Construction & assessment of a unified curated reference database for improving the taxonomic classification of bacteria using 16S rRNA sequence data.

Background & objectives: For bacterial community analysis, 16S rRNA sequences are subjected to taxonomic classification through comparison with one of the three commonly used databases [Greengenes, SILVA and Ribosomal Database Project (RDP)]. It was hypothesized that a unified database containing fully annotated, non-redundant sequences from all the three databases, might provide better taxonomic classification during analysis of 16S rRNA sequence data. Hence, a unified 16S rRNA database was constructed and its performance was assessed by using it with four different taxonomic assignment methods, and for data from various hypervariable regions (HVRs) of 16S rRNA gene. Methods: We constructed a unified 16S rRNA database (16S-UDb) by merging non-ambiguous, fully annotated, full-length 16S rRNA sequences from the three databases and compared its performance in taxonomy assignment with that of three original databases. This was done using four different taxonomy assignment methods [mothur Naïve Bayesian Classifier (mothur-nbc), RDP Naïve Bayesian Classifier (rdp-nbc), UCLUST, SortMeRNA] and data from 13 regions of 16S rRNA [seven hypervariable regions (HVR) (V2-V8) and six pairs of adjacent HVRs]. Results: Our unified 16S rRNA database contained 13,078 full-length, fully annotated 16S rRNA sequences. It could assign genus and species to larger proportions (90.05 and 46.82%, respectively, when used with mothur-nbc classifier and the V2+V3 region) of sequences in the test database than the three original 16S rRNA databases (70.88-87.20% and 10.23-24.28%, respectively, with the same classifier and region). Interpretation & conclusions: Our results indicate that for analysis of bacterial mixtures, sequencing of V2-V3 region of 16S rRNA followed by analysis of the data using the mothur-nbc classifier and our 16S-UDb database may be preferred.

Biochemical regulation and structural analysis of copper-transporting ATPase in a human hepatoma cell line for Wilson disease.

Hepatic copper levels differ among patients with Wilson disease (WD) and normal individuals depending on the dietary intake, copper bioavailability, and genetic factors. Copper chloride (CuCl2 ) caused dose-dependent reduction in cell viability of human teratocarcinoma (HepG2) cell line, measured using the 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Cells were exposed to different concentrations of CuCl2 in log doses and maximum cell viability reduction was recorded at 15 µg/mL. Toxic dose of CuCl2 is potent inducer of reactive oxygen species (ROS). Apoptosis as a pattern of cell death was confirmed through sub-G1 fraction and morphological changes such as mitochondrial depolarization, endoplasmic reticulum and lysosomal destabilization, phosphatidylserine translocation, and DNA damage. Our transcriptional and translational results strongly support apoptotic cell death. Using the available data present in dbSNP and bioinformatics tools, three nonsynonymous single nucleotide polymorphisms (nsSNPs) were identified as deleterious, reducing the stability of protein ATP7B. Structural analysis of native and mutant ATP7B proteins was investigated using molecular dynamics simulation (MDS) approach. Mutation in ATP7B gene might disturb the structural conformation and catalytic function of the ATP7B protein may be inducing WD. Hence, excess dietary intake of copper chloride must be avoided for safety of health to prevent from WD.

Spectrum of genomic variations in Indian patients with progressive familial intrahepatic cholestasis.

BACKGROUND: Progressive familial intrahepatic cholestasis (PFIC) is caused by variations in ATP8B1, ABCB11 or ABCB4 genes. Data on genetic variations in Indian patients with PFIC are lacking. METHODS: Coding and splice regions of the three genes were sequenced in unrelated Indian children with PFIC phenotype. The variations identified were looked for in parents, 30 healthy persons and several variation databases, and their effect was assessed in-silico. RESULTS: Among 25 children (aged 1-144 months), nine (36%) had unique major genomic variations (ATP8B1: 4, ABCB11: 3 and ABCB4: 2). Seven had homozygous variations, which were assessed as 'pathogenic' or 'likely pathogenic'. These included: (i) four amino acid substitutions (ATP8B1: c.1660G > A/p.Asp554Asn and c.2941G > A/p.Glu981Lys; ABCB11: c.548 T > C/p.Met183Thr; ABCB4: c.431G > A/p.Arg144Gln); (ii) one 3-nucleotide deletion causing an amino acid deletion (ATP8B1: c.1587_1589delCTT/p.Phe529del); (iii) one single-nucleotide deletion leading to frame-shift and premature termination (ABCB11: c.1360delG/p.Val454Ter); and (iv) a complex inversion of 4 nucleotides with a single-nucleotide insertion leading to frame-shift and premature termination (ATP8B1: c.[589_592inv;592_593insA]/p.Gly197LeufsTer10). Two variations were found in heterozygous form: (i) a splice-site variation likely to cause abnormal splicing (ABCB11: c.784 + 1G > C), and (ii) a nucleotide substitution that created a premature stop codon (ABCB4: c.475C > T/p.Arg159Ter); these were considered as variations of uncertain significance. Three of the nine variations were novel. CONCLUSIONS: Nine major genomic variations, including three novel ones, were identified in nearly one-third  of Indian children with PFIC. No variation was identified in nearly two-thirds of patients, who may have been related to variations in promoter or intronic regions of the three PFIC genes, or in other bile-salt transport genes.

Relationship of Severity of Hepatitis A with Polymorphisms in Hepatitis A Virus Cellular Receptor 1 (HAVCR1) Gene.

INTRODUCTION AND AIM: HAVCR1 protein is the cellular receptor for hepatitis A virus (HAV). Genetic polymorphism in this gene may alter the outcome of HAV infection. In a previous study, a 6-amino acid insertion (157insMTTTVP) in HAVCR1 gene was associated with more severe disease. We decided to investigate this association further. MATERIAL AND METHODS: We sequenced exon 4 of the HAVCR1 gene in patients with clinical hepatitis A attending our institution, and a group of healthy controls in a disease-endemic setting in India. Frequencies of different haplotypes of a genomic region with two overlapping insertion-deletion polymorphisms (indels; rs141023871 and rs139041445) were compared between patients and controls, as well as between patients with and without a severe form of disease (liver failure). RESULTS: The gene had three haplotypes in the region of interest - a short form, an intermediate-form with a 5-amino acid 157insMTTVP insertion and a long-form with a 6-amino acid 157insMTTTVP insertion. The allele frequency (29/150 [19%] vs. 43/146 [29%]; p = ns) and haplotype frequency (29/75 [39%] vs. 39/73 [53%]; p = ns) of the 157insMTTTVP variant were similar in hepatitis A patients and healthy controls (30%). Further, the allele frequency (12/58 [21%] vs. 17/92 [18%]; p = ns) and haplotype frequency (12/29 [41%] vs.17/46 [37%]; p = ns) of the longest variant were also similar in patients with severe and mild disease. DISCUSSION: In the study population, the 157insMTTTVP variant of HAVCR1 gene was not associated with more severe outcome of HAV infection. Further studies in other populations around the world are needed to assess the relation of this genetic variation with disease outcome.

Genotyping of hepatitis B virus isolates from Lahaul and Spiti district in Himachal Pradesh, India.

Hepatitis B virus (HBV) has several genotypes. In the Indian population, genotypes A and D are the most frequent. HBV infection is hyper-endemic in the Lahaul and Spiti district in Himachal Pradesh; however, the virus genotype in this area is not known. We sequenced a 398-nucleotide segment of HBV genome that included parts of pre-S1/S2 and polymerase genes from 17 specimens from this district, and assigned a viral genotype to these. Of the 17 specimens studied, 13 (76% [95% confidence interval = 50-92%]) showed the presence of genotype C HBV; the remaining four were genotype D (n = 4; 24%) HBV. Prevalence of genotype C HBV was much higher in the district than in other parts of India. This may reflect the historical mixing of this population with that in China. Since genotype C has a higher risk of chronicity and mother-to-child transmission, prevention of HBV infection may need particular emphasis in this area.

A Novel Truncation Mutation in ATP8B1 Gene in Progressive Familial Intrahepatic Cholestasis.

BACKGROUND: Progressive familial intrahepatic cholestasis has been only infrequently reported from India. CASE CHARACTERISTICS: An Indian girl with progressive cholestatic liver disease beginning during infancy, normal gamma-glutamyl transpeptidase levels, parental consanguinity, positive family history and a fatal outcome. OBSERVATION: A novel, homozygous mutation (c.[589_592inv;592_593insA]) in ATP8B1 gene, with a markedly truncated protein (p.[Gly197LeufsTer10]) was found. MESSAGE: The novel mutation found expands the spectrum of genetic variations associated with progressive familial intrahepatic cholestasis.