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Development of a new DHPLC assay for genotyping UGT1A1(TA)n polymorphism associated with Gilbert's syndrome
Jurković Mlakar, Simona (Author), Ostanek, Barbara (Author)

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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 a 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. Materials and methods: Fifty DNA samples with commongenotypes ((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.

Language:Serbian
Keywords:hiperbilirubinemija, Gilbertov sindrom, mikrosatelit, farmakogenomika, SSCP, UGT1A1
Work type:Not categorized (r6)
Tipology:1.01 - Original Scientific Article
Organization:FFA - Faculty of Pharmacy
Year:2011
Number of pages:str. 167-173
Numbering:Vol. 21, no. 2
UDC:577
ISSN on article:1330-0962
COBISS.SI-ID:3031153 Link is opened in a new window
Views:676
Downloads:214
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Record is a part of a journal

Title:Biochemia medica
Shortened title:Biochem. med.
Publisher:Hrvatsko društvo medicinskih biokemičara
ISSN:1330-0962
COBISS.SI-ID:3502345 This link opens in a new window

Secondary language

Language:English
Title:Razvoj nove metode DHPLC za genotipizaciju polimorfizma UGT1A1(TA)n povezanog s Gilbertovim sindromom

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