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Combined Effect of Regulatory Polymorphisms on Transcription of UGT1A1 as a Cause of Gilbert Syndrome

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Combined Effect of Regulatory Polymorphisms on Transcription of UGT1A1 as a Cause of Gilbert Syndrome

Katsuyuki Matsui et al. BMC Gastroenterol.

Abstract

Background: Gilbert syndrome is caused by defects in bilirubin UDP-glucuronosyltransferase (UGT1A1). The most common variation believed to be involved is A(TA)7TAA. Although several polymorphisms have been found to link with A(TA)7TAA, the combined effect of regulatory polymorphisms in the development of Gilbert syndrome remains unclear.

Methods: In an analysis of 15 patients and 60 normal subjects, we detected 14 polymorphisms and nine haplotypes in the regulatory region. We classified the 4-kbp regulatory region of the patients into: the TATA box including A(TA)7TAA; a phenobarbital responsive enhancer module including c.-3275T>G; and a region including other ten linked polymorphisms. The effect on transcription of these polymorphisms was studied.

Results: All haplotypes with A(TA)7TAA had c.-3275T>G and additional polymorphisms. In an in-vitro expression study of the 4-kbp regulatory region, A(TA)7TAA alone did not significantly reduce transcription. In contrast, c.-3275T>G reduced transcription to 69% of that of wild type, and the linked polymorphisms reduced transcription to 88% of wild type. Transcription of the typical regulatory region of the patients was 56% of wild type. Co-expression of constitutive androstane receptor (CAR) increased the transcription of wild type by a factor of 4.3. Each polymorphism by itself did not reduce transcription to the level of the patients, however, even in the presence of CAR.

Conclusions: These results imply that co-operation of A(TA)7TAA, c.-3275T>G and the linked polymorphisms is necessary in causing Gilbert syndrome.

Figures

Figure 1
Figure 1
List of vectors constructed. UGT1A1 regulatory regions (c.-4076 to c.-1, Nos. 1 - 8), with differing combinations of polymorphic variations, were inserted into the luciferase reporter plasmid PGV-B2. The wild-type vector is No.1, and No.2 is a typical vector with regulatory regions showing Gilbert syndrome. The open box is a wild-type region between gtPBREM and the TATA box, including ten linked polymorphisms: c.-3152G>A, c.-2951A>G, c.-2743T>C, c.-2737T>C, c.-2726G>A, c.-2724AT[8], c.-2473T>G, c.-1352A>C, c.-689A>C and c.-364C>T. The gray box is a mutant-type region having mutant-type sequence corresponding to the wild-type region. Luc is luciferase cDNA. The name of the expression vector corresponds to the polymorphisms included and the type of vector. In the first part of the name, T and G respectively denote c.-3275T and c.-3275G. In the second part of the name, w and m refer to wild type and mutant-type region between gtPBREM and the TATA box. In the third part, (TA)6 and (TA)7 denote A(TA)6TAA and A(TA)7TAA. In the fourth part, wild, Gilbert and combination indicate that each vector has wild-type sequence, or typical mutant-type sequence in subjects suffering from Gilbert syndrome, or an artificial sequence constructed for this study.
Figure 2
Figure 2
Transcriptional activity of the continuous 4-kbp regulatory region (c.-4076 to c.-1) with/without the CAR expression plasmid. The combination of variations in each expression vector is shown in Figure 1. Name of expression vector represents polymorphisms included and vector type. In the first part of the name, T and G respectively denote c.-3275T and c.-3275G. In the second part of the name, w and m refer to wild type and mutant-type region between gtPBREM and the TATA box. In the third part, (TA)6 and (TA)7 denote A(TA)6TAA and A(TA)7TAA. In the fourth part, wild, Gilbert and combination indicate that each vector has wild-type sequence, or typical mutant-type sequence in subjects suffering from Gilbert syndrome, or an artificial sequence constructed for this study. The mean transcriptional activity of the wild-type vector, T-w-(TA)6_wild, without the CAR expression plasmid was chosen as 1.00. Each column and bar represents the mean and SD of four independent determinations. The vector with typical regulatory region showing Gilbert syndrome is G-m-(TA)7_Gilbert. The open box is a wild-type region between gtPBREM and the TATA box, including ten linked polymorphisms: c.-3152G>A, c.-2951A>G, c.-2743T>C, c.-2737T>C, c.-2726G>A, c.-2724AT[8], c.-2473T>G, c.-1352A>C, c.-689A>C and c.-364C>T. The gray box is a mutant-type region having mutant-type sequence corresponding to the wild-type inner region. Luc is luciferase cDNA.

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