Sunil Kumar Polipalli

Hepatitis B virus BCP, Precore/core, X gene mutations/genotypes and the risk of hepatocellular carcinoma in India.

The study aims to characterize mutations of the HBV genome involving BCP, Precore/core and X regions and also defines HBV genotypes in patients of hepatocellular carcinoma (HCC). The study involved 150 HBV‐related HCC cases and 136 HBV‐related chronic liver disease patients without HCC as controls. HBV DNA was subjected to mutational analysis using SSCP technique, genotyping by RFLP, and direct nucleotide sequencing. HBV DNA was found in 58.7% (88/150) of the HCC cases and 74.3% (101/136) of controls. HBV mutants were observed in 44.3% of HCC cases and 43.2% of controls. HBV/D was prevalent amongst the patients and controls, followed by HBV/A. The prevalence of the TT1504 mutation in the X gene, the V1753 and T1762/A1764 mutations in the BCP region, and G1914 mutation in the core gene were significantly higher in the HCC group than in the non‐HCC group. Multivariate analyses showed that the TT1504, V1753, A1762T/G1764A, and the G1914 mutations and the patients age, sex, and HBeAg status increased the risk of HCC development significantly. Also, patients with HCC had lower levels of serum albumin, viral load, and platelet counts but higher values of alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, bilirubin, and Alpha feto‐protein than those of controls (P < 0.001 for all comparisons). HBV/D was the predominant genotype associated with HCC cases seen in India. The presence of different types of HBV mutations, age, sex, HBeAg status, and viral load was found to increase significantly the risk of HCC development in India. J. Med. Virol. 82: 1115–1125, 2010.

Diagnostic utility of hepatitis C virus core antigen in hemodialysis patients

BACKGROUND Patients undergoing hemodialysis are at high risk for Hepatitis C virus infection. Anti-HCV antibody detection is widely used for screening this infection but is not sensitive for window period detection. An ELISA to detect the HCV Core Antigen has recently become available. OBJECTIVES To investigate the utility of the HCV core Antigen ELISA in the detection of HCV infection in hemodialysis patients and to compare with 3rd generation ELISA validated by real-time PCR. METHODS Two hundred fifty hemodialysis patients were included in the study. Anti-HCV antibodies and Total HCVcAg was determined by third generation ELISA kits. HCV RNA was determined using Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and sensitivity of the two assays was confirmed by estimating viral load using real-time PCR. RESULTS Forty-three out of 250 (17.2%) patients were positive for anti-HCV antibodies and HCVcAg. 13/250 (5.2%) were positive for HCVcAg but anti-HCV negative, which is statistically significant (P<0.05). All 13 were confirmed viremic by in-house nested RT-PCR leading to specificity of 100%. Viral load of 49,258+/-28,682 copies/mL were detected in HCVcAg positive cases in comparison to 239,383+/-107,805 copies/mL in the only anti-HCV positive group (P<0.001). False negative cases for HCVcAg assay accounted for 2/250 (0.8%) in which the viral load was 306+/-461 copies/mL which was significantly lower in comparison to HCVcAg positive group (P<0.001, t-test=9.982). CONCLUSIONS Total HCVcAg ELISA is an accurate serological marker for early identification of HCV infection, than is possible by currently used serological assay. It will be useful for patients undergoing hemodialysis who have a longer window period due to immunosuppressed state. It is both a cost-effective and a less labor-intensive alternative to PCR, enhancing its clinical utility.

IL-18 polymorphisms in hepatitis B virus related liver disease.

Interleukine-18 (IL-18) was originally called interferon (INF-γ) inducing factor and plays a critical dual role in Th1 polarization and viral clearance. We aimed to explore whether single-nucleotide promoter polymorphisms (SNPs) are associated with the outcome of hepatitis B virus (HBV) infection. 271 HBV infected patients were recruited in this study out of these 109 were spontaneously recovered and 162 were diagnosed to be having persistent HBV infection which includes 48 chronic hepatitis, 84 liver cirrhosis, 30 HCC cases and were compared with 280 healthy controls. IL-18 promoter genotyping was performed with sequence-specific primers. The results demonstrated the significant involvement of genotype AA at position -607 in healthy controls (38.6%) when compared to cases (26.0%) (OR=0.54 (0.385-0.797)) and also associated with spontaneous clearance (37.6%) compared to persistent HBV infections (17.9%) (OR=2.76 (1.582-4.832)). Whereas, genotype CC at position -607 in cases (18.0%) when compared to healthy controls (6.7%) (OR=3.03 (1.734-5.303)) also associated with persistent HBV infections (24.1%) compared to spontaneous clearance (9.2%) (OR=0.31 (0.151-0.67)). And genotype GC at position -137 in cases (49.5%) compared to healthy controls (38.5%) (OR=1.55 (1.11-2.18)). Whereas, genotype GG at position -137 in healthy controls (56.8%) compared to cases (45.4%) (OR=0.63 (0.451-0.885)). No significant difference at position -137 was observed between spontaneous clearance and persistent HBV infections. These polymorphisms of the IL-18 gene promoter region at position -607 and -137 could be associated with different outcomes of HBV infection. The people with allele A at position -607 may be protected against HBV infection; moreover AA genotype is associated with spontaneous clearance.

Prevalence of Hepatitis B Virus Genotype D in Precore Mutants Among Chronic Liver Disease Patients from New Delhi, India

Hepatitis B is one of the most important causes of chronic viral hepatitis world wide. Mutations in the precore region of the hepatitis B virus (HBV) genome are frequently found in hepatitis B envelope antigen-negative cases. Data from India on the HBV genotype-associated distribution of precore mutations are limited. Our objective in this study was to genotype and detect the precore mutant with a point mutation from G to A at nucleotide 1896 using ligase chain reaction (LCR) and direct sequencing. A total of 115 cases of chronic liver disease were screened. The cases were evaluated on the basis of history, clinical examination, liver function profile, and serological test for HBV infection, which includes HBsAg, anti HBcIgG, HBeAg using commercially available ELISA kits. The cases, which were HBeAg+, HBeAg–, and HBV DNA+, were subjected to LCR and confirmed by direct sequencing. Of 115 chronic liver disease cases, 50 (43.5%) cases were HBV DNA positive. All cases were subjected to LCR; 11 (22%) cases confirmed the presence of precore mutants, while the remaining 39 (78%) were classified as the wild form of the virus. HBV genotyping by direct sequencing revealed that genotype D was predominant in both wild and mutant forms of the virus. We conclude that the HBV genotype distribution was not significantly different between precore mutants and the wild form of the virus (P>0.05). North Indian patients with genotype D were more likely to have persistent HBV infection with precore mutants. HBV genotypes correlate with the clinical outcome of chronic HBV infection.

Genetic Polymorphism of Interleukin-18 Gene Promoter Region in Rheumatoid Arthritis Patients from Southern India

BACKGROUND Interleukin-18 (IL-18) is a pro inflammatory cytokine which plays a key role in the acute and chronic inflammatory phases of Rheumatoid Arthritis (RA). The Single Nucleotide Polymorphisms (SNPs) of IL-18 gene promoter region at positions -137 and -607, are postulated to be associated with RA. To test this, this study aimed to identify the association between these SNPs of the IL-18 gene promoter region of RA in south Indian patients. MATERIALS AND METHODS This study was carried on 190 subjects among which 90 were RA patients and 100 were age and sex matched controls. Genomic DNA was extracted by Salting out method. IL 18 gene promotor region SNPs, IL 18 - 607 and IL 18 -137 were amplified by using sequence specific primers. The amplified products of different samples were separated by using a 1.5% agarose gel, stained with ethidium bromide and photographed. All statistical analyses were carried out by using SYSTAT 12 software. RESULTS At position 607, the frequencies of C allele, CC genotype, A allele and AA genotype were found to be significantly higher in patients and controls respectively and there was no significant difference in CA genotype. At position 137, there was no significant difference between the two groups with regard to G and C allelles but there was a significant increase in GG genotype of patients and CC genotype of controls. There was no association between duration of morning stiffness, rheumatoid factor positivity or negativity, age of onset and gender with distribution of genotypes and alleles. CONCLUSION C allele, CC genotype at position-607 and GG genotype at position-137 are risk factors and A allele, AA genotype at position-607 and CC genotype at position-137 have protective effect for RA.

Does Mutation of Hepatitis A Virus Exist in North India

BackgroundHuman hepatitis A, a widespread infectious disease that is hyperendemic in vast areas of the world, results in the infection of the liver. Different human HAV strains of diverse geographic origin are remarkably closely related. HAV exploits all known mechanisms of genetic variation to ensure survival, including mutation and genetic recombination.ObjectivesThe aim of the study was to undertake an in-depth analysis of the mutation in three groups: (i) mild acute hepatitis (m-AH), (ii) severe acute hepatitis (s-AH), and (iii) fulminant hepatitis (FHF) A patients, who were tested positive for HAV RNA.Materials and MethodsA total of 500 patients of acute viral hepatitis (AVH) were screened for HAV-IgM positivity from January 2003 to December 2004. HAV RNA positivity was subject to reverse transcription of RNA followed by polymerase chain reaction (RT-PCR) for the detection of HAV RNA. The HAV RNA positive cases were subject to single-stranded conformational polymorphism (SSCP).ResultsOut of 500 acute cases of hepatitis, 80 (16%) were positive for HAV-IgM. HAV RNA was detected in 34 (42.5%) cases by RT-PCR. Twenty-four (70.5%) were m-AH, seven (20.5%) were s-AH, and three (8.8%) were FHF. All the positive samples were subject to SSCP. No mobility shift was observed with respect to any screened samples by PCR-SSCP. Four (m-AHI-54, m-AHI-80, s-AHI-341 and FHFI-195 suspected cases were directly sequenced to prove that there was no point mutation.ConclusionSSCP demonstrates no mobility shift in the VP1/P2A region of the HAV genome. No point mutation was observed in the four suspected cases by sequencing. However a large study from different geographical locations is needed to achieve a logical conclusion about the existence of HAV mutation in the Indian population.

Association of Methylene Tetrahydrofolate Reductase Polymorphism with BMD and Homocysteine in Premenopausal North Indian Women.

BACKGROUND AND AIM Osteoporosis (OP) is a common nutrigenomic disease associated with various genetic components. Observational studies have indicated that mildly elevated homocysteine was a strong risk factor for osteoporotic fractures. Yet there is no clear biologic mechanism for an effect of homocysteine on bone.The aim of this study was to investigate the association of MTHFR C677T and A1298C polymorphisms, and to verify the association of these polymorphisms with bone mineral density and homocysteine in premenopausal women of northern India. MATERIAL AND METHODS We included 402 north Indian patients with altered BMD, both Osteopenic (OPN) and Osteoporosis, and normal controls. Genotype identification for MTHFR C677T and A1298C polymorphisms were analyzed by PCR-RFLP method, correlated with Bone Mineral Density (BMD), Homocysteine (Hcy), Folate and Vitamin B12. RESULTS The study groups did not differ in terms of age, weight and body mass indices. Prevalence of Genotype frequencies (GFs) for MTHFRC677T OP were (n: 402): CC 361 (89.8%), CT 25 (6.22%), TT 16 (3.98%) and that for MTHFR A1298C were (n: 402) AA 353(87.81%), AC 29(7.21%), CC 20(4.98%). Folate was significantly lower in the OP group than those in both the other groups, while there was no significant difference in Hcy in the OP group relative to OPN, as compared to controls. CONCLUSION The GFs for MTHFR C677T and A1298C polymorphisms were not different between both groups. In conclusion, polymorphism of the MTHFR 677T is associated with small differences in BMD with folate levels. Further, more investigations should be done in larger studies for other epigenetic pathways, that may increase the risk of Osteoporosis.

Cytogenetic Analysis for Suspected Chromosomal Abnormalities; A Five Years Experience

INTRODUCTION Chromosomal abnormalities are the results of alterations in the number or structure of chromosomes causing significant human morbidity and mortality. They are responsible for a large proportion of miscarriages, developmental delay, disorders of sexual development, congenital malformations and mental retardation. AIM The aim of this study was to describe the prevalence of different chromosomal abnormalities in North Indian patients referred for cytogenetic analysis. MATERIALS AND METHODS Total of 859 patients ranging from newborn to 37 years of age were referred to the division of genetics, Department of Paediatrics between 2010 and 2015, with a variety of clinical disorders; Down syndrome (DS), Turners syndrome (TS) and Klinefelter syndrome; amenorrhea; ambiguous sex and multiple congenital malformations. Chromosomal analysis was performed on lymphocyte culture according to standard methods. RESULTS Of the 859 cases studied, 371 (43.1%) had chromosomal abnormalities. The most common autosomal abnormalities were DS 302 (81.4%) and sex chromosomal abnormalities were TS 51 (13.7%). Numerical abnormalities were accounted for 353 (41.0%) and structural abnormalities 18 (2.0%), respectively. Various other chromosomal anomalies were also reported. CONCLUSION We have reviewed the incidence and distribution of chromosomal abnormalities and found higher rate of chromosomal abnormalities 43.1% in the referred cases. Our data suggest that chromosomal analysis is important tool in the evaluation of genetic disorders and helps clinicians to provide accurate diagnosis and proper genetic counselling.

AB142. Clinical, biochemical and growth hormone receptor polymorphism profile of children with short stature presenting to a tertiary care centre

Background and objective To evaluate the clinical and biochemical profile and growth hormone receptor polymorphism of children presenting with short stature to a tertiary care centre. This would thus be directing resources to patients with GH deficiency who would respond best to it.

AB152. Inborn errors of metabolism spectrum in symptomatic children of north India: 5-year prospective data from tertiary care centre.

Background Children with high suspicion of IEM is a more effective screening strategy in a resource limited country like India. We present a prospective analysis of symptomatic children with red flag signs suggestive of IEM referred for analysis by LCMSMS. This study investigated the spectrum of IEM in symptomatic children over a period of 5 years (1st June 2010 to May 31st 2015).