Mol Biol Rep (2014) 41:799–807 DOI 10.1007/s11033-013-2919-2
Cytotoxic T lymphocyte antigen 4 (CTLA4) gene polymorphism with bladder cancer risk in North Indian population Praveen Kumar Jaiswal • Vibha Singh Rama Devi Mittal
•
Received: 11 October 2012 / Accepted: 18 December 2013 / Published online: 4 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Cytotoxic T Lymphocyte antigen 4 (CTLA4) is a potent immunoregulatory molecule that suppresses antitumor response by down-regulating T cell activation. We examined candidate disease-susceptibility single nucleotide polymorphism (SNPs) of CTLA4 at ?49A/G, CT60A/G and -318C/T genes in bladder cancer (BC) patients of North Indian population. Histopathologically confirmed 200 patients of BC and 200 unrelated, healthy controls of similar ethnicity were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP) and amplification refractory mutation specific (PCR–ARMS) methods. In present study SNP CTLA4 ?49A/G, variant genotype showed 3.74-fold risks for BC. While looking at G allele carrier level, risk for BC was high (OR = 1.54). The risk for BC was also evident in case G allele (OR = 1.58). CTLA4 CT60A/G gene polymorphism variant genotype showed 1.36-fold risks for BC. While at G allele carrier and with variant G allele it showed significantly reduced risk for BC. CTLA4 ?49A/G genotype exhibited 1.57-fold risks with smoking in BC patients in homozygous mutant condition. In silico analysis further supports the results of SNP at CTLA4 ?49A/G and CTLA4 CT60A/G. None of the above SNPs of CTLA4 demonstrated association with tumor stage/grade for BC severity and progression. BCG immunotherapy had no impact on CTLA4 gene polymorphism revealing no significant association. Haplotype GAC showed high risk for BC while other haplotype AGT showed reduced risk for BC. Our results
Praveen Kumar Jaiswal and Vibha Singh have contributed equally to this study. P. K. Jaiswal V. Singh R. D. Mittal (&) Department of Urology, SGPGIMS, Raebareli Road, Lucknow 226014, India e-mail: [email protected]; [email protected]
indicated that genetic variations in CTLA4 gene (?49A/G, CT60A/G) play role in susceptibility to BC. Haplotype GAC showed high risk for BC. An association study utilizing a larger sample size and different ethnicity warrant further investigation through replication and advance techniques. Keywords Haplotype
CTLA4 Bladder cancer risk RFLP BCG
Introduction Bladder cancer (BC) is one of the most common forms of urogenital cancer. As a multifactorial disease, both environmental and genetic factors are involved in its development and progression of bladder cancer. BC is nearly three times more common in men than in women [1]. The incidence in India is comparatively low and it is 3.2 in males and 0.7 in females/hundred thousand people [2]. Cytotoxic T lymphocyte antigen 4 (CTLA4) genes is present on chromosome 2q33, that encodes an immunoregulatory molecule. It is expressed on the surface of activated T cells, and act via interaction with the B7 molecule which further down regulates T-cell activation. Three different polymorphisms have been described in the CTLA4 gene: one in the promoter region at position -318 from the ATG start Codon consisting of a C/T change [3]; a second in position 49 of exon 1, which lies in an A/G transition resulting in a threonine (Thr) or alanine (Ala) dimorphism [4]; and third within the 30 UTR at CT60A/G [5]. CTLA4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA4 and contributes to the pathogenesis of Graves’ disease [6]. The CTLA46230G[A (CT60) polymorphism was shown to be associated with variations in the mRNA level of soluble CTLA4 [7].
123
800
Cytotoxic T lymphocyte antigen 4 (CTLA4) plays an important role in the negative regulation of T cell proliferation and activation, and thus participates in antitumor immunity and cancer. Cytotoxic T cell antigen (CTLA4) is a homologous molecule of CD28 which plays an inhibitory role in the early and late stages of T cell activation [8]. CTLA4 ligation provides a negative signal for cell cycle and inhibits the activity of the transcriptional factors such as nuclear factor-jB (NF-jB), nuclear factor of activated T cells (NF-AT) and activator protein 1 (AP-1). Moreover, CTLA4 binds to CD28 ligands (CD80 and CD86) with higher 3 affinity and avidity and thereby also inhibits T cell activation [9]. It is reported that T effecter activity could be determined by CTLA4 single nucleotide polymorphisms (SNPs). Previous investigations revealed significant associations of polymorphisms in CTLA4 gene and the susceptibility to various types of cancers such as cervical cancer [3], Breast cancer [10], non-Hodgkin’s lymphoma [11] and multiple myeloma [12]. Incidentally no study has been reported till date showing association of polymorphic marker of the CTLA4 gene and bladder cancer. Hence we selected these polymorphic markers as they have been reported to be functional and associated with altered immune responses. Polymorphism at CTLA4 ?49A/G was associated with reduction in inhibitory function of CTLA4. A49G dimorphism (Thr/Ala exchange in a peptide) leads to the expression of defective receptor, and, as a result, the inhibitory effect of CTLA4 on lymphocyte T cell activation is impaired [6]. Furthermore CTLA4 CT60A/G polymorphism had associated with differential expression of mRNA level of CTLA4 protein [7]. SNP CTLA4 -318C/T lies in promoter region of CTLA4; had a higher promoter activity and which may affect the protein formation by increased transcription of CTLA-4 mRNA and expression of CTLA-4 protein in non-stimulated and activated T cells [3]. On the basis of these findings we have proposed the hypothesis and we have taken these SNPs in our study to see whether these are associated with bladder cancer susceptibility or not. In the present study we have investigated the genotype frequencies in BC and control and evaluated the polymorphisms at three positions in CTLA4 gene at -318C/T (rs5742909), ?49A/G(rs231775) and 30 UTR at CT60A/ G(rs 3087243) in North Indian population. Further the functional effects were determined in regions of CTLA4 ?49A/G, CTLA4 CT60A/G and CTLA4 -318C/T gene by different bioinformatics prediction tool.
Materials and methods Population characteristics DNA samples used in present study were from an on-going case–control study of bladder cancer, which started patient
123
Mol Biol Rep (2014) 41:799–807
recruitment in 2008. All enrolled patients were incident cases of histological confirmed invasive or superficial bladder cancer and were recruited from the department of Urology at Sanjay Gandhi Postgraduate Institute of Medical Sciences, a tertiary care centre, from May 2009 to June 2011. A total of 200 patients with histological confirmed transitional urothelial BC (mean age 58.5 years; 175 men and 25 women) were recruited for the study. Patients having previous history of other cancer and those who have cancer metastasized to the bladder from another origin, and previous radiotherapy were excluded from the study. Healthy and genetically unrelated individuals visiting the hospital for a routine check up or health awareness camps and hospital employees were recruited as the controls (n = 200). All the healthy individuals (controls) were age and sex matched with similar ethnicity and did not show any signs of malignancy or chronic disease. The mean age of the controls was 56.8 years, and M:F ratio as 179:21. Blood samples were available for all subjects. Ethnicity was based on self-report and categorized as North Indian. An epidemiologic questionnaire was designed for study participants to collect data on demographic characteristics, smoking history, occupation history, and other lifestyle factors were employed. At the end after filling the epidemiologic questionnaire, a 5-ml blood sample was drawn into coded tubes (giving some no. for identification of samples). Informed and written consent was taken from all subjects when interviewing for the demographic details and blood sample collection. The Ethical Review Board of the Institute approved the study. Epidemiology data collection The demographic details were obtained by interviewing each individual in cases and controls. The response rate for the interview was 75 % for the subjects. Individuals who smoked once a day for more than 5 years were defined as smokers. The individuals who had never smoked in their lifetime were regarded as non smokers. At the conclusion of the interview, a 5 ml of blood sample was drawn into coded vials. Clinical data collection The demographic and clinical characteristics of the patients are presented in Table 1. The clinical information about tumor size, number, stage and tumor grade, intravesical therapy and dates of recurrence, chemotherapy, radical cystectomy and pathological findings at cystectomy were provided by the Urologist in our department. The tumor stages were classified as per American Joint Committee on Cancer’s TNM staging system [13]. Of the 200 total patients enrolled in the study, 149 patients had non muscle
Mol Biol Rep (2014) 41:799–807
801
Table 1 Demographical details of urinary bladder cancer patients and healthy controls Variable
Cases (200) n (%)
Controls (200) n (%)
v2 p-value
0.531
Sex Female
25(12.5)
21(10.5)
Male Age (years)
175(87.5)
179(89.5)
58.5 ± 12.4
56.8 ± 10.8
0.117$
Non smokers
47(30.1)
155(77.5)
Cytotoxic T lymphocyte antigen 4 (CTLA4) gene polymorphism with bladder cancer risk in North Indian population Praveen Kumar Jaiswal • Vibha Singh Rama Devi Mittal
•
Received: 11 October 2012 / Accepted: 18 December 2013 / Published online: 4 January 2014 Ó Springer Science+Business Media Dordrecht 2014
Abstract Cytotoxic T Lymphocyte antigen 4 (CTLA4) is a potent immunoregulatory molecule that suppresses antitumor response by down-regulating T cell activation. We examined candidate disease-susceptibility single nucleotide polymorphism (SNPs) of CTLA4 at ?49A/G, CT60A/G and -318C/T genes in bladder cancer (BC) patients of North Indian population. Histopathologically confirmed 200 patients of BC and 200 unrelated, healthy controls of similar ethnicity were genotyped by polymerase chain reaction and restriction fragment length polymorphism (PCR–RFLP) and amplification refractory mutation specific (PCR–ARMS) methods. In present study SNP CTLA4 ?49A/G, variant genotype showed 3.74-fold risks for BC. While looking at G allele carrier level, risk for BC was high (OR = 1.54). The risk for BC was also evident in case G allele (OR = 1.58). CTLA4 CT60A/G gene polymorphism variant genotype showed 1.36-fold risks for BC. While at G allele carrier and with variant G allele it showed significantly reduced risk for BC. CTLA4 ?49A/G genotype exhibited 1.57-fold risks with smoking in BC patients in homozygous mutant condition. In silico analysis further supports the results of SNP at CTLA4 ?49A/G and CTLA4 CT60A/G. None of the above SNPs of CTLA4 demonstrated association with tumor stage/grade for BC severity and progression. BCG immunotherapy had no impact on CTLA4 gene polymorphism revealing no significant association. Haplotype GAC showed high risk for BC while other haplotype AGT showed reduced risk for BC. Our results
Praveen Kumar Jaiswal and Vibha Singh have contributed equally to this study. P. K. Jaiswal V. Singh R. D. Mittal (&) Department of Urology, SGPGIMS, Raebareli Road, Lucknow 226014, India e-mail: [email protected]; [email protected]
indicated that genetic variations in CTLA4 gene (?49A/G, CT60A/G) play role in susceptibility to BC. Haplotype GAC showed high risk for BC. An association study utilizing a larger sample size and different ethnicity warrant further investigation through replication and advance techniques. Keywords Haplotype
CTLA4 Bladder cancer risk RFLP BCG
Introduction Bladder cancer (BC) is one of the most common forms of urogenital cancer. As a multifactorial disease, both environmental and genetic factors are involved in its development and progression of bladder cancer. BC is nearly three times more common in men than in women [1]. The incidence in India is comparatively low and it is 3.2 in males and 0.7 in females/hundred thousand people [2]. Cytotoxic T lymphocyte antigen 4 (CTLA4) genes is present on chromosome 2q33, that encodes an immunoregulatory molecule. It is expressed on the surface of activated T cells, and act via interaction with the B7 molecule which further down regulates T-cell activation. Three different polymorphisms have been described in the CTLA4 gene: one in the promoter region at position -318 from the ATG start Codon consisting of a C/T change [3]; a second in position 49 of exon 1, which lies in an A/G transition resulting in a threonine (Thr) or alanine (Ala) dimorphism [4]; and third within the 30 UTR at CT60A/G [5]. CTLA4 gene polymorphism at position 49 in exon 1 reduces the inhibitory function of CTLA4 and contributes to the pathogenesis of Graves’ disease [6]. The CTLA46230G[A (CT60) polymorphism was shown to be associated with variations in the mRNA level of soluble CTLA4 [7].
123
800
Cytotoxic T lymphocyte antigen 4 (CTLA4) plays an important role in the negative regulation of T cell proliferation and activation, and thus participates in antitumor immunity and cancer. Cytotoxic T cell antigen (CTLA4) is a homologous molecule of CD28 which plays an inhibitory role in the early and late stages of T cell activation [8]. CTLA4 ligation provides a negative signal for cell cycle and inhibits the activity of the transcriptional factors such as nuclear factor-jB (NF-jB), nuclear factor of activated T cells (NF-AT) and activator protein 1 (AP-1). Moreover, CTLA4 binds to CD28 ligands (CD80 and CD86) with higher 3 affinity and avidity and thereby also inhibits T cell activation [9]. It is reported that T effecter activity could be determined by CTLA4 single nucleotide polymorphisms (SNPs). Previous investigations revealed significant associations of polymorphisms in CTLA4 gene and the susceptibility to various types of cancers such as cervical cancer [3], Breast cancer [10], non-Hodgkin’s lymphoma [11] and multiple myeloma [12]. Incidentally no study has been reported till date showing association of polymorphic marker of the CTLA4 gene and bladder cancer. Hence we selected these polymorphic markers as they have been reported to be functional and associated with altered immune responses. Polymorphism at CTLA4 ?49A/G was associated with reduction in inhibitory function of CTLA4. A49G dimorphism (Thr/Ala exchange in a peptide) leads to the expression of defective receptor, and, as a result, the inhibitory effect of CTLA4 on lymphocyte T cell activation is impaired [6]. Furthermore CTLA4 CT60A/G polymorphism had associated with differential expression of mRNA level of CTLA4 protein [7]. SNP CTLA4 -318C/T lies in promoter region of CTLA4; had a higher promoter activity and which may affect the protein formation by increased transcription of CTLA-4 mRNA and expression of CTLA-4 protein in non-stimulated and activated T cells [3]. On the basis of these findings we have proposed the hypothesis and we have taken these SNPs in our study to see whether these are associated with bladder cancer susceptibility or not. In the present study we have investigated the genotype frequencies in BC and control and evaluated the polymorphisms at three positions in CTLA4 gene at -318C/T (rs5742909), ?49A/G(rs231775) and 30 UTR at CT60A/ G(rs 3087243) in North Indian population. Further the functional effects were determined in regions of CTLA4 ?49A/G, CTLA4 CT60A/G and CTLA4 -318C/T gene by different bioinformatics prediction tool.
Materials and methods Population characteristics DNA samples used in present study were from an on-going case–control study of bladder cancer, which started patient
123
Mol Biol Rep (2014) 41:799–807
recruitment in 2008. All enrolled patients were incident cases of histological confirmed invasive or superficial bladder cancer and were recruited from the department of Urology at Sanjay Gandhi Postgraduate Institute of Medical Sciences, a tertiary care centre, from May 2009 to June 2011. A total of 200 patients with histological confirmed transitional urothelial BC (mean age 58.5 years; 175 men and 25 women) were recruited for the study. Patients having previous history of other cancer and those who have cancer metastasized to the bladder from another origin, and previous radiotherapy were excluded from the study. Healthy and genetically unrelated individuals visiting the hospital for a routine check up or health awareness camps and hospital employees were recruited as the controls (n = 200). All the healthy individuals (controls) were age and sex matched with similar ethnicity and did not show any signs of malignancy or chronic disease. The mean age of the controls was 56.8 years, and M:F ratio as 179:21. Blood samples were available for all subjects. Ethnicity was based on self-report and categorized as North Indian. An epidemiologic questionnaire was designed for study participants to collect data on demographic characteristics, smoking history, occupation history, and other lifestyle factors were employed. At the end after filling the epidemiologic questionnaire, a 5-ml blood sample was drawn into coded tubes (giving some no. for identification of samples). Informed and written consent was taken from all subjects when interviewing for the demographic details and blood sample collection. The Ethical Review Board of the Institute approved the study. Epidemiology data collection The demographic details were obtained by interviewing each individual in cases and controls. The response rate for the interview was 75 % for the subjects. Individuals who smoked once a day for more than 5 years were defined as smokers. The individuals who had never smoked in their lifetime were regarded as non smokers. At the conclusion of the interview, a 5 ml of blood sample was drawn into coded vials. Clinical data collection The demographic and clinical characteristics of the patients are presented in Table 1. The clinical information about tumor size, number, stage and tumor grade, intravesical therapy and dates of recurrence, chemotherapy, radical cystectomy and pathological findings at cystectomy were provided by the Urologist in our department. The tumor stages were classified as per American Joint Committee on Cancer’s TNM staging system [13]. Of the 200 total patients enrolled in the study, 149 patients had non muscle
Mol Biol Rep (2014) 41:799–807
801
Table 1 Demographical details of urinary bladder cancer patients and healthy controls Variable
Cases (200) n (%)
Controls (200) n (%)
v2 p-value
0.531
Sex Female
25(12.5)
21(10.5)
Male Age (years)
175(87.5)
179(89.5)
58.5 ± 12.4
56.8 ± 10.8
0.117$
Non smokers
47(30.1)
155(77.5)
