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Abstract
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. 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. 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. 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.
Highlights
Gilbert’s syndrome is the most common hereditary disorder of bilirubin metabolism
Our results show that sizing denaturing high-performance liquid chromatography (DHPLC) assay is more efficient compared to classical single-strand conformation polymorphism (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 UDP-glucuronosyltransferase 1A1 (UGT1A1)(TA)n genotypes
Fifty-seven samples were genotyped for the UGT1A1(TA)n polymorphism twice, using DHPLC and SSCP assays
Summary
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. Sequence variations in the UGT1A1 encoding gene (UGT1A1) that decrease the enzyme activity are responsible for inherited unconjugated hyperbilirubinemias ranging from Gilbert's syndrome (GS) (30% activity) to Crigler-Najjar syndrome type II (10% activity) and Crigler-Najjar syndrome type I (0% activity) [2,3]. While both Crigler-Najjar syndromes are very rare, GS is quite common [4]. The causative sequence variation in Caucasians is almost exclusively a (TA) dinucleotide insertion in the TATA-box of the UGT1A1 promoter.
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