and PFS (HR = 1.99, 95% CI: 1.15-3.45, p = 0.014) compared to the common GG genotype. However, no signi cant associations were identi ed between the other eight SNPs and OS or PFS of glioma patients. The multivariate Cox regression analysis did not found the effects the nine SNPs in the ANXA6 gene on the prognosis of glioma patients.

Discussion
This study was rstly to investigate the associations between ANXA6 polymorphisms and glioma risk and prognosis of glioma patients in the Chinese Han population. Our results indicated that the three SNPs (rs11960458, rs4958892, and rs3762993) of ANXA6 were signi cantly associated with the risk of glioma. Interestingly, rs11960458 and rs888988 were found to be correlated with poor prognosis of glioma patients. Furthermore, those clinical factors including age, extent of resection and chemotherapy were found to be key prognostic factors in survival of glioma patients. The three SNPs (rs11960458, rs4958892, and rs3762993) of ANXA6 were found to be signi cantly associated with the risk of glioma; rs11960458 and rs888988 were found to be correlated with poor prognosis of glioma patients in this study. The 52 SNPs from three genes of the ANXA family were selected from public databases and genotyped in 443 osteonecrosis of the femoral head patients and 273 control subjects using the Affymetrix Targeted Genotyping 3 K Chip array, results found that SNPs rs11960458 and rs9324677 of the ANXA6 gene were signi cantly associated with the risk of osteonecrosis of the femoral head in the Korean population, but no association was found between rs4958892 and rs888988 in ANXA6 and osteonecrosis of the femoral head risk [25]. This study was rstly to investigate the associations between ANXA6 polymorphisms and glioma risk and prognosis of glioma patients in the Chinese Han population. Therefore, we need to con rm the conclusion of this study in multi-ethnic population with larger scale.
In this study, those clinical factors including age, extent of resection and chemotherapy were found to be key prognostic factors in survival of glioma patients The comprehensive analysis of multicentric glioma patients revealed that age younger than 54 years, surgical resection, and radiotherapy were signi cantly associated with improved survival and were independent prognostic factors for OS [26]. Gui et al [27] also demonstrated that age, surgical resection and chemotherapy in uenced the survival rates of glioma patients. A systematic review and meta-analysis study showed that GTR substantially improves overall and progression-free survival compared with STR [28]. Our results did not found any signi cant association between radiotherapy and prognosis of glioma patients. However, Keime-Guibert et al [29] study indicated that radiotherapy resulted in a modest improvement in survival in elderly patients with glioblastoma. Therefore, we need to con rm the results in the further study with a large sample.
Several limitations should be addressed in this study. The sample in this study limited to Chinese population, therefore, the conclusion of this study warrants further con rmation in multi-ethnic population with larger scale. Additionally, this study did not elucidate the molecular mechanism under which the ANXA6 polymorphisms affect the glioma risk and the prognosis in glioma patients. Further functional studies will be required to elucidate these speci c mechanisms of the ANXA6 polymorphisms affect the prognosis in glioma patient.
In conclusion, the results indicated that age, extent of resection and chemotherapy were found to be key prognostic factors in survival of glioma patients.
Moreover, the present study is the rst time to nd that ANXA6 polymorphisms are associated with the glioma risk and affect the progression of glioma patients. These observations may be potentially valuable prognostic markers for glioma patients. The protocol of this study was approved by the Ethics Committee of Tangdu Hospital and was conducted in accordance with the principles of the Declaration of Helsinki. All participants gave written informed consent prior to blood samples collection in the study.

Consent for publication
Not applicable.

Competing interests
The authors declare that there are no con icts of interest.

Funding
Not applicable.

Authors' contributions
Yuan Shao contributed to the design of the study and performed the statistical analysis. Tuo Wang was responsible for manuscript preparation. Yao Sun and Zichao Xiong performed the experiments. Jiamin Wu and Xiaoying Ding contributed to collect the data. Xiaoye Guo was responsible for checking the data. All authors were responsible for drafting the manuscript, read and approved the nal version.    Adjust OR (95%CI) were calculated by logistic regression analysis with adjustments for age and gender. p < 0.05 indicates statistical significance. Adjust OR (95%CI) was calculated by logistic regression analysis with adjustments for age and gender.
p < 0.05 indicates statistical significance.   Figure 1 Haplotype block map for the SNPs in the ANXA6 gene Standard color frame is used to show LD pattern. Red squares display statistically signi cant associations between a pair of SNPs, as measured by D'; darker shades of red indicate higher D'.