Rapid proteasomal degradation of translocation-deficient UDP-glucuronosyltransferase 1A1 proteins in patients with Crigler-Najjar type II

UDP-glucuronosyltransferase form 1A1 (UGT1A1) is the only bilirubin-glucuronidating isoform of this protein, and genetic deficiencies of UGT1A1 cause Crigler-Najjar syndrome, a disorder resulting from nonhemolytic unconjugated hyperbilirubinemia. Here we have focused on the instability of a translocation-deficient UGT1A1 protein, which has been found in patients with Crigler-Najjar type II, to elucidate the molecular basis underlying the deficiency in glucuronidation of bilirubin. A substitution of leucine to arginine at position 15 (L15R/1A1) is predicted to disrupt the hydrophobic core of the signal peptide of UGT1A1. L15R/1A1 was synthesized in similar amounts to wild-type UGT1A1 protein (WT/1A1) in transfected COS cells. However, L15R/1A1 did not translocate across the endoplasmic reticulum membrane and was degraded rapidly with a half-life of about 50min, in contrast to the much longer half-life of about 12.8h for WT/1A1. Our findings demonstrate that L15R/1A1 was rapidly degraded by the proteasome owing to its mislocalization in the cell.     

Ezetimibe: A biomarker for efficacy of liver directed UGT1A1 gene therapy for inherited hyperbilirubinemia

As recently demonstrated in patients with factor IX deficiency, adeno-associated virus (AAV)-mediated liver-directed therapy is a viable option for inherited metabolic liver disorders. Our aim is to treat Crigler-Najjar syndrome type I (CN I), an inherited severe unconjugated hyperbilirubinemia, as a rare recessive inherited disorder. Because the number of patients eligible for this approach is small, the efficacy can only be demonstrated by a beneficial effect on the pathophysiology in individual patients. Serum bilirubin levels in potential candidates have been monitored since birth, providing an indication of their pathophysiology. Adjuvant phototherapy to prevent brain damage reduces serum unconjugated bilirubin (UCB) levels in CN I patients to the level seen in the milder form of the disease, CN type II. This therapy increases the excretion of UCB, thereby complicating the use of UCB and conjugated bilirubin levels in serum as biomarkers for the gene therapy we try to develop. Therefore, a suitable biomarker that is not affected by phototherapy is currently needed. To this end, we have investigated whether estradiol, ethinylestradiol or ezetimibe could be used as markers for uridine 5'-di-phospho-glucuronosyltransferase isoform 1A1 (UGT1A1) activity restored by AAV gene therapy in Gunn rats, a relevant animal model for CN I. Of these compounds, ezetimibe appeared most suitable because its glucuronidation rate in untreated control Gunn rats is low. Subsequently, ezetimibe glucuronidation was studied in both untreated and AAV-treated Gunn rats and the results suggest that it may serve as a useful serum marker for restored hepatic UGT1A1 activity.     

Inhibition of rat liver UDP-glucuronosyltransferase by silymarin and the metabolite silibinin-glucuronide

The inhibitory effects of silymarin, its main constituent silibinin and the metabolite silibinin-glucuronide on UDP-glucuronosiltransferase (UGT) were evaluated in rat hepatic microsomes. Three substrates were chosen to cover both UGT1A and UGT2B family isozymes: bilirubin (substrate of UGT1A1), p-nitrophenol (UGT1A6) and ethinylestradiol (UGT2B1 and 2B3 for position C17 and UGT1A1 for position C3). The study of p-nitrophenol and bilirubin glucuronidation indicated that silymarin (SM) and silibinin glucuronide (SB-G) were enzyme inhibitors. The kinetic analysis showed that the type of inhibition was competitive in all cases and the Ki obtained were: for p-nitrophenol glucuronidation, KiSB-Gapp: 14+/-1 microg/ml and KiSMapp: 51+/-10 microg/ml and for bilirubin glucuronidation, KiSB-Gapp: 16+/-3 microg/ml. In turn, ethinylestradiol glucuronidation was not affected by any of the compounds studied suggesting that the inhibitory effect was restricted to UGT1A isozymes. Similar studies performed using human hepatic microsomes showed that SM and SB-G were also inhibitors of human UGT1A isozymes. In conclusion, administration of silymarin or its main constituent silibinin could lead to the decrease in the glucuronidation of substrates whose conjugation depends on UGT1A isozymes in a process mediated by silibinin-glucuronide, though their effect in humans needs further investigation.     

Evolution of the activity of UGT1A1 throughout the development and adult life in a rat

Biliary excretion is the main route of disposal of bilirubin and impaired excretion results in jaundice, a well recognisable symptom of liver disease. Conjugation of bilirubin in the liver is essential for its clearance. The glucuronidation of bilirubin is catalysed by the microsomal UDP-glucuronosyltransferase UGT1A1. Patients with Crigler-Najjar syndrome type 1 and Gunn rats, mutant strain of the Wistar rats, bear an autosomal recessive disorder resulting in hyperbilirubinemia. The aim of this work is to add new data about activity of UGT1A1 during the perinatal period and adult life. The results showed that activity of UGT1A1 is detectable from day 22 of the gestation. After birth, activity of UGT1A1 gradually increases and reaches the levels of adult life. Furthermore, bilirubin azopigments have been separated and characterized by thin layer chromatography. We have found that concentration of samples by evaporation and ulterior storing at -20 degrees C seemed to be suitable for the maintenance of samples.     

Rescue of bilirubin-induced neonatal lethality in a mouse model of Crigler-Najjar syndrome type I by AAV9-mediated gene transfer

Crigler-Najjar type I (CNI) syndrome is a recessively inherited disorder characterized by severe unconjugated hyperbilirubinemia caused by uridine diphosphoglucuronosyltransferase 1A1 (UGT1A1) deficiency. The disease is lethal due to bilirubin-induced neurological damage unless phototherapy is applied from birth. However, treatment becomes less effective during growth, and liver transplantation is required. To investigate the pathophysiology of the disease and therapeutic approaches in mice, we generated a mouse model by introducing a premature stop codon in the UGT1a1 gene, which results in an inactive enzyme. Homozygous mutant mice developed severe jaundice soon after birth and died within 11 d, showing significant cerebellar alterations. To rescue neonatal lethality, newborns were injected with a single dose of adeno-associated viral vector 9 (AAV9) expressing the human UGT1A1. Gene therapy treatment completely rescued all AAV-treated mutant mice, accompanied by lower plasma bilirubin levels and normal brain histology and motor coordination. Our mouse model of CNI reproduces genetic and phenotypic features of the human disease. We have shown, for the first time, the full recovery of the lethal effects of neonatal hyperbilirubinemia. We believe that, besides gene-addition-based therapies, our mice could represent a very useful model to develop and test novel technologies based on gene correction by homologous recombination.     

Generation of Ugt1-Deficient Murine Liver Cell Lines Using TALEN Technology

The Crigler-Najjar Syndrome Type I (CNSI) is a rare genetic disorder caused by mutations in the Ugt1a1 gene. It is characterized by unconjugated hyperbilirubinemia that may result in severe neurologic damage and death if untreated. To date, liver transplantation is the only curative treatment. With the aim of generating mutant cell lines of the Ugt1 gene, we utilized the TALEN technology to introduce site-specific mutations in Ugt1 exon 4. We report a fast and efficient method to perform gene knockout in tissue culture cells, based on the use of TALEN pairs targeting restriction enzyme (RE) sites in the region of interest. This strategy overcame the presence of allele-specific single nucleotide polymorphisms (SNPs) and pseudogenes, conditions that limit INDELs'' detection by Surveyor. We obtained liver-derived murine N-Muli cell clones having INDELs with efficiency close to 40%, depending on the TALEN pair and RE target site. Sequencing of the target locus and WB analysis of the isolated cell clones showed a high proportion of biallelic mutations in cells treated with the most efficient TALEN pair. Ugt glucuronidation activity was reduced basal levels in the biallelic mutant clones. These mutant liver-derived cell lines could be a very useful tool to study biochemical aspects of Ugt1 enzyme activity in a more natural context, such as substrate specificity, requirement of specific co-factors, the study of inhibitors and other pharmacological aspects, and to correlate enzyme activity to the presence of specific mutations in the gene, by adding back to the mutant cell clones specific variants of the Ugt1 gene. In addition, since genome editing has recently emerged as a potential therapeutic approach to cure genetic diseases, the definition of the most efficient TALEN pair could be an important step towards setting up a platform to perform genome editing in CNSI.     

Age-dependent pattern of cerebellar susceptibility to bilirubin neurotoxicity in vivo in mice

Neonatal jaundice is caused by high levels of unconjugated bilirubin. It is usually a temporary condition caused by delayed induction of UGT1A1, which conjugates bilirubin in the liver. To reduce bilirubin levels, affected babies are exposed to phototherapy (PT), which converts toxic bilirubin into water-soluble photoisomers that are readily excreted out. However, in some cases uncontrolled hyperbilirubinemia leads to neurotoxicity. To study the mechanisms of bilirubin-induced neurological damage (BIND) in vivo, we generated a mouse model lacking the Ugt1a1 protein and, consequently, mutant mice developed jaundice as early as 36 hours after birth. The mutation was transferred into two genetic backgrounds (C57BL/6 and FVB/NJ). We exposed mutant mice to PT for different periods and analyzed the resulting phenotypes from the molecular, histological and behavioral points of view. Severity of BIND was associated with genetic background, with 50% survival of C57BL/6‑Ugt1^−/− mutant mice at postnatal day 5 (P5), and of FVB/NJ-Ugt1^−/− mice at P11. Life-long exposure to PT prevented cerebellar architecture alterations and rescued neuronal damage in FVB/NJ-Ugt1^−/− but not in C57BL/6-Ugt1^−/− mice. Survival of FVB/NJ-Ugt1^−/− mice was directly related to the extent of PT treatment. PT treatment of FVB/NJ-Ugt1^−/− mice from P0 to P8 did not prevent bilirubin-induced reduction in dendritic arborization and spine density of Purkinje cells. Moreover, PT treatment from P8 to P20 did not rescue BIND accumulated up to P8. However, PT treatment administered in the time-window P0–P15 was sufficient to obtain full rescue of cerebellar damage and motor impairment in FVB/NJ-Ugt1^−/− mice. The possibility to modulate the severity of the phenotype by PT makes FVB/NJ-Ugt1^−/− mice an excellent and versatile model to study bilirubin neurotoxicity, the role of modifier genes, alternative therapies and cerebellar development during high bilirubin conditions.KEY WORDS: Neonatal jaundice, Ugt1, Phototherapy, BIND, Mouse model     

Identification of the deletions in the UGT1A1 gene of the patients with Crigler-Najjar syndrome type I from Slovakia

Crigler-Najjar syndrome type I (CN I) is a rare autosomal recessive disorder due to hepatic dysfunction of uridine diphospho-glucuronosyltransferase (UGT) activity toward bilirubin. Complete inactivation of this enzyme causing CN I lead to accumulation of unconjugated bilirubin in serum and bile. Here we report the results of the molecular characterization of the uridine diphospho-glucuronosyltransferase 1A1 (UGT1A1) gene in a consanguineous family of Slovak Roms and an unrelated non-Romany family with CN I. Sequence analysis of UGT1A1 gene in all four Romany patients showed mutation in exon 4, a deletion of an A at codon 407 (1220delA), not yet described in homozygous status. All analysed patients were homozygous for 1220delA mutation and their 3 healthy sibs were heterozygous. The non-Romany patient was a compound heterozygote for two different deletions, 1220delA and 717-718delAG at codon 239. In the family of his cousin a son was born affected with CN I, who was homozygote for 717-718delAG mutation. His other niece affected with CN II was heterozygote for mutation 717-718delAG but homozygote for TA insertion and enhancer substitution T-3279G. Haplotype analysis suggests that the 1220delA mutation is identical by descent in both families, though they originate from two ethnically different populations (Slovaks vs. Roms).     

Cosegregation of intragenic markers with a novel mutation that causes Crigler-Najjar syndrome type I: implication in carrier detection and prenatal diagnosis.

Crigler-Najjar syndrome type 1 (CN-1) is a familial disorder characterized by severe unconjugated hyperbilirubinemia and jaundice and leads to kernicterus, neurological damage, and eventual death unless treated with liver transplantation. Previous reports identified mutations in the UGT1 gene complex to be the cause of the disease. The total absence of all phenol/bilirubin UGT proteins and their activities in liver homogenate of a CN-1 patient was determined by enzymological and immunochemical analysis. A novel homozygous nonsense mutation (CGA-->TGA) was identified in the patient by the combined techniques of PCR and direct sequencing. This mutation was located in exon 3 of the constant region in the gene complex which is common to all phenol and bilirubin UGTs. The segregation of the mutation in the patient's family was analyzed and confirmed the recessive nature of the disease. Newly developed intragenic polymorphic probes (UGT1* 4 and UGT-Const) were used on Southern blots of MspI-digested genomic DNA of the patient and his family. The segregation of individual alleles within the family was observed from haplotypes generated. Comparison of the segregation of haplotypes with the mutation for the patient and his family revealed the allele identified by the A1-B1-C2 haplotype to be carrying the mutation. The risk of recombination occurring is negligible, because of the intragenic nature of the probes. This study demonstrates the potential usefulness of these probes in carrier detection and prenatal/presymptomatic diagnosis.     

Crigler-Najjar syndrome type 2: Novel UGT1A1 mutation

Crigler-Najjar syndrome type 2 is a rare cause for persistent unconjugated hyperbilirubinemia, inherited in an autosomal recessive manner. Even though it is compatible with normal life span, in the absence of prompt suspicion and intensive management it can prove fatal not only in the neonatal period but also during adult life. Here, we describe a case with a novel homozygous UGT1A1 p.Pro176Leu mutation.     

Tissue-specific, inducible, and hormonal control of the human UDP-glucuronosyltransferase-1 (UGT1) locus

The human UDP-glucuronosyltransferase 1 (UGT1) locus spans nearly 200 kb on chromosome 2 and encodes nine UGT1A proteins that play a prominent role in drug and xenobiotic metabolism. Transgenic UGT1 (Tg-UGT1) mice have been created, and it has been demonstrated that tissue-specific and xenobiotic receptor control of the UGT1A genes is influenced through circulating humoral factors. In Tg-UGT1 mice, the UGT1A proteins are differentially expressed in the liver and gastrointestinal tract. Gene expression profiles confirmed that all of the UGT1A genes can be targeted for regulation by the pregnane X receptor activator pregnenolone-16alpha-carbonitrile (PCN) or the Ah receptor ligand 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). In addition, the selective induction of glucuronidation activity toward lamotrigine, ethinyl estradiol, chenodeoxycholic acid, and lithocholic acid by either PCN or TCDD in small intestine from Tg-UGT1 mice corresponded to expression of the locus in this tissue. Induction of UGT1A1 by PCN and TCDD is believed to be highly dependent upon glucocorticoids, because submicromolar concentrations of dexamethasone actively promote PCN and TCDD induction of UGT1A1 in Tg-UGT1 primary hepatocytes. The role of hormonal control of the UGT1 locus was further verified in pregnant and nursing Tg-UGT1 mice. In maternal 14-day post-conception Tg-UGT1mice, liver UGT1A1, UGT1A4, and UGT1A6 were induced, with the levels returning to near normal by birth. However, maternal liver UGT1A4 and UGT1A6 were dramatically elevated and maintained after birth, indicating that these proteins may play a critical role in maternal metabolism during lactation. With expression of the UGT1 locus confirmed in a variety of mouse tissues, these results suggested that the Tg-UGT1 mice will be a useful model to examine the regulatory and functional properties of human glucuronidation.     

Stimulation of transcriptional expression of human UDP-glucuronosyltransferase 1A1 by dexamethasone

Human UDP-glucuronosyltransferase (UGT) 1A1 is only enzyme in the conjugation of bilirubin for prevention of hyperbilirubinemia and jaundice. Deletion or mutation of the UGT1A1 gene causes Crigler-Najjar syndrome or Gilbert's syndrome. We previously reported the functional promoter region for expression of UGT1A1 [Hepatology Research 9, 152-163 (1997)]. We investigated the influence of some drugs on the transient transfection assay of the luciferase reporter gene containing the 5'-promoter region -3174/+14 of UGT1A1 in HepG2 cells. Among drugs investigated, dexamethasone was the most effective at the range of concentration of 10-100 microM, whereas stimulation by beta-estradiol was not found. We also could not find stimulation by bilirubin of the endogenous main substrate for UGT1A1. Stimulation by dexamethasone was continued for 48 hr. The luciferase reporter gene containing the 5'-region of -97/+14 was induced by dexamethasone but the gene of the 5'-region -53/+14 was not. The region -97/-53 is essential for induction by dexamethasone. This region contains HNF1 element, therefore, we speculated that dexamethasone directly and/or indirectly stimulates UGT1A1 expression through this HNF1 region in the promoter region of UGT1A1. Thus, we clarified that UGT1A1 was induced by dexamethasone and the key position was the region (-97/-53) in UGT1A1 promoter.     

Targeted inhibition of glucuronidation markedly improves drug efficacy in mice - a model

Finding UDP-glucuronosyltransferases (UGT) require protein kinase C-mediated phosphorylation is important information that allows manipulation of this critical system. UGTs glucuronidate numerous aromatic-like chemicals derived from metabolites, diet, environment and, inadvertently, therapeutics to reduce toxicities. As UGTs are inactivated by downregulating PKCs with reversibly-acting dietary curcumin, we determined the impact of gastro-intestinal glucuronidation on free-drug uptake and efficacy using immunosuppressant, mycophenolic acid (MPA), in mice. Expressed in COS-1 cells, mouse GI-distributed Ugt1a1 glucuronidates curcumin and MPA and undergoes irreversibly and reversibly dephosphorylation by PKC-specific inhibitor calphostin-C and general-kinase inhibitor curcumin, respectively, with parallel effects on activity. Moreover, oral curcumin-administration to mice reversibly inhibited glucuronidation in GI-tissues. Finally, successive oral administration of curcumin and MPA to antigen-treated mice increased serum free MPA and immunosuppression up to 9-fold. Results indicate targeted inhibition of GI glucuronidation in mice markedly improved free-chemical uptake and efficacy using MPA as a model.     

Effects on extrahepatic UDP-glucuronosyltransferases in hypophysectomized rat

The effects of hypophysectomy on hepatic and extrahepatic UDP-glucuronosyltransferase activities in adult male rats were observed. UDP-glucuronosyltransferase activities toward 1-naphthol decreased to 20-30% of control in the liver, kidney, lung, and testis. The mRNA of UGT1A6, which is an isoform contributing to the glucuronidation of various phenolic xenobiotics such as 1-naphthol, were decreased drastically in the liver, kidney, and testis by hypophysectomy. However, while bilirubin UDP-glucuronosyltransferase activity in the liver intensified, there was only a slight increase in the activity in the kidney and no alteration in the lung. The mRNA of UGT1A1, which is an isoform contributing to the glucuronidation of bilirubin, increased significantly in the liver and slightly in the kidney after hypophysectomy. These inductions and reductions in enzymatic activities and mRNA levels in each tissue were restored to control levels by intermittent injections of rat growth hormone. Interestingly, while hepatic UGT activity toward bisphenol A remained constant in hypophysectomized rats, the testicular UGT activity declined to 10-15% of control but returned to normal levels following growth hormone treatment, suggesting that an unknown UGT isoform (s) mediates bisphenol A glucuronidation in the testis. These results indicate that the expression of extrahepatic UGT is isoform-specific and regulated differentially in tissues by the pituitary gland.     

Glucuronidation of carboxylic acid containing compounds by UDP-glucuronosyltransferase isoforms

Glucuronide conjugation of xenobiotics containing a carboxylic acid moiety represents an important metabolic pathway for these compounds in humans. Several human UDP-glucuronosyltransferases (UGTs) have been shown to catalyze the formation of acyl-glucuronides, including UGT2B7, UGT1A3, and UGT1A9. In this study, recombinant expressed UGT isoforms were investigated with many structurally related carboxylic acid analogues, and the UGT rank order for catalyzing the glucuronidation of carboxylic acids was UGT2B7?UGT1A3 approximately UGT1A9. Despite being a poor substrate with UGT1A3, coumarin-3-carboxylic acid was not a substrate for any other UGT isoform tested in this study, suggesting that it could be a specific substrate for UGT1A3. Interestingly, UGT1A7 and UGT1A10 also react with several carboxylic acid aglycones. Kinetic analysis showed that UGT2B7 exhibits much higher glucuronidation efficiency (Vmax/Km) with ibuprofen, ketoprofen, and others, compared to UGT1A3. These data indicate that UGT2B7 could be the major isoform involved in the glucuronidation of carboxylic acid compounds in humans.     

Homodimerization of human bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) and its functional implications

Genetic lesions of bilirubin-uridine-diphosphoglucuronate glucuronosyltransferase-1 (UGT1A1) completely or partially abolish hepatic bilirubin glucuronidation, causing Crigler-Najjar syndrome type 1 or 2, respectively. Clinical observations indicate that some mutant forms of human UGT1A1 (hUGT1A1) may be dominant-negative, suggesting their interaction with the wild-type enzyme. To evaluate intermolecular interaction of hUGT1A1, Gunn rat fibroblasts were stably transduced with hUGT1A1 cDNA. Gel permeation chromatography of solubilized microsomes suggested dimerization of hUGT1A1 in solution. Nearest-neighbor cross-linking analysis indicated that, within microsomal membranes, hUGT1A1 dimerized more efficiently at pH 7.4 than at pH 9. Two-hybrid analysis in yeast and mammalian systems demonstrated positive interaction of hUGT1A1 with itself, but not with another UGT isoform, human UGT1A6, which differs only in the N-terminal domain. Dimerization was abolished by deletion of the membrane-embedded helix from the N-terminal domain of hUGT1A1, but not by substitution of several individual amino acid residues or partial deletion of the C-terminal domain. A C127Y substitution abolished UGT1A1 activity, but not its dimerization. Coexpression of mutagenized and wild-type hUGT1A1 in COS-7 cells showed that the mutant form markedly suppressed the catalytic activity of wild-type hUGT1A1. Homodimerization of hUGT1A1 may explain the dominant-negative effect of some mutant forms of the enzyme.     

Selective and potent inhibition of different hepatic UDP-glucuronosyltransferase activities by omega,omega,omega-triphenylalcohols and UDP derivatives.

A homologous series of omega,omega,omega-triphenylalcohols and corresponding omega,omega,omega-triphenylalkyl-UDP derivatives was synthesized and tested as inhibitors of UDP-glucuronosyltransferase (UGT) activity in rat liver microsomes, with 1-naphthol, testosterone and bilirubin as substrates. Introduction of the UDP moiety in the triphenylalcohols increased their inhibition potency markedly toward the isoforms which glucuronidate 1-naphthol and testosterone, but strongly decreased that toward bilirubin. The inhibiting potency of the UDP-derivatives increased as a function of the length of the hydrocarbon chain. The best inhibitor 7,7,7-triphenylheptyl-UDP showed an I50 of 30 and 10 microM for 1-naphthol and testosterone glucuronidation, respectively; even a 1 mM concentration of the compound had little, if any, effect on bilirubin glucuronidation. The inhibition by 7,7,7-triphenylheptyl-UDP was mixed-type toward 1-naphthol, and non competitive toward testosterone (apparent K(i) 30 microM and 1.7 microM, respectively); on the other hand, the inhibition was competitive toward the common substrate UDP-glucuronic acid (apparent K(i) 1.9-1.2 microM). In addition, 7,7,7-triphenylheptyl-UDP (0.25-0.50 mM) almost inhibited glucuronidation of 1-naphthol and testosterone catalyzed by the recombinant rat liver UGT-2B1 and human liver UGT-1A1, whose cDNA has been expressed in V79 cells. In conclusion, the data indicate that 7,7,7-triphenyheptyl-UDP interacted competitively with the UDP binding site of UGT. The results also indicate that it is possible to design transition state analogue inhibitors with specificity for different UGT forms.     

Spectrum of UGT1A1 Variations in Chinese Patients with Crigler-Najjar Syndrome Type II

Crigler–Najjar Syndrome type II (CNS-II) is an autosomal recessive hereditary condition of unconjugated hyperbilirubinemia without hemolysis, with bilirubin levels ranging from 102.6 μmol/L to 342 μmol/L. CNS-II is caused by a deficiency of UDP-glucuronyl transferase (UGT), which is encoded by the UDP-glucuronyl transferase 1A1 gene (UGT1A1). In East Asian populations, the compound homozygous UGT1A1 G71R and Y486D variants are frequently observed in cases with bilirubin levels exceeding 200 μmol/L. In this study, we investigated the spectrum of UGT1A1 variations in Chinese CNS-II patients. We sequenced the enhancer, promoter, and coding regions of UGT1A1 in 11 unrelated Chinese CNS-II patients and 80 healthy controls. Nine of these patients carried variations that are here reported for the first time in CNS-II patients, although they have been previously reported for other types of hereditary unconjugated hyperbilirubinemia. These individual variations have less influence on UGT activity than do the compound homozygous variation (combination of homozygous G71R variant and Y486D variant). Therefore, we propose that the spectrum of UGT1A1 variations in CNS-II differs according to the bilirubin levels.     

Gender difference in serum bisphenol A levels may be caused by liver UDP-glucuronosyltransferase activity in rats

Gender difference in human bisphenol A (BPA) concentrations was revealed by determining serum BPA. We studied the serum concentrations and the metabolism of BPA in rats by an HPLC system. Rat serum BPA concentrations were significantly higher in males (24.9+/-7.38 ng/ml, P=0.026, n=10) than in females (8.27+/-3.11 ng/ml, n=10), as in humans. The resultant enzyme reaction products of BPA glucuronidation in the rat liver microsomes fraction were analyzed by an HPLC system. The ratio of BPA glucuronidation in the microsome reaction was significantly higher (P=0.015) in female than in male rats. The mRNA expression of UDP-glucuronosyltransferase 2B1 (UGT2B1), an isoform of UGT related to BPA glucuronidation, in the rat liver was analyzed by a real-time quantitative RT-PCR. The relative expression level of UGT2B1 mRNA was significantly higher (P<0.001) in female than in male rat livers. The gender difference in serum BPA concentrations may be explained by the difference in clearance based on the UGT activities.     

A genome-wide association study of total bilirubin and cholelithiasis risk in sickle cell anemia

Serum bilirubin levels have been associated with polymorphisms in the UGT1A1 promoter in normal populations and in patients with hemolytic anemias, including sickle cell anemia. When hemolysis occurs circulating heme increases, leading to elevated bilirubin levels and an increased incidence of cholelithiasis. We performed the first genome-wide association study (GWAS) of bilirubin levels and cholelithiasis risk in a discovery cohort of 1,117 sickle cell anemia patients. We found 15 single nucleotide polymorphisms (SNPs) associated with total bilirubin levels at the genome-wide significance level (p value <5 × 10(-8)). SNPs in UGT1A1, UGT1A3, UGT1A6, UGT1A8 and UGT1A10, different isoforms within the UGT1A locus, were identified (most significant rs887829, p = 9.08 × 10(-25)). All of these associations were validated in 4 independent sets of sickle cell anemia patients. We tested the association of the 15 SNPs with cholelithiasis in the discovery cohort and found a significant association (most significant p value 1.15 × 10(-4)). These results confirm that the UGT1A region is the major regulator of bilirubin metabolism in African Americans with sickle cell anemia, similar to what is observed in other ethnicities.