Linkage between A(TA)7TAA and −3279T>G mutations in UGT1A1 is not essential for pathogenesis of Gilbert syndrome

To the editor: Homozygous mutation A(TA)7TAA (briefly (TA)7) in the promoter of the UGT1A1 gene encoding the bilirubin UDP-glucuronosyl transferase is considered to be the cause of the Gilbert syndrome (OMIM 143500) in the vast majority of affected Caucasians (1). An enhancer polymorphism 3279T/G responsible for additional lowering of the transcriptional activity of UGT1A1 was identified in a subset of patients with Gilbert syndrome (2). Maruo et al. (3) suggested that the linkage of 3279G with (TA)7 is essential for the pathogenesis of Gilbert syndrome. The authors investigated 63 Caucasian and Japanese subjects. All 23 patients with Gilbert syndrome (11 Caucasians) were homozygous for (TA)7 and 3279G alleles. In contrast, none of the 40 control subjects (10 Caucasians) was homozygous for either of the mutations (Table 1). These findings may have important clinical implications because in case of perfect linkage, the genetic diagnosis of Gilbert syndrome could be easily established by PCRRFLP genotyping of the 3279T/G locus. We investigated a group of 25 Caucasians with Gilbert syndrome and 179 subjects with normal bilirubin levels (Table 1). Genotyping of the TATAA-box of UGT1A1 gene promoter was performed by fragment analysis of PCR products amplified from genomic DNA using a Cy5-labeled forward primer. The fragments were analyzed on an automated DNA sequencer. The phenobarbitalresponsive enhancer module of the UGT1A1 gene was checked for the presence of the 3279T/G polymorphism by PCR-Hpy8I RFLP (4) using 50CACCTCCTCCTTATTCTCTT and 50-CTCAT TCCTCCTCTCTAGCC forward and reverse primer, respectively. Consistent with the findings of Maruo et al. (3), all but one of our patients with Gilbert syndrome were double homozygotes for (TA)7 and 3279G. Interestingly, the exceptional patient, homozygous for (TA)7 and heterozygous for 3279G, had repeatedly elevated levels of unconjugated bilirubin around 100 mmol/l. Another 15 cases of Gilbert syndrome heterozygous for either (TA)7 or 3279G have been documented by Costa et al. (5) (Table 1). Seventeen (9.5%) of our controls were simultaneously heterozygous for (TA)7 and homozygous for 3279G whereas none of the control subjects studied by Maruo et al. (3) had this genotype. The strength of linkage disequilibrium is expressed by coefficients D0 and r, ranging from 0 to 1. The value of D05 1 indicates a complete linkage disequilibrium if one haplotype is not observed and r 5 1 indicates a perfect linkage disequilibrium in the case where two haplotypes out of four possible in two biallelic variants are not observed. The linkage disequilibrium between (TA)7 and 3279G allele was almost complete (D05 0.99; Po0.001) but not perfect (r 5 0.66) in our series. Similar values were calculated from the allelic frequencies published by Innocenti et al. (6) (133 healthy Caucasians, D05 0.99, r 5 0.65; Po0.001) and Costa et al. (5) (D05 0.88, r 5 0.53; Po0.001). These data provide evidence that the linkage between 3279G and (TA)7 is not essential for the pathogenesis of Gilbert syndrome in Caucasians, and genotyping of the 3279T/G locus instead of the TATAA-box of the UGT1A1 gene promoter may be misleading.