Robert Hume

Genetic variation in bilirubin UDP-glucuronosyltransferase gene promoter and Gilbert's syndrome

BACKGROUND The genetic basis of Gilberts syndrome is ill-defined. This common mild hyperbilirubinaemia sometimes presents as an intermittent jaundice. A reduced hepatic bilirubin UPD- glucuronosyltransferase (UGT) is associated with this syndrome. We have examined variation in the gene encoding the UGT1*1 enzyme and serum bilirubin levels in a Scottish population. METHODS Blood was collected from 12 patients with confirmed or suspected Gilberts syndrome, from 6 members of a family with 4 Gilbert members, and from 77 non-smoking, alcohol-free, drug-free volunteers recruited from the staff of a teaching hospital in Dundee. Polymerase chain reaction amplification was used to examine sequence variation of the promoter upstream of the UGT1*1 exon I. Genotypes were assigned as follows: 6/6 (homozygous for a common allele bearing the sequence [TA](6)TAA), 7/7 (homozygous for a rarer allele with the sequence [TA](7)TAA), and 6/7 (heterozygous with one of each allele). FINDINGS Individuals in the population with the 7/7 genotype had significantly higher bilirubin concentrations than those who had the 6/7 or 6/6 genotype. 14 volunteers underwent a 24 h fasting test to see if they had Gilberts syndrome, and all four positives had the 7/7 genotype. One confirmed Gilberts patient, two recurrent jaundice patients (with suspected Gilberts syndrome), and nine clinically diagnosed cases had the 7/7 genotype. Segregation of the 7/7 genotype with the Gilbert phenotype was also demonstrated in the family with four affected members. The frequency of the 7/7 genotype in this eastern Scottish population was 10-13%. INTERPRETATION In a healthy population there was an association between variation in bilirubin concentration and a mutation within the gene encoding the enzyme bilirubin UGT. This and other findings suggest the existence of a mild and a more severe form of Gilberts syndrome, depending on whether the gene defect lies in the promoter sequence upstream of UGT1*I exon I, as here (mild), or in the coding sequence (severe) of the gene.

A new microtechnique for the analysis of the human hepatic microsomal glucose-6-phosphatase system

A microtechnique has been developed which enables a complete kinetic analysis of the human hepatic microsomal glucose-6-phosphatase system to be carried out in microsomes isolated from very small liver samples. Complete or partial deficiencies of any of the proteins of the glucose-6-phosphatase system resulting in Type 1a, 1b, 1c or 1d glycogen storage disease can be therefore be diagnosed using hepatic needle biopsy samples, whereas previous methods of diagnosis needed large wedge biopsy samples requiring laparotomy.

The human glutathione S-transferases: studies on the tissue distribution and genetic variation of the GST1, GST2 and GST3 isozymes

1. Three sets of isozymes of glutathione‐S‐transferase (GST) have been identified in human tissues. They differ in their tissue distribution, incidence of genetic variation, susceptibility to inactivation by N‐ethylmaleimide and in their electrophoretic mobilities.

Genetic defects of the UDP-glucuronosyltransferase-1 (UGT1) gene that cause familial non-haemolytic unconjugated hyperbilirubinaemias.

Congenital familial non-haemolytic hyperbilirubinaemias are potentially lethal syndromes caused by genetic lesions that reduce or abolish hepatic bilirubin UDP-glucuronosyltransferase activity. Here we describe genetic defects that occur in the UGT1 gene complex that cause three non-haemolytic unconjugated hyperbilirubinaemia syndromes. The most severe syndrome, termed Crigler-Najjar syndrome type I, is mainly associated with mutations in exons 2 to 5 that affect all UGT1 enzymes and many of the mutations result in termination codons and frameshifts. Crigler-Najjar type II syndrome which is treatable with phenobarbital therapy is associated with less dramatic missense mutations or heterozygous expression of mutant and normal alleles. Gilberts syndrome, the most prevalent (2-19% in population studies) and mildest of the three syndromes is principally caused by a TA insertion at the TATA promoter region upstream of the UGT1A1 exon. Current methods used for the diagnosis and treatment of these diseases are discussed.

The development of glutathione S-transferase and glutathione peroxidase activities in human lung

The development of glutathione S-transferase and glutathione peroxidase activities has been studied in human lung cytosols. Whilst no clear change in glutathione peroxidase activity was identified, expression of the acidic glutathione S-transferase isoenzyme decreased markedly after 15 weeks of gestation so that at birth the level of activity of this isoenzyme was only about 20% of that in samples obtained during the first trimester. Basic glutathione S-transferase isoenzymes were weakly expressed during development and usually comprised less than 10% of cytosolic activity. Ion-exchange studies identified several basic isoenzymes that may correspond to the alpha, beta, gamma, delta and epsilon set previously identified in liver. Weak expression of apparently near-neutral isoenzymes was also detected; they were detected in only a few cytosols.

Separation of two forms of cytochrome P450 adrenal cortex mitochondria

The separation of two distinct forms of cytochrome P450 from adrenal cortex mitochondria has been achieved by the following steps; (1) lyophilisation (2) iso‐octane extraction, (3) (NH4)2SO4 fractionation in the presence of sodium cholate. The fraction precipitating between 25–35 percent (NH4)2SO4 gave a difference spectrum with 11‐deoxycorticosterone (11‐DOC) but not with 20α‐hydroxycholesterol (20α‐HOC). This fraction showed high 11β‐hydroxylase activity but low activity for side chain cleavage of cholesterol (S.C.C.). The fraction precipitating between 45–60 percent (NH4)2SO4 gave a difference spectrum with 20α‐HOC but not with 11‐DOC and exhibited high S.C.C. activity but low 11β‐hydroxylase activity. The absorption spectrum of the 45–60 percent fraction indicated a preponderance of high spin hemoprotein (λmax 395 nm).

Expression profiling of human fetal cytosolic sulfotransferases involved in steroid and thyroid hormone metabolism and in detoxification

Protection against chemical insult is essential for normal development of the fetus, however many detoxification enzymes are poorly expressed during fetal development. A major exception is the sulfotransferase (SULT) family, which appears to be widely expressed in the developing human. These enzymes also play a key role in biosynthesis and homeostasis of a number of hormones, including estrogens and iodothyronines. We therefore examined the enzyme activity, protein and mRNA expression of SULT 1A, 1B, 1C, 1E and 2A families in a variety of human fetal and adult tissues. Our results show that these SULTs are expressed in the human fetus, with most present at levels equivalent to or higher than the adult. As there are no isoform-selective substrates for SULTs 1B1 and 1C2 we used immunoblot analysis to show for the first time expression of SULT1B1 at high levels in fetal small intestine, and expression of SULT1C2 in fetal liver, kidney and small intestine. SULT1C2 was not expressed in adult liver or colon. Sulfotransferase expression in the developing fetus is therefore more widespread than in the adult, and this has significant implication for our understanding of human developmental physiology.

Glutathione S-transferase in human brain.

The glutathione S‐transferases are a complex group of multifunctional enzymes which may detoxify a wide range of toxic substances including drugs and carcinogens. Different isoenzymes vary in substrate specificity, tissue distribution and level of expression during development. Following reports of cell‐specific and age‐dependent expression in rat brain we have studied, immunohistochemically, expression of the Pi and Alpha class isoenzymes in 10 adult and 21 human fetal brains. Whilst Alpha isoenzyme is expressed only in adult brain, and then only focally, Pi isoenzyme is strongly expressed from as early as 12 weeks gestation. In the adult, expression is localized to choroid plexus, vascular endothelium, ventricular lining cells, pia‐arachnoid and astrocytes. In fetal brain, expression is also strong in cells with the morphology of tanycytes and in the cell bodies of radial glia. Neurons are consistently negative. Pi isoenzyme thus localizes to the sites of the blood‐CSF barrier, blood‐brain barrier, CSF‐brain barrier and pia‐arachnoid‐brain barrier. It is ideally placed to regulate neuronal exposure to potentially toxic substances derived from blood or cerebrospinal fluid. Expression so early in gestation is of significance and may imply a role in protection of the developing human brain.

Development of Human Liver UDP-Glucuronosyltransferases

The development of multiple UDPGT activities towards eight substrates has been studied in fetal term and adult post-mortem (less than 5 h after death) liver samples. Most fetal and term liver activities were less than 14% of adult values, except that towards 5-hydroxytryptamine which was present in fetal and term liver at adult levels. The majority of UDPGT activities develop to adult levels within 10-20 weeks postnatally, and even premature (30 weeks) which survive for up to 10 weeks will develop these enzyme activities. Immunoblot analysis of human liver microsomes and cDNA cloning of human UDPGT shows the existence of the family of isoenzymes in man, and it is important to determine the developmental pattern of individual drug glucuronidating enzymes in liver. Immunoblot analysis of developing liver shows the presence of two major UDPGT polypeptides in fetal liver, whereas more than five are observed in adult liver. The investigation of substrate specificity of individual UDPGTs by expression of cloned genes in COS-7 cells and the use of antibodies will facilitate the identification of enzymes present in perinatal liver.

The human glutathione S-transferases: Developmental aspects of the GST1, GST2, and GST3 loci

The expression of the GST1, GST2, and GST3 loci in fetal, neonatal, and infant tissues has been studied using starch gel electrophoresis and chromatofocusing. Each locus demonstrated developmental changes in expression, some of which were specific to a single tissue while others occurred in several tissues. GST1 was not usually expressed in any of the tissues studied before 30 weeks of gestation but steadily increased thereafter until adult levels were reached in late infancy. In neonates and older infants the frequencies of the GST1*0, GST1*1, and GST1*2 alleles were 0.79, 0.07, and 0.14, respectively. GST2 was always expressed in liver and adrenal but was only weakly expressed in spleen, cardiac muscle, and diaphragm. In kidney this locus was not usually expressed until nearly 1 year after birth. The GST3 isoenzymes were present in all fetal, neonatal, and infant tissues, although their expression in liver decreased after 30 weeks of gestation. Other isoenzymes with fast anodal mobilities were also identified in several tissues; these are believed to be GST3 isoenzymes that have undergone posttranslational modification rather than products of the putative GST4 locus. No specifically fetal isoenzymes were detected.