GNAS1 (Gαs) Gene T393C Polymorphism and Renal Cell Carcinoma Risk in a North Indian Population: A Case-Control Study

Abstract

Aim: Heterotrimeric G protein α-subunit Gαs is required for activation of adenylyl cyclase and generation of cyclic adenosine monophosphate (cAMP) in cells and plays a key role in multiple signal transduction pathways, linked to proapoptotic processes in cancer cells. This study investigated whether Gαs gene polymorphism was associated with increased renal cell carcinoma (RCC) risk in the North Indian population. In the present study, genotyping of GNAS1 gene in 196 RCC cases and 250 healthy controls was performed via polymerase chain reaction-restriction fragment length polymorphism. Results: The frequency of homozygous genotype CC was 29.6%, heterozygous TC was 51.5%, and homozygous TT was 18.9% in cases, whereas in controls it was 16.8%, 50.8%, and 32.4%, respectively; thus, there was a significant difference between the two groups (p=0.0001) in the univariate model. Further, multivariate analysis also demonstrated significant association of CC genotype with RCC risk (p=0.002). The high-risk genotype CC of GNAS1 gene showed threefold increase in risk to RCC relative to the TT genotype (unadjusted odds ratio [OR]=3.023, 95% confidence interval [CI]: 1.734-5.270). Whereas multivariate analysis showed a twofold increase in RCC risk among the CC genotype compared with the TT genotype (adjusted OR=2.181, 95% CI: 1.344-3.538). The C allele frequency was found to be significantly higher in RCC patients (55.3%) than in controls (42.2%) as compared with the T allele frequency that was 44.64% in RCC cases and 57.8% in controls. Moreover, patients with the CC genotype showed the worst prognosis in terms of the highest frequency of individuals having higher stages of RCC, but did not show any association with histological grade. Conclusion: Our results suggest that a T393C SNP could be considered as a genetic marker implicated in the pathogenesis of RCC.

Introduction

The Gαs is a protein that in humans is encoded by the GNAS1 gene that is located on chromosome 20q13 and consists of 13 exons (Weinstein et al., 2001). Somatic activating mutations of this gene have been implicated in the etiology of McCune Albright Syndrome (Collins et al., 2001) and sporadic, isolated endocrine tumors (Lyons et al., 1990), which chiefly supports a role of GNAS1 in tumor initiation and/or progression. The heterotrimeric G proteins are key signal transducers, composed of three distinct subunits Gα, Gβ, and Gγ, according to their decreasing molecular weights, which are encoded by separate genes. Gα is a GTPase, and Gβ and Gγ are stable subunits of inactive G protein (Wilson et al., 2010). Gα binds directly to adenylyl cyclase and stimulates or inhibits its activity, leading to an increase or decrease in cyclic adenosine monophosphate (cAMP) levels, respectively (Peeters et al., 2006). On activation by ligand-bound G-protein coupled receptors, the GDP is released and replaced by GTP on Gαs, which results in dissociation from the receptor and the βγ complex. The free Gαs subunit interacts with adenylate cyclase to stimulate the formation of cAMP. Rapid termination of the cellular response is required once the concentration of the stimulating ligand decreases. GTP bound to Gα is hydrolyzed rapidly to GDP; Gα is converted to its inactive form and re-associates with the βγ subunits returning to the “off” state, to enter a new cycle (Weinstein et al., 2004). The activated Gαs or cAMP has been shown to suppress Ras-dependent activation of Raf (Chen and Iyengar, 1994; Thompson and Lyons, 2005). The Ras/Raf signaling pathway is a significant intermediary of tumor cell proliferation and angiogenesis and a potent Raf inhibitor shows promising results in patients with renal cell carcinoma (RCC) (Klenke and Siffert, 2011). The outcome of the present study and earlier reports (Frey et al., 2006a) strongly suggests a role of the GNAS1 T393C polymorphism in tumor progression.

We have investigated whether a genetic host factor, the common T393C polymorphism in the GNAS1 gene, which encodes the G protein subunit, may be predictive for the clinical outcome in patients with RCC. We investigated a potential association between genotypes of the T393C polymorphism of GNAS1 gene and clinical outcome in a series of 196 North Indian patients with RCC surgically treated by nephrectomy (with curative intent) at different tumor stages and 250 healthy Indian volunteers (age and sex matched) serving as controls. To the best of our knowledge, this study is the first to investigate the distribution of GNAS1 T393C polymorphism among RCC patients in North Indian populations. Moreover, association of allele frequency and Fuhrman's grade and tumor-node-metastasis (TNM) staging was also evaluated. The principle aim of the present study was to provide further evidence that the T393C-dependent altered Gαs expression is associated with the prediction of outcome in patients with RCC, and represents a more general progression marker.

Materials and Methods

Subjects

The present case-control study was carried out from December 2008 to February 2011, in which 196 (138 men and 58 women) newly diagnosed RCC patients from a North Indian population (Delhi, Haryana, Uttar Pradesh, Rajasthan, and Punjab) registered in the Department of Urology, All India Institute of Medical Sciences (AIIMS), New Delhi, India, were recruited. Patients were recruited after radiologic and histologic diagnosis of RCC. The histological subtype distribution was 175 clear cell RCC, 11 papillary RCC, 8 chromophobe RCC, and 3 unclassified RCC. In total, 250 healthy volunteers were chosen who were free of any chronic diseases, having no history of any cancer and living in the same geographical area. They were matched with cases in sex and age. Smoking and hypertension status were also evaluated during the recruitment of cases and controls by means of a patient case record form and a questionnaire, respectively. Histopathologic staging and grading followed the TNM classification of the American Joint Committee on Cancer (AJCC). All patients received a patient information sheet and signed a consent form approved by the Institutional Committee on Human Ethics (ICHE proposal No. P-18/1.09.08). Likewise, controls received a volunteer information sheet and signed consent. About 5 mL of peripheral blood samples was collected from the patient and healthy volunteers for DNA extraction and henceforth processed for genotyping. To validate the findings of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP), the analysis of all mutant homozygous samples and 20% of heterozygotes was duplicated and confirmed by direct sequencing with 100% concordance.

DNA isolation

Genomic DNA samples were obtained from blood lymphocytes using a genomic DNA extraction kit (Bioserve Biotechnologies Pvt. Ltd.). Isolated DNA was resuspended in Tris EDTA buffer (pH 8.0) and stored at −20°C until use.

GNAS1 genotyping

The GNAS1 T393C polymorphism was determined by PCR-RFLP according to the method of Harries et al. (1997) with slight modifications. Genotypes of the T393C polymorphism were determined by PCR-RFLP using the following primers: forward primer 5′-TCCTAACTGACATGGTGCAA-3′, and reverse primer, 5′-TAAGGCCACACAAGTCGGGGT-3′. Reaction mixtures of 25 μL were used in PCR for the GNAS1 T393C polymorphism. The reaction mixture consisted of 2 μL of genomic DNA, 200 ng of forward and reverse primers, 1× Taq polymerase buffer (1.5 mM MgCl₂), dNTPs (0.3 mM), and 1.0 units of Taq DNA polymerase (Fermentas Life Sciences, Inc.). The PCR amplification was carried out with cycling parameters as follows: denaturation at 94°C; 35 cycles of DNA amplification were done at 94°C for 45 s, 58°C for 40 s, and an extension at 72°C for 45 s. The GNAS1 gene was detected by RFLP using the restriction endonuclease FokI. The PCR product of the 345-bp band was obtained and digested with 5 units of FokI restriction enzyme (New England Biolabs) in 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl₂, and 1 mM dithiothreitol, and incubated at 37°C for 16 h. About 10 μL of the digested reaction mixture was then loaded using 2% agarose gels containing ethidium bromide and visualized under short-wave UV light. The sizes were determined using 100-bp ladder (Fermentas Life Sciences, Inc.). The unrestricted products (345 bp) represent the TT genotype; the completely restricted products (259 and 86 bp) represent the CC genotype.

Statistical analysis

Differences in genotype of T393C polymorphism of GNAS1 gene between the RCC cases and the control subjects were studied. The Pearson's chi-square test was used to check whether control population followed the Hardy-Weinberg law. The associations between the genotypes and the clinicopathological characteristics of the RCC patients were also assessed for statistical significance using the chi-square test and Fisher's exact test. Multivariate logistic regression model was developed to adjust the effect of covariates like sex, hypertension, smoking, and body mass index (BMI) on the RCC risk among patients and healthy controls. Odds ratio (OR) was found at 95% confidence intervals (95% CIs), and p-values <0.05 were considered to indicate statistical significance in these tests. The Student's unpaired t-test was applied to compare continuous variables among cases and controls. All analyses were performed using the statistical package SPSS version 16 (SPSS).

Results

We have studied single-nucleotide polymorphisms in the GNAS1 gene among 196 RCC cases (138 men and 58 women) and compared them with 250 normal healthy controls (172 men and 78 women) for genotyping. We performed Pearson's chi-square test of our control population for genotypic distribution. The GNAS1 genotypic distribution was in Hardy-Weinberg equilibrium, with a p-value of 0.513 and a chi-square value of 0.427, respectively.

Association of various clinicopathological variables with RCC

Table 1 provides a description of genotyped study participants and known RCC risk factors. Cases and controls were matched on the basis of age and sex. The mean age was 54.21±0.967 and 54.01±0.916 years, in cases and controls, respectively. The sex ratio was similar in cases and controls (p=0.714). Hypertension was strongly associated with cases, having a frequency of 66 (33.7%) than that of 130 (16.0%) in controls, which was significant (p<0.001) with an OR of 2.665 (95% CI: 1.70-4.178). Smoking was associated strongly with RCC having 2.591 times more risk of developing the disease than that of healthy volunteers. The frequency of individuals with smoking habits was 54 (27.6%), less in cases than in controls [142 (72.4%)], with a p-value of 0.000 and an OR of 2.591 (95% CI: 1.594-4.210). RCC cases were more likely to have higher BMI than healthy controls. Table 1 shows significant difference (p=0.008) in mean BMI.

Table 1.

Associations Between Cigarette Smoking, Body Mass Index, and Hypertension in Renal Cell Carcinoma

Parameter	Cases (n=196)	Control (n=250)	OR	95% (CI)	p-Value^a
Age (years) (mean±SE)	54.21±0.967	54.01±0.916	—	—	0.883^b
Sex
Male	138 (70.4%)	172 (68.8%)	1.079	0.718-1.621	0.714
Female	58 (29.6%)	78 (31.2%)	1.0 (reference)
Hypertension
Present	66 (33.7%)	130 (16.0%)	2.665	1.700-4.178	0.000
Absent	40 (66.1%)	210 (84.0%)	1.0 (reference)
Smoking
Present	54 (27.6%)	32 (12.8%)	2.591	1.594-4.210	0.000
Absent	142 (72.4%)	218 (87.2%)	1.0 (reference)
BMI (mean±SE)	23.67±0.164	22.76±0.126	—	—	0.000^b
Low	69 (35.2%)	115 (46.0%)	1.0 (reference)
Middle	61 (31.1%)	82 (32.8%)	1.239	0.793-1.936	0.363
High	66 (33.7%)	53 (21.2%)	2.075	1.298-3.316	0.003

p-Value (two sided) Pearson chi-square test.

p-Value of t-test for independent samples.

OR, crude odds ratio calculated at 95% confidence interval (95% CI); BMI, body mass index.

Association of GNAS1 gene T393C polymorphism and RCC risk

The frequencies of the GNAS1 CC, TC, and TT genotypes were 29.6%, 51.5%, and 18.9% in the RCC patients, compared with 16.8%, 50.8%, and 32.4% in the controls. Strong association between the GNAS1 CC genotype and RCC risk was apparent (OR=3.023; 95% CI: 1.734-5.270; p=0.0001) in an unadjusted model. Further, multivariate logistic regression analysis also showed that there was a significant association between CC genotype and RCC (OR=2.181; 95% CI: 1.344-3.538; p=0.002). The genotype percentage of TT and combined CC/TC was 18.9% and 81.1% in cases, and 32.4% and 67.6% in controls, respectively, and this difference was statistically significant (OR=2.0597; 95% CI: 1.319-3.214; p=0.001), as evident from Table 2. Similarly, multivariate logistic regression shows significant association between the C carriers and RCC (CC/TC vs. TT, OR=2.138; 95% CI: 1.440-3.730; p=0.001). The incidence of C and T alleles of GNAS1 polymorphism was 175 (44.64%) and 217 (55.35%) in RCC group and 289 (57.8%) and 211 (42.2%) in the control group. The difference in the allele frequency was found to be statistically significant (p=0.0001, C vs. T allele, OR=1.698; 95% CI: 1.30-2.217) between the RCC cases and controls.

Table 2.

Multivariate Logistic Regression Model for Association of GNAS1 (T393C) Polymorphisms and Renal Cell Carcinoma Risk

Genotype	Cases (%)	Control (%)	OR ^a	95% CI	p-Value^b	OR ^c	95% CI	p-Value^d
GNAS1
CC	58 (29.6%)	42 (16.8%)	3.023	1.734-5.270	0.0001	2.181	1.344-3.538	0.002
TC	101 (51.5%)	127 (50.8%)	1.741	1.089-2.781	0.020	1.102	0.739-1.644	0.632
TT	37 (18.9%)	81 (32.4%)	1.0 (reference)			1.0 (reference)
CC/TC	159 (81.1%)	169 (67.6%)	2.0597	1.319-3.214	0.001	2.138	1.440-3.730	0.001
TT	37 (18.9%)	81 (32.4%)	1.0 (reference)			1.0 (reference)
T Allele	175 (44.64%)	289 (57.8%)	1.0 (reference)
C Allele	217 (55.35%)	211 (42.2%)	1.698	1.30-2.217	0.0001

Crude odds ratio calculated at 95% CI of unadjusted multivariate model.

p-Value (two sided) Fisher's exact test of unadjusted multivariate model.

Crude odds ratio calculated at 95% CI.

p-Value (two sided) Fisher's exact test.

Association of GNAS1 genotypes of RCC patients categorized with AJCC cancer stages

We also found the association between the genotypes of GNAS1 gene with the stage of RCC. Among the various genetic risk factors, presence of CC genotype (22.4%, 29.3%, and 48.3% for stage I, II, and III+IV, respectively) was significantly (p=0.005) associated with higher stages of RCC. The combined TT/TC genotype (41.3%, 31.3%, and 27.5% for stage I, II, and III+IV, respectively) showed significant trend (0.002) with lower cancer stages of RCC, as per the data in Table 3.

Table 3.

Association of Genotypes of GNAS1 (T393C) with Different Cancer Stages

Parameters	Stage I	Stage II	Stage III+IV	p-Value^a
CC	13 (22.4%)	17 (29.3%)	28 (48.3%)
TC	36 (35.6%)	33 (32.7%)	32 (31.7%)	0.005
TT	21 (56.8%)	10 (27.0%)	6 (16.2%)
TT/TC	57 (41.3%)	43 (31.2%)	38 (27.5%)
CC	13 (22.4%)	17 (29.3%)	28 (48.37%)	0.002

p-Value for chi-square test for trend.

Frequency of the GNAS1 genotypes of RCC patients categorized with different cancer grades

We also found the association between the genotypes of GNAS1 genotypes with RCC grades. The GNAS1 genotypes of RCC patients did not show any association with the Fuhrman's grades of RCC, as per the data in Table 4.

Table 4.

Association of Genotypes of GNAS1 (T393C) with Fuhrman's Grade

Parameters	Fuhrman's grade 1	Fuhrman's grade 2	Fuhrman's grade 3+4	p-Value^a
CC	12 (20.7%)	37 (63.8%)	9 (15.5%)
TC	18 (17.8%)	66 (65.3%)	17 (16.8%)	0.457
TT	12 (32.4%)	19 (51.4%)	6 (16.2%)
TT/TC	30 (21.7%)	85 (61.6%)	23 (16.7%)
CC	12 (20.7%)	37 (63.8%)	9 (15.5%)	0.992

p-Value for chi-square test for trend.

Discussion

During the last two decades until recently, there has been an annual increase of about 2% in RCC incidence worldwide (Gupta et al., 2008). Present efforts in cancer research are focused on discovery of biomarkers and genetic markers, which will ultimately enhance our knowledge to stratify patients into more distinguished risk categories, ultimately better predicting the clinical course and survival of the individual patient, especially in RCC. The main aim of the present study was to investigate whether a genetic host factor, the common T393C polymorphism in the gene GNAS1, may be predictive for survival in patients with RCC. The homozygous CC genotype was significantly associated with a decreased overall survival, while TT genotype was associated with increased survival. Multivariate analysis, including AJCC stage, confirmed the T393C polymorphism to be an independent prognostic factor for clinical outcome. Our results support earlier studies in which TT genotypes of the GNAS1 T393C polymorphism display increased transcription of G-alpha and a more favorable clinical course in bladder and clear cell RCC patients compared with both TC or CC genotypes (Frey et al., 2005b, 2006a). The analysis of the importance of GNAS1 polymorphisms for various diseases has proven difficult. As a result, only a few polymorphisms of this large gene have been studied. Our study is the first of its kind to report the association of GNAS1 genotypes with RCC progression in terms of AJCC stage and nuclear grade in a North Indian population. Cancer-specific data evaluated for each patient included stage at presentation and tumor grade.

The GNAS1 T393C polymorphism is located in exon 5 within a recombination hotspot that is in linkage equilibrium with two haplotype blocks, one located 5′ and one 3′ of the T393C polymorphism. The GNAS1 TT genotype is associated with increased Gαs mRNA expression in different tissues (Frey et al., 2005a). Therefore, genotype-dependent differences in mRNA decay due to transformed secondary structure might ultimately cause differences in Gαs mRNA expression, and increased expression of Gαs is associated with enhanced apoptosis (Frey et al., 2006a). Secondary messenger cyclic AMP, which is generated after the activation of Gαs, plays a major role in this proapoptotic process. An increased concentration of cAMP promotes apoptosis in several cell types, including leukemic cells (Myklebust et al., 1999), ovarian cancer cells (Srivastava et al., 1999), and lymphoma cells (Yan et al., 2000), and suppresses Ras-dependent activation of Raf (Chen and Iyengar, 1994). Raf inhibitor shows potential results in patients with RCC (Thompson and Lyons, 2005). It would, therefore, be of interest to investigate whether patients with specific T393C genotypes would especially benefit from novel anticancer drugs. Since we know that the C allele exerts less transcriptional activity, with the T variant being ∼1.6 folds more active, our hypothesis was that any individual having the C polymorphism will be at higher risk of developing RCC. This was in accordance to the findings of our study as well as the previous study (Frey et al., 2006b) that revealed that GNAS1 CC polymorphism was associated with higher RCC risks. The results of our study did not find any significant correlation between RCC and GNAS1 polymorphism when adjusted for tumor grade; however, significant correlation between cancer stages was seen. The limitation of this study was a relatively small sample size.

Conclusions

In conclusion, the data showed that the GNAS1 CC polymorphism was associated with higher RCC risk among the North Indian population. The main aim of this study was to understand the potential role of GNAS1 T393C gene polymorphism in the etiology of RCC in Indian subcontinent. Additional studies on a larger population size are warranted to elucidate the role of genetic variation in GNAS1 and RCC risk.

Footnotes

Acknowledgment

Dr. Sarwat Sultana is thankful to the Indian Council of Medical Research (ICMR), New Delhi, India, for providing the funds to carry out this research work.

Author Disclosure Statement

The authors of the present research work do not have any conflict of interest to present this research work.

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