P53 tumor suppressor gene mutations in hepatocellular carcinoma patients in India

Background: Specific mutations of the p53 tumor suppressor gene in hepatocellular carcinoma (HCC) have been reported from several parts of the world, but to the authors' knowledge to date the status of this gene has not been studied in HCC patients in India, where HCC is one of the major cancers and the frequency of chronic hepatitis B virus (HBV) as well as hepatitis C virus (HCV) infection and exposure to dietary aflatoxin B1 is very high. The most frequent mutation of the p53 gene in HCC is an AGGArg to AGTSer missense mutation at codon 249 of exon 7. Methods: Liver biopsy specimens from 21 HCC patients and 10 healthy controls were obtained through surgery or by needle biopsy technique. Phenol-chloroform-extracted DNA specimens were employed for the detection of HBV infection and p53 gene mutations. Nucleotide mutations of exons 4-9 of the p53 gene were analyzed by polymerase chain reaction (PCR), single strand confirmation polymorphism, and direct sequencing. Third-generation sandwich enzyme-linked immunosorbent assay (ELISA) was used for the serologic detection of HBV and HCV infection. Results: Analysis of exons 4-9 of the p53 gene revealed only 3 mutations (3 of 21 specimens, 14.28%; 95% confidence interval, -0.7-29.3), 2 mutations at codon 249 showing G→T transversions, and 1 mutation (4.7%) at codon 250 with a C→T transition. The base substitutions at the third base of codon 249 resulted in a missense mutation leading to a change in amino acid from arginine to serine whereas at codon 250 it caused a change from proline to serine. Dot blot hybridization and PCR for HBV DNA from HCCs revealed 58.8% (10 of 17 specimens) and 90.47% (19 of 21 specimens), positivity, respectively. ELISA for hepatitis B virus surface antigen in serum showed a positivity of 71.42% (15 of 21 specimens), but there was only 40% positivity (8 of 20 specimens) for hepatitis B virus envelope antigen whereas 6 of 17 patients (35.29%) showed the presence of antibodies against hepatitis B virus envelope protein. No patient was found to be positive for the HCV antibody. Conclusions: The very low frequency of p53 mutations and the extremely high frequency of HBV infection (> 90%) in HCC indicate that the mutations in the p53 gene frequently found in HCC reported from different endemic areas of the world may not play a direct role in the development of HCC in India. HBV infection and, possibly, exposure to the dietary aflatoxin B1 appear to play major roles in the molecular pathogenesis of HCC in India

Hepatitis B virus genotypes in acute and fulminant hepatitis patients from North India using two different molecular genotyping approaches

Data from India on hepatitis B virus (HBV) genotype related differences in clinical progression and outcome of acute and fulminant hepatitis B are limited. Sera from patients with acute hepatitis B (AHB) (n = 80), fulminant hepatitis B (FHB) (n = 40) and asymptomatic HBsAg carriers (ASC) (n = 40) were tested for HBV genotype using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and type-specific primers-based PCR (TSP-PCR). The genotype distribution for 160 patients with HBV related hepatitis/carriers were as follows: A, 3/80 (3.7%) in AHB, 2/40 (5%) in FHB and 7/40 (17.5%) in ASC; D, 77/80 (96.2%) in AHB, 38/40 (95%) in FHB and 33/40 (82.5%) in ASC. C, 0; B, 0; E, 0; F, 0 (p < 0.01, genotype D versus A). Compared with genotype D, genotype A patients had no significant clinical or biochemical differences (p > 0.05). HBV genotypes A and D were found to be prevalent in patients with HBV related acute and fulminant hepatitis from New Delhi, India. Genotype D was the dominant genotype prevalent in all patient categories while genotype A was solely responsible for AHB leading to chronic hepatitis B in 3.7% of the cases from this region

Association of core promoter mutations with viral breakthrough in chronic hepatitis B patients on long-term lamivudine therapy

Background and Aim: Virologic breakthrough (VBTH) during long-term lamivudine therapy is believed to be due to the emergence of rtYM204I/VDD mutants. We observed VBTH in 17 of 67 patients receiving long-term lamivudine therapy. The YMDD mutant at the onset of VBTH and/or subsequently was seen in eight (47%) of 17 such patients. We investigated other potential loci in the viral genome contributing to VBTH. Methods: Chronic hepatitis B (CHB) patients (n = 17) on long-term (=12 months) lamivudine therapy who had at least one episode of VBTH were selected (group I). Age, sex, serology and baseline viral load matched patients on long-term lamivudine without VBTH (n = 12) served as controls (group II). Hepatitis B virus (HBV) DNA sequences were analyzed for pre-S, surface, polymerase, core promoter, precore and core regions and were compared with sequences of respective genotypes. Results: Group I patients with VBTH (n = 17) either had rtYM204I/VDD mutations (n = 8, group Ia YMDD) or no rtYM204I/VDD mutations (n = 9, group Ib non-YMDD). Group Ia patients had median baseline HBV DNA of 2794 pg/mL (range 3-9166 pg/mL) and a mean alanine aminotransferase (ALT) level of 86 ± 33 IU/L. In group Ib, the median baseline viral DNA was 916 pg/mL (range 8.3-5787 pg/mL) and the mean ALT was 61 ± 38 IU/L. The first VBTH in group Ia and Ib patients was noted at 21 ± 9 months and 15.2 ± 5.9 months, respectively, with a rise in HBV DNA levels from undetectable limits to 952 pg/mL (range 4.3-4875 pg/mL) and 571 pg/mL (range 1.2-1970 pg/mL), respectively. Core promoter mutations were seen in five of eight (62.5%) and in six of nine (66.6%); classic double mutations (A1762T/G1764A) of core promoter region were detected in two and three patients and novel double mutations of core promoter (G1764T/C1766G) in one patient each of group Ia and Ib patients, respectively. Compared to 11 (68%) of 17 group I patients, only three (25%) of 12 patients in group II had core promoter mutations (P < 0.05). No patient in group II had double mutations of the core promoter region. No significant difference in viral mutations was seen in any other region of the viral genome between group I and group II patients. In group I, none of the 15 patients (two died of hepatocellular carcinoma), but five (42%) group II patients achieved hepatitis B e antigen (HBeAg) seroconversion and sustained response by month 24 (P <0.05). Conclusion: Both core promoter and YMDD motif mutation(s) are associated with VBTH in patients on long-term lamivudine therapy. Whether or not these promoter mutations in the absence of YMDD mutations confer drug resistance needs to be studied in an in vitro cell culture system, as they could create novel and stronger binding sites for hepatocyte nuclear factors

Prevalence of transfusion-transmitted virus infection in patients on maintenance hemodialysis from New Delhi, India

Transfusion-transmitted virus (TTV) has been reported from a number of hemodialysis (HD) units from various countries throughout the world. TTV has been associated with liver diseases, viral hepatitis B, and C. Clinical details and information regarding TTV prevalence from India are insufficient. The prevalence and clinical significance of TTV infection were studied in New Delhi, India in HD patients. Serum samples were derived from 75 patients on maintenance HD, and 75 age- and sex-matched voluntary blood donors were examined for TTV viremia by nested polymerase chain reaction (PCR) using primers derived from UTR (A) region of the TTV genome. The prevalence of TTV DNA in patients on HD (83%) was significantly (p<0.05) higher than in blood donors (43%). Clinical background including the mean age, sex, mean duration of HD, and mean alanine aminotransferase (ALT) levels did not differ significantly between TTV DNA-positive and -negative HD patients. Fifty-four (72%) TTV-positive HD patients and 7 (56%) TTV-negative HD patients had blood transfusion histories (p>0.05). Among TTV-positive patients, Hepatitis B virus (HBV) co-infection was present in 14.2% cases while hepatitis C virus (HCV) co-infection was absent. Persistent elevation of ALT levels was observed in 7(9.3%) HD patients; 3 (43%) of them were TTV positive and 4 (57%) were TTV negative (p>0.05). All 3 TTV-positive patients with elevated ALT levels were co-infected with HBV. Patients with TTV infection alone showed normal ALT levels. Prevalence of TTV infection is high in North Indian patients on maintenance HD. Also, none of the exclusively TTV DNA-positive patients had clinical or biochemical signs of liver disease. TTV seems to spread through parenteral routes. More often, TTV seems to be associated with parenterally transmitted virus HBV, indicating a parenteral mode of TTV transmission. The pathogenicity of TTV remains unclear from the present study

Level of phospho-STAT3 (Tyr705) correlates with copy number and physical state of human papillomavirus 16 genome in cervical precancer and cancer lesions.

Our earlier studies indicated an important role of inducible transcription factor STAT3 in the establishment of persistent infection of human papillomavirus (HPV) type 16 and promotion of cervical carcinogenesis. Since HPV load and its physical state are two potential determinants of this virally-induced carcinogensis, though with some exceptions, we extended our study to examine the role of active STAT3 level in cervical precancer and cancer lesions and it's association with HPV viral load and physical state. An elevated level of active STAT3 was measured by assessing phospho-STAT3-Y705 (pSTAT3), in tumor tissues harboring higher viral load irrespective of the disease grade. Physical state analysis of HPV16 by assessing the degree of amplification of full length E2 and comparing it with E6 (E2:E6 ratio), which predominantly represent episomal form of HPV16, revealed low or undetectable pSTAT3. A strong pSTAT3 immunoreactivity was found in tissues those harbored either mixed or predominantly integrated form of viral genome. Cumulative analysis of pSTAT3 expression, viral load and physical state demonstrated a direct correlation between pSTAT3 expression, viral load and physical state of HPV. The study suggests that there exists a strong clinical correlation between level of active STAT3 expression and HPV genome copy number, and integrated state of the virus that may play a pivotal role in promotion/maintanence of tumorigenic phenotype

Higher prevalence of human papillomavirus infection in adolescent and young adult girls belonging to different Indian tribes with varied socio-sexual lifestyle.

Despite high prevalence of human papillomavirus (HPV) infection and cervical cancer in Indian women, no study has been done in tribal populations whose socio-sexual lifestyle is different. Therefore, HPV screening has been carried out in pre-adolescent, adolescent and young adult tribal girls using self-collected urine samples.20-35 ml self-collected midstream urine samples were obtained from a total of 2278 healthy tribal girls (9-25 years) comprising pre-adolescent, adolescent and young adults from three Indian states: Madhya Pradesh, Jharkhand and Chhattisgarh. β-globin positive 2034 samples were employed for HPV detection and genotyping.The overall prevalence of HPV infection in tribal girls was 12.9% (262/2034). More than 65% (172/262) of them were infected with HR-HPV types of which HPV16 was the most predominant type (54%). Young adult girls aged 18-25 years showed a significantly higher prevalence of HPV infection (19.2%; OR = 3.36; 95% CI 2.97-6.34, P<0.001) as compared to that in adolescent (11.4%; OR = 1.82; 95% CI 1.20-2.76, P<0.01) or pre-adolescent girls (6.6%).This is a first study showing significantly a very high prevalence of HPV infection in adolescent and young adult tribal girls possibly due to different socio-sexual behavior, indicating a serious health concern for Indian tribal women

Alterations in microRNAs miR-21 and let-7a correlate with aberrant STAT3 signaling and downstream effects during cervical carcinogenesis

BACKGROUND: Present study provides clinical evidence of existence of a functional loop involving miR-21 and let-7a as potential regulators of aberrant STAT3 signaling recently reported by our group in an experimental setup (Shishodia et al. BMC Cancer 2014, 14:996). The study is now extended to a set of cervical tissues that represent natural history of human papillomavirus (HPV)-induced tumorigenic transformation. MATERIALS AND METHODS: Cervical tissues from histopathologically-confirmed pre-cancer (23) and cancer lesions (56) along with the normal control tissues (23) were examined for their HPV infection status, expression level of miR-21 &amp; let-7a and STAT3 &amp; pSTAT3 (Y705) by PCR-based genotyping, quantitative real-time PCR and immunoblotting. RESULTS: Analysis of cancer tissues revealed an elevated miR-21 and reduced let-7a expression that correspond to the level of STAT3 signaling. While miR-21 showed direct association, let-7a expression was inversely related to STAT3 expression and its activation. In contrast, a similar reciprocal expression kinetics was absent in LSIL and HSIL tissues which overexpressed let-7a. miR-21 was found differentially overexpressed in HPV16-positive lesions with a higher oncoprotein E6 level. Overexpression of miR-21 was accompanied by elevated level of other STAT3-regulated gene products MMP-2 and MMP-9. Enhanced miR-21 was found associated with decreased level of STAT3 negative regulator PTEN and negative regulator of MMPs, TIMP-3. CONCLUSION: Overall, our study suggests that the microRNAs, miR-21 and let-7a function as clinically relevant integral components of STAT3 signaling and are responsible for maintaining activated state of STAT3 in HPV-infected cells during cervical carcinogenesis

Role of polymorphic N-acetyl transferase2 (NAT2) and cytochrome P4502E1 (CYP2E1) gene in ATT induced hepatitis

Background and Aim: Antituberculosis drugs isoniazid and rifampicin in combination are known to develop drug induced hepatotoxicity (DIH). A higher risk of DIH during antituberculosis treatment (ATT) has been reported in Indian subcontinent compared to the western counterparts. The role of genetic factors in higher incidence of ATT hepatotoxicity in Indian population is still unclear. The present study was aimed at investigating the role of N-acetyltransferase2 (NAT2) and Cytochrome P4502E1 (CYP2E1) gene polymorphism in ATT hepatotoxicity. Design: The study population included 218 pulmonary tuberculosis patients who were started on ATT and followed up for the occurrence of ATT induced hepatitis. Genetic polymorphism of NAT2 and CYP2E1 gene was studied by PCR-RFLP. Results: Occurrence of DIH was 18.80% (41/218). There was a higher prevalence of NAT2 slow acetylator genotypes in DIH (70.73%) compared to non-DIH (44.63%), P &lt; 0.05. The frequency of NAT2<SUP>&#8727;</SUP>5/<SUP>&#8727;</SUP>7 and NAT2<SUP>&#8727;</SUP>6/<SUP>&#8727;</SUP>7 genotypes was significantly higher in DIH than non-DIH (19.51% vs. 6.78% and 19.51% vs. 5.08%). No association of CYP2E1 RsaI polymorphism could be demonstrated with DIH. However, DraI C/D genotype of CYP2E1 gene was mostly prevalent in DIH (85.37%) compared to non-DIH (64.41%), P &lt; 0.05. Slow acetylator status and CYP2E1 C/D or C/C genotype together showed higher frequency in DIH (65.85%) compared to non-DIH (28.81%), P &lt; 0.0001. Conclusion: The study demonstrates for the first time a possible association between DraI polymorphism of CYP2E1 gene and the risk of ATT hepatotoxicity. Genotyping of NAT2 and CYP2E1 gene could possibly identifying the high risk group for developing ATT induced hepatitis prior to medication