- Email: [email protected]
Association of human liver bilirubin UDP-glucuronyltransferase activity with a polymorphism in the promoter region of the UGT1A1 gene
Journul of Hepatology 2000; 33: 348-35 Printed in Denmark All rigkt.~ reserved Mmksgamd Copenhagen
I
Association of human liver bilirubin UDP-glucuronyltransferase activity with a polymorphism in the promoter region of the UGTlAl gene Maarten ’ Depumenrs
T. M. Raijmakers
‘, Peter
L. M. Jansen2,
See Editorial,
is a chronic mild form of unconjugated hyperbilirubinaemia in the absence of liver disease or overt haemolysis (14). The most consistent feature in Gilbert’s syndrome is an elevated serum unconjugated bilirubin level due to a deficiency in bilirubin glucuronidation. Serum levels may be elevated up to 3 or 4 times the upper normal reference level ILBERT'S SYNDROME
Received
13 August;
Correspondence:
rrvi.red 2 December
1999; nrcrpted
10 Junuar~~ NO0
Wilbert H. M. Peters, Department Gastroenterology, University Hospital St. Radboud, Box 9101. 6500 HB Nijmegen, The Netherlands. Tel: 31 24 3615123. Fax: 31 24 3540103.
348
and Wilbert
of’ Gastroenterology. ‘Obstetrics und Gprecology, University Hospitul St. Radboud, Nijmego~. Gmtroenterology, C’niwrsit~ Hospitul Groningen, The Nutherlund~
Backgvound/Aims: Gilbert’s syndrome is a benign form of a deficiency in bilirubin glucuronidation. It is associated with a homozygous polymorphism, A(TA),TAA instead of A(TA),TAA, in the TATA-box of the promoter region of the bilirubin UDP-glucuronyltransferase gene. In this study the correlation between this promoter region polymorphism and in vitro human liver bilirubin UDP-glucuronyltransferase enzyme activity was investigated. Me&&: Liver samples from organ transplant donors (n=39) and two known Gilbert’s syndrome patients were used for measuring bilirubin UDP-glucuronyltransferase enzyme activity and for isolation of DNA followed by detection of the promoter region polymorphism by polymerase chain reaction. Genotypes were assigned as follows; 6/6: homozygous for the A(TA),TAA-allele, 7/7: homozygous for the A(TA)-ITAA-allele, and 617: heterozygous with one of each alleles. Results: Seventeen out of 39 subjects (44%) had the homozygous 6/6 genotype, 18 subjects (46%) had the heterozygous 617 genotype, whereas four individuals (10%) and the two individuals with Gilbert’s syndrome
G
Eric A. I? Steegers3
of PO
H. M. Peters] md ‘Dqmtrwnt
q/’
had the 7/7 genotype correlated with Gilbert’s syndrome. This resulted in an allele frequency of 0.33 for the A(TA),TAA-allele. The median bilirubin UDPglucuronyltransferase enzyme activity of the 17 subjects with the 6/6 genotype (1565 nmol/g liver/h) was significantly higher than the activity of the 18 subjects with the 617 genotype (985 nmollg liver/h; pCO.05) and the six individuals with the 7/7 genotype (749 nmol/g liver/h; p
Key words: Bilirubin UDP-glucuronyltransferase; Gilbert’s syndrome; Human liver; Polymorphism; UGTl Al. pages
476-479
with episodes of manifest jaundice (3-5). The metabolism of some drugs, such as paracetamol and the anaesthetic propofol, may also be affected (5-8). The syndrome is benign and prevalent: 3 to 13% of the general population has been estimated to have Gilbert’s syndrome (2,3,9). Its mode of inheritance is unclear since many people do not have a clear family history because the syndrome often remains undiagnosed. Two different modes of inheritance have been postulated: autosomal dominant with incomplete penetrance (3) or an autosomal recessive (2). Recently, the genetic basis of Gilbert’s syndrome was reported to result from a polymorphism in the pro-
UGTIAI:
moter region of the UGTlAl gene encoding the bilirubin UDP-glucuronyltransferase (2,3). A variant TATAA element, which contains two extra nucleotides, A(TA),TAA instead of A(TA),TAA, in the upstream promoter region was associated with Gilbert’s syndrome. Homozygosity for the A(TA)7TAA allele (7/7 genotype) was associated with the clinical manifestations of Gilbert’s syndrome, i.e. a normal to elevated serum bilirubin level under baseline conditions and hyperbilirubinaemia upon fasting (2,3). The direct association between this polymorphism in the promoter region of the UGTlAl gene and the hepatic bilirubin UDP-glucuronyltransferase enzyme activity has not been studied. Here we have investigated the effect of the A(TA)7TAA-allele in the promoter region of the bilirubin UDP-glucuronyltransferase gene by correlating the hepatic bilirubin UDP-glucuronyltransferase enzyme activity to the genotype of the promoter region.
Materials and Methods Liver samples (n=39) from organ transplant donors were obtained shortly after death and were immediately frozen in liquid nitrogen and stored at -80°C until use. For practical reasons or reasons of privacy, no data on age, sex, lifestyle, medication, drug use, dietary habits or serum bilirubin levels of the donors of these liver samples were available. In addition, two liver samples of patients with the Gilbert’s syndrome were used in the study. The local medical review committee approved the study. Homogenates (20% w/v) were prepared by grinding small pieces of liver tissue in a glass/glass Potter (5 strokes) in sterile homogenisation buffer (0.25 M saccharose, 1 mM dithiothreitol, 1 mM EDTA and 30 mM Tris-HCl pH 7.5) and were stored in small aliquots at -80°C until use. Homogenates were used for isolation of DNA by the Wizard@ DNA Purification Kit (Promega, Madison, USA) according to the instructions of the manufacturer. Bilirubin UDP glucuronyltransferase assay Liver homogenates (lo%, w/v) were activated by preincubation with 1% (w/v) digitonin (Sigma Chemicals) for 10 min at room temperature. Activated liver homogenates were subsequently incubated with unconjugated bilirubin (Sigma Chemicals) under dim red light and an argon atmosphere for 15 min at 37°C. The reaction mixture contained (final concentrations): 5% (w/v) activated liver homogenate, 70 mM Tris-HCl pH 7.8, 3.3 mM MgC12, 1 mM D-saccharic acid 1,4lactone (Sigma Chemicals), 3.5 mM UDP-glucuronic acid (Sigma) and 40 PM unconjugated bilirubin. Reaction was stopped, reaction mixture was handled and bilirubin and its conjugates were determined exactly as described before by Cuypers et al. (10). Genotyping The number of TA-repeats in the promoter region of the UGTlAl gene was studied using the polymerase chain reaction conditions and primers (C and D) as described by Monaghan et al. (3). Amplification was confirmed by agarose electrophoresis before fragments were resolved on 11% polyacrylamide gels (382 acrylamide/bisacrylamide; Biorad) in Tris-Borate-EDTA buffer (11). Gels (20X20X0.075 cm) were run at 300 Volts for 3 h and stained with ethidiumbromide (Omnilabo) for 45 min. Genotypes were assigned as follows; 6/6: homozygous for the A(TA)sTAA-allele, 7/7: homozygous for the A(TA),. TAA-allele, and 6/7: heterozygous with one of each allele.
Statistics Kruskal-Wallis ANOVA and the Wilcoxon-Mann-Whitney used to assess statistical significance between groups. 0.05 was considered significant.
tests were A p-value of
Results Examples of the analysis of the polymorphism in the promoter region of the UGTlAl gene are shown in Fig. 1. The two known Gilbert’s syndrome patients both showed the 7/7 genotype. Of the 39 transplant donor liver samples tested, 17 (44%) showed the 6/6 genotype, 18 (46%) showed the 617 genotype and 4 (10%) had the 7/7 genotype (Table 1). This resulted in allele frequencies of 0.33 and 0.67 for the A(TA);ITAA- and A(TA)6_ TAA-alleles, respectively Fig. 2 shows the hepatic bilirubin UDP-glucuronyltransferase enzyme activities of all samples, separated into three groups selected on promoter region genotype. The two samples of the patients with Gilbert’s syndrome showed enzyme activities of 424 and 515 nmol/g liver/h. Median bilirubin UDP-glucuronyltransferase activity (see Table 1) of the 6/6-genotypes (1565, range 741-2666 nmol/g liver/h) was significantly higher as compared to that of the 7/7 genotypes (749 [424-11721 nmol/g liver/h, ~KO.005) and of the 6/7
Fig. 1. Genotyping of the polymorphism in the promoter region of the UGTlAl gene. Results shown are the polymerase chain reaction (PCR) products of 12 randomly chosen samples after analysis on 11% polyacrylamide gel. Presence of an A(TA)eTAA allele results in a PCR product of 98 bp, whereas presence of the A(TA)7TAA allele yields a 100 bp product. Genotypes are assigned as follows: 616, homozygous for A(TA)eTAA allele; 717, homozygous for the A(TA)7TAA allele; 617, heterozygous (both alleles present); M, 100 bp marker.
TABLE
1
Hepatic grouped
bilirubin according
UDP-glucuronyltransferase enzyme to the promoter region genotypes
Genotype
n
Enzyme
616 617 717
17 18 6
1565 [741-26661 985 [57@-2464](l) 749 [424-l 172]@)
activities
activity
Genotypes are denoted as given in the legend of Fig. 1. Enzyme activity is given as median [range] in nmoYg liver/h. Wilcoxon-MannWhitney:l) p
349
M. T. M. Ruijmukers et al. 3000
ences
c= f g
2500
2000
-
E .c
1500
-
,000
-
.z 8 E E 5
:
F
3d,
5w-
0 --&$ f*
0
6/6
6”
7/7
Genotype
Fig. 2. Hepatic hilirubin UDP-glucuronyltransferase enzyme activities plotted against the genotype of the UGTI Al promoter region. Median activities are indicated by a horizontal line. Samples are divided into three groups selected on genotype. Open diamonds indicate the samples qf’ the two knobvn patients with Gilbert’s syndrome.
genotypes (985 [570-24641 nmol/g liver/h, ~~0.05). Although the median enzyme activity of the 7/7 genotypes was lower than that of the heterozygous 617 genotypes, the difference did not reach significance (p= 0.14).
Discussion An occurrence of 10% of the 717 genotype in 39 random samples was shown in this study. This is comparable to the incidences reported by several other groups; 15% by Bosma et al. (2), lo-13% by Monaghan et al. (3), 16% by Biondi et al. (12) 13% by Bancroft et al. (13) and 11% by Beutler et al. (14). The allele frequency of the A(TA),TAA-allele (0.33) was similar to frequencies found by Bosma et al. (0.40) (2) Sampietro et al. (0.44) (1 l), Biondi et al. (0.34) (12) Beutler et al. (0.39) (14) and Galanello et al. (0.31) (15). Median hepatic bilirubin UDP-glucuronyltransferase activity in samples from donors with the 6/7 or 71 7 genotype were significantly lower as compared to that of the 6/6 genotypes. Earlier studies correlated the UGTl Al promoter genotype with the level of serum total bilirubin (2,3,11,12,15,16). Significantly higher bilirubin levels were found in the 717 genotypes as compared to the 616 genotypes. However, reports are contradictory with respect to total bilirubin levels in the 6/ 7 as compared to the 6/6 genotypes. Bosma et al. (2) Sampietro et al. (1 l), Biondi et al. (12) and Ishihara et al. (16) found significantly higher total serum bilirubin levels in the group with the 617 genotype as compared to the 6/6 genotypes. However, Monaghan et al. (3) and Galanello et al. (15) reported no significant differ-
350
in the levels of total
serum
bilirubin
groups with these two genotypes. Unfortunately not have serum bilirubin levels from organ
between we do donors,
since most subjects died after traffic accidents. and sampling blood to assay serum bilirubin level was either not possible or had very low priority at that time. Black & Billing (4) Aono et al. (17), and Peters & Jansen (18) reported lower hepatic bilirubin UDP-glucuronyltransferase activities in individuals with Gilbert’s syndrome as compared to controls. However, hepatic bilirubin UDP-glucuronyltransferase enzyme activities were not yet correlated with the promoter region genotype of the bilirubin UDP-glucuronyltransferase gene. Previous studies have shown that the 7/7 genotype is associated with Gilbert’s syndrome (2,3,11,12). As shown by Bosma et al. (2) and Ciotti et al. (19) the transcriptional activity of genes with A(TA),TAA in the promoter region was lo-15% of the activity of the genes with the wild-type A(TA&TAA. Transfection of the polymorphic gene in HepG2 cells resulted in reduced expression and lower enzyme activity (19). This may explain the lower median bilirubin UDP-glucuronyltransferase enzyme activity for the group with the 7/7 genotype and the intermediate activity for the group with the 6/7 genotype found in this study. The higher serum bilirubin levels in patients with Gilbert’s syndrome may directly result from this lower hepatic bilirubin UDP-glucuronyltransferase activity. However, the wide range of activities found, especially in the 6/6 and 6/7 groups, may indicate that additional factors apart from variations in the TATA-box may contribute to UDP bilirubin glucuronyltransferase enzyme activities. However, Aono et al. (17) reported that in a Japanese population missense mutations caused by a single nucleotide substitution in the coding region of the bilirubin UDP-glucuronyltransferase gene may also be associated with higher serum levels of bilirubin and the aetiology of Gilbert’s syndrome. Sato et al. (20) showed that none of seven patients with Gilbert’s syndrome with a missense mutation in the coding region was homozygous for the 717 genotype. In accordance with these findings, Beutler et al. (14) reported that different ethnic groups have different genotypes related to Gilbert’s syndrome and serum total bilirubin levels. They suggested that the polymorphism in the promoter region might serve as a “fine-regulator” of bilirubin levels within populations. The discrepancy found with respect to the genetic basis of Gilbert’s syndrome between the Japanese and Western researchers may thus be explained by differences between ethnic populations. In summary, we found a clear association between
UGTlAI: genotype versus enzyme activity
UGTlAl promoter region polymorphism and hepatic bilirubin UDP-glucuronyltransferase enzyme activity. We also noted a median UGTIAI enzyme activity in the heterozygous 6/7 group that was significantly lower than in the wild-type 616 group. This suggests that Gilbert’s syndrome may be associated not only with the 7/7 homozygous genotype but also with the 6/7 genotype. Studies in Caucasian populations have shown a consistent correlation between clinically manifest Gilbert’s syndrome and the 7/7 genotype, but do not rule out the possibility of an increased frequency of the 6/7 genotype among persons with a normal baseline but an elevated fasting bilirubin level.
9.
10.
11.
12.
13.
References 1. Gilbert A, Lereboullet I? Travaux orginaux. Semaine Medicale 1901; 21: 241-3. 2. 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. 3. 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: 578-81. 4. Black M, Billing BH. Hepatic bilirubin UDP-glucuronyltransferase activity in liver disease and Gilbert’s syndrome. N Engl J Med 1969; 280: 126671. 5. De Morais SM, Uetrecht JP Wells PG. Decreased glucuronidation and increased bioactivation of acetaminophen in Gilbert’s syndrome. Gastroenterology 1992; 102: 577-86. 6. McGurk KA, Brierly CH, Burchell B. Drug glucuronidation by human renal UDP-glucuronosyltransferases. Biochem Pharmac01 1998; 55: 1005-12. 7. Jansen PLM, Bosma PJ, Chowdurry JR. Molecular biology of bilirubin metabolism. Prog Liver Dis 1995; 13: 125-50. 8. Iyer L, King CD, Roy SK, Whitington PR, Tephly TR, Coffman B, et al. Genetic predisposition to the metabolism of irinotecan: role of UGTl Al in the glucuronidation of its active metabolite
14.
15.
16.
17.
18.
19.
20.
(SN-38) in human liver microsomes. J Clin Invest 1998; 101: 84754. Sieg A, Arab L, Schlierf G, Stiehl A, Kommerell B. Prevalence of Gilbert’s syndrome in Germany. Dtsch Med Wochenschr 1987; 112: 1206-8. Cuypers HTM, Ter Haar, EM, Jansen PLM. Microsomal conjugation and oxidation of bilirubin. Biochim Biophys Acta 1983; 758: 13543. Sampietro M, Lupica L, Perrero L, Comino A, Martinez di Montemuros F, Cappellini MD, et al. The expression of uridine diphosphate glucuronyltransferase gene is a major determinant of bilirubin level in heterozygous S-thalassaemia and in glucose6-phosphate dehydrogenase deficiency. Br J Haematol 1997; 99: 437-9. Biondi ML, Turri 0, Dilillo D, Stival G, Guagnellini E. Contribution of the TAT&box genotype (Gilbert syndrome) to serum bilirubin concentrations in the Italian population. Clin Chem 1999; 45: 89778. Bancroft JD, Kreamer B, Gourley GR. Gilbert syndrome accelerates development of neonatal jaundice. J Pediatr 1998; 132: 656 60. Beutler E, Gelbart T, Demina A. Racial variability in the UDPglucuronosyltransferase 1 (UGTlAl) promoter: a balanced polymorphism for regulation of bilirubin metabolism? Proc Nat1 Acad Sci USA 1998; 95: 817&l. Galanello R, Perseu L, Cipollina ML, Barella S, Giagu N, Turco MP et al. Hyperbilirubinaemia in heterozygous B-thalassaemia is related to co-inherited Gilbert’s syndrome. Br J Haematol 1997; 99: 433-6. Ishihara T, Gabazza EC, Adachi Y, Sato H, Maruo Y. Genetic basis of fasting hyperbilirubinemia. Gastroenterology 1999; 116: 1272. Aono S, Adachi Y, Uyama E, Yamada Y, Keino H, Nanno T, et al. Analysis of genes for bilirubin UDP-glucuronosyltransferase in Gilbert’s syndrome. Lancet 1995; 345: 958-9. Peters WHM, Jansen PLM. Microsomal UDP-glucuronyltransferase catalyzed bilirubin diglucuronide formation in human liver. J Hepatol 1986; 2: 182-94. 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: 413-50. Sato H, Adachi Y, Koiwai 0. The genetic basis of Gilbert’s syndrome. Lancet 1996; 347: 557-8.
3.51
I
Association of human liver bilirubin UDP-glucuronyltransferase activity with a polymorphism in the promoter region of the UGTlAl gene Maarten ’ Depumenrs
T. M. Raijmakers
‘, Peter
L. M. Jansen2,
See Editorial,
is a chronic mild form of unconjugated hyperbilirubinaemia in the absence of liver disease or overt haemolysis (14). The most consistent feature in Gilbert’s syndrome is an elevated serum unconjugated bilirubin level due to a deficiency in bilirubin glucuronidation. Serum levels may be elevated up to 3 or 4 times the upper normal reference level ILBERT'S SYNDROME
Received
13 August;
Correspondence:
rrvi.red 2 December
1999; nrcrpted
10 Junuar~~ NO0
Wilbert H. M. Peters, Department Gastroenterology, University Hospital St. Radboud, Box 9101. 6500 HB Nijmegen, The Netherlands. Tel: 31 24 3615123. Fax: 31 24 3540103.
348
and Wilbert
of’ Gastroenterology. ‘Obstetrics und Gprecology, University Hospitul St. Radboud, Nijmego~. Gmtroenterology, C’niwrsit~ Hospitul Groningen, The Nutherlund~
Backgvound/Aims: Gilbert’s syndrome is a benign form of a deficiency in bilirubin glucuronidation. It is associated with a homozygous polymorphism, A(TA),TAA instead of A(TA),TAA, in the TATA-box of the promoter region of the bilirubin UDP-glucuronyltransferase gene. In this study the correlation between this promoter region polymorphism and in vitro human liver bilirubin UDP-glucuronyltransferase enzyme activity was investigated. Me&&: Liver samples from organ transplant donors (n=39) and two known Gilbert’s syndrome patients were used for measuring bilirubin UDP-glucuronyltransferase enzyme activity and for isolation of DNA followed by detection of the promoter region polymorphism by polymerase chain reaction. Genotypes were assigned as follows; 6/6: homozygous for the A(TA),TAA-allele, 7/7: homozygous for the A(TA)-ITAA-allele, and 617: heterozygous with one of each alleles. Results: Seventeen out of 39 subjects (44%) had the homozygous 6/6 genotype, 18 subjects (46%) had the heterozygous 617 genotype, whereas four individuals (10%) and the two individuals with Gilbert’s syndrome
G
Eric A. I? Steegers3
of PO
H. M. Peters] md ‘Dqmtrwnt
q/’
had the 7/7 genotype correlated with Gilbert’s syndrome. This resulted in an allele frequency of 0.33 for the A(TA),TAA-allele. The median bilirubin UDPglucuronyltransferase enzyme activity of the 17 subjects with the 6/6 genotype (1565 nmol/g liver/h) was significantly higher than the activity of the 18 subjects with the 617 genotype (985 nmollg liver/h; pCO.05) and the six individuals with the 7/7 genotype (749 nmol/g liver/h; p
Key words: Bilirubin UDP-glucuronyltransferase; Gilbert’s syndrome; Human liver; Polymorphism; UGTl Al. pages
476-479
with episodes of manifest jaundice (3-5). The metabolism of some drugs, such as paracetamol and the anaesthetic propofol, may also be affected (5-8). The syndrome is benign and prevalent: 3 to 13% of the general population has been estimated to have Gilbert’s syndrome (2,3,9). Its mode of inheritance is unclear since many people do not have a clear family history because the syndrome often remains undiagnosed. Two different modes of inheritance have been postulated: autosomal dominant with incomplete penetrance (3) or an autosomal recessive (2). Recently, the genetic basis of Gilbert’s syndrome was reported to result from a polymorphism in the pro-
UGTIAI:
moter region of the UGTlAl gene encoding the bilirubin UDP-glucuronyltransferase (2,3). A variant TATAA element, which contains two extra nucleotides, A(TA),TAA instead of A(TA),TAA, in the upstream promoter region was associated with Gilbert’s syndrome. Homozygosity for the A(TA)7TAA allele (7/7 genotype) was associated with the clinical manifestations of Gilbert’s syndrome, i.e. a normal to elevated serum bilirubin level under baseline conditions and hyperbilirubinaemia upon fasting (2,3). The direct association between this polymorphism in the promoter region of the UGTlAl gene and the hepatic bilirubin UDP-glucuronyltransferase enzyme activity has not been studied. Here we have investigated the effect of the A(TA)7TAA-allele in the promoter region of the bilirubin UDP-glucuronyltransferase gene by correlating the hepatic bilirubin UDP-glucuronyltransferase enzyme activity to the genotype of the promoter region.
Materials and Methods Liver samples (n=39) from organ transplant donors were obtained shortly after death and were immediately frozen in liquid nitrogen and stored at -80°C until use. For practical reasons or reasons of privacy, no data on age, sex, lifestyle, medication, drug use, dietary habits or serum bilirubin levels of the donors of these liver samples were available. In addition, two liver samples of patients with the Gilbert’s syndrome were used in the study. The local medical review committee approved the study. Homogenates (20% w/v) were prepared by grinding small pieces of liver tissue in a glass/glass Potter (5 strokes) in sterile homogenisation buffer (0.25 M saccharose, 1 mM dithiothreitol, 1 mM EDTA and 30 mM Tris-HCl pH 7.5) and were stored in small aliquots at -80°C until use. Homogenates were used for isolation of DNA by the Wizard@ DNA Purification Kit (Promega, Madison, USA) according to the instructions of the manufacturer. Bilirubin UDP glucuronyltransferase assay Liver homogenates (lo%, w/v) were activated by preincubation with 1% (w/v) digitonin (Sigma Chemicals) for 10 min at room temperature. Activated liver homogenates were subsequently incubated with unconjugated bilirubin (Sigma Chemicals) under dim red light and an argon atmosphere for 15 min at 37°C. The reaction mixture contained (final concentrations): 5% (w/v) activated liver homogenate, 70 mM Tris-HCl pH 7.8, 3.3 mM MgC12, 1 mM D-saccharic acid 1,4lactone (Sigma Chemicals), 3.5 mM UDP-glucuronic acid (Sigma) and 40 PM unconjugated bilirubin. Reaction was stopped, reaction mixture was handled and bilirubin and its conjugates were determined exactly as described before by Cuypers et al. (10). Genotyping The number of TA-repeats in the promoter region of the UGTlAl gene was studied using the polymerase chain reaction conditions and primers (C and D) as described by Monaghan et al. (3). Amplification was confirmed by agarose electrophoresis before fragments were resolved on 11% polyacrylamide gels (382 acrylamide/bisacrylamide; Biorad) in Tris-Borate-EDTA buffer (11). Gels (20X20X0.075 cm) were run at 300 Volts for 3 h and stained with ethidiumbromide (Omnilabo) for 45 min. Genotypes were assigned as follows; 6/6: homozygous for the A(TA)sTAA-allele, 7/7: homozygous for the A(TA),. TAA-allele, and 6/7: heterozygous with one of each allele.
Statistics Kruskal-Wallis ANOVA and the Wilcoxon-Mann-Whitney used to assess statistical significance between groups. 0.05 was considered significant.
tests were A p-value of
Results Examples of the analysis of the polymorphism in the promoter region of the UGTlAl gene are shown in Fig. 1. The two known Gilbert’s syndrome patients both showed the 7/7 genotype. Of the 39 transplant donor liver samples tested, 17 (44%) showed the 6/6 genotype, 18 (46%) showed the 617 genotype and 4 (10%) had the 7/7 genotype (Table 1). This resulted in allele frequencies of 0.33 and 0.67 for the A(TA);ITAA- and A(TA)6_ TAA-alleles, respectively Fig. 2 shows the hepatic bilirubin UDP-glucuronyltransferase enzyme activities of all samples, separated into three groups selected on promoter region genotype. The two samples of the patients with Gilbert’s syndrome showed enzyme activities of 424 and 515 nmol/g liver/h. Median bilirubin UDP-glucuronyltransferase activity (see Table 1) of the 6/6-genotypes (1565, range 741-2666 nmol/g liver/h) was significantly higher as compared to that of the 7/7 genotypes (749 [424-11721 nmol/g liver/h, ~KO.005) and of the 6/7
Fig. 1. Genotyping of the polymorphism in the promoter region of the UGTlAl gene. Results shown are the polymerase chain reaction (PCR) products of 12 randomly chosen samples after analysis on 11% polyacrylamide gel. Presence of an A(TA)eTAA allele results in a PCR product of 98 bp, whereas presence of the A(TA)7TAA allele yields a 100 bp product. Genotypes are assigned as follows: 616, homozygous for A(TA)eTAA allele; 717, homozygous for the A(TA)7TAA allele; 617, heterozygous (both alleles present); M, 100 bp marker.
TABLE
1
Hepatic grouped
bilirubin according
UDP-glucuronyltransferase enzyme to the promoter region genotypes
Genotype
n
Enzyme
616 617 717
17 18 6
1565 [741-26661 985 [57@-2464](l) 749 [424-l 172]@)
activities
activity
Genotypes are denoted as given in the legend of Fig. 1. Enzyme activity is given as median [range] in nmoYg liver/h. Wilcoxon-MannWhitney:l) p
349
M. T. M. Ruijmukers et al. 3000
ences
c= f g
2500
2000
-
E .c
1500
-
,000
-
.z 8 E E 5
:
F
3d,
5w-
0 --&$ f*
0
6/6
6”
7/7
Genotype
Fig. 2. Hepatic hilirubin UDP-glucuronyltransferase enzyme activities plotted against the genotype of the UGTI Al promoter region. Median activities are indicated by a horizontal line. Samples are divided into three groups selected on genotype. Open diamonds indicate the samples qf’ the two knobvn patients with Gilbert’s syndrome.
genotypes (985 [570-24641 nmol/g liver/h, ~~0.05). Although the median enzyme activity of the 7/7 genotypes was lower than that of the heterozygous 617 genotypes, the difference did not reach significance (p= 0.14).
Discussion An occurrence of 10% of the 717 genotype in 39 random samples was shown in this study. This is comparable to the incidences reported by several other groups; 15% by Bosma et al. (2), lo-13% by Monaghan et al. (3), 16% by Biondi et al. (12) 13% by Bancroft et al. (13) and 11% by Beutler et al. (14). The allele frequency of the A(TA),TAA-allele (0.33) was similar to frequencies found by Bosma et al. (0.40) (2) Sampietro et al. (0.44) (1 l), Biondi et al. (0.34) (12) Beutler et al. (0.39) (14) and Galanello et al. (0.31) (15). Median hepatic bilirubin UDP-glucuronyltransferase activity in samples from donors with the 6/7 or 71 7 genotype were significantly lower as compared to that of the 6/6 genotypes. Earlier studies correlated the UGTl Al promoter genotype with the level of serum total bilirubin (2,3,11,12,15,16). Significantly higher bilirubin levels were found in the 717 genotypes as compared to the 616 genotypes. However, reports are contradictory with respect to total bilirubin levels in the 6/ 7 as compared to the 6/6 genotypes. Bosma et al. (2) Sampietro et al. (1 l), Biondi et al. (12) and Ishihara et al. (16) found significantly higher total serum bilirubin levels in the group with the 617 genotype as compared to the 6/6 genotypes. However, Monaghan et al. (3) and Galanello et al. (15) reported no significant differ-
350
in the levels of total
serum
bilirubin
groups with these two genotypes. Unfortunately not have serum bilirubin levels from organ
between we do donors,
since most subjects died after traffic accidents. and sampling blood to assay serum bilirubin level was either not possible or had very low priority at that time. Black & Billing (4) Aono et al. (17), and Peters & Jansen (18) reported lower hepatic bilirubin UDP-glucuronyltransferase activities in individuals with Gilbert’s syndrome as compared to controls. However, hepatic bilirubin UDP-glucuronyltransferase enzyme activities were not yet correlated with the promoter region genotype of the bilirubin UDP-glucuronyltransferase gene. Previous studies have shown that the 7/7 genotype is associated with Gilbert’s syndrome (2,3,11,12). As shown by Bosma et al. (2) and Ciotti et al. (19) the transcriptional activity of genes with A(TA),TAA in the promoter region was lo-15% of the activity of the genes with the wild-type A(TA&TAA. Transfection of the polymorphic gene in HepG2 cells resulted in reduced expression and lower enzyme activity (19). This may explain the lower median bilirubin UDP-glucuronyltransferase enzyme activity for the group with the 7/7 genotype and the intermediate activity for the group with the 6/7 genotype found in this study. The higher serum bilirubin levels in patients with Gilbert’s syndrome may directly result from this lower hepatic bilirubin UDP-glucuronyltransferase activity. However, the wide range of activities found, especially in the 6/6 and 6/7 groups, may indicate that additional factors apart from variations in the TATA-box may contribute to UDP bilirubin glucuronyltransferase enzyme activities. However, Aono et al. (17) reported that in a Japanese population missense mutations caused by a single nucleotide substitution in the coding region of the bilirubin UDP-glucuronyltransferase gene may also be associated with higher serum levels of bilirubin and the aetiology of Gilbert’s syndrome. Sato et al. (20) showed that none of seven patients with Gilbert’s syndrome with a missense mutation in the coding region was homozygous for the 717 genotype. In accordance with these findings, Beutler et al. (14) reported that different ethnic groups have different genotypes related to Gilbert’s syndrome and serum total bilirubin levels. They suggested that the polymorphism in the promoter region might serve as a “fine-regulator” of bilirubin levels within populations. The discrepancy found with respect to the genetic basis of Gilbert’s syndrome between the Japanese and Western researchers may thus be explained by differences between ethnic populations. In summary, we found a clear association between
UGTlAI: genotype versus enzyme activity
UGTlAl promoter region polymorphism and hepatic bilirubin UDP-glucuronyltransferase enzyme activity. We also noted a median UGTIAI enzyme activity in the heterozygous 6/7 group that was significantly lower than in the wild-type 616 group. This suggests that Gilbert’s syndrome may be associated not only with the 7/7 homozygous genotype but also with the 6/7 genotype. Studies in Caucasian populations have shown a consistent correlation between clinically manifest Gilbert’s syndrome and the 7/7 genotype, but do not rule out the possibility of an increased frequency of the 6/7 genotype among persons with a normal baseline but an elevated fasting bilirubin level.
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