Priyanka Srivastava

Association of Promoter Polymorphisms in MMP2 and TIMP2 with Prostate Cancer Susceptibility in North India

BACKGROUND AND AIMS The importance of matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) in tumor progression is well documented. MMP2/TIMP2 system has a significant impact on the development and progression of cancer and genetic polymorphisms in the promoters of MMP2 (-1306C/T, 735C/T) and TIMP2 (-418G/A, -303C/T) are correlated with decreased enzyme activity. We sought to determine whether genetic polymorphisms in MMP2 and TIMP2 polymorphisms may be associated with varying risk of prostate cancer (PCa) in men in North India. METHODS Genotyping was done by PCR-restriction fragment length polymorphism method in 190 histologically confirmed PCa patients and 200 unrelated, healthy, age-matched individuals of similar ethnicity. RESULTS Patients with MMP2 (-1306) CT genotype as well as T allele were at higher risk of PCa (p = 0.018; OR = 1.68 and p = 0.015; OR = 1.52). This effect was even more evident in the case of the T allele carrier (CT + TT) (p = 0.011; OR = 1.71). MMP2 (735) C>T, TIMP2 (-418) G>C and TIMP2 (-303) C>T polymorphism demonstrated no association. However, TIMP2 (-418) GC was found to be involved in progression of PCa but not in initiation. Haplotype results demonstrated that MMP2 (1306T-735C) and TIMP2 (418G-303T) were associated with a 1.5- and 1.8-fold increased risk, respectively. CONCLUSIONS Our data indicated that MMP2-1306C>T gene polymorphism contributes to PCa susceptibility. These findings suggested MMP2 variants as a predictor of PCa progression risk among North Indian men. We assume that analysis of these gene polymorphisms can help identify patient subgroups at high risk of poor disease outcome.

Association of IL-12, IL-18 variants and serum IL-18 with bladder cancer susceptibility in North Indian population.

IL-12 and IL-18 are immunomodulatory cytokines that play important roles in host immune response against cancers. Variation in DNA sequence in gene promoter may lead to altered IL-18 production and/or activity, and hence can modulate an individuals susceptibility to BC. To test this hypothesis, we investigated the relationship of IL-18 gene promoter -137 G/C and -607C/A polymorphisms and IL12 (-16974) A/C with the risk of BC in North Indian population. Polymorphisms in IL-18 and IL-12 genes were analyzed in 200 BC patients and 200 age, ethnicity and sex-matched controls, using restriction fragment length polymorphism-polymerase chain reaction (PCR-RFLP) and amplification refractory mutation specific-polymerase chain reaction (ARMS) method. The concentrations of IL-18 in serum were determined by ELISA. Significant association was observed with IL18 (-137) G/C heterozygous genotype (GC) with 1.96 folds risk of BC as well at C allele carrier and variant C allele having 2 fold and 1.6 fold risk for BC respectively. IL18 (-607) C/A, heterozygous CA genotype also showed a high risk (OR=1.59) for BC. While IL12 (-16974) A/C heterozygote genotype and C allele carrier demonstrated reduced risk of BC. Hetero genotype of IL18 (-137) G/C was associated with risk of recurrence (HR=2.35) in superficial BC patients receiving BCG treatment thus showing least survival. The distributions of IL-18 gene haplotypes were not significantly different between patients and controls. Serum IL-18 levels were significantly higher in BC patients than in the healthy subjects (p=0.025). Serum IL-18 levels was also significantly associated with IL18 (-137) G/C in heterozygous genotype (GC) (p=0.048). Our results suggest that IL-18 gene polymorphism contributes to bladder cancer risk whereas IL-12 is protective. A relation between IL18 (-137) G/C in heterozygous genotype with elevated IL-18 serum level and bladder cancer risk has been registered in the present study.

Gene variants of XRCC4 and XRCC3 and their association with risk for urothelial bladder cancer

The DNA double strand break repair gene XRCC4, an important caretaker of genome stability and XRCC3 are suggested to play an imperative role in the development of carcinogenesis. However, no evidence has been provided showing that these genes are associated with risk of urinary bladder cancer (UBC). The study was designed to examine the polymorphisms associated with two genes namely XRCC4 G1394T (rs6869366), intron 3 (rs28360317), intron 7 rs1805377 and rs2836007 and XRCC3 (rs861539 and rs1799796), respectively and investigate their role as susceptible markers for UBC risk in North Indian cohort. In this hospital-based case–control study histologically confirmed 211 UBC patients and 244 age and gender matched controls of similar ethnicity were genotyped by means of PCR-RFLP. Significant different distributions in the frequency of the XRCC4 intron 3 genotype, but not the XRCC4 G1394T or intron 7 genotypes, between the UBC and control groups were observed. XRCC4 intron 7 Del/Del conferred enhanced risk (OR 1.94; P 0.017) in UBC. Interestingly, XRCC −1394 G>T variant genotype GG was associated with reduced risk (OR 0.27; P 0.020). However, none of the four polymorphisms in XRCC4 were associated with tobacco smoking and risk of recurrence in patients treated with BCG immunotherapy. Similarly, none of the XRCC3 polymorphisms were associated with UBC susceptibility. Our results suggested that the XRCC4 intron 3 rs6869366 genotype and intron 7 rs28360317 may be associated with UBC risk and may be a novel useful marker for primary prevention and anticancer intervention.

Bladder cancer risk associated with genotypic polymorphism of the matrix metalloproteinase-1 and 7 in North Indian population

Matrix metalloproteinases (MMPs) contribute to tumor invasion and microenvironment, hence are associated with bladder cancer risk. We therefore, tested whether polymorphisms in MMP genes modify the risk of bladder cancer (BC) and whether smoke exposure modifies this risk. Genotyping was performed in 200 BC patients and 200 controls by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). MMP1-1607 2G/2G and MMP7-181 GG genotype were associated with increased risk of BC (p <0.001; OR, 3.04; 95% CI1.71–5.39 and p, 0.005; OR, 2.38; 95% CI1.30–4.34) respectively. Smokers in BC patients showed significant increased risk for the same SNPs (p, 0.006; OR, 3.20; 95% CI1.40–7.31 and p, 0.009; OR, 2.85; 95% CI1.30–6.23 respectively). Haplotype analysis too revealed significant association with G/2G of MMP1-519-1607 (p < 0.001; OR, 2.62; 95% CI1.68–4.09). The 2G allele carrier (1G/2G + 2G/2G) of MMP1-1607 showed a protective effect and high recurrence free survival in Bacillus Calmette-Guerin (BCG) treated non muscle invasive BC (NMIBC) patients (log rank p, 0.030). Our datasuggested that MMP11607 2G and MMP7181 G allele were associated with high risk of BC, which was quite evident amongst smokers too. BCG treated NMIBC patients reflected protective effect for 2G allele carrier (1G/2G+2G/2G) of MMP1-1607. This study provided new support for the association of MMP1-1607 and MMP7-181 in bladder cancer development, the tumorigenic effect of which was observed to be more enhanced in case of tobacco exposure.

Matrix metalloproteinase (MMP-9 and MMP-2) gene polymorphisms influence allograft survival in renal transplant recipients

BACKGROUND Matrix metalloproteinases (MMPs), especially basement membrane-degrading MMP-9 and MMP-2, play an important role in immune-mediated tissue destruction of the allograft by allowing influx of leucocytes and mononuclear cells into the graft. The present study was, therefore, undertaken to investigate the association of functional polymorphisms in MMP-9 (836A>G, 1721G>C and 2003G>A) and MMP-2 (-735C>T) genes with risk of allograft rejection in renal transplant recipients of North India. METHODS Three hundred and six renal transplant recipients categorized into 228 non-rejecters and 78 rejecters were genotyped for MMP-9 and MMP-2 polymorphisms by polymerase chain reaction-restriction fragment length polymorphism methodology. RESULTS A significant difference in genotype/allele frequencies was observed between healthy individuals in comparison to non-rejecters/rejecters for MMP-9 836A>G, MMP-9 2003G>A and MMP-2 -735C>T. Mutant allele carriers for MMP-9 2003G>A [GA+AA-; odds ratio (OR) = 0.45, 95% confidence intervals (95% CI) = 0.24-0.85, P = 0.014] and MMP-2 -735C>T (CT+TT-; OR = 0.40, 95% CI = 0.18-0.91, P = 0.029) demonstrated significantly reduced risk for allograft rejection. The mean time to first rejection episode was significantly higher in GA/AA and CT/TT genotype recipients for MMP-9 2003G>A (log P = 0.026) and MMP-2 -735C>T (log P = 0.003), respectively. Haplotypes with mutant alleles for MMP-9 1721C>G-2003G>A and mutant allele genotype combinations for both MMP-9 2003G>A and MMP-2 -735C>T conferred significantly reduced risk for allograft rejection. CONCLUSION Mutant alleles for MMP-9 2003G>A and MMP-2 -735C>T are associated with reduced risk for allograft rejection and improved allograft survival in North Indian transplant recipients and could serve as an ideal marker to predict pre-transplant allograft outcome.

Mutations in patients with osteogenesis imperfecta from consanguineous Indian families

Osteogenesis imperfecta (OI) is a spectrum of genetic disorders with decreased bone density and bone fragility. Most of the cases of OI are inherited in autosomal dominant fashion with mutations in COL1A1 or COL1A2 genes. Over last few years, twelve genes for autosomal recessive OI have been identified. In this study we have evaluated seven patients with OI from consanguineous Indian families. Homozygosity mapping using SNP microarray was done and selected candidate genes were sequenced. Candidate genes were identified in four out of seven patients studied. Four mutations, namely; a homozygous non-sense (p.Q178*) and a deletion (p.F277del) mutations in SERPINF1 gene, a missense mutation (p.M101K) in PPIB gene and a nonsense mutation (p.E45*) in CRTAP gene were identified. In three patients for whom the regions of homozygosity did not reveal any known autosomal recessive OI genes, exome sequencing was performed and we identified a known missense mutation (p.G1012S) in COL1A2 gene in one of the patients. As WNT1 gene was not properly covered in exome sequencing in one patient, the gene was sequenced and a homozygous in-frame deletion of four amino acids (p.Phe176_Leu179del) was identified. In one of the three cases the exome sequencing did not reveal a mutation in any known OI genes, suggesting the possibility of mutations in an unidentified gene. The phenotypes of all the cases are described. This work proves the power of homozygosity mapping followed by candidate gene sequencing approach for clinical application in consanguineous families.

Association of Common Variants of Vascular Endothelial Growth Factor and Interleukin-18 Genes with Allograft Survival in Renal Transplant Recipients of North India

Increased vascular endothelial growth factor (VEGF) production promotes enhanced endothelial permeability, enhanced leukocyte migration into the allograft, thereby leading to a clinically recognized rejection episode. Interleukin-18 (IL-18), a potent proinflammatory cytokine, may also be involved in mechanisms of kidney allograft rejection. The present study was, therefore, undertaken to investigate the association of functional polymorphisms in VEGF (2578C>A, 1154A>G) and IL-18 (607C>A, 137G>C) genes with risk of allograft rejection in renal transplant recipients of North India. Two hundred renal transplant recipients, 150 matched recipients-donors, and 200 unrelated healthy individuals were genotyped by amplification refractory mutation specific polymerase chain reaction and by polymerase chain reaction-restriction fragment length polymorphism. Variant allele VEGF 1154A>G (p = 0.56; odds ratio [OR] = 1.32) and variant allele (p = 0.004, OR = 1.54) and variant genotype (p = 0.007, OR = 3.26) of IL-18 607C>A, GC of IL-18 137G>C (p = 0.043, OR = 0.63) were significantly different in healthy individuals as compared with the patients with renal transplant. When 114 nonrejectors were compared with 36 rejectors (150 recipients) for association with allograft rejection, significant association was observed in heterozygous genotype of VEGF 2578C>A (p = 0.033), VEGF 1154A>G (p = 0.024). In IL-18 137G>C, CC genotype, C allele showed protective association with allograft rejection. Kaplan-Meier analysis indicated a higher mean time for first rejection episode in CA genotype carriers (31 months) as compared with AA (29 months) for VEGF 2578C>A (log p = 0.035). In VEGF, the haplotypes A-A and A-G (2578-1154) were associated with reduced risk and in IL-18 607A-137G, they were associated with high risk for allograft rejection. This observation suggests these polymorphisms are an ideal marker for prediction of pretransplant allograft outcome.

Role of p53 gene polymorphism and bladder cancer predisposition in northern India

p53 is a major orchestrator of cellular response to a broad array of stress types by regulating apoptosis, cell cycle arrest, etc. A few polymorphic sites, one at codon72 of exon4, intron3 16bpdel/ins, intron6G>A have been studied with regard to Bladder cancer (BC) risk in North Indians. Genotypes were assessed in hospital-based case-control study comprising of 200BC cases, 265healthy controls. After extraction of genomic DNA from blood, genotyping was done using PCR Restriction Fragment Length Polymorphism. Individuals with p53R72P G>C, CC genotype demonstrated marginally reduced risk of BC (p=0.053, OR=0.29, 95% 16bp-ins/del.

Association studies of Toll-like receptor gene polymorphisms with allograft survival in renal transplant recipients of North India

Srivastava P, Singh A, Kesarwani P, Jaiswal PK, Singh V, Mittal RD. Association studies of Toll‐like receptor gene polymorphisms with allograft survival in renal transplant recipients of North India.

Novel mutations in the transmembrane natriuretic peptide receptor NPR-B gene in four Indian families with acromesomelic dysplasia, type Maroteaux

Acromesomelic dysplasia, type Maroteaux is a disorder characterized by disproportionate short stature predominantly affecting the middle and distal segments of the upper and lower limbs. It is an autosomal recessive disorder due to mutation in NPR2 gene which impairs skeletal growth. To screen the mutations in the gene NPR2, all of its coding exons and splice junction sites were PCR amplified from genomic DNA of affected individuals of four families and sequenced. Four homozygous mutations in four different families were identified. These include three novel mutations including a deletion frameshift mutation (p.Cys586Ter), one nonsense mutation (p.Arg479Ter), one missense mutation (p.Val187Asp) and one reported missense mutation (p.Tyr338Cys). The study describes phenotypes of Indian patients and expands the mutation spectrum of the disorder.