Association of genetic variants in the CART gene with glioma susceptibility in a Chinese population

Glioma, which is a rare and highly fatal brain cancer, has been studied for many decades. However, only a few etiological factors have been established. Genetic factors play an essential roles in the development of gliomas and are key component of preventive oncology. However, only a small proportion of the genetic effect has been yet established. In current study, we systematically evaluated whether genetic variants of CART gene, which generates multiple biologically active peptides, contribute to susceptibility of gliomas among Chinese people with a two-stage, case– control study. In stage I, we found rs2239670 (Allele A vs G: OR = 1.33; 95% CI = 1.03-1.70; P = 0.026) and rs11575893 (Allele T vs C: OR = 1.29; 95% CI = 1.01-1.65; P = 0.040) were significantly associated with increased glioma susceptibility. Then the two SNPs were significantly replicated in an independent stage. When pooled together, both rs2239670 (Allele A vs G: OR = 1.27; 95% CI = 1.10-1.46; P = 0.001) and rs11575893 (Allele T vs C: OR = 1.25; 95% CI = 1.09-1.45; P = 0.002) were significant associated with increased glioma susceptibility. Our data suggest that the genetic variants in the CART gene potentially predispose their carriers to gliomas.


INTRODUCTION
Gliomas make up approximately 80% of all malignant brain tumors [1].Although relatively uncommon in the general population, gliomas owns high morbidity and mortality [2][3][4][5].In China, the estimated incidence and mortality numbers of brain tumors, which were mainly composed by gliomas, are 101.6 and 61.0 thousands in 2015, respectively [6], while the correspondent numbers in United States are 27,770 and 16,050 in 2016, respectively [7].Gliomas were composes of several subtypes, mainly including Glioblastomas, which account for approximately 60 to 70%, anaplastic astrocytomas, which account for 10 to 15%, and anaplastic oligodendrogliomas and anaplastic oligoastrocytomas, which account for 10% [3,[8][9][10][11].To present, only a small proportion of the genetic factors for gliomas has been yet established, including results of familial studies, candidate gene studies, and genome-wide association studies (GWASs) [12][13][14][15][16]. Understanding of the genetic basis is a key component of preventive oncology.
Cocaine and amphetamine regulated transcript (CART) gene encodes cocaine and amphetamine regulated transcript prepropeptide (CARTPT), which generates multiple biologically active peptides [17][18][19].These peptides play an important role in cancers, schizophrenia, alcohol use disorders, nicotine dependence, methamphetamine dependence, and obesity [20][21][22][23][24][25].Genetic association studies could provide solid evidence for the potential role of CART gene in human malignant gliomas.However, no studies have evaluated that whether genetic variants of CART contribute to risk of gliomas.In current, we performed a two-stage, case-control study to investigated the association between tag SNPs in the CART gene and gliomas susceptibility among Chinese population.

Demographic characteristics of the subjects
In stage I, a total of 400 gliomas patients and 400 healthy control were recruited, while 800 gliomas patients and 800 healthy control were recruited in stage II.Table 1 shows the comparison of gliomas patients and controls by selective characteristics in both stage I and stage II.No significant difference in the distribution of age, gender, family history of cancer, and smoking status was found between the gliomas patients and healthy controls in two stages (P > 0.05).Only the gliomas patients are more likely to be drinkers in stage II (P < 0.001).

Associations between CART gene polymorphisms and glioma susceptibility in the discovery stage
According to the selection criteria, four tagSNPs (rs2239670, rs3846659, rs11575893, and rs6894772) are selected using SNPinfo.Table 2 presents the genotype frequencies of the selected SNPs and their associations with glioma susceptibility.None of the genotype distributions for the four tag SNPs departed from the HWE (P > 0.05).Our results showed that rs2239670 (Allele A vs G: OR = 1.33; 95% CI = 1.03-1.70;P = 0.026) and rs11575893 (Allele T vs C: OR = 1.29; 95% CI = 1.01-1.65;P = 0.040) were significantly associated with increased glioma susceptibility.Compared with the carriers of genotype GG of rs2239670, those of genotype AA (OR = 1.91; 95% CI = 1.02-3.57)had significantly increased glioma susceptibility.However, no significant associations were found for rs3846659 and rs6894772.

Validation analysis of the association between selected CART SNPs and glioma susceptibility
To validate the results above, we evaluated the associations of CART rs2239670 and rs11575893 with glioma susceptibility was evaluated in stage II (Table 3).The genotype distribution of rs2239670 and rs11575893 in controls were also consistent with the HWE (P > 0.05).The positive trend for rs2239670 (OR trend = 1.24; 95% CI = 1.04-1.47;P = 0.015) and rs11575893 (OR trend = 1.23; 95% CI = 1.04-1.47;P = 0.018) was significantly replicated in Stage II.When pooled the results of the two stages together, both rs2239670 (Allele A vs G: OR = 1.27; 95% CI = 1.10-1.46;P = 0.001) and rs11575893 (Allele T vs C: OR = 1.25; 95% CI = 1.09-1.45;P = 0.002) were significant associated with increased glioma susceptibility.For rs2239670, compared with the carriers of genotype We also explored the effect modification of alcohol status and smoking status (Table 4), however, the results didn't change materially.

DISCUSSION
In this study, a two-stage, case-control study was applied to systematically evaluated whether genetic variants of CART gene contribute to susceptibility of gliomas among Chinese population.We identified that both rs2239670 and rs11575893 were significant associated with increased glioma susceptibility.To our knowledge, this should be the first study which aims to evaluated the association between genetic variants of CART gene and susceptibility of gliomas.
Except that exposure to ionizing radiation has been established as a risk factor, most underlying cause for gliomas has not been identified [26][27][28][29][30]. Genetic factors, which have been explored for many decades, play an essential roles in the development of gliomas and are key component of preventive oncology, however, only a small proportion of the genetic effect has been yet established [1,14,[31][32][33][34].CART gene is located at 5q13.2 in humans, and is expressed in diverse brain structures, as well as endocrine tissues [35].In 1999, Echwald et al first evaluated the potential effect of sequence variants CART gene in subjects with early onset obesity [25].It was also found that hypothalamic CART expression could be involved a variety of neuroendocrine functions including food-intake [36].Furthermore, CART gene was identified to be related with type 2 diabetes mellitus, hormone release, breast cancer, small bowel carcinoid tumors, neuroendocrine tumor, and so on [37][38][39][40][41][42][43].
While for rs11575893, motifs GCNF,Hand1,Myc could be changed, and it also could bind NRSF, ZNF263, and SIN3AK20.NRSF and ZNF263 are important modulator of malignant progression [47,48].Overall, our findings provided evidence for the important role of SNPs in CART gene in the tumorigenesis of gliomas.
Our study has several strengths.First, the two-stage study design provides a solid and trustable conclusion; second, the large sample size provides enough statistical power to detect such moderate associations.When interpreting the results of this study, several limitations should also be considered.First, selection and information bias might be unavoidable due to the natural of casecontrol study design, although the controls have been matched by age, gender, and race.Second, we still have not enough sample size to detect gene-environment interactions in current study.Third, it is uncertain whether our findings can be generalized to other populations due to the samples recruited in this study was restricted to Chinese population.
In conclusion, this study found that both CART rs2239670 and rs11575893 were significant associated with increased glioma risk using a two-stage, case-control study with a large sample size.The results suggest that the rs2239670 and rs11575893 may be used as biomarkers for prediction and screening of lung cancer.Validations with larger population-based studies in different ethnic groups and mechanism studies are warranted to further interpret the findings.

Table 3 : Genotype frequencies of CART rs2239670, rs11575893 and association with risk of gliomas in stage II and the merged results
1 adjusted for Age, gender, family history of cancer, smoking status and alcohol status