Uridine Diphosphate-glucuronosyltransferase Gene Research Articles

UGT2B13 and UGT2C1 are involved in lambda-cyhalothrin resistance in Rhopalosiphum padi

Uridine diphosphate-glucuronosyltransferases (UGTs) are major multifunctional detoxification phase II enzymes involved in the metabolic detoxification of xenobiotics. However, their roles in insecticides resistance are still unclear. In this study, we identified two UGTs genes (UGT2B13 and UGT2C1) in Rhopalosiphum padi, a serious insect pest of wheat worldwide. Bioassays results showed that the resistance ratio of R. padi resistance strain (LC-R) to lambda-cyhalothrin (LC) was 2963.8 fold. The roles of UGT2B13 and UGT2C1 in lambda-cyhalothrin resistance were evaluated. Results indicated that the UGTs contents were significantly increased in the LC resistant strain of R. padi. UGT2B13 and UGT2C1 were significantly overexpressed in the LC-R strain. Transcription levels of UGT2B13 and UGT2C1 were relatively higher in the gut of LC-R strain. RNA interference (RNAi) of UGT2B13 or UGT2C1 significantly decreased the UGTs contents of the LC-R aphids and increased mortality of R. padi exposure to the LC50 concentration of LC. This study provides a new view that UGTs are involved in LC resistance of R. padi. The findings will promote further work to detailed the functions of UGTs in the metabolism resistance of insects to insecticides.

Chlorpyrifos and chlorfenapyr resistance in Spodoptera frugiperda (Lepidoptera: Noctuidae) relies on UDP-glucuronosyltransferases.

Fall armyworm, Spodoptera frugiperda (J. E. Smith), has become an important agricultural pest worldwide. S. frugiperda is mainly controlled by the chemical insecticides, whereas the frequent application of insecticides would result in the resistance development. Insect uridine diphosphate-glucuronosyltransferases (UGTs), as phase II metabolism enzymes, play vital roles in the breakdown of endobiotic and xenobiotics. In this study, 42 UGT genes were identified by RNA-seq, including 29 UGT genes were elevated compared to the susceptible population, and the transcript levels of 3 UGTs (UGT40F20, UGT40R18, and UGT40D17) were increased by more than 2.0-fold in the field populations. Expression pattern analysis revealed that S. frugiperda UGT40F20, UGT40R18, and UGT40D17 were increased by 6.34-, 4.26-, and 8.28-fold, compared the susceptible populations, respectively. The expression of UGT40D17, UGT40F20, and UGT40R18 was affected after exposure to phenobarbital, chlorpyrifos, chlorfenapyr, sulfinpyrazone, and 5-nitrouracil. The induced expression of UGT genes may have improved UGT enzymatic activity, while the inhibition of UGTs genes expression may decreased UGT enzymatic activity. Sulfinpyrazone, and 5-nitrouracil, significantly increased the toxicity of chlorpyrifos and chlorfenapyr, as well as phenobarbital significantly reduced the toxicity of chlorpyrifos and chlorfenapyr against the susceptible populations and field populations of S. frugiperda. The suppression of UGTs (UGT40D17, UGT40F20, and UGT40R18) significantly increased the insensitivity of the field populations to chlorpyrifos and chlorfenapyr. These findings strongly supported our viewpoint that UGTs may play a critical role in insecticide detoxification. This study provides a scientific basis for the management of S. frugiperda.

Gilbert or Crigler\u2013Najjar syndrome? Neonatal severe unconjugated hyperbilirubinemia with P364L UGT1A1 homozygosity

BackgroundSeveral mutations of bilirubin uridine diphosphate-glucuronosyltransferase gene (UGT1A1) have been reported in patients with unconjugated hyperbilirubinemia. Few reports are available about the p.Pro364Leu mutation (P364L, c.1091C > T) in homozygous newborns. We describe the clinical, laboratory and therapeutic approach in two Chinese neonates with severe jaundice, homozygous for the P364L mutation.Case presentationTwo Chinese breastfed female infants presented prolonged unconjugated hyperbilirubinemia at the age of 1 month. Total bilirubin was higher than 15 mg/dl (D < 1). An exhaustive etiological work-up to detect possible causes of hyperbilirubinemia (notably hemolytic ones) was negative.The promoter and coding regions of UGT1A1 were amplified by polymerase chain reaction (PCR) from genomic DNA isolated from leukocytes. Both patients resulted homozygous for a variant site within the coding region of the gene in the 4 exon, c.1091C > T, p.Pro364Leu.In front of the persistently high level of unconjugated bilirubin, phototherapy was performed without persistent results. A treatment with phenobarbital was then begun and bilirubin level progressively decreased, with a complete and persistent normalization. The therapy was stopped.ConclusionUGT1A1 enzyme activity associated with the P364L mutation has been described as 35.6% of the wild-type enzyme activity. Photo-therapy and phenobarbital can be useful in front of persistently high level of unconjugated bilirubin.Our cases presented high bilirubin values, overlapping between Gilbert syndrome (GS) and Crigler-Najjar syndrome type II (CNS), but the complete normalization of bilirubin makes GS more likely. Homozygous P364L variant can be associated with severe neonatal unconjugated hyperbilirubinemia in Chinese infants, but jaundice can completely resolve in a few months, contrary to what happens in Crigler-Najjar syndrome type II.

Доброкачественная гипербилирубинемия типа Жильбера

INTRODUCTION: Benign hyperbilirubinemia of Gilbert type affects 10% of population, most commonly men of 20–30 years of age. In the article, description of a classic clinical case illustrating the course of Gilbert syndrome, is given. After an intercurrent infection, a 32-year old man presented with jaundice with elevation of the level of unconjugated bilirubin against the normal background level of enzymes. Bearing in mind a probability of being excluded from the flight crew, the patient agreed to the puncture biopsy of the liver, and the morphological examination of the hepatic tissue confirmed the diagnosis of benign hyperbilirubinemia of Gilbert type. A molecular genetic examination also showed the existence of tyrosine-arginine repeats in the promotor region of uridine diphosphate–glucuronosyltransferase gene. Adequately selected treatment with phenobarbital stimulating the activity of the enzyme, led to normalization of bilirubin level and to improvement of the patient’s well-being. CONCLUSION: Chronic familial non-hemolytic hyperbilirubinemias are often given insufficient attention not only due to the fact that their outcomes do not pose a serious threat to the patient’s health, and, according to the erroneous opinion, are rare, but to a larger extent due to the fact that the diagnosis presents certain difficulties for a therapist or a general practitioner.

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Announcement of the 2022 winners of the Spychala and Women in Science Awards

Species differences in phenobarbital-mediated UGT gene induction in rat and human liver microtissues.

Species differences in hepatic metabolism of thyroxine (T4) by uridine diphosphate glucuronosyl transferase (UGT) and susceptibility to thyroid hormone imbalance could underlie differences in thyroid carcinogenesis caused by hepatic enzyme inducers in rats and humans. To investigate this hypothesis we examined profiles of hepatic UGT induction by the prototypical CAR activator phenobarbital (PB) in rat and human liver 3D microtissues. The rationale for this approach was that 3D microtissues would generate data more relevant to humans. Rat and human liver 3D microtissues were exposed to PB over a range of concentrations (500 u M - 2000 u M) and times (24-96 hr). Microarray and proteomics analyses were performed on parallel samples to generate integrated differentially expressed gene (DEG) datasets. Bioinformatics analysis of DEG data, including CAR response element (CRE) sequence analysis of UGT promoters, was used to assess species differences in UGT induction relative to CAR-mediated transactivation potential. A higher proportion of human UGT promoters were found to contain consensus CREs compared to the rat homologs. UGTs 1a6, 2b17 and 2b37 were upregulated by PB in rat liver 3D microtissues, but unaltered in human liver 3D microtissues. By contrast, human UGTs 1A8, 1A10 and 2B10 showed higher levels of induction (RNA and /or protein) compared to the rat homologs. There was general concordance between the presence of CREs and the induction of UGT RNA. As UGT1A and 2B isoforms metabolise T4, these results suggest that differences in UGT induction could contribute to differential susceptibility to CAR-mediated thyroid carcinogenesis in rats and humans.

Genome-Wide Identification and Expression Analysis of Udp-Glucuronosyltransferases in the Whitefly Bemisia Tabaci (Gennadius) (HemipterA: Aleyrodidae).

Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is an important agricultural pest worldwide. Uridine diphosphate (UDP)-glucuronosyltransferases (UGTs) are one of the largest and most ubiquitous groups of proteins. Because of their role in detoxification, insect UGTs are attracting increasing attention. In this study, we identified and analyzed UGT genes in B. tabaci MEAM1 to investigate their potential roles in host adaptation and reproductive capacity. Based on phylogenetic and structural analyses, we identified 76 UGT genes in the B. tabaci MEAM1 genome. RNA-seq and real-time quantitative PCR (RT-qPCR) revealed differential expression patterns of these genes at different developmental stages and in association with four host plants (cabbage, cucumber, cotton and tomato). RNA interference results of selected UGTs showed that, when UGT352A1, UGT352B1, and UGT354A1 were respectively silenced by feeding on dsRNA, the fecundity of B. tabaci MEAM1 was reduced, suggesting that the expressions of these three UGT genes in this species may be associated with host-related fecundity. Together, our results provide detailed UGTs data in B. tabaci and help guide future studies on the mechanisms of host adaptation by B. tabaci.

Characterization of UDP-glucuronosyltransferase genes and their possible roles in multi-insecticide resistance in Plutella xylostella (L.).

Uridine diphosphate-glucuronosyltransferases (UGTs), as multifunctional detoxification enzymes, play important roles in the biotransformation of various compounds. However, their roles in insecticide resistance are still unclear. This study presents a genome-wide identification of the UGTs in diamondback moth, Plutella xylostella (L.), a notorious insect pest of cruciferous crops worldwide. The possible roles of these UGTs in insecticide resistance were evaluated. A total of 21 putative UGTs in P. xylostella were identified. Quantitative real-time polymerase chain reaction (PCR)-based analyses showed that all the UGT genes were expressed in all tested developmental stages and tissues. Bioassay results indicated that a field-collected population (BL) was resistant to 9 of 10 commonly used insecticides, and 10 of 21 UGT mRNAs were upregulated in the BL population. Exposure to the LC50 of each insecticide affected the expression of most UGT genes. Among these, the expression levels of UGT40V1, UGT45B1 and UGT33AA4 were induced by more than five insecticides, whereas indoxacarb and metaflumizone significantly repressed the expression of most UGT genes. UGTs may play important roles in the metabolism of commonly used insecticides in P. xylostella. These findings provide valuable information for further research on the physiological and toxicological functions of specific UGT genes in P. xylostella. © 2017 Society of Chemical Industry.

Correlation between UGT1A1 polymorphism and neonatal hyperbilirubinemia of neonates in Wuhan.

This study attempts to discuss the correlation between UGT1A1*28 as uridine diphosphate glucuronosyltransferase gene promoter and coding region Gly71Arg gene polymorphism with neonatal hyperbilirubinemia of neonates in Wuhan. A total of 168 neonates were divided into the hyperbilirubinemia group (case group, n=108) and healthy neonates group (control group, n=60). Their DNA was obtained through blood extraction. The gene exon mutation of UGT1A1 was detected by Sanger sequencing, which revealed the relationship between UGT1A1*28 and Gly71Arg polymorphism with neonatal hyperbilirubinemia of neonates. The results showed that: (1) The frequency of UGT1A1*28 allele mutation in the case group and the control group was 9.3% and 10% respectively, with the difference being not significant between the two groups (P>0.05). (2) The frequency of Gly71Arg allele mutation in the case group and the control group was 35.1% and 21.7% respectively, with the difference being significant between the two groups (P<0.01). (3) The serum bilirubin level of Gly71Arg mutant homozygous and heterozygous subgroups (n=66) in the case group was 302.7±31.4 μmol/L, which was significantly higher than 267.3±28.5 μmol/L of the wild subgroup (n=42) (P<0.01). It was suggested that the occurrence of neonatal hyperbilirubinemia of neonates in Wuhan was not associated with UGT1A1*28 gene polymorphism, but closely with the Gly71Arg gene polymorphism. Meanwhile, the Arg allele mutation was related to the degree of jaundice.

Bilirubin Uridine Diphosphate-glucuronosyltransferase Polymorphism as a Risk Factor for Prolonged Hyperbilirubinemia in Japanese Preterm Infants

To determine whether a variant of the bilirubin uridine diphosphate-glucuronosyltransferase gene (UGT1A1*6) is a risk factor for prolonged hyperbilirubinemia in preterm infants. UGT1A1 genotypes in 46 Japanese preterm infants (<37 weeks of gestation) were compared with UGT1A1 genotypes in 38 control infants, using polymerase chain reaction-direct sequencing. Prolonged unconjugated hyperbilirubinemia was defined as serum total bilirubin concentration of >150 µmol/L (8.77 mg/dL) beyond 14 days of life. In the case group, 41 of 46 infants (89.1%) had a polymorphic variant, c.211G>A, p.G71R (UGT1A1*6). In the control group, 7 of 38 (18.4%) had UGT1A1*6. The allele frequency of UGT1A1*6 was 0.641 in the prolonged hyperbilirubinemia group, which was significantly higher than in the control group (0.092; P < .001). In total, 39 of 46 infants in the case group were breast fed, and only 10 infants in the control group were breast fed. These data suggest that UGT1A1*6 is a risk factor for prolonged unconjugated hyperbilirubinemia in preterm infants in Japan. Given the different rate of breast feeding in this study, additional data are necessary for drawing a definitive conclusion.

Population pharmacokinetics and Bayesian estimation of mycophenolic acid concentrations in Chinese adult renal transplant recipients.

Mycophenolate mofetil (MMF) is an important immunosuppressant used in renal transplantation, and mycophenolic acid (MPA) is the active component released from the ester prodrug MMF. The objective of this study was to investigate the population pharmacokinetics of mycophenolic acid (MPA) following oral administration of MMF in Chinese adult renal transplant recipients and to identify factors that explain MPA pharmacokinetic variability. Pharmacokinetic data for MPA and covariate information were retrospectively collected from 118 patients (79 patients were assigned to the group for building the population pharmacokinetic model, while 39 patients were assigned to the validation group). Population pharmacokinetic data analysis was performed using the NONMEM software. The pharmacokinetics of MPA was best described by a two-compartment model with a first-order absorption rate with no lag time. Body weight and serum creatinine level were positively correlated with apparent clearance (CL/F). The polymorphism in uridine diphosphate glucuronosyltransferase gene, UGT2B7, significantly explained the interindividual variability in the initial volume of distribution (V1/F). The estimated population parameters (and interindividual variability) were CL/F 18.3 L/h (34.2%) and V1/F 27.9 L (21.3%). The interoccasion variability was 13.7%. These population pharmacokinetic data have significant clinical value for the individualization of MMF therapy in Chinese adult renal transplant patients.

Uridine Diphosphate-Glucuronosyltransferase (UGT) Xenobiotic Metabolizing Activity and Genetic Evolution in Pinniped Species.

There are various interspecies differences in xenobiotic-metabolizing enzymes. It is known that cats show slow glucuronidation of drugs such as acetaminophen and strong side effects due to the UGT1A6 pseudogene. Recently, the UGT1A6 pseudogene was found in the Northern elephant seal and Otariidae was suggested to be UGT1A6-deficient. From the results of measurements of uridine diphosphate-glucuronosyltransferase (UGT) activity using liver microsomes, the Steller sea lion, Northern fur seal, and Caspian seal showed UGT activity toward 1-hydroxypyrene and acetaminophen as low as in cats, which was significantly lower than in rat and dog. Furthermore, UGT1A6 pseudogenes were found in Steller sea lion and Northern fur seal, and all Otariidae species were suggested to have the UGT1A6 pseudogene. The UGT1 family genes appear to have undergone birth-and-death evolution based on a phylogenetic and synteny analysis of the UGT1 family in mammals including Carnivora. UGT1A2-1A5 and UGT1A7-1A10 are paralogous genes to UGT1A1 and UGTA6, respectively, and their numbers were lower in cat, ferret and Pacific walrus than in human, rat, and dog. Felidae and Pinnipedia, which are less exposed to natural xenobiotics such as plant-derived toxins due to their carnivorous diet, have experienced fewer gene duplications of xenobiotic-metabolizing UGT genes, and even possess UGT1A6 pseudogenes. Artificial environmental pollutants and drugs conjugated by UGT are increasing dramatically, and their elimination to the environment can be of great consequence to cat and Pinnipedia species, whose low xenobiotic glucuronidation capacity makes them highly sensitive to these compounds.

Gender matters: Estrogen receptor alpha (ERα) and histone deacetylase (HDAC) 1 and 2 control the gender-specific transcriptional regulation of human uridine diphosphate glucuronosyltransferases genes (UGT1A)

Gender influences incidence, progression, and therapy of hepatogastrointestinal diseases. The aim of this study was to elucidate the molecular mechanism of gender-specific UDP-glucuronosyltransferases (UGT1A) regulation, representing important hepatogastrointestinal detoxification enzymes for xenobiotics, drugs, and endobiotics. UGT1A-gene activation was studied by reporter gene experiments and estrogen receptor alpha (ESR1/ERα) co-transfection using KYSE70- and HepG2 cells (male origin), and SW403 cells (female origin). Cell lines, and humanized transgenic UGT1A (htgUGT1A) mice (female/male) were treated with the ERα inhibitor tamoxifen. UGT1A mRNA expression was analyzed by TaqMan PCR, the recruitment of ERα, histone deacetylases (HDAC), and the aryl hydrocarbon receptor (AhR) by chromatin immunoprecipitation (ChIP), and ERα expression in gastrointestinal mouse tissues by Western blot and immunofluorescence. In KYSE70 cells (male), UGT1A gene expression was induced 5-10 fold, and inhibited in the presence of ERα by 55-77%. In SW403 (female) cells, absent inducibility was restored after tamoxifen treatment. In the jejunum and colon of tgUGT1A mice, UGT1A induction that was exclusively detected in male mice could be restored in female mice after tamoxifen pre-treatment. ChIP assays demonstrated the recruitment of ERα and HDACs to the xenobiotic response elements of UGT1A promoters during gene repression. Western blot showed higher ERα expression in the female jejunum and colon. We show gender-specific transcriptional control of UGT1A genes in jejunum and colon, which is repressed by ERα and the recruitment of HDCAs to the UGT1A promoter sequence in females. A molecular mechanism controlling gender-specific drug metabolism and its therapeutic reversal is demonstrated.

Development of icterus gravis in a preterm infant with G71R UGT1A1 polymorphism

BackgroundUridine diphosphate-glucuronosyltransferase (UGT) gene family is involved in the detoxification of biomaterials and drugs in the liver. Among the UGT gene family members, only UGT1A1 is involved in bilirubin conjugation. As a result, deficient UGT1A1 activity causes jaundice. One disease that is characterized by reduced UGT1A1 activity is Gilbert’s syndrome. Two prevalent UGT1A1 polymorphisms responsible for Gilbert’s syndrome have been identified: G71R in exon 1 and A(TA)7TAA in the TATA box of the promoter region. Recently, the G71R polymorphism has been associated with breastfeeding jaundice and neonatal hyperbilirubinemia in term infants. However, its association with jaundice in very low birth weight infants (VLBWIs) has never been reported.Case presentationThe patient was a female born at 28 weeks, 4 days gestation with a birth weight of 1172 g. On day 21, intense yellowing of the skin and eyes was noted, and the patient’s total bilirubin level was 23.7 mg/dL (her direct bilirubin level was 2.1 mg/dL). Therefore, an exchange transfusion was conducted. She had neither blood type incompatibility nor a family history of constitutional jaundice. Metabolic screens for amino and organic acids were negative. No elevation of any of the examined antibody titers was noted, and no evidence of an inflammatory reaction was observed. In addition, no hematological abnormalities were detected. The direct/indirect Coombs test, irregular antibody test and red blood cell antibody dissociation test were all negative, and her thyroid function was normal. We performed sequence analysis of the UGT1A1 gene after the patient’s parents provided written informed consent. Exon 1 of the UGT1 gene on chromosome 2 was analyzed by direct sequencing. A heterozygous substitution from G to A (211G→A: G71R) in base 211 was noted.ConclusionWe speculated that this preterm infant with carrying the G71R polymorphism reduced UGT1A1 activity and developed severe jaundice that was likely triggered by factors such as breast feeding and medications. The polymorphism appears at some frequency among VLBWIs, which would necessitate adequate care of severe jaundice even after the acute phase.

Compound Heterozygote of a Novel Missense Mutation (p.K402T) and a Double Missense Mutation (p.[G71R;Y486D]) in Type II Crigler‐Najjar Syndrome

362 C rigler-Najjar syndrome type II (CN-II; # Mendelian Inheritance in Man [MIM] 606785) is a moderate type of hereditary unconjugated hyperbilirubinemia caused by mutations of the bilirubin uridine diphosphate-glucuronosyltransferase gene UGT1A1 (1). Diagnosis of this syndrome is important in distinguishing it from the severe phenotype of hereditary unconjugated hyperbilirubinemia, known as Crigler-Najjar syndrome type I (CNI; MIM #218800). The serum bilirubin concentration of CN-I is more than 20 mg/dL (343mmol/L) and can reach 30 to 50 mg/dL (513–855mmol/L). Patients with CN-I are in danger of kernicterus and must receive daily phototherapy right from the neonatal period (2). They should undergo liver transplantation to avoid kernicterus. Hyperbilirubinemia in patients with CN-II is less severe than CN-I (1). The serum bilirubin concentration of CN-II is seldom harmful after the neonatal period. The serum bilirubin concentration of patients with CN-I or CN-II is often intermediate between the values specifying the 2 syndromes. Accurate diagnosis of the 2 syndromes is necessary for appropriate treatment. A phenobarbitalloading test is effective in differential diagnosis (3), but the results are not always clear, in which case genetic diagnosis is necessary. The cDNA of UGT1A1 was first cloned in 1991, and a large number of mutations have been found in patients with CN-I, CN-II, and Gilbert syndrome (GS; MIM #143500) (4,5). Mutations of UGT1A1 differ according to ethnic grouping. East Asians display many unique mutations; in particular, a major polymorphic mutation is c.211G>A: p.G71R in exon 1 of UGT1A1 (6). p.G71R reduces glucuronidation ability to less than 32% of wild type and is an important cause of Gilbert syndrome (7). One of the most important causes of CN-II in Asians is a double missense mutation, p.[G71R;Y486D] (8). This double mutation has hardly ever been detected in white and African populations.

Pharmacogenetic Impact of UDP‐Glucuronosyltransferase Metabolic Pathway and Multidrug Resistance—Associated Protein 2 Transport Pathway on Mycophenolic Acid in Thoracic Transplant Recipients: An Exploratory Study

To assess the contribution of polymorphisms in the uridine diphosphate glucuronosyltransferase gene (UGT) and the multidrug resistance-associated protein 2 gene (ABCC2) to mycophenolic acid (MPA) pharmacokinetics and clinical outcomes in thoracic transplant recipients. Open-label, cross-sectional study. Transplant clinic in Vancouver, British Columbia, Canada. Sixty-eight thoracic (36 lung, 32 heart) transplant recipients who were receiving steady-state oral mycophenolate mofetil. Eleven blood samples were obtained from each patient over a 12-hour dosing period. Plasma concentrations of MPA (active metabolite of mycophenolate mofetil), the MPA metabolites 7-Omycophenolic acid glucuronide (MPAG) and acyl glucuronide (AcMPAG), and free MPA were measured, and dose-normalized conventional pharmacokinetic parameters were determined by noncompartmental methods. Genetic polymorphisms in UGT and ABCC2 were determined by sequencing, and their contributions to pharmacokinetic variability were investigated by using multivariate analysis. For both the lung and heart transplant groups, the UGT2B7 variant 802T (Tyr(268) or UGT2B7*2, rs7439366) and the UGT2B7 variant -138A modified AcMPAG exposure (2.5-3.7-fold and 9.3-12.3-fold higher AcMPAG area under the concentration-time curve [AUC] and AcMPAG:MPA ratio, respectively). In an exploratory analysis, occurrences of rejection, infection, anemia, and leukopenia were associated with an AcMPAG AUC greater than 50 μg·hour/ml and an AcMPAG:MPA ratio greater than 2. UGT2B7 is a promising gene candidate that may influence MPA pharmacokinetics clinically; however, larger clinical pharmacogenetic studies in thoracic transplant subpopulations are warranted to corroborate the role of AcMPAG and UGT2B7 variants in optimizing mycophenolate mofetil therapy.

Prediction of Deleterious Non-synonymous Single-Nucleotide Polymorphisms of Human Uridine Diphosphate Glucuronosyltransferase Genes

UDP glucuronosyltransferases (UGTs) are an important class of Phase II enzymes involved in the metabolism and detoxification of numerous xenobiotics including therapeutic drugs and endogenous compounds (e.g. bilirubin). To date, there are 21 human UGT genes identified, and most of them contain single-nucleotide polymorphisms (SNPs). Non-synonymous SNPs (nsSNPs) of the human UGT genes may cause absent or reduced enzyme activity and polymorphisms of UGT have been found to be closely related to altered drug clearance and/or drug response, hyperbilirubinemia, Gilbert's syndrome, and Crigler-Najjar syndrome. However, it is unlikely to study the functional impact of all identified nsSNPs in humans using laboratory approach due to its giant number. We have investigated the potential for bioinformatics approach for the prediction of phenotype based on known nsSNPs. We have identified a total of 248 nsSNPs from human UGT genes. The two algorithms tools, sorting intolerant from tolerant (SIFT) and polymorphism phenotyping (PolyPhen), were used to predict the impact of these nsSNPs on protein function. SIFT classified 35.5% of the UGT nsSNPs as "deleterious"; while PolyPhen identified 46.0% of the UGT nsSNPs as "potentially damaging" and "damaging". The results from the two algorithms were highly associated. Among 63 functionally characterized nsSNPs in the UGTs, 24 showed altered enzyme expression/activities and 45 were associated with disease susceptibility. SIFT and Polyphen had a correct prediction rate of 57.1% and 66.7%, respectively. These findings demonstrate the potential use of bioinformatics techniques to predict genotype-phenotype relationships which may constitute the basis for future functional studies.

Combined Test for UGT1A1 –3279T→G and A(TA)nTAA Polymorphisms Best Predicts Gilbert's Syndrome in Italian Pediatric Patients

Gilbert's syndrome is a common hereditary chronic or recurrent, mild unconjugated hyperbilirubinemia. Polymorphisms in the bilirubin uridine diphosphate glucuronosyl transferase gene (UGT1A1) causing a decreased enzyme activity are associated with susceptibility to the syndrome. Homozygosity for TA(7) allele of the A(TA)(n)TAA promoter polymorphism is found in the majority of Caucasian patients. We sought to investigate the role of three UGT1A1 polymorphisms (A[TA](n)TAA, -3279T-->G, and G71R) in the susceptibility to Gilbert's syndrome in 53 Italian pediatric subjects compared to 83 unaffected controls. Carriage of two TA(n) risk alleles (TA(7) and TA(8)) and -3279G homozygosity were similarly associated with hyperbilirubinemia (odds ratio [OR] = 11.59, 95% confidence interval [CI] = 4.80-27.98; p < 0.001, and OR = 11.51, 95% CI = 5.06-26.19; p < 0.001, respectively). Homozygosity for both TA7 and -3279G was associated with the highest relative risk estimate (OR = 19.23, 95% CI = 7.34-50.4; p < 0.001), but a significant association was found also for TA7 heterozygosity combined with -3279G/G genotype (OR = 7.98, 95% CI = 2.54-25.06; p < 0.001). The G71R variant was found only in two controls. Our results demonstrate that genotyping of both UGT1A1 A(TA)(n)TAA and -3279T-->G polymorphisms best defines genetic susceptibility to Gilbert's syndrome in Caucasian pediatric patients, and the TA7 heterozygous genotype combined with homozygosity for the -3279G allele can also be associated with pediatric mild hyperbilirubinemia.

The Role of UGT1A1*28 Mutation in Jaundiced Infants with Hypertrophic Pyloric Stenosis

Hypertrophic pyloric stenosis (HPS) may be accompanied by jaundice, a condition referred to as the icteropyloric syndrome (IPS). It has long been suspected that the etiology of IPS is an early manifestation of Gilbert's syndrome (GS). Clinical features common to both GS and IPS include jaundice precipitated by fasting and improved with feeding. Prevalence of jaundice in HPS is similar to that of clinically apparent GS in the general population. Discovery of a mutation in the promoter region of the bilirubin uridine diphosphate glucuronosyl transferase gene (UGT1A1*28) as the most common cause of GS has provided a tool to determine the role of GS in IPS. The aims of this study were to determine 1) the prevalence of IPS in a large group of infants with HPS, 2) whether disease severity contributed to the manifestation of IPS, and 3) whether GS played a role in IPS. Radioactive PCR and sequencing were used to determine the presence of UGT1A1*28 mutations. We determined a prevalence of IPS of 14.3% in HPS. Infants with IPS had significantly higher levels of alkalosis than infants with HPS alone. GS mutations were 4-fold higher in IPS (43.8%) than HPS (10.7%). In conclusion, the frequency of jaundice in HPS is similar to that of clinically apparent GS in the general population. Manifestation of IPS results from a more severe degree of metabolic disturbance and the presence of GS mutations.

Genetic polymorphisms of bilirubin uridine diphosphate-glucuronosyltransferase gene in Japanese patients with Crigler-Najjar syndrome or Gilbert's syndrome as well as in healthy Japanese subjects.

Numerous mutations of bilirubin uridine diphosphate-glucuronosyltransferase gene (UGT1A1) have been reported in patients with familial unconjugated hyperbilirubinemia. The UGT1A1 mutation appears to be considerably different among ethnic groups. To clarify the incidence of this gene mutation in the Japanese population, the presence of UGT1A1 mutation was investigated in a group of Japanese patients with Crigler-Najjar syndrome type 2 (CNS2) and Gilbert's syndrome (GS), as well as in healthy anicteric subjects. Four patients with CNS2, 63 patients with GS, and 71 healthy subjects were enrolled in the study. The promoter and coding regions of UGT1A1 were amplified by polymerase chain reaction (PCR) from genomic DNA isolated from leukocytes. The PCR products were directly sequenced by a dye terminating method. The UGT1A1 enzyme activity was determined in COS7 cells transfected with wild or P364L (1091 C > T) mutant DNA. Homozygous Y486D was observed in all four patients with CNS2. The GS patients had UGT1A1 mutations with 13 different genotypes in the promoter and coding region. Homozygous TA insertion in the TATA box (TA7) of the promoter region (TA7/7; 33%), homozygous G71R (9%), and combination of TA7/6 and heterozygous G71R (17%) were the most frequent findings in GS patients. Homozygous or heterozygous Y486D (8%) and P229Q (8%) were also observed in GS. A novel mutation, heterozygous P364L, was also identified in a GS patient. In addition to GS patients, homozygous or heterozygous TA7, G71R, and heterozygous Y486D were also observed in healthy subjects. The allele frequency of G71R and TA7 was 0.183 and 0.113 in healthy subjects, respectively. The P364L UGT1A1 enzyme activity was 64.4% lower than the wild-type enzyme activity. Polymorphisms in the coding region of UGT1A1 were commonly observed in Japanese patients with GS and in healthy subjects. The genetic basis of hyperbilirubinemia appears to be different between the Japanese and Caucasian populations.