title: Identification of Promotor and Exonic Variations, and Functional Characterization of a Splice Site Mutation in Indian Patients with Unconjugated Hyperbilirubinemia.
abstract: BACKGROUND
  Mild unconjugated hyperbilirubinemia (UH), due to reduced activity of the enzyme uridine diphosphoglucuronate-glucuronosyltransferase family, polypeptide 1 (UGT1A1), is a common clinical condition. Most cases are caused by presence in homozygous form of an A(TA)7TAA nucleotide sequence instead of the usual A(TA)6TAA sequence in promoter region of the UGT1A1 gene. In some cases, other genetic variations have been identified which differ between populations. There is need for more data on such genetic variations from India.
  
  
  METHODS
  DNA from subjects with unexplained persistent or recurrent UH was tested for the presence of TA promoter insertions. In addition, all five exons and splicing site regions of UGT1A1 gene were sequenced. Several bioinformatics tools were used to determine the biological significance of the observed genetic changes. Functional analysis was done to look for effect of a splice site mutation in UGT1A1.
  
  
  RESULTS
  Of 71 subjects with UH (68 male; median age [range], 26 [16-63] years; serum bilirubin 56 [26-219] μM/L, predominantly unconjugated) studied, 65 (91.5%) subjects were homozygous for A(TA)7TAA allele, five (7.0%) were heterozygous, and one (1.4%) lacked this change. Fifteen subjects with UH had missense exonic single nucleotide changes (14 heterozygous, 1 homozygous), including one subject with a novel nucleotide change (p.Thr205Asn). Bioinformatics tools predicted some of these variations (p.Arg108Cys, p.Ile159Thr and p.Glu463Val) to be deleterious. Functional characterization of an exonic variation (c.1084G>A) located at a splice site revealed that it results in frameshift deletion of 31 nucleotides and premature truncation of the protein.
  
  
  CONCLUSIONS
  Our study revealed several single nucleotide variations in UGT1A1 gene in Indian subjects with UH. Functional characterization of a splice site variation indicated that it leads to disordered splicing. These variations may explain UH in subjects who lacked homozygous A(TA)7TAA promoter alleles.
authors.affiliation: Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, 226014, India.
authors.forename: Neha
authors.initials: N
authors.lastname: Gupta
authors.affiliation: Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, 226014, India.
authors.forename: Mercilena
authors.initials: M
authors.lastname: Benjamin
authors.affiliation: Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, 500001, India.|Graduate Studies, Manipal University, Manipal, 576104, Karnataka, India.
authors.forename: Anjana
authors.initials: A
authors.lastname: Kar
authors.affiliation: Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, 226014, India.
authors.forename: Sachin Dev
authors.initials: SD
authors.lastname: Munjal
authors.affiliation: Biomedical Informatics Center, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, 226014, India.
authors.forename: Aditya N
authors.initials: AN
authors.lastname: Sarangi
authors.affiliation: Diagnostics Division, Centre for DNA Fingerprinting and Diagnostics, Hyderabad, 500001, India.
authors.forename: Ashwin
authors.initials: A
authors.lastname: Dalal
authors.affiliation: Department of Gastroenterology, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, 226014, India.|Biomedical Informatics Center, Sanjay Gandhi Postgraduate Institute of Medical Sciences, Lucknow, 226014, India.
authors.forename: Rakesh
authors.initials: R
authors.lastname: Aggarwal
mesh_terms: D000293:Adolescent; D000328:Adult; D001663:Bilirubin / Q000097:blood; D005091:Exons / Q000235:genetics*; D005260:Female; D014453:Glucuronosyltransferase / Q000235:genetics*; D006579:Heterozygote; D006720:Homozygote; D006801:Humans; D006932:Hyperbilirubinemia / Q000235:genetics*; D007194:India; D008297:Male; D008875:Middle Aged; D009154:Mutation; D020125:Mutation, Missense / Q000235:genetics; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D011401:Promoter Regions, Genetic / Q000235:genetics*; D022821:RNA Splice Sites / Q000235:genetics*; D055815:Young Adult; D000099148:UGT1A1 Enzyme
chemical_list: D001663:Bilirubin; D014453:Glucuronosyltransferase; D022821:RNA Splice Sites; D000099148:UGT1A1 Enzyme