Clin Chem Lab Med 2008;46(2):174–178  2008 by Walter de Gruyter • Berlin • New York. DOI 10.1515/CCLM.2008.035 2007/249 Article in press - uncorrected proof Rare TA repeats in promoter TATA box of the UDP glucuronosyltranferase (UGT1A1) gene in Croatian subjects Nora Nikolac 1, *, Ana-Maria Simundic 1 , Elizabeta Topic 1 , Zvonko Jurcic 2 , Mario Stefanovic 1 , Jerka Dumic 3 and Sandra Supraha Goreta 3 1 University Department of Chemistry, Sestre Milosrdnice University Hospital, Zagreb, Croatia 2 Department of Pediatrics, Sestre Milosrdnice University Hospital, Zagreb, Croatia 3 Department of Biochemistry and Molecular Biology, Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia Abstract Background: Gilbert’s syndrome is a chronic or recur- rent mild unconjugated hyperbilirubinemia caused by decreased activity of UDP glucuronosyltranferase (UGT1A1). The most common cause of Gilbert’s syn- drome in Caucasians is homozygous variant of the A(TA) 7 TAA promoter polymorphism. Alleles with five or eight TA repeats have also been described, but they are very rare in Caucasian populations. Methods: Over a 6-year period (2001–2006), 1109 sub- jects with suspected Gilbert’s syndrome were includ- ed in this study. Genotyping of (TA) 6 and (TA) 7 alleles was performed using high-resolution electrophoretic separation of amplified PCR products on Spreadex EL300 gels. In seven subjects, aberrant electrophor- etic patterns were observed and additionally sequenced on an ABI Prism 310 Genetic Analyzer. Results: Genotype distributions for 1102 subjects with (TA) 6 or (TA) 7 alleles were as follows: 54.10%, 26.33% and 18.94% for the (TA) 7 /(TA) 7 , (TA) 6 /(TA) 7 and (TA) 6 / (TA) 6 , respectively. Sequencing of seven samples that could not be identified as one of these alleles ident- ified four subjects with the (TA) 5 /(TA) 7 , two with the (TA) 7 /(TA) 8 and one with the (TA) 6 /(TA) 8 genotype. Conclusion: Genotyping of TA repeats in the promoter region of the UGT1A1 gene revealed the presence of rare alleles with five or eight TA repeats, with a very high frequency of the (TA) 7 allele in subjects suspect- ed of having Gilbert’s syndrome. Clin Chem Lab Med 2008;46:174–8. Keywords: genetic polymorphism; Gilbert’s syn- drome; hyperbilirubinemia; neonatal jaundice. *Corresponding author: Nora Nikolac, B.Sc., University Department of Chemistry, Sestre Milosrdnice University Hospital, Vinogradska 29, 10000 Zagreb, Croatia Phone/Fax: q385-1-3768-280, E-mail: nora.nikolac@gmail.com Received May 28, 2007; accepted September 11, 2007 Introduction Gilbert’s syndrome (GS) is an inherited, chronic, inter- mittent, mild, unconjugated hyperbilirubinemia with- out liver disease and overt hemolysis, caused by decreased activity of UDP glucuronosyltranferase (UGT1A1) (1–3). Bilirubin concentration typically fluc- tuates with time, ranging from 20 to 50 mmol/L w rarely over 85 mmol/L (3)x , with an increase after stress, infection or starving. Other liver functions are normal, and GS does not require any special medical treat- ment. However, the utmost importance of recognizing this condition lies in differential diagnostics from oth- er liver dysfunctions associated with hyperbilirubin- emia. UGT1A1 genotyping can also be useful in neonatal diagnostics, as it was found to be associated with prolonged neonatal hyperbilirubinemia (4). Although very rarely, GS may coincide with some hereditary anemia, such as hereditary spherocytosis (5), congenital dyserythropoietic anemia (6) and glucose-6-phosphate dehydrogenase deficiency (7), and increase the risk of gallstone formation. The decreased activity of UGT1A1 may be caused by numerous polymorphisms in the UGT1A1 gene (8). The most common polymorphism in Caucasian pop- ulations is a TA insertion in TA(TA) 6 TAA sequence in the promoter region (9). This allele, (TA) 7 , is respon- sible for a decreased transcription rate and results in 30% lower activity compared to the wild type. Because bilirubin conjugation catalyzed by UGT1A1 appears to be the rate-limiting step in bilirubin elim- ination, decreased enzymatic activity in GS leads to lower bilirubin conjugation and its elevated blood concentrations. Promoter TATA box is highly polymorphic and var- iants with five or eight TA repeats were also described in association with GS. The number of the repeats inversely correlates with transcriptional activity (10), thus allele (TA) 8 is found to be associated with the lowest and allele (TA) 5 with the highest enzyme activity. The distribution of TATA box polymorphisms shows a large ethnic variability (9). In comparison to other ethnic groups, the frequency of the (TA) 7 allele in Caucasian populations is intermediate (38.7%). Asian populations show the lowest frequency of the (TA) 7 allele (16%), yet it is still the most common cause of GS. The highest frequency was found in Africans (49.5%), although their serum bilirubin concentration was in general 15%–20% lower than in Caucasians (11). Interestingly, in Africans the alleles (TA) 5 and (TA) 8 were found to be 3.5% and 6.9%, respectively (9, 12), while these alleles are very rare in Caucasian populations. However, the literature