Rapid Method for Detection of Extra (TA) in the Promoter of the Bilirubin-UDP-Glucuronosyl Transferase 1 Gene Associated with Gilbert Syndrome

Abstract

Gilbert syndrome (GS) is an inherited form of chronic mild unconjugated hyperbilirubinemia (1)(2)(3), although many patients do not have a clear family history (4). Hepatic glucuronidation of bilirubin is catalyzed by isoenzyme 1A1 of UDP-glucuronosyl transferase (UGT1A1). The majority of GS subjects were found to be homozygous for an extra TA in the TATA-box in the promoter region of UGT1A1 (5)(6)(7). Transcription of the (TA)7 allele is reduced by at least 70% compared with the wild-type (TA)6 allele. Because bilirubin UGT1A1 is the only enzyme with substantial bilirubin glucuronidating activity in humans (8), the presence of this extra TA in both alleles can explain the impaired conjugation of bilirubin found in Caucasoid GS patients (6). A previous study of a large population found that the prevalence of the “abnormal” bilirubin UGT1A1 allele was 35–40% (9), leading to an expected frequency of homozygotes of ∼16%; however, only 5% had increased serum concentrations of unconjugated bilirubin. Thus, a reduced expression of bilirubin UGT1A1, attributable to the (TA)7 abnormality in the promoter region, appears to be necessary, but not sufficient, for GS to be manifested clinically. To date, the TA polymorphism has been detected by PCR amplification of the TATA-box element and high resolution polyacrylamide gel electrophoresis (9) or by direct sequencing (6). Recently, a new technique for sensitive, relatively inexpensive and automated high-throughput screening of mutations, denaturing HPLC (DHPLC), was introduced (10)(11)(12)(13). In this report, we evaluate the feasibility of applying DHPLC for the detection of TATA-box variants in the promoter region of the UGT1A1 gene in subjects with GS. The UGT1A1 promoter was analyzed by both DHPLC and direct sequencing in 20 unrelated GS patients (16 males and 4 females; age range, 16–40 years) and 20 …