- Email: [email protected]
Jaundice, genes and promoters
Journal of Hepntology 2000; 33: 476419 Printed in Denmark All rights wsrrwd Munks~aard Copmhagm
Journalof Hepatology ISSN01txw27K
Editorial
Jaundice, genes and promoters Christian Depurtmrnt
of Gastroenterologv
P Strassburg und Heputology,
and Michael Hannow
P Manns
Medical Shol,
Hunnover. Gerrnq
See Article, pages 348--351
A
N ATTRACTIVE model
in medicine is the identification of a genetic defect which explains the etiology of a disease phenotype. The past decade has seen significant progress by the Human Genome Project and other studies in identifying countless candidate genes, which await their functional characterization. In liver diseases, genetic polymorphisms have been demonstrated for conditions such as hemochromatosis, Wilson’s disease and cx,-antitrypsin deficiency. However, as the example of a,-antitrypsin deficiency illustrates, the presence of the homozygous PiZZ genotype leads to clinically important liver disease in only 15-20’s of affected individuals. The significant part of the scientific work beyond the identification of a candidate gene is the establishment of a conclusive link between gene and function, which may lead to the eventual identification of a molecular mechanism. This link is frequently established by assembling a complex puzzle of individual findings. A genetic condition of the liver which falls into this category is unconjugated, non-hemolytic hyperbilirubinemia. The implicated gene in unconjugated hyperbilirubinemia belongs to the uridine diphosphate 5’ glucuronosyltransferase (UGT) supergene family. UGTs perform a simple biochemical reaction leading to the conjugation of target substrates with glucuronic acid and facilitating their subsequent biliary or renal elimination as water-soluble glucuronides (1). With the exception of few examples, i.e. the analgesic properties of the morphine-6-glucuronide, compounds conjugated with glucuronic acid are biologically inactive. Over 50 UGTs have been identified in different vertebrate species, indicating that glucuronidation represents a
Covrespondencr: Christian P Strassburg, Department of Gastroenterology and Hepatology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel: 49 511 532 2853. Fax: 49 511 532 2093. e-mail: [email protected]
476
highly conserved metabolic pathway. Hundreds of chemically diverse compounds, including dietary conpharmaceutical drugs, endogenous horstituents, mones, bile acids and the heme catalytic byproduct bilirubin, as well as aromatic hydrocarbons, flavonoids and environmental mutagens have been found to undergo glucuronidation. This promiscuity in target substrate choice is a result of the UGTs ability to catalyze glucuronide formation using hydroxy, carbonyl, sulfuryl, carboxyl and amine groups. In humans, 15 UGT transcripts have been identified and divided into two families based upon sequence homology (1). UGT2 genes are located on chromosome 4q13 and 4q28, consist of 6 exons, and preferentially glucuronidate endobiotic substrates including steroids and bile acids. In contrast, the UGTlA gene locus has been mapped to chromosome 2q37 and enables the transcription of individual gene products sharing a common carboxy terminal portion of 280 amino acids and an exon 1 encoded divergent amino terminal portion of approximately 250 amino acids (2) (Fig. 1). Utilizing a strategy of exon sharing, potentially 9 functional exon 1 sequences can be combined with the constant exons 2-5 to form individual UGTlA transcripts. The tissue specific and individual regulation of the UGTlA first exons has recently been identified in the human hepato-gastrointestinal tract. In human liver, transcripts of UGTlAl, UGTlA3, UGTlA4, UGTIA6 and UGTlA9 have been detected and cloned, whereas the novel UGTlA7 (esophagus, stomach), UGTlAX (esophagus, colon) and UGTlAlO (esophagus, bile ducts. stomach, colon) transcripts have been identified exclusively in extrahepatic epithelial tissues (3,4). The genetic organization of the UGTlA gene locus therefore enables a tissue specific gene expression of hepatic and extrahepatic UGTs and most likely ensures that a broad array of differing substrates can undergo glucuronidation in man (3,4). This is also a result of the considerable overlap of substrate
Jaundice, genes and promoters
A(TA),TAA
UGTlAl
(bilirubin UGT)
UGTlA3 UGTlA4
chromosome 2q37 Fig. I. The UGTlA gene locus on chromosome 2q37 covers approximately 1.50 kilo base pairs, and combines unique exon I sequences and common exon 2-5 sequences to form individual transcripts. UGTlAl represents the human bilirubin UGT with an upstream wild-type A(TA)eTAA promoter element. Mutant alleles of this element or of the UGTlAl first exon sequence exclusively affect UGTIAI, while polymorphisms of exons 2-5 affect all UGTlA gene products. In the unconjugated hyperbilirubinemias, polymorphisms have been iden@ed in all 5 exons and the promoter sequence. UGTlA2 represents a pseudogene. Arrows represent putative promoter elements driving exon 1 expression (Schematic not drawn to scale).
specificities, which has been demonstrated by studies with recombinant UGT proteins (1). Fatal unconjugated hyperbilirubinemia in CriglerNajjar’s syndrome and the search for the responsible metabolic pathway has been a driving force for the discovery of the human UGTlA gene locus. The hydrophobic tetrapyrrole bilirubin represents the endpoint of heme catabolism. Only 5% exists as the excretable bilirubin(di)glucuronide. In 199 1, the bilirubin conjugating transferase, UGTlAl, was identified and cloned (5). Although the UGTlA4 cDNA, which was cloned at the same time, encoded a protein that had a low bilirubin UGT activity in vitro, it has become evident that UGTlAl is the major bilirubin UGT in humans. This is surprising in light of the aforementioned number of different human UGTs and their considerable overlap of substrate specificities. The initial studies of unconjugated hyperbilirubinemia in patients with Crigler-Najjar type 1 disease, which are characterized by a complete loss of bilirubin glucuronidation, provided evidence for coding region mutations within exons 2, 3 and 4 of the UGTlA gene locus (Fig. 1) (6-S). Mutant alleles of the common UGTZA exons 2-5 affect all transcripts generated by the UGTl A locus, both hepatic and extrahepatic, and this was viewed as the reason for the complete absence of bilirubin giucuronide formation in Crigler-Najjar type 1 disease. In addition, the Gunn rat represented a model with the analogous features (9). The detection of exon 1 mutations in Crigler-Najjar type 2 disease, characterized by a decrease of bilirubin conjugation activity to below 30%, but not its complete absence, appeared to confirm this
hypothesis (10). However, the discovery of mutations in the first exon of UGTlAl in patients with CriglerNajjar type 1 disease (11) has demonstrated that this can result in the selective inactivation of UGTlAl and a complete absence of bilirubin conjugation in humans despite the considerable redundancy of catalytic activity and tissue-specific expression of human UGT gene products. This finding is significant for two reasons: (i) it confirms that UGTlAl is the only biologically relevant bilirubin UGT and that none of the unaffected UGTIA or UGT2B genes can substitute for this activity, and (ii) no alternative pathway exists for the detoxification and elimination of bilirubin, with perhaps the exception of the much less effective generation of hydrophilic lumirubin by photoisomeration. Consequently, the UGTlAl gene has moved to the center of attention in unconjugated hyperbilirubinemia. To date, 33 differing alleles of this gene have been identified (UGTlAl*l-UGTlA1*3 (reviewed in (1)). It has been realized that in Crigler-Najjar type 1 as well as in type 2 disease mutant alleles can affect all 5 exons. For the hyperbilirubinemic phenotype, homozygous or compound heterozygous mutant alleles are required (12). The paper by Raijmakers et al. published in this issue of the Journal, analyzes the activity of UGTlAl with relevance to a third condition of hereditary unconjugated hyperbilirubinemia: Gilbert’s (GilbertMeulengracht) syndrome. In this disease, affecting as much as 5-10% of the general population, unconjugated bilirubin levels typically fluctuate in response to stress, fasting or nicotinic acid provocation. Hepatic bilirubin UGT activity is only mildly reduced by ap477
C. P. Strassburg and A4 P. Manns
for this disproximately 30%. As a genetic mechanism ease variant, a polymorphism of the UGTlAl promoter TATA element leading to a TA insertion into the wild-type A(TA)6TAA promoter has been identified (UGTlA1*28) and implicated in its etiology (13,14). Studies on the effect and prevalence of UGTIAI promoter polymorphisms were able to show that individuals heterozygous or homozygous for the UGTlAl”28 polymorphism had higher serum bilirubin levels when compared to individuals with the wildtype A(TA)6TAA UGTIAl promoter element. In vitro experiments have confirmed that the UGTI Al *28 promoter activity is only lo-33% of the wild-type promoter (13,1.5). However, the frequency of the A(TA),_ TAA allele was found to be 3440% in Caucasians, resulting in a homozygous Gilbert genotype in 9916% of the general population (13,14,16). These data indicate that polymorphisms of the promoter driving expression of the only human bilirubin UGT are common and affect almost half of the Caucasian population. Since only about 5% of the population are diagnosed with Gilbert’s disease, the question remains to be answered whether additional factors contribute to this conjugation defect. To complicate matters, UGTlAZ promoter polymorphisms are not exclusive to patients with Gilbert’s disease. In a recent study, it was demonstrated that the combination of a coding sequence missense mutation in exon 3 (UGTlAl*32) and the UGTlAl promoter polymorphism can lead to a complete abrogation of UGTl Al catalytic activity and therefore cause Crigler-Najjar type 1 disease (15). On the other hand, patients suffering from Gilbert’s disease have been found to exhibit heterozygous missense mutations in exon 1 of the UGTIAI coding sequence (17), and it appears questionable whether UGTlAl promoter polymorphisms are required for the Gilbert phenotype (18). In addition, the combination of heterozygous UGTIAI exon 1 and promoter mutations has also been demonstrated in patients with Gilbert’s disease (18). From all the information gathered on the genetic basis of unconjugated hyperbilirubinemia in humans, a picture emerges, in which the homozygous, heterozygous or compound heterozygous mutation of any of the 5 UGTlAl exons and/or the promoter region can lead to varying degrees of bilirubin UGT activity reduction, and, as a consequence, to the clinical syndromes that we classify as Crigler-Najjar type 1, Crigler-Najjar type 2 or Gilbert’s disease. Hence, the hereditary unconjugated hyperbilirubinemias share a common etiology determining the severity of disease, most likely as a result of the combination of autosomal recessively inherited polymorphisms functioning as genetic risk factors. 478
In the study by Raijmakers et al. (12) another piece of the puzzle is presented linking hepatic bilirubin UGT activity to the presence of a mutant homozygous, heterozygous or wild-type UGTI Al promoter genotype. The correlation of serum bilirubin concentration and the UGTlAl*28 genotype has been documented in the original descriptions of the promoter polymorphism (13,14) and other papers. Here. the authors examine 39 organ transplant donors for in vitro hepatic bilirubin UGT activity and their respective UGTI Al promoter genotype in addition to two subjects with diagnosed Gilbert’s disease. They demonstrate a correlation of the heterozygous A(TA),TAA genotype with a 37% reduction, as well as of the homozygous A(TA),. TAA genotype with a 52% reduction of mean enzymatic activity in bilirubin activity assays using liver tissue homogenates from the same patients. Although the authors were unable to present data on the bilirubin levels in their study population allowing a direct correlation of the biological effects of these ill vitro lindings, evidence is provided that UGTlAl promoter polymorphisms form a biochemical basis of variations of bilirubin conjugation activity in human liver. The the homozygous determined frequency of UGTlAl*28 genotype in 10% of the studied organ donors falls well within the results obtained by other studies. An interesting finding of this study is the considerable interindividual variation of bilirubin UGT activity presented in Fig. 2. The authors report that coding region mutations have been detected in patients with Gilbert’s disease; however, the factors contributing to the marked interindividual variations of hepatic bilirubin UGT activity remain unclear. In the group of 17 patients identified as exhibiting the wild-type promoter genotype, seven subjects show bilirubin UGT activities around 800-1000 nmol/g/h. which represents approximately the range of the mean of bilirubin UGT activity of the heterozygous UGTZAI promoter group. Conversely, the highest activities measured in the heterozygous promoter group are similar to the highest activities in the wild-type group. These data clearly indicate that additional factors contribute to hepatic bilirubin UGT activity. This is further substantiated by the two patients included with known Gilbert’s disease, who show the lowest bilirubin conjugation activity out of the homozygous mutant promoter group and out of the entire study population, which was determined to be around 250 nmol/g/h. Apart from potential differences in tissue sample quality, the presence of coding region polymorphisms, in particular in the wild-type promoter group, may account for some of the low activities documented for these subjects. Other mechanisms may include transporter abnormalities, which are
Jaundice, genes and promoters
implicated by abnormal bromosulphthaleine and indocyanine green clearances in some patients with Gilbert’s syndrome. The presented data prove the important point that promoter polymorphisms are indeed capable of modulating hepatic bilirubin glucuronidation activity and are therefore responsible for the published findings of elevated unconjugated hyperbilirubinemia in most of the patients. The difficulty of predicting an effect attributable to an individual coding region polymorphism and/or promoter polymorphism, however, remains and can only be conclusively answered by recombinant expression and characterization of the mutant genotype. The broad interindividual variation of hepatic bilirubin UGT activity demonstrated in this study is an invitation to search for additional pieces of this puzzle. Biochemical mechanisms contributing to hepatic bilirubin UGT activity in particular include the identification of UGTl Al gene polymorphisms, their biological effects alone and in differing combinations. Fortunately, Gilbert’s disease is a benign condition which does not require treatment. However, UGTZA promoter polymorphisms have a high prevalence in the Caucasian population. They have now been demonstrated to modulate hepatic bilirubin UGT activity. UGTl A promoter polymorphisms can be viewed as a predisposing factor for the development of Crigler-Najjar’s disease upon combination with additional polymorphic UGTZAl alleles and are therefore a component of the biological relationship of jaundice, genes and promoters.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
References 1. Tukey RH, Strassburg CP Human UDP-glucuronosyltransferase: metabolism, expression and disease. Annu Rev Pharmacol Toxic01 2000; 40: 581619. 2. Ritter JK, Chen E Sheen YY, Tran HM, Kimura S, Yeatman MT, et al. A novel complex locus UGTl encodes human bilirubin, phenol, and other UDP-glucuronosyltransferase isozymes with identical carboxyl termini. J Biol Chem 1992; 267: 3257-61. 3. Strassburg CP, Oldhafer K, Manns MP Tukey RH. Differential expression of the UGTlA locus in human liver, biliary, and gastric tissue: identification of UGTlA7 and UGTlAlO transcripts in extrahepatic tissue. Mol Pharmacol 1997; 52: 212-20. 4. Strassburg CE Manns ME Tukey RH. Expression of the UDP-
16.
17.
18.
glucuronosyltransferase IA locus in human colon. Identification and characterization of the novel extrahepatic UGTlA8. J Biol Chem 1998; 273: 8719-26. Ritter JK, Crawford JM, Owens IS. Cloning of two human liver bilirubin UDP-glucuronosyltransferase cDNAs with expression in COS-1 cells. J Biol Chem 1991; 266: 1043-7. Bosma PJ, Chowdhury JR, Huang TJ, Lahiri E Oude Elferink RP, Van Es HH, et al. Mechanisms of inherited deficiencies of multiple UDP-glucuronosyltransferase isoforms in two patients with Crigler-Najjar syndrome, type I. Faseb J 1992; 6: 2859-63. Bosma PJ, Chowdhury NR, Goldhoorn BG, Hofker MH, Oude Elferink RE Jansen PL, et al. Sequence of exons and the flanking regions of human bilirubin-UDP-glucuronosyltransferase gene complex and identification of a genetic mutation in a patient with Crigler-Naijar syndrome, type I. Hepatology 1992; 15: 941-7. Ritter JK, Yeatman MT, Ferreira P Owens IS. Identification of a genetic alteration in the code for bilirubin UDP-glucuronosyltransferase in the UGTl gene complex of a Crigler-Najjar type I patient. J Clin Invest 1992; 90: 150-5. Iyanagi T. Molecular basis of multiple UDP-glucuronosyltransferase isoenzyme deficiencies in the hyperbilirubinemic rat (Gunn rat). J Biol Chem 1991; 266: 24048852. Bosma PJ, Goldhoorn B, Oude Elferink RP Sinaasappel M, Oostra BA, Jansen PL. A mutation in bilirubin uridine 5’-diphosphate-glucuronosyltransferase isoform 1 causing CriglerNajjar syndrome type II. Gastroenterology 1993; 105: 21620. Ritter JK, Yeatman MT, Kaiser C, Gridelli B, Owens IS. A phenylalanine codon deletion at the UGTl gene complex locus of a Crigler-Najjar type I patient generates a pH-sensitive bilirubin UDP-glucuronosyltransferase. J Biol Chem 1993; 268: 23573-9. Raijmakers MTM, Jansen PLM, Steegers EAP, Peters WHM. Association of human liver bilirubin UDP-glucuronyl-transferase activity with a polymorphism in the promoter region of the UGTIAI gene. J Hepatol 2000; 33: 348-5 1. Bosma PJ, Chowdhury JR, Bakker C, Gantla S, de Boer A, Oostra BA, 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. Monaghan G, Ryan M, Seddon R, Hume R, Burchell B. Genetic variation in bilirubin UPD-glucuronosyltransferase gene promoter and Gilbert’s syndrome. Lancet 1996; 347: 578-81. Ciotti M, Chen F, Rubaltelli FF, Owens IS. Coding defect and a TATA box mutation at the bilirubin UDP-glucuronosyltransferase gene cause Crigler-Najjar type I disease. Biochim Biophys Acta 1998; 1407: 4&50. Lampe JW, Bigler J, Horner NK, Potter JD. UDP-glucuronosyltransferase (UGTlA1*28 and UGTlA6*2) polymorphisms in Caucasians and Asians: relationships to serum bilirubin concentrations. Pharmacogenetics 1999; 9: 341-9. Koiwai 0, Nishizawa M, Hasada K, Aono S, Adachi Y, Mamiya N, et al. Gilbert’s syndrome is caused by a heterozygous missense mutation in the gene for bilirubin UDP-glucuronosyltransferase. Hum Mol Genet 1995; 4: 11836. Sato H, Adachi Y, Koiwai 0. The genetic basis of Gilbert’s syndrome. Lancet 1996; 347: 557-8.
479
Journalof Hepatology ISSN01txw27K
Editorial
Jaundice, genes and promoters Christian Depurtmrnt
of Gastroenterologv
P Strassburg und Heputology,
and Michael Hannow
P Manns
Medical Shol,
Hunnover. Gerrnq
See Article, pages 348--351
A
N ATTRACTIVE model
in medicine is the identification of a genetic defect which explains the etiology of a disease phenotype. The past decade has seen significant progress by the Human Genome Project and other studies in identifying countless candidate genes, which await their functional characterization. In liver diseases, genetic polymorphisms have been demonstrated for conditions such as hemochromatosis, Wilson’s disease and cx,-antitrypsin deficiency. However, as the example of a,-antitrypsin deficiency illustrates, the presence of the homozygous PiZZ genotype leads to clinically important liver disease in only 15-20’s of affected individuals. The significant part of the scientific work beyond the identification of a candidate gene is the establishment of a conclusive link between gene and function, which may lead to the eventual identification of a molecular mechanism. This link is frequently established by assembling a complex puzzle of individual findings. A genetic condition of the liver which falls into this category is unconjugated, non-hemolytic hyperbilirubinemia. The implicated gene in unconjugated hyperbilirubinemia belongs to the uridine diphosphate 5’ glucuronosyltransferase (UGT) supergene family. UGTs perform a simple biochemical reaction leading to the conjugation of target substrates with glucuronic acid and facilitating their subsequent biliary or renal elimination as water-soluble glucuronides (1). With the exception of few examples, i.e. the analgesic properties of the morphine-6-glucuronide, compounds conjugated with glucuronic acid are biologically inactive. Over 50 UGTs have been identified in different vertebrate species, indicating that glucuronidation represents a
Covrespondencr: Christian P Strassburg, Department of Gastroenterology and Hepatology, Hannover Medical School, Carl-Neuberg-Str. 1, 30625 Hannover, Germany. Tel: 49 511 532 2853. Fax: 49 511 532 2093. e-mail: [email protected]
476
highly conserved metabolic pathway. Hundreds of chemically diverse compounds, including dietary conpharmaceutical drugs, endogenous horstituents, mones, bile acids and the heme catalytic byproduct bilirubin, as well as aromatic hydrocarbons, flavonoids and environmental mutagens have been found to undergo glucuronidation. This promiscuity in target substrate choice is a result of the UGTs ability to catalyze glucuronide formation using hydroxy, carbonyl, sulfuryl, carboxyl and amine groups. In humans, 15 UGT transcripts have been identified and divided into two families based upon sequence homology (1). UGT2 genes are located on chromosome 4q13 and 4q28, consist of 6 exons, and preferentially glucuronidate endobiotic substrates including steroids and bile acids. In contrast, the UGTlA gene locus has been mapped to chromosome 2q37 and enables the transcription of individual gene products sharing a common carboxy terminal portion of 280 amino acids and an exon 1 encoded divergent amino terminal portion of approximately 250 amino acids (2) (Fig. 1). Utilizing a strategy of exon sharing, potentially 9 functional exon 1 sequences can be combined with the constant exons 2-5 to form individual UGTlA transcripts. The tissue specific and individual regulation of the UGTlA first exons has recently been identified in the human hepato-gastrointestinal tract. In human liver, transcripts of UGTlAl, UGTlA3, UGTlA4, UGTIA6 and UGTlA9 have been detected and cloned, whereas the novel UGTlA7 (esophagus, stomach), UGTlAX (esophagus, colon) and UGTlAlO (esophagus, bile ducts. stomach, colon) transcripts have been identified exclusively in extrahepatic epithelial tissues (3,4). The genetic organization of the UGTlA gene locus therefore enables a tissue specific gene expression of hepatic and extrahepatic UGTs and most likely ensures that a broad array of differing substrates can undergo glucuronidation in man (3,4). This is also a result of the considerable overlap of substrate
Jaundice, genes and promoters
A(TA),TAA
UGTlAl
(bilirubin UGT)
UGTlA3 UGTlA4
chromosome 2q37 Fig. I. The UGTlA gene locus on chromosome 2q37 covers approximately 1.50 kilo base pairs, and combines unique exon I sequences and common exon 2-5 sequences to form individual transcripts. UGTlAl represents the human bilirubin UGT with an upstream wild-type A(TA)eTAA promoter element. Mutant alleles of this element or of the UGTlAl first exon sequence exclusively affect UGTIAI, while polymorphisms of exons 2-5 affect all UGTlA gene products. In the unconjugated hyperbilirubinemias, polymorphisms have been iden@ed in all 5 exons and the promoter sequence. UGTlA2 represents a pseudogene. Arrows represent putative promoter elements driving exon 1 expression (Schematic not drawn to scale).
specificities, which has been demonstrated by studies with recombinant UGT proteins (1). Fatal unconjugated hyperbilirubinemia in CriglerNajjar’s syndrome and the search for the responsible metabolic pathway has been a driving force for the discovery of the human UGTlA gene locus. The hydrophobic tetrapyrrole bilirubin represents the endpoint of heme catabolism. Only 5% exists as the excretable bilirubin(di)glucuronide. In 199 1, the bilirubin conjugating transferase, UGTlAl, was identified and cloned (5). Although the UGTlA4 cDNA, which was cloned at the same time, encoded a protein that had a low bilirubin UGT activity in vitro, it has become evident that UGTlAl is the major bilirubin UGT in humans. This is surprising in light of the aforementioned number of different human UGTs and their considerable overlap of substrate specificities. The initial studies of unconjugated hyperbilirubinemia in patients with Crigler-Najjar type 1 disease, which are characterized by a complete loss of bilirubin glucuronidation, provided evidence for coding region mutations within exons 2, 3 and 4 of the UGTlA gene locus (Fig. 1) (6-S). Mutant alleles of the common UGTZA exons 2-5 affect all transcripts generated by the UGTl A locus, both hepatic and extrahepatic, and this was viewed as the reason for the complete absence of bilirubin giucuronide formation in Crigler-Najjar type 1 disease. In addition, the Gunn rat represented a model with the analogous features (9). The detection of exon 1 mutations in Crigler-Najjar type 2 disease, characterized by a decrease of bilirubin conjugation activity to below 30%, but not its complete absence, appeared to confirm this
hypothesis (10). However, the discovery of mutations in the first exon of UGTlAl in patients with CriglerNajjar type 1 disease (11) has demonstrated that this can result in the selective inactivation of UGTlAl and a complete absence of bilirubin conjugation in humans despite the considerable redundancy of catalytic activity and tissue-specific expression of human UGT gene products. This finding is significant for two reasons: (i) it confirms that UGTlAl is the only biologically relevant bilirubin UGT and that none of the unaffected UGTIA or UGT2B genes can substitute for this activity, and (ii) no alternative pathway exists for the detoxification and elimination of bilirubin, with perhaps the exception of the much less effective generation of hydrophilic lumirubin by photoisomeration. Consequently, the UGTlAl gene has moved to the center of attention in unconjugated hyperbilirubinemia. To date, 33 differing alleles of this gene have been identified (UGTlAl*l-UGTlA1*3 (reviewed in (1)). It has been realized that in Crigler-Najjar type 1 as well as in type 2 disease mutant alleles can affect all 5 exons. For the hyperbilirubinemic phenotype, homozygous or compound heterozygous mutant alleles are required (12). The paper by Raijmakers et al. published in this issue of the Journal, analyzes the activity of UGTlAl with relevance to a third condition of hereditary unconjugated hyperbilirubinemia: Gilbert’s (GilbertMeulengracht) syndrome. In this disease, affecting as much as 5-10% of the general population, unconjugated bilirubin levels typically fluctuate in response to stress, fasting or nicotinic acid provocation. Hepatic bilirubin UGT activity is only mildly reduced by ap477
C. P. Strassburg and A4 P. Manns
for this disproximately 30%. As a genetic mechanism ease variant, a polymorphism of the UGTlAl promoter TATA element leading to a TA insertion into the wild-type A(TA)6TAA promoter has been identified (UGTlA1*28) and implicated in its etiology (13,14). Studies on the effect and prevalence of UGTIAI promoter polymorphisms were able to show that individuals heterozygous or homozygous for the UGTlAl”28 polymorphism had higher serum bilirubin levels when compared to individuals with the wildtype A(TA)6TAA UGTIAl promoter element. In vitro experiments have confirmed that the UGTI Al *28 promoter activity is only lo-33% of the wild-type promoter (13,1.5). However, the frequency of the A(TA),_ TAA allele was found to be 3440% in Caucasians, resulting in a homozygous Gilbert genotype in 9916% of the general population (13,14,16). These data indicate that polymorphisms of the promoter driving expression of the only human bilirubin UGT are common and affect almost half of the Caucasian population. Since only about 5% of the population are diagnosed with Gilbert’s disease, the question remains to be answered whether additional factors contribute to this conjugation defect. To complicate matters, UGTlAZ promoter polymorphisms are not exclusive to patients with Gilbert’s disease. In a recent study, it was demonstrated that the combination of a coding sequence missense mutation in exon 3 (UGTlAl*32) and the UGTlAl promoter polymorphism can lead to a complete abrogation of UGTl Al catalytic activity and therefore cause Crigler-Najjar type 1 disease (15). On the other hand, patients suffering from Gilbert’s disease have been found to exhibit heterozygous missense mutations in exon 1 of the UGTIAI coding sequence (17), and it appears questionable whether UGTlAl promoter polymorphisms are required for the Gilbert phenotype (18). In addition, the combination of heterozygous UGTIAI exon 1 and promoter mutations has also been demonstrated in patients with Gilbert’s disease (18). From all the information gathered on the genetic basis of unconjugated hyperbilirubinemia in humans, a picture emerges, in which the homozygous, heterozygous or compound heterozygous mutation of any of the 5 UGTlAl exons and/or the promoter region can lead to varying degrees of bilirubin UGT activity reduction, and, as a consequence, to the clinical syndromes that we classify as Crigler-Najjar type 1, Crigler-Najjar type 2 or Gilbert’s disease. Hence, the hereditary unconjugated hyperbilirubinemias share a common etiology determining the severity of disease, most likely as a result of the combination of autosomal recessively inherited polymorphisms functioning as genetic risk factors. 478
In the study by Raijmakers et al. (12) another piece of the puzzle is presented linking hepatic bilirubin UGT activity to the presence of a mutant homozygous, heterozygous or wild-type UGTI Al promoter genotype. The correlation of serum bilirubin concentration and the UGTlAl*28 genotype has been documented in the original descriptions of the promoter polymorphism (13,14) and other papers. Here. the authors examine 39 organ transplant donors for in vitro hepatic bilirubin UGT activity and their respective UGTI Al promoter genotype in addition to two subjects with diagnosed Gilbert’s disease. They demonstrate a correlation of the heterozygous A(TA),TAA genotype with a 37% reduction, as well as of the homozygous A(TA),. TAA genotype with a 52% reduction of mean enzymatic activity in bilirubin activity assays using liver tissue homogenates from the same patients. Although the authors were unable to present data on the bilirubin levels in their study population allowing a direct correlation of the biological effects of these ill vitro lindings, evidence is provided that UGTlAl promoter polymorphisms form a biochemical basis of variations of bilirubin conjugation activity in human liver. The the homozygous determined frequency of UGTlAl*28 genotype in 10% of the studied organ donors falls well within the results obtained by other studies. An interesting finding of this study is the considerable interindividual variation of bilirubin UGT activity presented in Fig. 2. The authors report that coding region mutations have been detected in patients with Gilbert’s disease; however, the factors contributing to the marked interindividual variations of hepatic bilirubin UGT activity remain unclear. In the group of 17 patients identified as exhibiting the wild-type promoter genotype, seven subjects show bilirubin UGT activities around 800-1000 nmol/g/h. which represents approximately the range of the mean of bilirubin UGT activity of the heterozygous UGTZAI promoter group. Conversely, the highest activities measured in the heterozygous promoter group are similar to the highest activities in the wild-type group. These data clearly indicate that additional factors contribute to hepatic bilirubin UGT activity. This is further substantiated by the two patients included with known Gilbert’s disease, who show the lowest bilirubin conjugation activity out of the homozygous mutant promoter group and out of the entire study population, which was determined to be around 250 nmol/g/h. Apart from potential differences in tissue sample quality, the presence of coding region polymorphisms, in particular in the wild-type promoter group, may account for some of the low activities documented for these subjects. Other mechanisms may include transporter abnormalities, which are
Jaundice, genes and promoters
implicated by abnormal bromosulphthaleine and indocyanine green clearances in some patients with Gilbert’s syndrome. The presented data prove the important point that promoter polymorphisms are indeed capable of modulating hepatic bilirubin glucuronidation activity and are therefore responsible for the published findings of elevated unconjugated hyperbilirubinemia in most of the patients. The difficulty of predicting an effect attributable to an individual coding region polymorphism and/or promoter polymorphism, however, remains and can only be conclusively answered by recombinant expression and characterization of the mutant genotype. The broad interindividual variation of hepatic bilirubin UGT activity demonstrated in this study is an invitation to search for additional pieces of this puzzle. Biochemical mechanisms contributing to hepatic bilirubin UGT activity in particular include the identification of UGTl Al gene polymorphisms, their biological effects alone and in differing combinations. Fortunately, Gilbert’s disease is a benign condition which does not require treatment. However, UGTZA promoter polymorphisms have a high prevalence in the Caucasian population. They have now been demonstrated to modulate hepatic bilirubin UGT activity. UGTl A promoter polymorphisms can be viewed as a predisposing factor for the development of Crigler-Najjar’s disease upon combination with additional polymorphic UGTZAl alleles and are therefore a component of the biological relationship of jaundice, genes and promoters.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
References 1. Tukey RH, Strassburg CP Human UDP-glucuronosyltransferase: metabolism, expression and disease. Annu Rev Pharmacol Toxic01 2000; 40: 581619. 2. Ritter JK, Chen E Sheen YY, Tran HM, Kimura S, Yeatman MT, et al. A novel complex locus UGTl encodes human bilirubin, phenol, and other UDP-glucuronosyltransferase isozymes with identical carboxyl termini. J Biol Chem 1992; 267: 3257-61. 3. Strassburg CP, Oldhafer K, Manns MP Tukey RH. Differential expression of the UGTlA locus in human liver, biliary, and gastric tissue: identification of UGTlA7 and UGTlAlO transcripts in extrahepatic tissue. Mol Pharmacol 1997; 52: 212-20. 4. Strassburg CE Manns ME Tukey RH. Expression of the UDP-
16.
17.
18.
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