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Genetic defects of the UDP-glucuronosyltransferase-1 (UGT1) gene that cause familial non-haemolytic unconjugated hyperbilirubinaemias
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Clinica Chimica Acta 266 (1997) 63-74
Genetic defects of the UDP-glucuronosyltransferase-1 (UGT1) gene that cause familial non-haemolytic unconjugated hyperbilirubinaemias Douglas J. Clarke*, Nabil Moghrabi, Gemma Monaghan, Andrew Cassidy, Maureen Boxer, Robert Hume, Brian Burchell Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Medical School, Dundee, DD1 9SY, Scotland, UK
Received 6 December 1996; accepted 23 July 1997
Abstract
Congenital familial non-haemolytic hyperbilimbinaemias are potentially lethal syndromes caused by genetic lesions that reduce or abolish hepatic bilirubin UDP-glucuronosyltransferase activity. Here we describe genetic defects that occur in the UGT1 gene complex that cause three non-haemolytic unconjugated hyperbilirubinaemia syndromes. The most severe syndrome, termed Crigler-Najjar syndrome type I, is mainly associated with mutations in exons 2 to 5 that affect all UGT1 enzymes and many of the mutations result in termination codons and frameshifts. Crigler-Najjar type II syndrome which is treatable with phenobarbital therapy is associated with less dramatic missense mutations or heterozygous expression of mutant and normal alleles. Gilbert's syndrome, the most prevalent (2-19% in population studies) and mildest of the three syndromes is principally caused by a TA insertion at the TATA promoter region upstream of the UGT1A1 exon. Current methods used for the diagnosis and treatment of these diseases are discussed. © 1997 Elsevier Science B.V. Keywords: Unconjugated hyperbilirubinaemia; Crigler-Najjar syndrome; Gilbert's syndrome; Bilirubin UDP-glucuronosyltransferase; UGT1 gene; Mutations
Corresponding author. Fax: + 44 1382 633952; e-mail: [email protected] 0009-8981/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0009-8981 (97)00167-8
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1. Introduction Bilirubin, the breakdown product of haem derived from haemoproteins such as haemoglobin, cytochromes, catalase and peroxidase is produced at - 2 5 0 mg/day mainly by the spleen due to erythrocyte turnover. This potentially toxic compound has long been associated with jaundice and hyperbilirubinaemia [1]. In man, bilirubin is efficiently transported to the liver, where the more polar water soluble bilirubin diglucuronide is synthesised in hepatic endoplasmic reticulum catalysed by bilirubin UDP-glucuronosyltransferase. The bilirubin diglucuronides are then excreted into the bile via an active transport mechanism in the bile canaliculus. Hyperbilirubinaemias may be classified into two types depending on whether the bilirubin is conjugated to glucuronic acid or not. Unconjugated hyperbilirubinaemias can be caused by mutations within the UDP-glucuronosyltransferase-1 (UGT1) gene, drug displacement of bilirubin from serum albumin, defects of bilirubin transport into the liver or defects of hepatic glucuronidation caused by liver damage [ 1]. Severe unconjugated hyperbilirubinaemia may result in neurological (kernicterus) and renal damage and prove to be fatal in childhood. Conjugated hyperbilirubinaemias such as Dubin Johnson syndrome are caused by genetic defects in canalicular bilimbin glucuronide transport into bile catalysed by the ATP-dependent multidrug resistance protein [2]. This mini-review will focus on defects of UGT1 gene expression that cause unconjugated hyperbilirubinaemias, namely Crigler-Najjar and Gilbert's syndromes.
2. Crigler-Najjar and Gilbert's syndromes Three types of unconjugated hyperbilirubinaemias have been characterised. Two of these are different subtypes of the congenital disease Crigler-Najjar syndrome (CN) which is characterised by high serum bilirubin levels ranging from 3 to 40-fold greater than normal levels ( < 17/zM) [1,3]. If patients with this syndrome with bilirubin levels greater than 200/zM are left untreated they usually succumb to bilirubin toxicity which eventually leads to death. Patients with CN type I (CN1) and type II (CN2) have serum bilirubin levels of 340-700 /zM and 50-500/zM, respectively. This is due to the absence or reduction of hepatic bilirubin UDP-glucuronosyltransferase activity in CN1 and CN2, respectively. A rough classification of the subtype may be made according to serum bilirubin concentrations, but since there is a considerable overlap in the ranges encountered this is often impossible. The CN subtypes may be subclassifted by their response to phenobarbital administration, where CN1 patients are
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refractive to this treatment, CN2 patients' serum bilirubin levels are generally reduced by 30-80%. Unfortunately however this test is not always successful in CN2 patients and it sometimes has to be repeated several times to provide a response. Altemative means of subclassifying the disease subtypes have therefore been sought. Although assay of bilirubin UGT activity in liver needle biopsies has proved a popular method in the past, obviously the viability of the sample should be tested to confirm any negative result. High pressure liquid chromatography analysis of patient bile has proved to be a definitive test in a number of laboratories and is now becoming the method of choice for the accurate diagnosis of disease subtypes. Data summarising bile pigment analysis are given in Table 1, where different types of familial hyperbilirubinaemia have been examined. In CN1 no glucuronides are produced or detected in bile, whereas mainly monoglucuronides are observed in CN2 bile (Table 1). Production of monoglucuronides may reflect the excess of bilirubin substrate for a reduced amount of enzyme and can be observed in vitro using cloned expressed bilirubin UGT; the percentage of monoglucuronides formed is related to the concentration of bilirubin in the incubation media [5]. The third type of unconjugated hyperbilirubinaemia is Gilbert's syndrome which unlike CN syndrome is benign. Serum bilirubin levels in this syndrome range from 1 7 - 5 0 / x M and bilirubin UGT activity is reduced down to 30-35% of normal levels in Gilberts patients [1]. As a result, more monoglucuronide and less diglucuronide are formed and excreted in bile, but the overall excretion of glucuronides is up to 90% effective (Table 1). As these three syndromes are inherited and the probable disease causing protein was known this led investigators to clone and examine the gene that encodes human bilirubin UGT.
Table 1 Percentage composition of bilirubin, its mono- and diglucuronides present in bile of normal, Gilbert's syndrome and Crigler-Najjar syndrome affected individuals Composition" Unconjugated bilirubin Bilirubin monoglucuronide Bilirubin diglucuronide
Clinical state Normal
Gilbert
CN2
CN1
1-3% 6-18% 76-90%
2-12% 25-32% 59-82%
10-81% b 12-63% b 1-56% b
100%c 0% 0%
aFrom reference [4] and Clarke and Burchell (unpublished work). bThe values do not take into account variation of percentage composition of each type of biliary pigment upon phenobarbital therapy. CThe biliary pigment composition of CN1 was determined from a patient receiving phototherapeutic treatment.
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3. UGT1 gene structure and function
The UGT1 gene, which encodes several UGT1 isoforms, has a complex structure consisting of four common (exons 2-5) and 13 variable exons encoding the different UGT isoforms [6]. The gene locus has been regionally assigned and physically mapped to human chromosome 2q37 [7]. Each single variable exon 1 is preceded by a regulatory 5' region and in response to a specific signal, transcription processing splices mRNA from each single exon to the four common exons to provide a template for synthesis of an individual isoform (Fig. 1). The UGT1 gene encodes several isoforms involved in the detoxification of compounds of diverse chemical structures, however there is only one physiologically important bilirubin UGT which is encoded by exon 1A1 and the common exons of the gene complex [8]. Exons 1A6 and 1A8 encode the substrate-specific binding domain for isoforms involved in the glucuronidation of phenolic compounds [9], whilst 1A4 encodes the substrate binding domain of an isoform that conjugates amines [10]. At present, the substrate specificity of the other isoforms is unknown, however expression of the relevant cDNAs in cell culture will reveal this in the near future. Examination of the UGT1 gene structure in hyperbilirubinaemic patients has revealed more than 30 different genetic defects in the Crigler-Najjar (CN) (a) Common Exons
Variable Exon l ' e of the UGT1 Gena Complex
A13
A12
All
A10
A9
A8
A7
AS
A5
A4
A3
2
A2~
A1
3
(b) Fig. 1. The human UGT1 gene locus. (a) Schematic representation of the genomic structure of the UGT1 gene complex. The complexity of the gene structure is denoted by the presence of multiple exon ls encoding the substrate binding domains for UGT isoforms. Each exon 1 is predicted to be differentially spliced to the exon 2-5 cluster corresponding to the constant domain, the process being initiated via promoters upstream of each exon 1. (b) Exploded view of exon 1A1 and exons 2-5 of the gene complex that have been identified as sites for genetic mutations that cause the unconjugated hyperbilirubinaemias described in this review.
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s y n d r o m e s and one genetic alteration that accounts for the majority of G i l b e r t ' s s y n d r o m e cases.
4.
Genetic defects in Crigler-Najjar syndrome type I
Inherited lesions that cause CN1 have been found in exons 1A1, 2, 3, 4 and 5 (Table 2) and are associated with a complete lack o f bilirubin U G T activity either in liver biopsy specimens or b y expression o f the mutant protein. In addition, mutations that occur in exons 2 - 5 in CN1 patients can also affect the functional activity of other U G T 1 isoforms causing it to be reduced considerably or totally absent in biopsy specimens. The glucuronidation of phenolic substrates such as 1-naphthol, 4-nitrophenol and propofol are all reduced in CN1 patients bearing mutations in the c o m m o n region (for references see Table 2). As 18 out o f the 23 CN1 mutations found to date are in exons 2 - 5 (Table 2) it is likely
Table 2 Genetic defects in Crigler-Najjar syndrome type I patients Exon
Mutation
Serum bilirubin (/xM)
Incidence
Reference
1A1 1A1 1A1/2 1A1 1A1 2 2/4 2 2 2 3/4 3/4 3 3 3 3/4 3/4 4 4 4 4/5
A80 ( + 4bp) A170 (-F) C177R/A294 ( - 13bp) G276R C280X Del. of exon A291V/K426E A294 (--13 bp) G308E Q331X W335X/A368T W335X/A408( + G) R341X Q357R Q357X Q357X/A401P Q361X/P387R $376F $381R A401P A401P/K437X
356-486 430 380 420 530 430 > 350 598 > 350, > 350, 408-459 430 > 350 > 350 270-365 > 350, > 350 410 > 350 NK 513, 684 >350, >350 > 350, > 350 > 350
1 1 1 1 1 1 1 1 3 1 1 1 1 3 1 1 1 2 2 2 1
UP [11] [12] [ 13] [14] [12] [15] [16] [15,17] [18] [15] [15] [ 19] [15] [20] [15] [21] [17,18] [15] [15] [15]
The numbering refers to the codon position in UGT1A1; X indicates a termination codon; a backslash indicates that these patients are heterozygous for two alleles. NK indicates that the serum bilirubin concentration was not quoted in the corresponding reference. UP indicates unpublished studies from this laboratory.
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that the majority of CN1 patients have diminished or absent activity for all their UGT1 proteins. As expected, the nonsense mutations at amino acids 280, 331,335, 341,357, 361 and 437, the frameshift mutations at amino acids 80, 294 and 408, the in-frame deletion at amino acid 170 and deletion of exon 2 cause a complete loss of bilirubin UGT activity. Interestingly the numerous missense mutations that are scattered in exons 1A1, 2, 3 and 4 of the gene complex that account for 48% of all the CN1 mutations identified so far indicate crucial amino acid residues that are important for the normal function of the enzyme. Recent examination of a CN1 patient's genomic DNA in this laboratory (unpublished data) has revealed that a novel 4 bp insertion in exon 1A1 at codon 80 also causes a catastrophic loss of all UGT1 proteins and activities, even though the lesion is present in the bilirubin UGT-specific exon 1A1. It is easy to understand how this frameshift mutation introduces a stop codon in the bilirubin UGT-specific exon, but how is the synthesis of other UGT isoforms disrupted? Apparently, the primary unspliced transcript from the UGT gene, no matter which UGT enzyme is needed, is a common product including all variable exons. The 4 bp insertional mutation may have an effect on the global secondary structure of the UGT1 primary transcripts resulting in abnormal splicing problems and/or the production of unstable mRNAs which are rapidly degraded.
5. Genetic defects in Crigler-Najjar syndrome type II The difference between CN1 and the less severe CN2 is that in the latter disease the mutant bilirubin UGT enzyme has some activity. Thus, CN2 hyperbilirubinaemia is classified by a response to phenobarbital treatment, which induces the production of poorly functional bilirubin UGT, and thereby reduces the serum bilirubin level. The nature of the genetic lesion determines the severity of the disease. The convenient clinical classification of Crigler-Najjar patients into type I and type II has no meaningful genetic basis (Table 3). In contrast to CN1 patients where the majority of disease causing mutations occur in exons 2-5, four out of the 9 mutations identified have been found in exon 1A1 in CN2 patients (Table 3). Interestingly, the same mutation, R209W, in exon 1A1 has been observed in four unrelated patients with severe CN2 (Table 3). Mutations at this residue obviously lead to production of small amounts of poorly functional protein, which can be revealed by transfection of the mutated cDNAs into COS cells where kinetic analysis can be performed [12]. Recently a Japanese team reported the identification of missense mutations in several hyperbilirubinaemic patients with a non-fasted serum bilirubin level of 53-86 /.tM that they propose have Gilbert's syndrome [24-26]. Here, the
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Table 3 Genetic defects in Crigler-Najjar syndrometype II patients Exon
Mutation
Serum bilirubin ( / z M )
Incidence
Reference
1A1 1A1 1A1 1AI / 5 1A1 1AI 2 2 2 4
L15R G71R G71R/normal G71R2/ Y486D2 R209W P229Q/normal Q331R Q331X/normal R336W R367G/normal
195 65 55, 86, NK 286 340, 400, 340, 333 56, 72 342 116 NK 53
1 1 6 1 4 2 1 1 1 1
[22] [23] [24-26] [28] [29,30], UP [24-26] [31] [32] [33] [24-26]
Abbreviations as in Table 2. G71R2/Y486D2 indicates a patient who is homozygousfor an allele bearing two mutations.
specific clinical definition of Gilbert's disease is being questioned. The well known text book definition is a mild jaundice when serum bilirubin does not increase above 50/.tM after a 400 calorie 24-h restricted diet [1,34]. Clearly as these patients have a non-fasted serum bilirubin concentration greater than 50 /.tM (Table 3) (which would become more elevated after a calorie restricted diet) they should be clinically and genetically classified as CN2. In addition, the incidence of these alleles in the population is too low to account for the high incidence of Gilbert's syndrome (2-19% in the population) [27]. Interestingly, seven of these patients have been described with a similar G71R mutation [23-26] which causes a relatively mild CN2 phenotype relative to others reported indicating that this is a less critical amino substitution in bilirubin UGT.
6. Genetic variation in the UGT1 gene associated with Gilbert's syndrome The recognition of the reduction in serum bilirubin in response to phenobarbital [1], indicated that human hepatic bilirubin UGT could be induced by drugs including alcohol [35]. Indeed, potential drug regulatory sites are observed in the 5' region immediately upstream of the UGT1A1 exon [36]. Recently, a TA insertion in the regulatory TATA box of the UGT1A1 promoter has been tightly associated with hyperbilirubinaemia in Gilbert's patients [37,38]. Surveys of the general population do not easily reveal an association between serum bilirubin and UGT1A1 TATA promoter genotype. However, when a volunteer population abstained from drug and alcohol use and were fasted, phenotype and genotype can be directly correlated [38]. Previously
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undiagnosed Gilbert's patients have been identified by genotyping the Eastern Scottish and Igloolik Inuit of Canada populations where 10-13% and 17-19% exhibit the TA insertion genotype, respectively [38,39]. In total, 4 5 - 5 1 % of these populations are heterozygotes. As well as the homozygous TA insertion which appears to account for the majority of cases of classical Gilbert's syndrome in the population, a rare TATA allele bearing a TA deletion has been identified in two unrelated Scottish subjects which have a more severe clinical phenotype than the insertion allele (Monaghan et al., unpublished work). In addition, a patient with a serum bilirubin level of 31 /xM has been identified as having a Y486D missense mutation present in one allele [26].
7. Genotype analysis of Crigler-Najjar patients Previously we reported two cDNA probes (UGT1A4, UGT-Const) which recognise polymorphic regions within the gene complex [19]. Southern blot analysis of Mspl-digested genomic DNA with these probes allowed the generation of haplotypes which were used to analyze the segregation of mutant alleles within a CN1 affected pedigree [19]. Fig. 2 demonstrates that this haplotyping technique has allowed us to identify the segregation of mutant
(a)
(b) ALIA1
ALIA1
C21C2
CllC2
BIlB2
i
ALIA1
BIIB2
$
ALIA1
ALIA2 S21Bi C21C2
C2
i t , d, C21C2
C21C2 ,C21C1
Fig. 2. Diagnosis of the inheritance of Crigler-Najjar syndrome carrying alleles by haplotype analysis using intragenic polymorphic UGT1 probes and Southern blotting. (a) and (b) represent pedigrees of CN1 and CN2 affected families, respectively. The filled symbols represent the affected individual; the open symbols represent a normal individual, the half-filled symbols represent carriers; squares and circles represent males and females, respectively and the double line in generation I indicates consanguinity. Below each symbol in the pedigree the haplotype derived from the genotype analysis using the three biallelic UGT1 polymorphisms previously described in this laboratory. The haplotypes of the offspring are divided by a vertical line, with the paternally and maternally derived haplotypes to the left and right, respectively.
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alleles in other CN affected families. Note that the haplotypes A1-B2-C2 and A2-B2-C2 cosegregate with the disease in family (a) and (b), respectively (Fig. 2). Such information can therefore be used for future prenatal and presymptomatic diagnosis in these families. This further illustrates the usefulness of this relatively rapid technique to predict the cartier status of an individual without the requirement to carry out tedious mutation analysis by sequencing the UGT1 gene. Although we have used this technique for the unequivocal diagnosis of disease inheritance in a number of families it cannot be used when one of the parents of a CN affected child is homozygous for a haplotype for obvious reasons. As a result we are currently investigating the use of further polymorphic markers within the UGT1 gene to allow the definitive diagnosis of mutant alleles in every CN family.
8. Current therapy and future treatment for Crigler-Najjar syndromes Phototherapy in infancy is an effective short-term means of lowering serum bilirubin levels, by converting bilirubin to the more readily excreted photobilirubin; however, as the skin thickens with age it becomes progressively less effective [1]. Although administration of haem oxygenase inhibitor, tinmesoporphyrin, to CN patients has been shown to reduce bilirubin levels [40], once again it is not an effective long-term treatment as it causes iron deficiency anaemia [41]. Phenobarbital administration has remained the most successful means of reducing bilirubin levels in CN2; indeed a well-documented CN2 patient lived without any obvious deleterious effect to his eighth decade [31]. By contrast the only current therapy for CN1 is described in a consensus report of a World Registry which was recently published. Partial orthotopic liver transplantation performed at a young age appears to be the best eventual current treatment for the more severe disorder [42]. In the future, CN1 is a prime target for development of hepatic gene therapy, being a lethal condition caused by a defect in the terminal stage of a metabolic pathway.
Acknowledgements We thank the Wellcome Trust, The Scottish Home and Health Department and Medical Research Council for funds supporting this work.
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[32] Koiwai O, Aono S, Adachi Y, Kamisako T, Yasui Y, Nishizawa M, Sato H. Crigler-Najjar syndrome type II is inherited both as a dominant and as a recessive trait. Hum Mol Genet 1996;5:645-7. [33] Rubaltelli D, Ciotti M, Rubaltelli FF, Owens IS. Different missense mutations at the UGT1 gene-complex locus that generate pH-sensitive activity of the major bilirubin UDPglucuronosyltransferase in Crigler-Najjar type-II patients. FASEB J 1995;9:A1267. [34] Lasceiles PT, Donaldson D. Calorie restriction test. In: Lascelles PT, Donaldson D, editors. Diagnostic function tests in chemical pathology. Lancaster: Kluwer, 1989:24-25. [35] Ideo G, De Franchis R, Del Ninno E, Dioguardi N. Ethanol increases liver uridinediphosphate-glucuronosyltransferase. Experientia 1971;27:24-5. [36] Brierley CH, Senafi SB, Clarke D, Hsu M-H, Johnson EF, Burchell B. Regulation of the human bilirubin UDP-glucuronosyltransferase gene. Adv Enz Regul 1996;36:85-97. [37] Bosma PJ, Roy Chowdhury J, Bakker C, Gantla S, de Boer A, Oostra BA, Lindhout D, Tytgat GN, Jansen PL, Oude Elferink RP et al. The genetic basis of the reduced expression of bilirubin UDP-glucuronosyltransferase 1 in Gilbert's syndrome. N Engl J Med 1995;333:1171-5. [38] Monaghan G, Ryan M, Seddon R, Hume R, Burchell B. Genetic variation in bilirubin UDP-glucuronosyltransferase gene promoter and Gilbert's syndrome. Lancet 1996;347:57881. [39] Monaghan G, Foster B, Jurima-Romet M, Hume R, Burchell B. UGTI*I genotyping in a Canadian Inuit population. Pharrnacogenetics 1997;7:153-6. [40] Landaw SA, Drummond GS, Kappas A. Targeting of heme oxygenase inhibitors to the spleen markedly increases their ability to diminish bilirubin production. Pediatrics 1989;84:1091-6. [41] Kappas A, Drummond GS, Galbraith RA. Prolonged clinical use of a heme oxygenase inhibitor: hematological evidence for an inducible but reversible iron-deficiency state. Pediatrics 1993;91:537-9. [42] van der Veere CN, Sinaasappel M, McDonagh AF, Rosenthal E Labrune E Odievre M, Fevery J, Otte JB, McClean P, Burk G, Masakowski V, Sped W, Mowat AE Vergani GM, Heller K, Wilson JE Shepherd R, Jansen PL. Current therapy for Crigler-Najjar syndrome type 1: report of a world registry. Hepatology 1996;24:311-5.
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Genetic defects of the UDP-glucuronosyltransferase-1 (UGT1) gene that cause familial non-haemolytic unconjugated hyperbilirubinaemias Douglas J. Clarke*, Nabil Moghrabi, Gemma Monaghan, Andrew Cassidy, Maureen Boxer, Robert Hume, Brian Burchell Department of Molecular and Cellular Pathology, University of Dundee, Ninewells Medical School, Dundee, DD1 9SY, Scotland, UK
Received 6 December 1996; accepted 23 July 1997
Abstract
Congenital familial non-haemolytic hyperbilimbinaemias are potentially lethal syndromes caused by genetic lesions that reduce or abolish hepatic bilirubin UDP-glucuronosyltransferase activity. Here we describe genetic defects that occur in the UGT1 gene complex that cause three non-haemolytic unconjugated hyperbilirubinaemia syndromes. The most severe syndrome, termed Crigler-Najjar syndrome type I, is mainly associated with mutations in exons 2 to 5 that affect all UGT1 enzymes and many of the mutations result in termination codons and frameshifts. Crigler-Najjar type II syndrome which is treatable with phenobarbital therapy is associated with less dramatic missense mutations or heterozygous expression of mutant and normal alleles. Gilbert's syndrome, the most prevalent (2-19% in population studies) and mildest of the three syndromes is principally caused by a TA insertion at the TATA promoter region upstream of the UGT1A1 exon. Current methods used for the diagnosis and treatment of these diseases are discussed. © 1997 Elsevier Science B.V. Keywords: Unconjugated hyperbilirubinaemia; Crigler-Najjar syndrome; Gilbert's syndrome; Bilirubin UDP-glucuronosyltransferase; UGT1 gene; Mutations
Corresponding author. Fax: + 44 1382 633952; e-mail: [email protected] 0009-8981/97/$17.00 © 1997 Elsevier Science B.V. All rights reserved. PII S0009-8981 (97)00167-8
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1. Introduction Bilirubin, the breakdown product of haem derived from haemoproteins such as haemoglobin, cytochromes, catalase and peroxidase is produced at - 2 5 0 mg/day mainly by the spleen due to erythrocyte turnover. This potentially toxic compound has long been associated with jaundice and hyperbilirubinaemia [1]. In man, bilirubin is efficiently transported to the liver, where the more polar water soluble bilirubin diglucuronide is synthesised in hepatic endoplasmic reticulum catalysed by bilirubin UDP-glucuronosyltransferase. The bilirubin diglucuronides are then excreted into the bile via an active transport mechanism in the bile canaliculus. Hyperbilirubinaemias may be classified into two types depending on whether the bilirubin is conjugated to glucuronic acid or not. Unconjugated hyperbilirubinaemias can be caused by mutations within the UDP-glucuronosyltransferase-1 (UGT1) gene, drug displacement of bilirubin from serum albumin, defects of bilirubin transport into the liver or defects of hepatic glucuronidation caused by liver damage [ 1]. Severe unconjugated hyperbilirubinaemia may result in neurological (kernicterus) and renal damage and prove to be fatal in childhood. Conjugated hyperbilirubinaemias such as Dubin Johnson syndrome are caused by genetic defects in canalicular bilimbin glucuronide transport into bile catalysed by the ATP-dependent multidrug resistance protein [2]. This mini-review will focus on defects of UGT1 gene expression that cause unconjugated hyperbilirubinaemias, namely Crigler-Najjar and Gilbert's syndromes.
2. Crigler-Najjar and Gilbert's syndromes Three types of unconjugated hyperbilirubinaemias have been characterised. Two of these are different subtypes of the congenital disease Crigler-Najjar syndrome (CN) which is characterised by high serum bilirubin levels ranging from 3 to 40-fold greater than normal levels ( < 17/zM) [1,3]. If patients with this syndrome with bilirubin levels greater than 200/zM are left untreated they usually succumb to bilirubin toxicity which eventually leads to death. Patients with CN type I (CN1) and type II (CN2) have serum bilirubin levels of 340-700 /zM and 50-500/zM, respectively. This is due to the absence or reduction of hepatic bilirubin UDP-glucuronosyltransferase activity in CN1 and CN2, respectively. A rough classification of the subtype may be made according to serum bilirubin concentrations, but since there is a considerable overlap in the ranges encountered this is often impossible. The CN subtypes may be subclassifted by their response to phenobarbital administration, where CN1 patients are
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refractive to this treatment, CN2 patients' serum bilirubin levels are generally reduced by 30-80%. Unfortunately however this test is not always successful in CN2 patients and it sometimes has to be repeated several times to provide a response. Altemative means of subclassifying the disease subtypes have therefore been sought. Although assay of bilirubin UGT activity in liver needle biopsies has proved a popular method in the past, obviously the viability of the sample should be tested to confirm any negative result. High pressure liquid chromatography analysis of patient bile has proved to be a definitive test in a number of laboratories and is now becoming the method of choice for the accurate diagnosis of disease subtypes. Data summarising bile pigment analysis are given in Table 1, where different types of familial hyperbilirubinaemia have been examined. In CN1 no glucuronides are produced or detected in bile, whereas mainly monoglucuronides are observed in CN2 bile (Table 1). Production of monoglucuronides may reflect the excess of bilirubin substrate for a reduced amount of enzyme and can be observed in vitro using cloned expressed bilirubin UGT; the percentage of monoglucuronides formed is related to the concentration of bilirubin in the incubation media [5]. The third type of unconjugated hyperbilirubinaemia is Gilbert's syndrome which unlike CN syndrome is benign. Serum bilirubin levels in this syndrome range from 1 7 - 5 0 / x M and bilirubin UGT activity is reduced down to 30-35% of normal levels in Gilberts patients [1]. As a result, more monoglucuronide and less diglucuronide are formed and excreted in bile, but the overall excretion of glucuronides is up to 90% effective (Table 1). As these three syndromes are inherited and the probable disease causing protein was known this led investigators to clone and examine the gene that encodes human bilirubin UGT.
Table 1 Percentage composition of bilirubin, its mono- and diglucuronides present in bile of normal, Gilbert's syndrome and Crigler-Najjar syndrome affected individuals Composition" Unconjugated bilirubin Bilirubin monoglucuronide Bilirubin diglucuronide
Clinical state Normal
Gilbert
CN2
CN1
1-3% 6-18% 76-90%
2-12% 25-32% 59-82%
10-81% b 12-63% b 1-56% b
100%c 0% 0%
aFrom reference [4] and Clarke and Burchell (unpublished work). bThe values do not take into account variation of percentage composition of each type of biliary pigment upon phenobarbital therapy. CThe biliary pigment composition of CN1 was determined from a patient receiving phototherapeutic treatment.
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3. UGT1 gene structure and function
The UGT1 gene, which encodes several UGT1 isoforms, has a complex structure consisting of four common (exons 2-5) and 13 variable exons encoding the different UGT isoforms [6]. The gene locus has been regionally assigned and physically mapped to human chromosome 2q37 [7]. Each single variable exon 1 is preceded by a regulatory 5' region and in response to a specific signal, transcription processing splices mRNA from each single exon to the four common exons to provide a template for synthesis of an individual isoform (Fig. 1). The UGT1 gene encodes several isoforms involved in the detoxification of compounds of diverse chemical structures, however there is only one physiologically important bilirubin UGT which is encoded by exon 1A1 and the common exons of the gene complex [8]. Exons 1A6 and 1A8 encode the substrate-specific binding domain for isoforms involved in the glucuronidation of phenolic compounds [9], whilst 1A4 encodes the substrate binding domain of an isoform that conjugates amines [10]. At present, the substrate specificity of the other isoforms is unknown, however expression of the relevant cDNAs in cell culture will reveal this in the near future. Examination of the UGT1 gene structure in hyperbilirubinaemic patients has revealed more than 30 different genetic defects in the Crigler-Najjar (CN) (a) Common Exons
Variable Exon l ' e of the UGT1 Gena Complex
A13
A12
All
A10
A9
A8
A7
AS
A5
A4
A3
2
A2~
A1
3
(b) Fig. 1. The human UGT1 gene locus. (a) Schematic representation of the genomic structure of the UGT1 gene complex. The complexity of the gene structure is denoted by the presence of multiple exon ls encoding the substrate binding domains for UGT isoforms. Each exon 1 is predicted to be differentially spliced to the exon 2-5 cluster corresponding to the constant domain, the process being initiated via promoters upstream of each exon 1. (b) Exploded view of exon 1A1 and exons 2-5 of the gene complex that have been identified as sites for genetic mutations that cause the unconjugated hyperbilirubinaemias described in this review.
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s y n d r o m e s and one genetic alteration that accounts for the majority of G i l b e r t ' s s y n d r o m e cases.
4.
Genetic defects in Crigler-Najjar syndrome type I
Inherited lesions that cause CN1 have been found in exons 1A1, 2, 3, 4 and 5 (Table 2) and are associated with a complete lack o f bilirubin U G T activity either in liver biopsy specimens or b y expression o f the mutant protein. In addition, mutations that occur in exons 2 - 5 in CN1 patients can also affect the functional activity of other U G T 1 isoforms causing it to be reduced considerably or totally absent in biopsy specimens. The glucuronidation of phenolic substrates such as 1-naphthol, 4-nitrophenol and propofol are all reduced in CN1 patients bearing mutations in the c o m m o n region (for references see Table 2). As 18 out o f the 23 CN1 mutations found to date are in exons 2 - 5 (Table 2) it is likely
Table 2 Genetic defects in Crigler-Najjar syndrome type I patients Exon
Mutation
Serum bilirubin (/xM)
Incidence
Reference
1A1 1A1 1A1/2 1A1 1A1 2 2/4 2 2 2 3/4 3/4 3 3 3 3/4 3/4 4 4 4 4/5
A80 ( + 4bp) A170 (-F) C177R/A294 ( - 13bp) G276R C280X Del. of exon A291V/K426E A294 (--13 bp) G308E Q331X W335X/A368T W335X/A408( + G) R341X Q357R Q357X Q357X/A401P Q361X/P387R $376F $381R A401P A401P/K437X
356-486 430 380 420 530 430 > 350 598 > 350, > 350, 408-459 430 > 350 > 350 270-365 > 350, > 350 410 > 350 NK 513, 684 >350, >350 > 350, > 350 > 350
1 1 1 1 1 1 1 1 3 1 1 1 1 3 1 1 1 2 2 2 1
UP [11] [12] [ 13] [14] [12] [15] [16] [15,17] [18] [15] [15] [ 19] [15] [20] [15] [21] [17,18] [15] [15] [15]
The numbering refers to the codon position in UGT1A1; X indicates a termination codon; a backslash indicates that these patients are heterozygous for two alleles. NK indicates that the serum bilirubin concentration was not quoted in the corresponding reference. UP indicates unpublished studies from this laboratory.
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that the majority of CN1 patients have diminished or absent activity for all their UGT1 proteins. As expected, the nonsense mutations at amino acids 280, 331,335, 341,357, 361 and 437, the frameshift mutations at amino acids 80, 294 and 408, the in-frame deletion at amino acid 170 and deletion of exon 2 cause a complete loss of bilirubin UGT activity. Interestingly the numerous missense mutations that are scattered in exons 1A1, 2, 3 and 4 of the gene complex that account for 48% of all the CN1 mutations identified so far indicate crucial amino acid residues that are important for the normal function of the enzyme. Recent examination of a CN1 patient's genomic DNA in this laboratory (unpublished data) has revealed that a novel 4 bp insertion in exon 1A1 at codon 80 also causes a catastrophic loss of all UGT1 proteins and activities, even though the lesion is present in the bilirubin UGT-specific exon 1A1. It is easy to understand how this frameshift mutation introduces a stop codon in the bilirubin UGT-specific exon, but how is the synthesis of other UGT isoforms disrupted? Apparently, the primary unspliced transcript from the UGT gene, no matter which UGT enzyme is needed, is a common product including all variable exons. The 4 bp insertional mutation may have an effect on the global secondary structure of the UGT1 primary transcripts resulting in abnormal splicing problems and/or the production of unstable mRNAs which are rapidly degraded.
5. Genetic defects in Crigler-Najjar syndrome type II The difference between CN1 and the less severe CN2 is that in the latter disease the mutant bilirubin UGT enzyme has some activity. Thus, CN2 hyperbilirubinaemia is classified by a response to phenobarbital treatment, which induces the production of poorly functional bilirubin UGT, and thereby reduces the serum bilirubin level. The nature of the genetic lesion determines the severity of the disease. The convenient clinical classification of Crigler-Najjar patients into type I and type II has no meaningful genetic basis (Table 3). In contrast to CN1 patients where the majority of disease causing mutations occur in exons 2-5, four out of the 9 mutations identified have been found in exon 1A1 in CN2 patients (Table 3). Interestingly, the same mutation, R209W, in exon 1A1 has been observed in four unrelated patients with severe CN2 (Table 3). Mutations at this residue obviously lead to production of small amounts of poorly functional protein, which can be revealed by transfection of the mutated cDNAs into COS cells where kinetic analysis can be performed [12]. Recently a Japanese team reported the identification of missense mutations in several hyperbilirubinaemic patients with a non-fasted serum bilirubin level of 53-86 /.tM that they propose have Gilbert's syndrome [24-26]. Here, the
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Table 3 Genetic defects in Crigler-Najjar syndrometype II patients Exon
Mutation
Serum bilirubin ( / z M )
Incidence
Reference
1A1 1A1 1A1 1AI / 5 1A1 1AI 2 2 2 4
L15R G71R G71R/normal G71R2/ Y486D2 R209W P229Q/normal Q331R Q331X/normal R336W R367G/normal
195 65 55, 86, NK 286 340, 400, 340, 333 56, 72 342 116 NK 53
1 1 6 1 4 2 1 1 1 1
[22] [23] [24-26] [28] [29,30], UP [24-26] [31] [32] [33] [24-26]
Abbreviations as in Table 2. G71R2/Y486D2 indicates a patient who is homozygousfor an allele bearing two mutations.
specific clinical definition of Gilbert's disease is being questioned. The well known text book definition is a mild jaundice when serum bilirubin does not increase above 50/.tM after a 400 calorie 24-h restricted diet [1,34]. Clearly as these patients have a non-fasted serum bilirubin concentration greater than 50 /.tM (Table 3) (which would become more elevated after a calorie restricted diet) they should be clinically and genetically classified as CN2. In addition, the incidence of these alleles in the population is too low to account for the high incidence of Gilbert's syndrome (2-19% in the population) [27]. Interestingly, seven of these patients have been described with a similar G71R mutation [23-26] which causes a relatively mild CN2 phenotype relative to others reported indicating that this is a less critical amino substitution in bilirubin UGT.
6. Genetic variation in the UGT1 gene associated with Gilbert's syndrome The recognition of the reduction in serum bilirubin in response to phenobarbital [1], indicated that human hepatic bilirubin UGT could be induced by drugs including alcohol [35]. Indeed, potential drug regulatory sites are observed in the 5' region immediately upstream of the UGT1A1 exon [36]. Recently, a TA insertion in the regulatory TATA box of the UGT1A1 promoter has been tightly associated with hyperbilirubinaemia in Gilbert's patients [37,38]. Surveys of the general population do not easily reveal an association between serum bilirubin and UGT1A1 TATA promoter genotype. However, when a volunteer population abstained from drug and alcohol use and were fasted, phenotype and genotype can be directly correlated [38]. Previously
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undiagnosed Gilbert's patients have been identified by genotyping the Eastern Scottish and Igloolik Inuit of Canada populations where 10-13% and 17-19% exhibit the TA insertion genotype, respectively [38,39]. In total, 4 5 - 5 1 % of these populations are heterozygotes. As well as the homozygous TA insertion which appears to account for the majority of cases of classical Gilbert's syndrome in the population, a rare TATA allele bearing a TA deletion has been identified in two unrelated Scottish subjects which have a more severe clinical phenotype than the insertion allele (Monaghan et al., unpublished work). In addition, a patient with a serum bilirubin level of 31 /xM has been identified as having a Y486D missense mutation present in one allele [26].
7. Genotype analysis of Crigler-Najjar patients Previously we reported two cDNA probes (UGT1A4, UGT-Const) which recognise polymorphic regions within the gene complex [19]. Southern blot analysis of Mspl-digested genomic DNA with these probes allowed the generation of haplotypes which were used to analyze the segregation of mutant alleles within a CN1 affected pedigree [19]. Fig. 2 demonstrates that this haplotyping technique has allowed us to identify the segregation of mutant
(a)
(b) ALIA1
ALIA1
C21C2
CllC2
BIlB2
i
ALIA1
BIIB2
$
ALIA1
ALIA2 S21Bi C21C2
C2
i t , d, C21C2
C21C2 ,C21C1
Fig. 2. Diagnosis of the inheritance of Crigler-Najjar syndrome carrying alleles by haplotype analysis using intragenic polymorphic UGT1 probes and Southern blotting. (a) and (b) represent pedigrees of CN1 and CN2 affected families, respectively. The filled symbols represent the affected individual; the open symbols represent a normal individual, the half-filled symbols represent carriers; squares and circles represent males and females, respectively and the double line in generation I indicates consanguinity. Below each symbol in the pedigree the haplotype derived from the genotype analysis using the three biallelic UGT1 polymorphisms previously described in this laboratory. The haplotypes of the offspring are divided by a vertical line, with the paternally and maternally derived haplotypes to the left and right, respectively.
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alleles in other CN affected families. Note that the haplotypes A1-B2-C2 and A2-B2-C2 cosegregate with the disease in family (a) and (b), respectively (Fig. 2). Such information can therefore be used for future prenatal and presymptomatic diagnosis in these families. This further illustrates the usefulness of this relatively rapid technique to predict the cartier status of an individual without the requirement to carry out tedious mutation analysis by sequencing the UGT1 gene. Although we have used this technique for the unequivocal diagnosis of disease inheritance in a number of families it cannot be used when one of the parents of a CN affected child is homozygous for a haplotype for obvious reasons. As a result we are currently investigating the use of further polymorphic markers within the UGT1 gene to allow the definitive diagnosis of mutant alleles in every CN family.
8. Current therapy and future treatment for Crigler-Najjar syndromes Phototherapy in infancy is an effective short-term means of lowering serum bilirubin levels, by converting bilirubin to the more readily excreted photobilirubin; however, as the skin thickens with age it becomes progressively less effective [1]. Although administration of haem oxygenase inhibitor, tinmesoporphyrin, to CN patients has been shown to reduce bilirubin levels [40], once again it is not an effective long-term treatment as it causes iron deficiency anaemia [41]. Phenobarbital administration has remained the most successful means of reducing bilirubin levels in CN2; indeed a well-documented CN2 patient lived without any obvious deleterious effect to his eighth decade [31]. By contrast the only current therapy for CN1 is described in a consensus report of a World Registry which was recently published. Partial orthotopic liver transplantation performed at a young age appears to be the best eventual current treatment for the more severe disorder [42]. In the future, CN1 is a prime target for development of hepatic gene therapy, being a lethal condition caused by a defect in the terminal stage of a metabolic pathway.
Acknowledgements We thank the Wellcome Trust, The Scottish Home and Health Department and Medical Research Council for funds supporting this work.
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References [1] Roy Chowdhury J, Wolkoff A, Roy Chowdhury N, Arias IM. Hereditary jaundice and disorders of bilirubin metabolism. In: Scriver CR, Beaudet AI, Sly WS, Valle D, editors. The metabolic and molecular basis of inherited diseases, 7th edition. New York: McGraw Hill, 1995;Chapter 67:2161-2208. [2] Buchler M, Konig J, Brom M, Kartenbeck J, Spring H, Hories T, Keppler D. cDNA cloning of the hepatocyte canalicular isoform of the multidrug resistance protein, cMrp, reveals a novel conjugate export pump deficient in hyperbilirubinemic mutant rats. J Biol Chem 1996;271:15091-8. [3] Arias IM, Gartner LM, Cohen M, Ezzer JB, Levi J. Chronic nonhemolytic unconjugated hyperbilirubinemia with glucuronyl transferase deficiency: clinical, biochemical, pharmacologic and genetic evidence for heterogeneity. Am J Med 1969;47:395-409. [4] Sinaasappel M, Jansen PL. The differential diagnosis of Crigler-Najjar disease, types 1 and 2, by bile pigment analysis. Gastroenterology 1991;100:783-9. [5] Senafi SB, Clarke D J, Burchell B. Investigation of the substrate specificity of a cloned expressed human bilirubin UDP-glucuronosyltransferase: UDP-sugar specificity and involvement in steroid and xenobiotic glucuronidation. Biochem J 1994;303:233-40. [6] Ritter JK, Chen F, Sheen YY, Tran HM, Kimma S, Yeatman MT, Owens IS. A novel complex locus UGT1 encodes human bilirubin, phenol, and other UDP-glucuronosyltransferase isozymes with identical carboxyl termini. J Biol Chem 1992;267:3257-61. [7] van Es HH, Bout A, Liu J, Anderson L, Duncan AM, Bosma P, Oude-Elferink R, Jansen PL, Roy Chowdhury J, Schurr E. Assignment of the human UDP glucuronosyltransferase gene (UGT1A1) to chromosome region 2q37. Cytogenet Cell Genet 1993;63:114-6. [8] Bosma PJ, Seppen J, Goldhoorn B, Bakker C, Oude ER, Roy Chowdhury J, Roy Chowdhury N, Jansen PL. Bilirubin UDP-glucuronosyltransferase 1 is the only relevant bilirubin glucuronidating isoform in man. J Biol Chem 1994;269:17960-4. [9] Ebner TE, BurcheU B. Substrate specificities of two stably expressed human liver UDPglucuronosyltransferases of the UGT1 family. Drug Metab Dispos 1993;21:50-5. [10] Green MD, Bishop WP, Tephly TR. Expressed human UGT1.4 Protein catalyses the formation of quaternary ammonium linked glucuronides. Drug Metab Dispos 1995;23:299302. [11] Ritter JK, Yeatman MT, Kaiser C, Gridelli B, Owens ISA. Phenylalanine codon deletion at the UGT1 gene complex locus of a Crigler-Najjar type I patient generates a pH-sensitive bilirubin UDP-glucuronosyltransferase. J Biol Chem 1993;268:23573-9. [12] Seppen J, Bosma PJ, Goldhoorn BG, Bakker CT, Roy Chowdhury J, Roy Chowdhury N, Jansen PL, Oude ER. Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate- glucuronosyltransferase. J Clin Invest 1994;94:238591. [13] Ciotti M, Yeatman MT, Sokol RJ, Owens IS. Altered coding for a strictly conserved di-glycine in the major bilirubin UDP-glucuronosyltransferase of a Crigler-Najjar type I patient. J Biol Chem 1995;270:3284-91. [14] Aono S, Yamada Y, Keino H, Sasaoka Y, Nakagawa T, Onishi S, Mimura S, Koiwai O, Sato H. A new type of defect in the gene for bilirubin uridine 5'- diphosphate-glucuronosyltransferase in a patient with Crigler-Najjar syndrome type I. Pediatr Res 1994;35:629-32. [15] Labrune P, Myara A, Hadchouel M, Ronchi F, Bernard O, Trivin F, Roy Chowdhury N, Roy Chowdhury J, Munnich A, Odievre M. Genetic heterogeneity of Crigler-Najjar syndrome type I: a study of 14 cases. Hum Genet 1994;94:693-7.
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