This study was conducted in 95 consecutive patients presenting to the Liver Clinic at the Department of Gastroenterology of the Institute of Postgraduate Medical Education & Research, Kolkata, India, for evaluation of persistent unconjugated hyperbilirubinemia (serum bilirubin greater than 1.2 mg/dL) documented at least twice over a period of one month. Serum total bilirubin and its unconjugated fraction were estimated in fasting condition. Each of these individuals had a normal finding on physical examination, normal hepatobiliary ultrasound, normal liver function test excluding hyperbilirubinemia and a normal reticulocyte count. A total of 95 adult healthy volunteers from the same ethnic population as the GS patients were included as controls. Each control also underwent the same set of investigations as the patients. Their fasting serum bilirubin level estimated twice at intervals of fifteen days was consistently less than 1.0 mg/dL. Patients were excluded if they were under any medication during the past one month or consumed alcohol regularly or had present or past history of hepatic/hematological disease. Additionally, quantitative estimation of hemoglobin fractions (A₀, A₂ and F) was carried out on each patient and control by cation-exchange HPLC using the Variant^TM Hemoglobin Testing System Beta Thalassemia Short Program (Bio-Rad Diagnostics, Hercules, USA). A 5 mL blood sample was collected from each patient by venipuncture and DNA was isolated using a standard protocol[25]. Data and samples were collected with written informed consent of the patients and controls, after the approval was obtained from the Human Research Ethics Committee of the Institute of Postgraduate Medical Education & Research, Kolkata.

DNA re-sequencing of the promoter and exon 1*1 encoding the substrate-specific region of bilirubin-UGT1 gene as well as the 4 common exons (exons 2-5) was carried out using an automated DNA sequencer (ABI-3100; Applied Bio-systems, Foster City, USA.). DNA samples were first amplified by the polymerase chain reaction technique using an ABI-9700 thermal cycler. PCR products were then cleaned using exonuclease-I (USB. Corporation, Cleveland, USA) and shrimp alkaline phosphatase (Amersham, Freiburg, Germany), and sequencing reactions were carried out. DNA resequencing was carried out in both forward and reverse directions. Raw DNA sequences were analyzed as previously described[26], and variant genotypes were identified.

UGT1A1 promoter fragments, 331-336 bp in length depending on the number of TA repeats and CAT insertion, were amplified using primers (F: 5’-tgtagatcttcctctctggtaacact-3’) and (R: 5’-atgaagctttgctcctgccagaggttc-3’) from genomic DNA of individuals homozygous for six and seven TA repeats, and CAT insertion on (TA)₇TAA background. DNA amplification was carried out with polymerase chain reaction (PCR) using FastStart Taq DNA polymerase (Roche, Mannheim, Germany) with an initial denaturation at 95 °C for 10 min, followed by 35 cycles at 94 °C for 1 min, at 56 °C for 45 s and at 72 °C for 30 s on an ABI-9 700 (Applied Bio-systems, Foster City, USA.) thermal cycler. To facilitate subcloning of the PCR products in the reporter gene construct, oligonucleotides F and R were designed with a Bgl II and a Hind III restriction enzyme site at the 5’ end, respectively. PCR products were doubly digested with Bgl II and Hind III, purified by Qiagen gel purification kit (Qiagen, Hilden, Germany) and subcloned into pGL3-basic vector (Promega, Madison, USA). The integrity of the resulting plasmids was confirmed by restriction mapping and sequencing analyses. Promoter activity of each construct was measured in HepG2 cell line. Cells were grown in MEM medium supplemented with 2 mmol/L L-glutamine, 0.1 mmol/L non-essential amino acids, 1 mmol/L sodium pyruvate and 10% fetal calf serum (GIBCO -BRL, Grand Island, USA) at 37 °C with 50 mL/L CO₂. Exponentially growing cells were trypsinized, seeded at 2.5×10⁵ cells, and incubated overnight prior to transfection. Transfection was carried out by lipofectamin (Invitrogen, Carlsbad, USA) using 2 μg of each of the constructs (namely (TA)₆ TAA, (TA)₇ TAA and CAT insertion on (TA)₇ TAA background), as well as the pGL3-basic and pGL3-control as positive controls (TA)₇TAA, the luciferase gene was under the control of SV40 promoter and enhancer. After 48 h of transfection, the cells were lysed and centrifuged in cell culture lysis buffer (Promega, Madison, USA). Luciferase activity was assayed in the cell lysate by measuring the photoluminescence in a Monolight 2010 single channel luminometer and the total cellular protein content was measured by the standard Bradford’s method. Luciferase activity was normalized to total cell protein concentration. Normalized luciferase activity of each construct was expressed as a ratio to that of the pGL3-basic vector. Three independent experiments were performed for each construct and all measurements were determined in duplicate.

Equality of proportions was statistically tested by the standard normal test procedure[27]. Equalities of mean values of various hematological parameters for individuals belonging to different genotypes at various polymorphic loci were statistically tested using the Student t-test or the analysis of variance (ANOVA) procedure, as appropriate. Regression analysis was performed to test the significance of dependence of un-conjugated serum bilirubin level on some relevant variables. Allele frequencies were estimated using the gene-counting method.

No statistically significant (P > 0.05) differences in the proportions of males and females were observed between the patients (87 males, 8 females) and controls (77 males, 18 females). The mean ages of male and female patients (30.1 ± 1.1 years of males, 26.8 ± 5.1 years of females) and controls (30.2 ± 1.1 years of males, 31.1  ± 2.9 years of females) were not significantly different (P > 0.05). Since elevated HbA₂ levels could potentially increase the unconjugated bilirubin level, we tested the significance of the regression coefficient of HbA₂ level on unconjugated bilirubin, separately for patients and controls. In both sets, there was no statistically significant impact of HbA₂ (P-values for patients and controls were 0.638 and 0.106, respectively). The difference in the mean values of HbA₂ between patients and controls was not significantly different (t = 1.76, d.f. = 128, P > 0.05). The 8 GS patients, but none of the normal controls, who were habitual smokers were asked to refrain from smoking for 24 h prior to blood collection. We tested whether the inclusion of these 8 GS patients had a significant impact on our findings. We therefore, examined whether the mean value of un-conjugated bilirubin among patients who were smokers (n = 8) was significantly higher than that among patients who were not smokers (n = 87). No significant difference was found (mean for smoker patients=2.67 ± 0.33, mean for non-smoker patients = 3.38 ± 0.30, t = 0.696, d.f. = 93, P = 0.488). We have performed all analyses that were reported below with and without the inclusion of the 8 GS patients who were habitual smokers. No differences in inferences were found (results not shown). Therefore, we presented all results including these 8 GS subjects.

Eleven sequence variants in UGT1A1 gene was observed, of which 3 each were in the promoter, 3 in exon 1, 2 in exon 2, 1 in exon 3 and 2 in exon 4. No variant was found in exon 5. The genotype and variant allele frequencies at these positions in patients and controls are given in Table 1. Of these 11 variant sites, 2 were non-polymorphic (frequency of the rarer allele < 1%), while the remaining 9 sites were polymorphic either among patients or among controls or in both. One of the polymorphic sites was the TA insertion [(TA)₇TAA allele] in the TATA box of the UGT1A1 promoter[2,3]. The frequencies of the (TA)₇TAA allele (0.879) and the 7/7 genotype (80%) among the patients were significantly higher (P < 0.005) than those among the controls (0.384 and 10%, respectively). The un-conjugated serum bilirubin values for GS patients belonging to the 6/6, 7/6 and 7/7 genotypes are presented in Figure 1. The mean values of un-conjugated bilirubin for GS patients and normal controls were 2.5 ± 0.25 mg/dL and 0.61 ± 0.09 mg/dL respectively. The mean+SE value of un-conjugated bilirubin among patients with the 7/6 genotype was 3.46 ± 0.54 mg/dL, which was not significantly different (P = 0.05) from patients with the 7/7 genotype (3.31 ± 0.33 mg/dL). The mean un-conjugated serum bilirubin values for these genotypes among controls were only 0.59 ± 0.10 mg/dL and 0.64 ± 0.15 mg/dL, respectively.

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Figure 1 Distributions of un-conjugated serum bilirubin values among Gilbert’s syndrome patients classified by the genotype of the common TA insertion in the TATA box of the UGT1A1 promoter.

No significant differences (P = 0.05) were found in the mean bilirubin levels among individuals belonging to the various genotypes at the remaining single nucleotide polymorphic loci. With respect to the C6844G polymorphism resulting in a non-synonymous amino acid change (A321G), all patients were CC homozygotes, while about 30% of the controls were CG heterozygotes (Table 1). We did not find any significant effect of the G71R polymorphism on un-conjugated bilirubin level. This polymorphism also showed no significant interaction with the TATA box insertion polymorphism (results not shown). These findings are discordant with those reported among the Japanese[19].

Among the polymorphic variants described in Table 1, aside from the familiar TA insertion, the most striking was the CAT insertion (nucleotide positions -85 to -83) in the CAAT box of UGT1*1 promoter. Normally, there is one copy of the CAT trinucleotide present in human Genbank (http://www.ncbi.nlm.nih.gov/Genbank/). We found two copies of this trinucleotide in some GS patients. To confirm that this was an insertion we searched the chimpanzee (gi|6456543|gb|AF135463.1|AF135463) and gorilla (gi|6456545|gb|AF135464.1|AF135464) databases. In both species there is only copy, confirming that the single copy is the ancestral state. This CAT insertion was found only among nine (10%) GS patients, who were all homozygous for the TA insertion. We found that GS patients with the CAT-insertion had a significantly (P < 0.001) elevated mean level (6.13 ± 1.61 mg/dL) compared to those without the insertion (2.93 ± 0.28 mg/dL). Individuals who possessed the CAT insertion did not consistently possess a variant allele at any of the other sites. One individual who was heterozygous for the CAT insertion was also heterozygous for the I322V variant, and other three CAT-insertion heterozygotes were also heterozygotes for the H376R variant.

Since about 10% of the (TA)₇TAA homozygotes carried the CAT insertion and the insertion significantly elevated the bilirubin level, we postulated that the insertion had a functional impact. To examine this, we studied the change in DNA-folding of the promoter region caused by this insertion. The most stable structures, namely those with lowest free energy (dG) values, are given in Figure 2 for the UGT1A1 promoter region without (dG = -14.4) and with (dG = -14.8) the additional CAT insertion. A striking structural change was found around the region of the insertion, the loops were converted to stems. It is well known that formation of stems in the promoter could reduce transcription of the gene. A detailed functional analysis of the UGT1A1 promoter by luciferase reporter assay confirmed that there was a two-fold decrease in transcription efficiency for the variant (TA)₇TAA promoter allele, and a 20-fold decrease when there was a CAT insertion in the promoter on the background of the (TA)₇TAA allele compared to the normal (TA)₆TAA promoter allele (Figure 3).

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Figure 2 Folded DNA structures of the UGT1A1 promoter region with one copy of the CAT trinucleotide (shaded region) (A) and two copies of the CAT trinucleotide (CAT insertion allele, shaded region) (B).

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Figure 3 Transcriptional activities of the (TA) ₆TAA, (TA)₇ TAA and CAT alleles as assessed by a luciferase reporter assay.