Original Article
Association of Interleukin‐10 Promoter Polymorphism (‐1082 G/A) and Gastric Cancer in Andhra Pradesh Population of South India Amar Chand‐Bhayal1, Devulapalli Krishnaveni1, Kondadasula Pandu‐Ranga‐Rao2, Boddu Prabhakar2, Abbagani Vidyasagar3, Bal Murali‐Krishna2, Penchikala Anita2, Akka Jyothy1, Pratibha Nallari4, Ananthapur Venkateshwari1
Abstract Background: Gastric Cancer (GC) is one of the most commonly diagnosed malignancies. Genetic variation in genes encoding cytokines and their receptors, determine the intensity of the inflammatory response, which may contribute to individual differences in the outcome and severity of the disease. Interleukin-10 (IL-10) is a multifunctional cytokine with both immunosuppressive and antiangiogenic functions. Polymorphisms in the IL-10 gene promoter genetically determine inter-individual differences in IL-10 production. In the present study, we investigated the association between the IL-10 –1082 G/A polymorphism and the susceptibility to gastric cancer in a South Indian population from Andhra Pradesh. Methods: We genotyped 100 patients diagnosed with gastric cancer and 132 healthy control subjects for -1082G/A single nucleotide polymorphism by Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR) method followed by agarose gel electrophoresis.
1. Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad, India 2. Dept. of Gastroenterology, Osmania General Hospital, Dr. NTR University of Health Sciences, Hyderabad, India 3. Dept. of Gastroenterology, Gandhi Hospital, Dr. NTR University of Health Sciences, Sec-bad, India 4. Dept. of Genetics, Osmania University, Hyderabad, India
Results: The distribution of IL-10 genotypes at -1082 G/A were GG 18 %, GA 35% and AA 47 % in gastric cancer patients and GG 31.82 %, GA 37.88 % and AA 30.3% in control subjects. The allelic frequencies of G and A were 0.355 and 0.645 in GC patients and 0.508 and 0.492 in control subjects respectively. The IL-10 -1082 A allele was associated with risk of gastric cancer (OR=1.873, 95%CI-1.285-2.73and P= 0.001048**). Conclusion: Our study indicates that allele A of IL-10-1082 G/A polymorphism may be considered as one of the important risk factor in the etiology of gastric cancer. Keywords: Cytokines; Interleukin-10; Gastric cancer; Polymorphism Please cite this article as: Bhayal AC, Krishnaveni D, Pandu Ranga Rao K, Prabhakar B, Vidyasagar A, Krishna BM, Anita P, Jyothy A, Nallari P, Venkateshwari A. Association of Interleukin-10 Promoter Polymorphism (-1082 G/A) and Gastric Cancer in Andhra Pradesh Population of South India. Iran J Cancer Prev. 2012;5(3):117-23.
Introduction Gastric Cancer (GC) is one of the most commonly diagnosed malignancies and remains a considerable public health problem worldwide. Gastric cancer is the fourth most common cancer and the second leading cause of death from cancer, after lung cancer causing nearly one million deaths worldwide per year [1]. Gastric cancer incidence shows considerable variation in geography, though a male preponderance is reported. Age standardized incidence is highest in countries in Eastern Asia,
Corresponding Author: Ananthapur Venkateshwari, Ph.D; Assistant Professor of Genetics Tel: (+91) 40 23 40 36 81 E-mail: [email protected] Received: 18 Jan. 2012 Accepted: 7 Apr. 2012 Iran J Cancer Prev 2012; 3:117-123
Eastern Europe, and in some countries in central South America (overall age standardized rates 2035/100,000). The incidence is low in North America, Western Europe (A) have been reported to influence IL-10 expression and to be associated with increased risk of gastric cancer. It has been reported that IL-101082G/A polymorphism is correlated with the expression of IL-10 and accordingly affects the susceptibility to some types of tumors, such as cervical cancer and prostate cancer. The association of -1082 alleles G and A with a low (AA), high (GG) and medium (GA) IL-10 production were shown by in vivo and in vitro studies [10- 12- 13]. In the present study, we investigated the association between the IL-10-1082 G/A (rs1800896) polymorphism and the susceptibility with gastric cancer in Andhra Pradesh population of South India.
118
Materials and Methods Subject A total of 100 endoscopically and histopathologically confirmed gastric cancer patients in the age group of 22-70 years, referred to the Department of Gastroenterology, Osmania General Hospital, Hyderabad and Department of Gastroenterology, Gandhi Hospital, Secunderabad were considered for the present study. One hundred thirty two healthy controls with no family history of gastric ulcer or cancer were selected randomly. A structured questionnaire was used to elicit information on epidemiological factors such as age, sex, dietary habits, weight, addictions; family history of cancer etc. All the patients were tested for Helicobacter pylori infectivity status on antral biopsies by urease test following the method of Vaira et al. [14]. The study was approved by the Institutional Ethical Committee and informed consent was obtained from all recruited subjects. DNA Extraction Five ml of blood was collected from each subject in vacutainers with anticoagulant Ethylenediamine Tetra Acetic Acid (EDTA). Genomic DNA was isolated from whole-blood samples of all the patients and control subjects, by the salting out procedure of Lahiri and Nurnberger [15]. IL-10-1082G/A Genotyping by ARMS-PCR IL-10 -1082 G/A polymorphism genotyping was carried out with Allele Refractory Mutation detection System–Polymerase Chain Reaction (ARMS-PCR) [16]. Allele A was amplified with Forward Sense Primer FSP-A (5' -AAC ACT ACT AAG GCT TCT TTG GGT A-3') and allele G was amplified with the primer FSP-G (5’-AAC ACT ACT AAG GCT TCT TTG GGT G -3'). The primer RP-CAS (5'-GTA AGC TTC TGT GGC TGG AGT C-3') was used as reverse primer (common antisense) in both the reactions. These reactions amplify allele specific sequence of 161 bp of the promoter of IL-10 gene. Internal control primers amplifying a 796-bp fragment from the third intron of the HLA-DRB1 gene were included in each reaction. Sequences of internal control primer are as follows: Internal Control 1: 5’ TGC CAA GTG GAG CAC CCA A 3’ and Internal Control 2: 5’ GCA TCT TGC TCT GTG CAG AT 3’. PCR was performed in a total volume of 10µl with 100 ng of total genomic DNA, 1X reaction buffer with1.5 mM MgCl2, 200µM of each dNTPs, and 0.5 µM of each primer and 0.8 IU of Taq polymerase. The cycling conditions were as follows: an initial denaturation at 950C for 5 minutes, followed by 35 cycles at 950C Iranian Journal of Cancer Prevention
Association of Interleukin-10 Promoter Polymorphism (-1082 G/A) and....
Table 1. Demographic Details of Gastric Cancer Patients and Controls GC cases (N=100) Controls (N=132) Variable N (%) N (%) OR (95%CI) P-value† ____________________________________________________________________________ Gender Male 68 (68) 96 (72.73) Female 32 (32) 36 (27.27) 0.797 (0.451-1.407) 0.433 Age (years) ≤ 40 Years 18 (18) 72 (54.45) >40 Years 82 (82) 60 (45.55) 0.182 (0.099-0.338)
Association of Interleukin‐10 Promoter Polymorphism (‐1082 G/A) and Gastric Cancer in Andhra Pradesh Population of South India Amar Chand‐Bhayal1, Devulapalli Krishnaveni1, Kondadasula Pandu‐Ranga‐Rao2, Boddu Prabhakar2, Abbagani Vidyasagar3, Bal Murali‐Krishna2, Penchikala Anita2, Akka Jyothy1, Pratibha Nallari4, Ananthapur Venkateshwari1
Abstract Background: Gastric Cancer (GC) is one of the most commonly diagnosed malignancies. Genetic variation in genes encoding cytokines and their receptors, determine the intensity of the inflammatory response, which may contribute to individual differences in the outcome and severity of the disease. Interleukin-10 (IL-10) is a multifunctional cytokine with both immunosuppressive and antiangiogenic functions. Polymorphisms in the IL-10 gene promoter genetically determine inter-individual differences in IL-10 production. In the present study, we investigated the association between the IL-10 –1082 G/A polymorphism and the susceptibility to gastric cancer in a South Indian population from Andhra Pradesh. Methods: We genotyped 100 patients diagnosed with gastric cancer and 132 healthy control subjects for -1082G/A single nucleotide polymorphism by Amplification Refractory Mutation System-Polymerase Chain Reaction (ARMS-PCR) method followed by agarose gel electrophoresis.
1. Institute of Genetics and Hospital for Genetic Diseases, Osmania University, Begumpet, Hyderabad, India 2. Dept. of Gastroenterology, Osmania General Hospital, Dr. NTR University of Health Sciences, Hyderabad, India 3. Dept. of Gastroenterology, Gandhi Hospital, Dr. NTR University of Health Sciences, Sec-bad, India 4. Dept. of Genetics, Osmania University, Hyderabad, India
Results: The distribution of IL-10 genotypes at -1082 G/A were GG 18 %, GA 35% and AA 47 % in gastric cancer patients and GG 31.82 %, GA 37.88 % and AA 30.3% in control subjects. The allelic frequencies of G and A were 0.355 and 0.645 in GC patients and 0.508 and 0.492 in control subjects respectively. The IL-10 -1082 A allele was associated with risk of gastric cancer (OR=1.873, 95%CI-1.285-2.73and P= 0.001048**). Conclusion: Our study indicates that allele A of IL-10-1082 G/A polymorphism may be considered as one of the important risk factor in the etiology of gastric cancer. Keywords: Cytokines; Interleukin-10; Gastric cancer; Polymorphism Please cite this article as: Bhayal AC, Krishnaveni D, Pandu Ranga Rao K, Prabhakar B, Vidyasagar A, Krishna BM, Anita P, Jyothy A, Nallari P, Venkateshwari A. Association of Interleukin-10 Promoter Polymorphism (-1082 G/A) and Gastric Cancer in Andhra Pradesh Population of South India. Iran J Cancer Prev. 2012;5(3):117-23.
Introduction Gastric Cancer (GC) is one of the most commonly diagnosed malignancies and remains a considerable public health problem worldwide. Gastric cancer is the fourth most common cancer and the second leading cause of death from cancer, after lung cancer causing nearly one million deaths worldwide per year [1]. Gastric cancer incidence shows considerable variation in geography, though a male preponderance is reported. Age standardized incidence is highest in countries in Eastern Asia,
Corresponding Author: Ananthapur Venkateshwari, Ph.D; Assistant Professor of Genetics Tel: (+91) 40 23 40 36 81 E-mail: [email protected] Received: 18 Jan. 2012 Accepted: 7 Apr. 2012 Iran J Cancer Prev 2012; 3:117-123
Eastern Europe, and in some countries in central South America (overall age standardized rates 2035/100,000). The incidence is low in North America, Western Europe (A) have been reported to influence IL-10 expression and to be associated with increased risk of gastric cancer. It has been reported that IL-101082G/A polymorphism is correlated with the expression of IL-10 and accordingly affects the susceptibility to some types of tumors, such as cervical cancer and prostate cancer. The association of -1082 alleles G and A with a low (AA), high (GG) and medium (GA) IL-10 production were shown by in vivo and in vitro studies [10- 12- 13]. In the present study, we investigated the association between the IL-10-1082 G/A (rs1800896) polymorphism and the susceptibility with gastric cancer in Andhra Pradesh population of South India.
118
Materials and Methods Subject A total of 100 endoscopically and histopathologically confirmed gastric cancer patients in the age group of 22-70 years, referred to the Department of Gastroenterology, Osmania General Hospital, Hyderabad and Department of Gastroenterology, Gandhi Hospital, Secunderabad were considered for the present study. One hundred thirty two healthy controls with no family history of gastric ulcer or cancer were selected randomly. A structured questionnaire was used to elicit information on epidemiological factors such as age, sex, dietary habits, weight, addictions; family history of cancer etc. All the patients were tested for Helicobacter pylori infectivity status on antral biopsies by urease test following the method of Vaira et al. [14]. The study was approved by the Institutional Ethical Committee and informed consent was obtained from all recruited subjects. DNA Extraction Five ml of blood was collected from each subject in vacutainers with anticoagulant Ethylenediamine Tetra Acetic Acid (EDTA). Genomic DNA was isolated from whole-blood samples of all the patients and control subjects, by the salting out procedure of Lahiri and Nurnberger [15]. IL-10-1082G/A Genotyping by ARMS-PCR IL-10 -1082 G/A polymorphism genotyping was carried out with Allele Refractory Mutation detection System–Polymerase Chain Reaction (ARMS-PCR) [16]. Allele A was amplified with Forward Sense Primer FSP-A (5' -AAC ACT ACT AAG GCT TCT TTG GGT A-3') and allele G was amplified with the primer FSP-G (5’-AAC ACT ACT AAG GCT TCT TTG GGT G -3'). The primer RP-CAS (5'-GTA AGC TTC TGT GGC TGG AGT C-3') was used as reverse primer (common antisense) in both the reactions. These reactions amplify allele specific sequence of 161 bp of the promoter of IL-10 gene. Internal control primers amplifying a 796-bp fragment from the third intron of the HLA-DRB1 gene were included in each reaction. Sequences of internal control primer are as follows: Internal Control 1: 5’ TGC CAA GTG GAG CAC CCA A 3’ and Internal Control 2: 5’ GCA TCT TGC TCT GTG CAG AT 3’. PCR was performed in a total volume of 10µl with 100 ng of total genomic DNA, 1X reaction buffer with1.5 mM MgCl2, 200µM of each dNTPs, and 0.5 µM of each primer and 0.8 IU of Taq polymerase. The cycling conditions were as follows: an initial denaturation at 950C for 5 minutes, followed by 35 cycles at 950C Iranian Journal of Cancer Prevention
Association of Interleukin-10 Promoter Polymorphism (-1082 G/A) and....
Table 1. Demographic Details of Gastric Cancer Patients and Controls GC cases (N=100) Controls (N=132) Variable N (%) N (%) OR (95%CI) P-value† ____________________________________________________________________________ Gender Male 68 (68) 96 (72.73) Female 32 (32) 36 (27.27) 0.797 (0.451-1.407) 0.433 Age (years) ≤ 40 Years 18 (18) 72 (54.45) >40 Years 82 (82) 60 (45.55) 0.182 (0.099-0.338)
