Susanta Roychoudhury

TP53 codon 72 polymorphism and cervical cancer: a pooled analysis of individual data from 49 studies.

BACKGROUND Cervical cancer is caused primarily by human papillomaviruses (HPV). The polymorphism rs1042522 at codon 72 of the TP53 tumour-suppressor gene has been investigated as a genetic cofactor. More than 80 studies were done between 1998 and 2006, after it was initially reported that women who are homozygous for the arginine allele had a risk for cervical cancer seven times higher than women who were heterozygous for the allele. However, results have been inconsistent. Here we analyse pooled data from 49 studies to determine whether there is an association between TP53 codon 72 polymorphism and cervical cancer. METHODS Individual data on 7946 cases and 7888 controls from 49 different studies worldwide were reanalysed. Odds ratios (OR) were estimated using logistic regression, stratifying by study and ethnic origin. Subgroup analyses were done for infection with HPV, ethnic origin, Hardy-Weinberg equilibrium, study quality, and the material used to determine TP53 genotype. FINDINGS The pooled estimates (OR) for invasive cervical cancer were 1.22 (95% CI 1.08-1.39) for arginine homozygotes compared with heterozygotes, and 1.13 (0.94-1.35) for arginine homozygotes versus proline homozygotes. Subgroup analyses showed significant excess risks only in studies where controls were not in Hardy-Weinberg equilibrium (1.71 [1.21-2.42] for arginine homozygotes compared with heterozygotes), in non-epidemiological studies (1.35 [1.15-1.58] for arginine homozygotes compared with heterozygotes), and in studies where TP53 genotype was determined from tumour tissue (1.39 [1.13-1.73] for arginine homozygotes compared with heterozygotes). Null results were noted in studies with sound epidemiological design and conduct (1.06 [0.87-1.29] for arginine homozygotes compared with heterozygotes), and studies in which TP53 genotype was determined from white blood cells (1.06 [0.87-1.29] for arginine homozygotes compared with heterozygotes). INTERPRETATION Subgroup analyses indicated that excess risks were most likely not due to clinical or biological factors, but to errors in study methods. No association was found between cervical cancer and TP53 codon 72 polymorphism when the analysis was restricted to methodologically sound studies. FUNDING German Research Foundation (DFG).

The Indian Genome Variation database (IGVdb): A project overview

Indian population, comprising of more than a billion people, consists of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, one needs to understand the diversity and relatedness at the genetic level in such a diverse population. In this backdrop, six constituent laboratories of the Council of Scientific and Industrial Research (CSIR), with funding from the Government of India, initiated a network program on predictive medicine using repeats and single nucleotide polymorphisms. The Indian Genome Variation (IGV) consortium aims to provide data on validated SNPs and repeats, both novel and reported, along with gene duplications, in over a thousand genes, in 15,000 individuals drawn from Indian subpopulations. These genes have been selected on the basis of their relevance as functional and positional candidates in many common diseases including genes relevant to pharmacogenomics. This is the first large-scale comprehensive study of the structure of the Indian population with wide-reaching implications. A comprehensive platform for Indian Genome Variation (IGV) data management, analysis and creation of IGVdb portal has also been developed. The samples are being collected following ethical guidelines of Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT), India. This paper reveals the structure of the IGV project highlighting its various aspects like genesis, objectives, strategies for selection of genes, identification of the Indian subpopulations, collection of samples and discovery and validation of genetic markers, data analysis and monitoring as well as the project’s data release policy.Indian population, comprising of more than a billion people, consists of 4693 communities with several thousands of endogamous groups, 325 functioning languages and 25 scripts. To address the questions related to ethnic diversity, migrations, founder populations, predisposition to complex disorders or pharmacogenomics, one needs to understand the diversity and relatedness at the genetic level in such a diverse population. In this backdrop, six constituent laboratories of the Council of Scientific and Industrial Research (CSIR), with funding from the Government of India, initiated a network program on predictive medicine using repeats and single nucleotide polymorphisms. The Indian Genome Variation (IGV) consortium aims to provide data on validated SNPs and repeats, both novel and reported, along with gene duplications, in over a thousand genes, in 15,000 individuals drawn from Indian subpopulations. These genes have been selected on the basis of their relevance as functional and positional candidates in many common diseases including genes relevant to pharmacogenomics. This is the first large-scale comprehensive study of the structure of the Indian population with wide-reaching implications. A comprehensive platform for Indian Genome Variation (IGV) data management, analysis and creation of IGVdb portal has also been developed. The samples are being collected following ethical guidelines of Indian Council of Medical Research (ICMR) and Department of Biotechnology (DBT), India. This paper reveals the structure of the IGV project highlighting its various aspects like genesis, objectives, strategies for selection of genes, identification of the Indian subpopulations, collection of samples and discovery and validation of genetic markers, data analysis and monitoring as well as the project’s data release policy.

Analysis of CAG repeats in SCA1, SCA2, SCA3, SCA6, SCA7 and DRPLA loci in spinocerebellar ataxia patients and distribution of CAG repeats at the SCA1, SCA2 and SCA6 loci in nine ethnic populations of eastern India

Abstract. To identify various subtypes of spinocerebellar ataxias (SCAs) among 57 unrelated individuals clinically diagnosed as ataxia patients we analysed the SCA1, SCA2, SCA3, SCA6, SCA7 and DRPLA loci for expansion of CAG repeats. We detected CAG repeat expansion in 6 patients (10.5%) at the SCA1 locus. Ten of the 57 patients (17.5%) had CAG repeat expansion at the SCA2 locus, while four had CAG expansion at the SCA3/MJD locus (7%). At the SCA6 locus there was a single patient (1.8%) with 21 CAG repeats. We have not detected any patient with expansion in the SCA7 and DRPLA loci. To test whether the frequencies of the large normal alleles in SCA1, SCA2 and SCA6 loci can reflect some light on prevalence of the subtypes of SCAs we studied the CAG repeat variation in these loci in nine ethnic sub-populations of eastern India from which the patients originated. We report here that the frequency of large normal alleles (>31 CAG repeats) in SCA1 locus to be 0.211 of 394 chromosomes studied. We also report that the frequency of large normal alleles (>22 CAG repeats) at the SCA2 locus is 0.038 while at the SCA6 locus frequency of large normal alleles (>13 repeats) is 0.032. We discussed our data in light of the distribution of normal alleles and prevalence of SCAs in the Japanese and white populations.

Alterations in candidate genes PHF2, FANCC, PTCH1 and XPA at chromosomal 9q22.3 region: Pathological significance in early- and late-onset breast carcinoma

IntroductionYounger women with breast carcinoma (BC) exhibits more aggressive pathologic features compared to older women; young age could be an independent predictor of adverse prognosis. To find any existing differences in the molecular pathogenesis of BC in both younger and older women, alterations at chromosomal (chr.) 9q22.32-22.33 region were studied owing to its association in wide variety of tumors. Present work focuses on comparative analysis of alterations of four candidate genes; PHF2, FANCC, PTCH1 and XPA located within 4.4 Mb region of the afore-said locus in two age groups of BC, as well as the interrelation and prognostic significance of alterations of these genes.MethodsDeletion analysis of PHF2, FANCC, PTCH1 and XPA were examined in a subset of 47 early-onset (group-A: ≤ 40 years) and 59 late-onset (group-B: > 40 years) breast carcinomas using both microsatellite and exonic markers. Methylation Sensitive Restriction analysis (MSRA) was done to check for promoter methylation. Quantitative real-time polymerase chain reaction (Q-PCR) and immunohistochemisty (IHC) was done in some genes to see their relative mRNA and protein expressions respectively. Clinico-pathological correlation of different parameters as well as patient survival was calculated using different statistical softwares like EpiInfo 6.04b, SPSS 10.0 etc.ResultsEither age group exhibited high frequency of overall alterations in PHF2, FANCC and PTCH1 compared to XPA. Samples with alteration (deletion/methylation) in these genes showed reduced level of mRNA expression as seen by Q-PCR. Immunohistochemical analysis of FANCC and PTCH1 also supported this observation. Poor patient survival was noted in both age groups having alterations in FANCC. Similar result was also seen with PTCH1 and XPA alterations in group-A and PHF2 alterations in group-B. This reflected their roles as prognostic tools in the respective groups in which they were altered.ConclusionOverall alterations of PHF2, FANCC and PTCH1 were comparatively higher than XPA. Differential association of alterations in FANCC and PTCH1 with that of PHF2, XPA and two breast cancer susceptibility genes (BRCA1/BRCA2) in the two age groups suggests differences in their molecular pathogenesis and dysregulation of multiple DNA repair pathways as well as hedgehog dependent stem cell renewal pathway.

Genetic and epigenetic changes of HPV16 in cervical cancer differentially regulate E6/E7 expression and associate with disease progression

OBJECTIVE The study was aimed at understanding the complex interactions of genetic and epigenetic events in expression of HPV16 E6/E7 and progression of cervical carcinoma. For this, expression of E6/E7 was done in 36 samples, along with the physical status, methylation and LCR sequence variations. Later, the genetic and epigenetic studies were extended to 239 samples to find out the association of these factors with progression of cervical cancer. METHODS E6/E7 expression was quantified by real-time PCR. Physical status of HPV16 was determined by mutiplex-PCR of whole E2 ORF using overlapping primers and E6 ORF and validated by real-time PCR. Methylation status of P97 promoter/enhancer was analyzed by methylation sensitive restriction analysis (MSRA). Viral lineage and variations in LCR was ascertained by sequencing LCR/E6/E7 ORFs. RESULTS Samples with episomal unmethylated virus showed comparatively high expression of E6/E7 than episomal methylated, integrated unmethylated and integrated methylated forms of HPV16. Variations in the LCR, particularly in the binding sites of negatively regulating transcription factors, also contribute to high expression of E6/E7. The integrated form significantly increases with decrease of episomal form during tumor progression. Methylation of the promoter/enhancer gradually decreased with tumor progression and is inversely correlated to integration. Two novel variants were observed in E6 gene in European- and North-American-1-lineages. Log-rank test revealed better prognosis of the patients with episomal methylated HPV16 compared to the other forms. CONCLUSION Our results show higher expression of E6/E7 in samples with episomal unmethylated virus having sequence variations in LCR.

Polymorphisms at p53, p73, and MDM2 loci modulate the risk of tobacco associated leukoplakia and oral cancer

Polymorphisms at loci controlling cellular processes such as cell cycle, DNA repair, and apoptosis may modulate the risk of cancer. We examined the association of two linked polymorphisms (G4C14–A4T14) at p73 and one polymorphism (309G > T) at MDM2 promoter with the risk of leukoplakia and oral cancer. The p73 and MDM2 genotypes were determined in 197 leukoplakia patients, 310 oral cancer patients and in 348 healthy control subjects. The p73 GC/AT genotype increased the risk of leukoplakia (OR = 1.6, 95% CI = 1.1–2.3) and oral cancer (OR = 2.4, 95% CI = 1.7–3.3) but the 309G > T MDM2 polymorphism independently could not modify the risk of any of the diseases. Stratification of the study population into subgroups with different tobacco habits showed that the risk of the oral cancer is not modified further for the individuals carrying p73 risk genotype. However, leukoplakia patients with smokeless tobacco habit showed increased risk with combined GC/AT and AT/AT (OR = 3.0, 95% CI = 1.3–7.0) genotypes. A combined analysis was done with our previous published data on p53 codon 72 pro/arg polymorphism. Analysis of pair wise genotype combinations revealed increase in risk for specific p73‐MDM2 and p73‐p53 genotype combinations. Finally, the combined three loci analyses revealed that the presence of at least one risk allele at all three loci increases the risk of both leukoplakia and oral cancer.

Frequent alterations of the candidate genes hMLH1, ITGA9 and RBSP3 in early dysplastic lesions of head and neck: Clinical and prognostic significance

To understand the association between candidate tumor suppressor genes (TSGs) human mismatch repair protein homologue 1 (hMLH1), AP20 region gene 1 (APRG1), integrin α RLC (ITGA9), RB1 serine phosphates from human chromosome 3 (RBSP3) at chromosomal 3p22.3 region and development of head and neck squamous cell carcinoma (HNSCC), alterations (deletion/promoter methylation/expression) of these genes were analyzed in 65 dysplastic lesions and 84 HNSCC samples. Clinicopathological correlations were made with alterations of the genes. In HNSCC, deletion frequencies of hMLH1, ITGA9, and RBSP3 were comparatively higher than APRG1. Overall alterations (deletion/methylation) of hMLH1, ITGA9, and RBSP3 were high (45–55%) in mild dysplasia and comparable in subsequent stages of tumor progression. Quantitative RT‐PCR analysis showed reduced expression of these genes in tumors concordant to their molecular alterations. An in vitro demethylation experiment by 5‐aza‐2′‐deoxycytidine confirmed the promoter hypermethylation of RBSP3 in Hep2 and UPCI:SCC084 cell lines. Functionally less‐active RBSP3A isoform was predominant in tumor tissues contrary to the adjacent normal tissue of tumors where more active RBSP3B isoform was prevalent. In immunohistochemical analysis, intense nuclear staining of hMLH1 and pRB (phosphorylated RB, the substrate of RBSP3) proteins were seen in the basal layer of normal epithelium. In tumors, concordance was seen between (i) low/intermediate level of hMLH1 expression and its molecular alterations; and (ii) intense nuclear staining of pRB and RBSP3 alterations. Poor patient outcome was seen with hMLH1 and RBSP3 alterations. Moreover, in absence of human papilloma virus (HPV) infection, tobacco‐addicted patients with hMLH1, RBSP3 alterations, and nodal invasions showed poor prognosis. Thus our data suggests that dysregulation of hMLH1, ITGA9, and RBSP3 associated multiple cellular pathways are needed for the development of early dysplastic lesions of the head and neck. (Cancer Sci 2010)

Alterations of 3p21.31 Tumor Suppressor Genes in Head and Neck Squamous Cell Carcinoma: Correlation With Progression and Prognosis

The aim of our study was to analyze the alterations of some candidate tumor suppressor genes (TSGs) viz. LIMD1, LTF, CDC25A, SCOTIN, RASSF1A and CACNA2D2 located in the chromosomal region 3p21.31 associated with the development of early dysplastic lesions of head and neck. In analysis of 72 dysplastic lesions and 116 squamous cell carcinoma of head and neck, both deletion and promoter methylation have been seen in these genes except for CDC25A and SCOTIN where no methylation has been detected. The alteration of LIMD1 was highest (50%) in the mild dysplastic lesions and did not change significantly during progression of tumor indicating its association with this stage of the disease. It was evident that alterations of LTF, CDC25A and CACNA2D2 were associated with development of moderate dysplastic lesions, while alterations in RASSF1A and CACNA2D2 were needed for progression. Novel somatic mutations were seen in exon 1 of LIMD1 (7%), intron 3/exon4 splice junction of LTF (2%) and exon 7 of cdc25A (10%). Quantitative RT‐PCR analysis revealed mean reduced expression of the genes in the following order: LTF (67.6 ± 16.8) > LIMD1 (53.2 ± 20.1) > CACNA2D2 (23.7 ± 7.1) > RASSF1A (15.1 ± 5.6) > CDC25A (5.3 ± 2.3) > SCOTIN (0.58 ± 0.54). Immunohistochemical analysis of CDC25A showed its localization both in cytoplasm and nucleus in primary lesions and oral cancer cell lines. In absence of HPV infection, LTF and RASSF1A alterations jointly have adverse impact on survival of tobacco addicted patients. Thus, our data suggested that multiple candidate TSGs in the chromosomal 3p21.31 region were differentially associated with the early dysplastic lesions of head and neck.

Cholera toxin (CTX) genetic element in Vibrio cholerae O139.

PFGE analysis of the NotI- and SfiI-digested genome of Vibrio cholerae O139 strains isolated from different epidemic regions of India showed that all the strains are of clonal origin and the genome size is about 2.2 Mb. An analysis of the electrophoretic profiles of the genome of O139 strains, the RFLP of the cholera toxin (ctx) gene and Southern blot hybridization of NotI-digested genomes of classical, El Tor and O139 with a NotI-linking clone of classical strain 569B, suggest that these strains closely resemble V. cholerae O1 biotype El Tor, but are widely different from the classical O1 vibrios. Using restriction enzymes which cleave a single site in either the core region or in the direct repeat sequence (RS) of the CTX genetic element, it has been shown that the genome of most of the O139 strains has two copies of the ctx gene in tandem connected by two RSs. The chromosomal location of the CTX genetic element in the O139 strain is the same as that reported for El Tor vibrios. The organization of the virulence gene cassettes in different O139 strains shows genetic heterogeneity in the population. Whilst most of the epidemic strains have two copies of the CTX genetic element, in some strains the number of elements has been amplified and in at least one strain a single copy of the element has been deleted.

SH3GL2 and CDKN2A/2B loci are independently altered in early dysplastic lesions of head and neck: correlation with HPV infection and tobacco habit

To understand the association of candidate tumour suppressor genes SH3GL2, p16INK4a, p14ARF, and p15INK4b in the pathogenesis of head and neck squamous cell carcinoma (HNSCC), we studied the deletion, mutation, and methylation of these genes in 61 dysplastic lesions and 94 HNSCC samples. In mild dysplasia, SH3GL2, p16INK4a, and p14ARF showed a higher frequency of overall alterations (60–70%) than in p15INK4b (40%). However, in subsequent stages of tumour progression, the alteration frequency of these genes did not change significantly. One novel mutation in common exon 2 of p16INK4a/p14ARF and three in exon 9 of SH3GL2 were seen. Concordance was seen in the expression of these genes with their molecular alterations. Deletions of INK4A‐ARF and p15INK4b have a significant poor patient outcome. The alterations of p16INK4a, p14ARF, and p15INK4b were positively correlated with tobacco and inversely with HPV, while SH3GL2 alterations were independent of these factors. Based on aetiological factors, four tumour subtypes were recognized: HPV−tobacco− (I), HPV+tobacco− (II), HPV−tobacco+ (III), and HPV+tobacco+ (IV). Groups III and IV showed a high frequency of p16INK4a/p14ARF/p15INK4b alterations with significant poor patient outcome in comparison to group II. Our findings suggest that deregulation of SH3GL2‐associated signalling and p16INK4a/p14ARF/p15INK4b‐mediated G1–S/G2–M checkpoints of cell cycle are independent pathways for the development of early dysplastic lesions of the head and neck. Copyright