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Coinheritance of Gilbert syndrome–associated UGT1A1 mutation increases gallstone risk in cystic fibrosis†
Article first published online: 23 MAR 2006
Copyright © 2006 American Association for the Study of Liver Diseases
Volume 43, Issue 4, pages 738–741, April 2006
How to Cite
Wasmuth, H. E., Keppeler, H., Herrmann, U., Schirin-Sokhan, R., Barker, M. and Lammert, F. (2006), Coinheritance of Gilbert syndrome–associated UGT1A1 mutation increases gallstone risk in cystic fibrosis. Hepatology, 43: 738–741. doi: 10.1002/hep.21105
Potential conflict of interest: Nothing to report.
- Issue published online: 23 MAR 2006
- Article first published online: 23 MAR 2006
- Manuscript Accepted: 11 JAN 2006
- Manuscript Received: 1 SEP 2005
The prevalence of “black” pigment gallstones is increased in patients with cystic fibrosis (CF). Bile acid malabsorption with augmented bilirubin uptake from the intestine and the development of “hyperbilirubinbilia” have been proposed as key factors in gallstone formation in CF patients. We have now tested the hypothesis that the coinheritance of the common UGT1A1 promoter mutation associated with Gilbert syndrome is an additional lithogenic risk factor for gallstone formation in CF. Our results show that patients with CF and gallstones are significantly more likely to carry at least one Gilbert UGT1A1 allele compared with stone-free patients (OR 7.3; P = .042) and that these carriers display significantly higher serum levels of unconjugated bilirubin (P = .002). In conclusion, the Gilbert UGT1A1 allele increases the risk of gallstone formation in CF. This genetic association supports the current concept for gallstone formation in CF and suggests that genetic and exogenous sources contributing to hyperbilirubinbilia might be lithogenic in CF patients. (HEPATOLOGY 2006;43:738–741.)
The prevalence of cholelithiasis in patients with cystic fibrosis (CF) is increased to 15% to 30% compared to less than 5% in an age-matched control population. Because bile salt malabsorption has been observed in human CF patients1 and mouse models,2 these gallstones were traditionally considered to be of the cholesterol type. However, this view was challenged by a systematic analysis demonstrating that the major component of gallstones in cystic fibrosis is calcium bilirubinate and not cholesterol.3 Accordingly, ursodeoxycholic acid has been shown to be ineffective as a litholytic agent in patients with CF.4 Experimental studies indicate that the formation of pigment gallstones in CF might result, at least in part, from chronic bile salt loss from the small intestine, which induces resorption of unconjugated bilirubin from the large intestine.1, 5 The subsequent enterohepatic cycling of bilirubin from the colon to the liver increases biliary secretion of conjugated bilirubin (“hyperbilirubinbilia”), which precipitates with calcium after deconjugation in the more alkaline bile of CF patients.5
Gilbert syndrome is characterized by decreased bilirubin conjugation due to a diminished activity of the enzyme UDP glucuronosyltransferase (UGT). The principal bilirubin conjugate in Gilbert syndrome is bilirubin monoglucuronide, not bilirubin diglucuronide.6, 7 Therefore, only a 1-step hydrolysis is required to produce unconjugated bilirubin, causing elevated levels of unconjugated bilirubin in blood and duodenal bile.8 Some Gilbert syndrome patients exhibit a variety of ill-defined abdominal complaints, including pain, which could be related to a higher prevalence of bilirubin gallstones in Gilbert syndrome.6
A common variant of the UGT1A1 gene promoter displays a 2-bp (TA) insertion within the A(TA)6TAA element.9 The homozygous state of this mutation [A(TA)7TAA] has been identified as the major cause of Gilbert syndrome.9 The heterozygous state of the UGT1A1 mutation [A(TA)6TAA/A(TA)7TAA] is found in a considerable proportion of apparently healthy individuals, but is also associated with slightly increased levels of unconjugated bilirubin compared with the homozygous wild-type allele.10 We tested the hypothesis that the coinheritance of gene mutations causing CF and Gilbert syndrome is associated with an increased prevalence of gallbladder stones.
Patients and Methods
Overall, 52 patients with CF (25 males, 27 females; mean age ± SD, 20.4 ± 8.6 yr) were included in the study. CF was diagnosed according to standard criteria,11 and most patients were tested for a panel of 29 mutations12; at least 1 chromosome carrying the common mutation ΔF508 was detected in 41 of 46 patients (89.1%). Informed consent was obtained from patients or parents if the patient was younger than 18 years. Details of the patients have been published recently.13 No patient presented with overt hemolytic anemia, as demonstrated by hemoglobin concentrations of 12.5 g/dL or more in females and 13.1 g/dL or more in males. Only 1 patient presented with elevated lactate dehydrogenase activity (range, 112-254 U/L) but normal bilirubin and hemoglobin levels. However, a minor influence of ineffective erythropoesis on bilirubin levels due to lipovitamin deficiency was not excluded. In 9 CF patients, gallstones were identified by abdominal ultrasound examinations.
Genotyping and Genetic Analysis.
The UGT1A1 promoter [(A(TA)xTAA] was genotyped by direct sequencing of polymerase chain reaction products (403 bp; forward primer 5′-GAGGTTCTGGAAGTACTTTGC-3′, reverse primer 5′-CCAAGCATGCTCAGCCAG-3′) with an automated sequencer (ABI PRISM 310; Applera Corporation, Norwalk, CT) using the dye-terminator method (Big Dye Terminator Cycle Sequencing-Ready Reaction kit, Applera Corporation).
Liver Chemistry Tests.
Conjugated (direct) bilirubin concentrations in serum were determined via a modified Jendrassik-Gróf assay with diazotized sulfanilic acid.14, 15 Total bilirubin levels were measured with 2,5-dichlorophenyl diazonium tetrafluoroborate as diazo reagent15 using standard protocols for the Hitachi 717 analyzer (Roche Diagnostics, Mannheim, Germany). Serum levels of unconjugated bilirubin, which were calculated by subtracting direct from total bilirubin, were correlated to UGT1A1 genotypes via ANOVA followed by a Newman-Keuls multiple comparison test.
In our cohort of CF patients, 26 patients were heterozygous for the A(TA)7TAA allele, while 5 patients were homozygous for the A(TA)7TAA allele. Compared with stone-free patients with CF, significantly more CF patients with gallstones either carried the heterozygous A(TA)7TAA allele (OR 7.7; P < .05) or the heterozygous and/or homozygous A(TA)7TAA allele (allele positivity, OR 7.3; P < .05). Only 1 patient with gallstones did not carry the mutated UGT1A1 allele. Homozygosity for the Gilbert mutation appeared to confer an increased gallstone risk (OR 5.3); however, the difference was not significant (P = .23), most likely due to the small number of patients with this genotype (n = 5).
The relative frequencies of the UGT1A1 genotypes of CF patients with and without gallstones were visualized using the classic triangular de Finetti diagram.16 This diagram (Fig. 1) illustrates that the frequency of heterozygous Gilbert mutations (vertical axis) is markedly higher compared with the expected frequency indicated by the Hardy-Weinberg parabola.16
Figure 2 shows that the serum levels of unconjugated bilirubin also correlated with UGT1A1 genotypes. Values were highest in patients with homozygous A(TA)7TAA alleles, followed by patients with the heterozygous mutation and those with homozygous wild-type alleles (P = .002). Other quantitative traits (including serum aminotransferase, γ-glutamyl transpeptidase, alkaline phosphatase, and lactate dehydrogenase) were not associated with UGT1A1 genotypes.
Our data suggest that the A(TA)7TAA allele of the UGT1A1 promoter is a lithogenic risk factor in patients with CF. Of note, an augmented enterohepatic cycling of bilirubin due to an increased absorption of unconjugated bilirubin has recently been proposed as the cause of pigment gallstones.1 It has been suggested that leakage of bile salts from the small intestine to the colon leads to solubilization of unconjugated bilirubin and promotes its absorption from the large intestine, thereby augmenting bilirubin secretion into bile (“hyperbilirubinbilia”) with increased lithogenicity.1 Consequently, any pathological condition with an augmented spillage of bile salts into the colon might represent a risk factor for gallstone formation, as has been demonstrated in patients with Crohn disease who have a lack of functional ileum.5
We propose that in CF patients, genetic factors that are associated with increased biliary secretion of bilirubin monoglucuronide further raise the hazards for gallstone formation. In our case–control study, we demonstrated that the A(TA)7TAA allele of the UGT1A1 promoter represents such a risk factor in CF patients. Whereas the A(TA)7TAA allele is not clearly associated with gallstone formation in healthy individuals, the coinheritance of two or more gene variations that predispose to bilirubin deposition in the biliary tree, either by decreased hepatic conjugation (e.g., UGT1A1 promoter mutations) or by increased secretion into more alkaline bile (e.g., cystic fibrosis transmebrane codnuctance regulator mutations), might lead to an increased prevalence of pigment gallstones. In support of this concept, an increased prevalence of the Gilbert mutation has been detected in gallstone patients with hereditary spherocytosis,17 sickle cell anemia,18 and thalassaemia,19 all of which increase the biliary supply of unconjugated bilirubin as a result of intermittent hemolysis.
What are the clinical consequences of our findings? Traditionally, patients with CF and elevated serum aminotransferases, reduced hepatic excretory functions, and poor nutritional status are treated with ursodeoxycholic acid (UDCA).20, 21 The rationale is that UDCA reduces the accumulation of hyperviscid secretion and cytotoxic hydrophobic bile salts in the biliary tree. In our group, 21 patients received UDCA ± taurine (40.4%; mean UDCA dose 10 ± 5 mg/kg/d). Because UDCA has also been shown to increase the enterohepatic cycling of bilirubin,22 it might even further increase the risk for pigment gallstone formation in CF patients, particularly in those carrying the Gilbert mutation—albeit in our study, the proportion of patients receiving treatment did not differ significantly between gallstone carriers (5/9) and stone-free controls (16/42) (56% vs. 38%, respectively). It remains to be studied whether norUDCA—the C23-nor derivative of UDCA that undergoes cholehepatic shunting, induces a bicarbonate-rich hypercholeresis,23 and might reduce bilirubin precipitation in bile2—is stone-protective in CF.
This article is dedicated to Dr. Hiltrud Döhmen, on the occasion of her 65th birthday, for her initiatives that have improved and institutionalized health care for patients with CF.
- 2Cystic fibrosis transmembrane conductance regulator gene mutations cause “black” pigment gallstone formation: new insights from mouse models and implications for therapeutic interventions in cystic fibrosis. In: AdlerG, BlumHE, FuchsM, StangeEF, eds. Gallstone Pathogenesis and Treatment. Lancaster: Kluwer Academic Publishers, 2004: 24–27., , , , , .
- 8Analysis of bilirubins in biological fluids by extraction and thin-layer chromatography of the intact tetrapyrroles: application to bile of patients with Gilbert's syndrome, hemolysis, or cholelithiasis. HEPATOLOGY 1983; 3: 177–183., , , , .
- 14Modification of the Malloy-Evelyn method for a simple, reliable determination of total bilirubin in serum. J Clin Lab Invest 1972; 29( Suppl 126): 11.12., , .
- 16Conziderazioni matematiche sul'ereditarieta mendeliana. Meiron 1926; 6: 3–41..
- 20Ursodeoxycholic acid for cystic fibrosis-related liver disease. Cochrane Database Syst Rev 2000; 2: CD000222., , .