Nisha Thakur

TNFα–308G/A Polymorphism as a Risk Factor for HPV Associated Cervical Cancer in Indian Population

Background: Investigation of the potential association of single nucleotide polymorphisms (SNPs) at –308 G/A and –238 G/A of Tumor necrosis factor α (TNFα) with susceptibility to HPV-16 associated cervical cancer in Indian women. Methods: The study included 165 histologically confirmed cases with 45 precancer and 120 cancer patients and an equal number (165) of healthy controls with normal cervical cytology. PCR-RFLP was employed to analyze TNFα promoter polymorphisms, which were confirmed by direct sequencing. Both patients and controls were screened for Human Papillomavirus (HPV) infection. Results: The frequency of –308 A allele in TNFα was significantly higher in cases compared with control subjects (21% in cases vs. 9% in controls; p < 0.01), with an odds ratio of 2.7 (95% CI = 1.41–5.15). Also, women carrying A allele for this locus presented 3 times increased susceptibility to HPV 16 infection as evident from carrier genotype distribution between HPV positive cases and control subjects (24% in HPV positive cases vs. 9% in controls; p < 0.01; OR = 3.1; 95% CI = 1.60–6.03). No such association was found for TNFα–238 (G/A) polymorphism with the risk of development of cervical cancer. Conclusion: It suggests that SNP at –308 (G/A) of TNFα promoter may represent an increased risk for HPV infection and development of cervical cancer in Indian women.

Association between human leukocyte antigen class II alleles and human papillomavirus-mediated cervical cancer in Indian women.

We investigated the association of human leukocyte antigen (HLA) II (DRB1 and DQB1) alleles with susceptibility to human papillomavirus (HPV)-associated cervical precancer and cancer cases in a hospital-based case-control study in a northern Indian population. A total of 202 subjects, including 100 patients comprising 31 cervical precancer (cervical intraepithelial neoplasia [CIN] 2/3) and 69 invasive cervical cancer cases, and 102 healthy controls participated in the study. Both patients and controls were screened for HPV infection using a polymerase chain reaction (PCR-based approach. Low-resolution PCR-sequence specific priming (PCR-SSP) was used to genotype HLA II (DRB1 and DQB1). Our results demonstrate that the DRB1*15 allele/DRB1*15-DQB1*06 haplotype may have a predisposition for HPV infection (p(c) < 0.05) or cervical cancer/precancer (p(c) < 0.05) development, whereas the DRB1*04 allele/DRB1*04-DQB1*03 haplotype might exhibit susceptibility to cervical precancerous lesions (p(c) < 0.05). The DRB1*13 allele/DRB1*13-DQB1*06 haplotype was strongly protective against risk to HPV infection (p(c) < 0.002) as well as cervical cancer (p(c) 0.01). Therefore, we have demonstrated that HLA DR-DQ polymorphisms are involved in genetic susceptibility to cervical cancer or HPV infection in a northern Indian population.

Effect of aberrant promoter methylation of FHIT and RASSF1A genes on susceptibility to cervical cancer in a North Indian population

Abstract As current evidence suggests the involvement of epigenetic modification of tumour suppressor genes in human cancer, we investigated the aberrant promoter methylation of FHIT and RASSF1A genes in human papillomavirus (HPV)-mediated cervical cancer in Indian women. We analysed 60 cervical cancer tissue biopsies of different clinical stage and histological grading and 23 healthy control samples with normal cervical cytology. Methylation-specific polymerase chain reaction (MSP) was performed to analyse the methylation status of FHIT and RASSF1A genes and confirmed by sequencing. Both patients and controls were screened for HPV infection and 98% of the HPV-infected cases showed positivity for HPV type 16. Aberrant promoter methylation of the FHIT gene was found in 28.3% (17/60) of cases and of the RASSF1A gene in 35.0% (21/60) of cases; promoter methylation of both the genes was found in 13.3% (8/60) of cervical cancer cases. Methylation was significantly (p<0.01) associated with the cervical cancer cases compared with controls. None of the 23 controls was found to be methylated in either of these genes. This is the first study indicating a correlation between the promoter methylation of FHIT and RASSF1A genes and the clinical stage and histological grading of cervical carcinoma in Indian women. Future studies are underway to examine the practical implications of these findings for use as a biomarker.

Association of MDM2 and p53 Polymorphisms with the Advancement of Cervical Carcinoma.

Cervical cancer is one of the most common gynecological malignancies that causes a serious health problem worldwide. The aim of the present study was to analyze the association of p53 codon72 (arginine/proline) polymorphism (rs1042522) and Murine Double Minute 2 (MDM2) SNP309 T/G (rs2279744) with the advancement of cervical cancer by using polymerase chain reaction-restriction fragment length polymorphism method followed by direct sequencing. The frequencies of GG genotype at 309 position in the second promoter (P2) of MDM2 and Arginine in codon72 of p53 were found to be 3.5 (odds ratio [OR]=3.51; 95% confidence interval [CI]=1.93-6.4; p<0.0001) and 5 (OR=4.978; 95% CI=2.7-9.2; p<0.0001) fold higher, respectively, in cases than in the control. On gene-gene interactions between MDM2 and p53 polymorphisms, the frequency of MDM2 G/G and p53 Arg/Arg together was found to be 6.5-fold higher in cervical cancer patients compared with healthy controls (OR=6.497; 95% CI=2.987-14.13; p<0.0001). We found an association of p53 codon72 arginine and MDM2 SNP309 GG genotype with different clinical and histological grades, human papillomavirus (HPV) infection, and age at the time of diagnosis of cervical cancer. In conclusion, Arginine at codon72 of p53 and GG genotype at 309 in P2 of MDM2 together reveal a direct proportionality with the tumor grade of cervical cancer along with HPV infection in postmenopausal women.

Homocysteine levels are associated with cervical cancer independent of methylene tetrahydrofolate reductase gene (MTHFR) polymorphisms in Indian population

Human papillomavirus is considered to be a major aetiological factor but is not sufficient for the development of cervical cancer. Other host factors, including altered homocysteine levels, a functional marker of folate inadequacy, might contribute to the carcinogenic process. Herein we investigated the potential association of homocysteine levels and MTHFR polymorphisms with cervical cancer in 203 histologically confirmed cases including 39 precancer cases and 231 healthy controls with normal cervical cytology. Both patients and controls were screened for human papillomavirus infection. We found that homocysteine and consequently cysteine levels were significantly higher in cases, both cancer and precancer (p < 0.001) than controls. However, polymorphisms in the MTHFR gene (677C/T and 1298A/C) that are reported to modulate homocysteine levels were not associated with disease. Thus, our study establishes an association of total homocysteine levels with the risk of developing carcinoma of the uterine cervix.

Association of cyclin D1 gene polymorphisms with risk of esophageal squamous cell carcinoma in Kashmir Valley : a high risk area

Investigation of potential association of SNPs (G870A, rs9344; G1722C, rs678653) of cyclin D1 gene (CCND1) with susceptibility to esophageal squamous cell carcinoma (ESCC) in Kashmir valley (India). The study included 302 subjects comprising 151 ESCC cases and 151 controls. PCR‐RFLP and direct sequencing were employed for genotyping. The G870A polymorphism, the individuals carrying GA + AA genotype was having 2.80‐fold increased risk for development of ESCC (OR 2.8, 95% CI = 1.77–4.4; P = 0.0001) compared to GG genotype. Further a significantly higher risk was observed in individuals who consume >3 cups per day of salted tea (OR = 5.1; 95% CI = 1.6–16.7; P = 0.0016) and had smoking habits (OR = 6.3; 95% CI = 2.9–13.9; P = 0.0005). We also demonstrate for the first time in CCND1 1722 locus, the CC genotype was strongly associated with increased risk of developing ESCC (OR = 2.58; 95% CI = 1.61–4.15; P = 0.0001). In addition, the frequency of polymorphic C allele was also found to be higher in cases (OR = 1.92; 95% CI = 1.37–2.69; P = 0.0002). There appears to be an influence of CCND1 G870A/G1772C genotypes on genetic susceptibility to ESCC. Mol. Carcinog. ©2011 Wiley‐Liss, Inc.

Genetic variant of CCND1: Association with HPV-mediated cervical cancer in Indian population

The potential association of single nucleotide polymorphisms (SNPs) (G870A and G1722C) of CCND1 with susceptibility to cervical cancer was investigated. The study included 200 cervical cancer cases along with an equal number of healthy controls. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and direct sequencing were employed for genotyping. We found that women carrying the 870AA genotype have a 2.49-fold increased risk for the development of cervical cancer (odds ratio (OR) 2.49; 95% confidence interval (CI) 1.51–4.09; p = 0.0004) compared with GG+GA genotypes. For the 1722 locus, the frequency of the polymorphic ‘C’ allele was strongly associated with a reduced risk of cervical cancer (p = 0.019; OR 0.71; 95% CI 0.54–0.94). Our data suggest that CCND1 G870A polymorphism could act as a risk factor for the development of cervical cancer. And G1722C polymorphism may play a protective role against the development of human papillomavirus-associated cervical cancer among Indian women.

Computational prediction of novel human miRNAs/miRNA target sites in correlation with SNP (rs678653) at 3’UTR of cyclin D1 gene

MicroRNAs (miRNAs) are an extensive class of small noncoding RNAs, having profound impact on many biological processes in development, differentiation, growth and metabolism by regulating gene expression. Polymorphism in 3’UTR showed to alter the miRNA binding site in several human genes and consequently affects the function of the resulting protein posttrancriptionally. To investigate the putative functional consequences of the Cyclin D1 (CCND1) G/C1722 SNP (rs678653) located on 3’UTR of the gene, in-silico approach was applied for the prediction of miRNA binding sites using miRDB version 3.0 & 5.0. We were able to predict 16 miRNA/miRNA target sites on 3’UTR of human CCND1 gene. Out of 16 predicted miRNAs 11 were novel miRNAs/miRNA target sites which target CCND1. However no miRNA target sites were predicted on the DNA/RNA sequence of 3’UTR of human CCND1 gene encompassing the SNP (rs678653). Hence, SNP (rs678653) is not likely to be of functional importance according to this algorithmic prediction. Further experimental analysis is required to validate this computational prediction for CCND1 variants. Abbreviations: miRNA: MicroRNA; SNP: Single nucleotide polymorphism; CCND1: Cyclin D1, UTR: Untranslated region; SVM: Support vector machine Introduction MicroRNAs (miRNAs) have emerged as an important class of short endogenous non-coding RNAs, 18-25 nucleotide (nt) that act as post-transcriptional regulators of gene expression. miRNAs are thought to be involved in diverse biological processes by regulating gene expression. Numerous miRNAs have been identified in various species and many more miRNAs remain to be detected [1]. Following the first documentation of miRNA in C. elegans about two decades ago by Lee et al. [2] to date more than 4000 distinct miRNAs have been annotated in vertebrates, invertebrates and plants, and many miRNAs that correspond to putative genes have also been identified. This discovery has introduced a totally new dimension in our understanding of how gene expression is regulated [3]. In the cytoplasm of the cell, miRNA is typically found in the mature form as 18-25 nt long. miRNAs are initially transcribed as part of much longer primary transcripts (termed pri-miRNAs) [4]. Pri-miRNA is ~100 bp in length and forms a stem-loop fold back structure. It is further processed into a mature miRNA (MIR) that is ~22 bp in size by RNAse III ribonucleases and binds to a specific target site on an mRNA for posttranscriptional repression. The critical region for MIR binding in animals is the “seed” region (nt 2-7 from 5’ end of MIR), which preferentially binds to a target site in the 3’UTR of the mRNA by Watson-Crick pairing [5]. SNPs associated with polygenetic disorders such as cancer can create, destroy or modify miRNA binding sites. Genome-wide bioinformatics analysis predicted ∼64% of transcribed SNPs as target SNPs that can modify (increase/decrease) the binding energy of putative miRNA:: mRNA duplexes by >90% [6]. Previously several reports have shown that miRNA signatures are emerging as a potential biomarker. Numerous miRNAs have been found to have links with some types of cancer [7,8]. These types of studies were mainly remained focussed on cancers including papillary thyroid and colorectal carcinoma [7,9,10], thereby showing the importance of exploring the function of miRNAs in the field of oncology. Various efforts have been made to evaluate the effect of SNPs on putative miRNA target sites on cancer susceptibility e.g. breast cancer [6,11] non-small cell lung cancer [12], esophageal squamous cell carcinoma [13], gastric cancer [14], colon cancer [15], head and neck cancer [16], glioma [17], hepatocellular carcinoma [18], thyroid cancer [19], prostate cancer [20], renal cell carcinoma [21] and bladder cancer [22]. A case-control population study has been conducted by Nicoloso et al. [6], for assessment of implication of target SNPs on breast cancer susceptibility and observed that germline occurrence of rs799917Correspondence to: Nisha Thakur, Division of Molecular Diagnostics, National Institute of Cancer Prevention and Research (ICMR), I-7, Sector-39, Noida201301, India, E-mail: nisha_icpo@yahoo.co.in, nisha.thakur@gov.in

Ceramic-polymer nanocomposites with increased dielectric permittivity and low dielectric loss

The use of lead free materials in device fabrication is very essential from environmental point of view. We have synthesized the lead free ferroelectric polymer nanocomposite films with increased dielectric properties. Lead free bismuth titanate has been used as active ceramic nanofillers having crystallite size 24nm and PVDF as the polymer matrix. Ferroelectric β-phase of the polymer composite films was confirmed by X-ray diffraction pattern. Mapping data confirms the homogeneous dispersion of ceramic particles into the polymer matrix. Frequency dependent dielectric constant increases up to 43.4 at 100Hz, whereas dielectric loss decreases with 7 wt% bismuth titanate loading. This high dielectric constant lead free ferroelectric polymer films can be used for energy density applications.

Genetic basis of HPV mediated cervical cancer in Indian women

Cervical cancer is multi-factorial disease comprising both genetic and environmental components. Human papillomavirus is considered to be a major etiological factor for the development of cervical cancer. HPV type 16 is the most prevalent HPV accounting for more than 70% of cervical cancer cases in India, followed by HPV type 18 and other high-risk types. However, an infection with HPV is essential but it is not sufficient for the development of cervical cancer, which implies the involvement of host genetic factors. The primary cause in the development and progression of cervical neoplasia has been shown to be dependent on a series of cellular genetic and epigenetic events including mutation, deletion, polymorphism and or methylation of various tumor suppressor genes, checkpoint genes of cell cycle machinery etc. Little is known about whether genetic variation in tumor suppressor genes and cell cycle regulatory genes among Indian women associate with susceptibility to cervical cancer. We showed that promoter region of tumor suppressor genes FHIT and RASSF1A were aberrantly methylated in 28.3% and 35.0% respectively in cancer cases but both genes were methylated for 13.3% cases only. We identified a novel mutation at nucleotide position 655, at codon 98 from CAT→CGT with ultimate replacement of amino acid Histidine by Arginine in cervical cancer cases. Molecular modeling was performed to predict the effect of this mutation in disease pathology. We predict that this change, His to Arg substitution in substrate binding domain may generate catalytically inactive protein with loss of tumor suppressor activity. We also showed a novel association of cell cycle regulatory gene, cyclin D1 (CCND1) variation with cervical cancer cases in Indians. Strong evidence of CCND1G870A as risk factor and G1722C as protective factor for cervical cancer was observed. Therefore, presently studies are underway to examine the practical implications of these findings. This would give us important insights into genetic predisposition to cervical cancer in our population.