Gilbert syndrome caused by a homozygousmissense mutation (Tyr486Asp) of bilirubinUDP-glucuronosyltransferase gene

doi:10.1016/S0022-3476(98)70408-1

The Journal of Pediatrics

Volume 132, Issue 6, June 1998, Pages 1045-1047

https://doi.org/10.1016/S0022-3476(98)70408-1 Get rights and content

Abstract

We report a case of Gilbert syndrome caused by a homozygous missense mutation (Tyr486Asp) of the bilirubin UDP-glucuronosyltransferase gene. Homozygous missense mutations of the gene have previously been recognized as responsible for Crigler-Najjar syndrome type II. We conclude that Gilbert syndrome in some patients results from homozygous missense mutations of the UDP-glucuronosyltransferase gene. (J Pediatr 1998;132:1045-7.)

Section snippets

Case Report

A 12-year-old Japanese girl with no family history of apparent jaundice periodically was visibly jaundiced from infancy. Physical examination revealed skin and conjunctiva that were icteric but not anemic. No hepatosplenomegaly was seen, and no congenital anomalies were defined. Her serum total bilirubin concentration was 44.4 μmol/L (2.6 mg/dl), and her indirect-reacting bilirubin was 30.7 μmol/L (1.8 mg/dl). She was not anemic. Her serum bile acid concentrations were normal (3.3 to 4.6

Methods

Amplification of exons and the promoter region of UGT1*1 was carried out by polymerase chain reaction from genomic DNA.³ Approximately 100 ng of total genomic DNA was amplified by three pairs of oligonucleotide primers. Exons 2, 3, and 4 and intervening introns were amplified simultaneously, as described elsewhere.² The 5' region of UGT1*1 including the TATA box to exon 1 and exon 5 were amplified separately. Conditions for polymerase chain reaction were as follows: an initial denaturation for

Results

Gene analysis revealed that the patient was homozygous for normal TATA box ([TA]₆TAA). However, a homozygous missense mutation of 1456 T→G; Tyr486Asp in exon 5 was detected (Figure).

Figure. Nucleotide sequences of a part of exon 5 of UGT1*1 gene amplified from genomic DNA of control, patient with Gilbert syndrome, and her parents. The mutation, a single nucleotide transversion of T(▿) by G(▾) at base position 1456 in UGT1*1 cDNA, generates an amino acid substitution of tyrosine by aspartic

Discussion

It is commonly recognized that Crigler-Najjar syndrome type II is caused by homozygous missense mutations of UGT1*1.1, 3 On the other hand, Gilbert syndrome has been observed in subjects with heterozygous missense mutations⁸ and a homozygous 2-base (TA) insertion mutation of the TATA box of the gene.4, 9 However, in this reporthomozygous missense mutation of Tyr486Asp was detected in a patient with Gilbert syndrome. Our previous in vitro expression study of a mutated gene (Pro229Gln obtained

Acknowledgements

We thank Dr. Yoshio Imai for his clinical assistance in treating and monitoring the patient.

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In Caucasians and Africans, most of the patients with GS are caused by the TATA box-promoter variants (Bosma et al., 1995; Monaghan et al., 1996; Maruo et al., 2004). Yet, in east Asians, the c.-3279 T > G and other missense mutations (c.211G > A and c.1456 T > G) are important causes of hyperbilirubinemia (Aono et al., 1993; Koiwai et al., 1995; Maruo et al., 1998; Maruo et al., 2000). The novel missense mutations p.(Ala471Gly) and p.(Ser530Phe) were detected in the transmembrane domain of the UGT1A1 protein in patients, P19 presenting with CNS type I, and P13 with GS, respectively.
Unconjugated hyperbilirubinemia (UCB) is a feature of Gilbert's syndrome (GS) and Crigler-Najjar's syndrome (CNS), which are two hereditary defects in bilirubin metabolism. Both syndromes are linked to mutations in the UGT1A1 gene, which cause either the decrease or the absence of the UGT1A1 enzymatic activity. Here, we investigated the molecular basis of the UGT1A1 gene in Tunisian patients presenting with unconjugated hyperbilirubinemia.
Twenty-four patients with UCB were investigated. The screening protocol for hemoglobinopathies, enzymopathies, and membrane defects was executed in all patients. Afterward, the molecular analysis of the entire UGT1A1 gene was performed by DNA Sanger sequencing. Several bioinformatic tools were used to explore the effects of novel mutations.
Fifteen different UGT1A1 variations were identified, among which four are described here for the first time. In exon 5, the c.1412C > G; p.(Ala471Gly) and c.1589C > T; p.(Ser530Phe) mutations were detected in patients presenting with CNS type I and GS, respectively. In the 3′UTR region of UGT1A1, the c.*90C > T mutation was detected in 3 patients with CNS type I. In the same region, the c.*388C > T defect was found in a GS patient. A deleterious and damaging effect on the UGT1A1 protein were predicted for both exonic mutations. Furthermore, novel microRNAs were identified as targetting the mutated sequences for the 3′UTR mutations.
Our study provides novel data on UCB among Tunisians. Furthermore, we report four novel mutations associated with both GS and CNS. The identification of these mutations increases the spectrum of the UGT1A1 mutations and contributes to an understanding of the molecular abnormalities associated with unconjugated hyperbilirubinemia.
Bile pigment metabolism and its disorders
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Bilirubin is the breakdown product of the heme moiety of hemoproteins, including hemoglobin, myoglobin, and several hepatic hemoproteins. About 80% of the 200–300 mg bilirubin is produced daily in reticuloendothelial cells from hemoglobin of senescent erythrocytes, the remainder being derived mostly from hepatic hemoproteins. Microsomal heme oxygenases cleave heme forming biliverdin, which is reduced to bilirubin by biliverdin reductase. At low concentrations, bilirubin is cytoprotective and provides metabolic benefits. However, bilirubin-induced neurological damage (BIND) caused by severe unconjugated hyperbilirubinemia is a major clinical concern. Physiologically, BIND is prevented by binding to plasma albumin, rapid uptake by hepatocytes, UGT1A1-mediated glucuronidation, and active canalicular excretion. A fraction of the bilirubin glucuronides is pumped out by ABCC3 through the sinusoidal surface of hepatocytes and undergoes reuptake by hepatocytes located downstream to sinusoidal blood flow. A small portion of bilirubin excreted in bile undergoes enterohepatic recycling, the remainder being degraded by bacteria to urobilinogen. Unconjugated hyperbilirubinemia can be caused by excessive bilirubin production, reduced hepatocellular uptake, or defective glucuronidation, whereas conjugated hyperbilirubinemia results from defective canalicular excretion or abnormal reuptake. Hyperbilirubinemia, which is universal in neonates, normally resolves in 1–2 weeks, but can be exacerbated or prolonged by breastfeeding or delayed UGT1A1 maturation (Lucey–Driscoll syndrome). In addition to acquired hepatobiliary diseases and hemolysis, a number of inherited liver disorders can cause lifelong hyperbilirubinemia. Coding region mutations of UGT1A1 can cause complete or partial loss of bilirubin glucuronidation (Crigler–Najjar syndrome, type 1 and type 2, respectively). A variant UGT1A1 promoter can reduce UGT1A1 expression, causing mild and innocuous unconjugated hyperbilirubinemia (Gilbert syndrome). Conjugated hyperbilirubinemia can result from mutations of the ABCC2 that reduce canalicular secretion of bilirubin glucuronides and many other organic anions causing Dubin–Johnson syndrome. Concurrent mutations of ABCB1 and ABCB3 disable reuptake of bilirubin glucuronides, causing mild conjugated hyperbilirubinemia (Rotor syndrome). Several other inherited defects of bile canalicular proteins cause four types of progressive familial intrahepatic cholestasis that cause mixed hyperbilirubinemia as well as liver injury.
Glucuronidated bilirubin: Significantly increased in hepatic encephalopathy
2019, Progress in Molecular Biology and Translational Science
Citation Excerpt :
The preferred substrate for UDP-glucuronosyltransferase family 1 member A1 is bilirubin, although it also has moderate activity with simple phenols, flavones, and steroids. Mutations in this gene result in Crigler–Najjar syndromes types I and II12–14 and in Gilbert syndrome,15–17 which are severe to mild liver disorders. A series of studies indicate that glucuronidation of bilirubin is a rate-limiting step for the disposal of bilirubin in animals and the cause of various diseases.18–20
Bilirubin is produced by the breakdown of hemoglobin in senescent erythrocytes by macrophages and carried by albumin from blood circulation to the liver for removal in normal physiology. Glucuronic acid modification of bilirubin by UDP-glucuronyltransferase in the liver is the key event for its subsequent elimination from human body. Conditions that accelerate the breakdown of erythrocytes may cause an elevated blood level of unconjugated bilirubin whereas the factors affect the glucuronidated bilirubin formation and subsequent elimination may cause decreased or increased blood level of glucuronidated bilirubin, the water soluble “direct bilirubin” measured by clinical blood test. Studies showed that increased total serum bilirubin has a protective effect on cardiovascular and other related diseases, but it is unknown how direct bilirubin levels were related to different diseases. By taking advantage of the data collected in the clinical laboratory of our hospital, the direct bilirubin data from 192,535 patients with 72 clinically defined diseases were compared to that of healthy controls (10,497). Based on the mean, median, and p values, we found that patients with hepatic encephalopathy had the highest serum direct bilirubin level, which resembled acute hepatic encephalopathy caused by increased serum direct bilirubin level in neonates. In contrast, patients with uremia, nephrotic syndrome, and preeclampsia had significantly lower levels of serum direct bilirubin. Taken together, our data revealed that serum direct bilirubin levels were either increased or decreased in a disease-dependent manner. The possible molecular mechanisms of increased direct bilirubin levels in patients suffering hepatic encephalopathy are discussed.
Bilirubin Metabolism and Its Disorders
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Bilirubin uridine diphosphate-glucuronosyltransferase variation is a genetic basis of breast milk jaundice
2014, Journal of Pediatrics
Citation Excerpt :
The components of breast milk clearly contribute to the onset of neonatal jaundice.37 Our in vitro investigation showed the inhibitory effect of 5α-pregnane-3α, 20β-diol on UGT1A1 activity.26 5α-pregnane-3α, 20β-diol does not inhibit transcriptional activity of either the UGT1A1*1 wild-type enhancer-promoter or the c.-3279T>G+A(TA)7TAA enhancer-promoter (UGT1A1*28).
To evaluate the role of bilirubin UDP-glucuronosyltransferase family 1, polypeptide A1 (UGT1A1) gene variations on prolonged unconjugated hyperbilirubinemia associated with breast milk feeding (breast milk jaundice [BMJ]).
UGT1A1 gene allelic variation was analyzed in 170 Japanese infants with BMJ with polymerase chain reaction-direct sequencing, and their genotypes compared with serum bilirubin concentrations. In 62 of 170 infants, serum bilirubin concentration was followed after 4 months of life. Genotypes were examined in 55 infants without BMJ.
Of 170 infants with BMJ, 88 (51.8%) were homozygous UGT1A1*6. Serum bilirubin concentrations (21.8 ± 3.65 mg/dL) were significantly greater than in infants with other genotypes (P < .0001). The Gilbert UGT1A1*28 allele was not detected in infants with BMJ, except in an infant who was compound heterozygous with UGT1A1*6. At 4 months of age, serum bilirubin concentration improved to >1 mg/dL, except in 2 infants who were homozygous UGT1A1*7. Homozygous UGT1A1*6 was not detected in the control group.
One-half of the infants with BMJ were homozygous UGT1A1*6 and exhibited a serum bilirubin concentration significantly greater than other genotypes. This finding indicates that UGT1A1*6 is a major cause of BMJ in infants in East Asia. Previous finding have demonstrated that 5β-pregnane-3α,20β-diol present in breast milk inhibits p.G71R-UGT1A1 bilirubin glucuronidation activity. Thus, prolonged unconjugated hyperbilirubinemia may develop in infants with UGT1A1*6 who are fed breast milk.
Bile Pigment Metabolism and Its Disorders
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^☆: From the Departments of Pediatrics and Biology, Shiga University of Medical Science, Otsu, Shiga, Japan.

^☆☆: Supported in part by grants-in-aid for scientific research from the Ministry of Education, Science and Culture of Japan (08557030 and 08670878).

^★: Submitted for publication April 15, 1997.

^★★: Reprint requests: Hiroshi Sato, PhD, Department of Biology, Shiga University of Medical Science, Seta Tsukinowa, Otsu, Shiga 520-21, Japan.

^♢: 9/22/89014

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Gilbert syndrome caused by a homozygousmissense mutation (Tyr486Asp) of bilirubinUDP-glucuronosyltransferase gene☆,☆☆,★,★★,♢

Abstract

Section snippets

Case Report

Methods

Results

Discussion

Acknowledgements

Int Hepatol Commun

Biochem Biophys Res Commun

Lancet

Lancet

Lancet