title: Development of a new DHPLC assay for genotyping UGT1A (TA)n polymorphism associated with Gilbert's syndrome.
abstract: BACKGROUND
  Gilbert's syndrome is the most common hereditary disorder of bilirubin metabolism. The causative mutation in Caucasians is almost exclusively a (TA) dinucleotide insertion in the UGT1A1 promoter. Affected individuals are homozygous for the variant promoter and have 7 TA repeats instead of 6. Promoters with 5 and 8 TA repeats also exist but are extremely rare in Caucasians. The aim of our study was to develop denaturing high-performance liquid chromatography (DHPLC) assay for genotyping UGT1A1(TA)n polymorphism and to compare it with a previously described single-strand conformation polymorphism (SSCP) assay.
  
  
  METHODS
  Fifty DNA samples with common genotypes ((TA)6/6, (TA)6/7, (TA)7/7) as well as 7 samples with one of the following rare genotypes- (TA)5/6, (TA)5/7, (TA)6/8 or (TA)7/8 were amplified by polymerase chain reaction (PCR) and genotyped by DHPLC using sizing mode. All samples were previously genotyped by SSCP assay which was validated by sequencing analysis.
  
  
  RESULTS
  All samples with either common or rare genotypes showed completely concordant results between DHPLC and SSCP assays. Our results show that sizing DHPLC assay is more efficient compared to classical SSCP assay due to shorter time of genotyping analysis, ability of genotyping increased number of samples per day, higher robustness, reproducibility and cost-effectiveness with no loss of accuracy in detection of all UGT1A1(TA)n genotypes.
  
  
  CONCLUSIONS
  We developed a new DHPLC assay which is suitable for accurate, automated, highthroughput, robust genotyping of all UGT1A1(TA)n polymorphism variants, compared to a labour intensive and time-consuming SSCP assay.
authors.affiliation: University of Ljubljana, Faculty of Pharmacy, Department of Clinical Biochemistry, Askerceva 7, Ljubljana, Slovenia.
authors.forename: Simona Jurkovic
authors.initials: SJ
authors.lastname: Mlakar
authors.forename: Barbara
authors.initials: B
authors.lastname: Ostanek
mesh_terms: D001663:Bilirubin / Q000235:genetics / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005819:Genetic Markers; D060005:Genotyping Techniques / Q000379:methods; D005878:Gilbert Disease / Q000175:diagnosis / Q000235:genetics; D014453:Glucuronosyltransferase / Q000235:genetics*; D006801:Humans; D017720:Molecular Epidemiology / Q000379:methods*; D016254:Mutagenesis, Insertional / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D011110:Polymorphism, Genetic / Q000235:genetics*; D015203:Reproducibility of Results; D016385:TATA Box / Q000235:genetics*; D044465:White People / Q000235:genetics; D000099148:UGT1A1 Enzyme
chemical_list: D001663:Bilirubin; D005819:Genetic Markers; D014453:Glucuronosyltransferase; D000099148:UGT1A1 Enzyme