Conny T. Bakker

Congenital Jaundice in Rats with a Mutation in a Multidrug Resistance-Associated Protein Gene

The human Dubin-Johnson syndrome and its animal model, the TR− rat, are characterized by a chronic conjugated hyperbilirubinemia. TR− rats are defective in the canalicular multispecific organic anion transporter (cMOAT), which mediates hepatobiliary excretion of numerous organic anions. The complementary DNA for rat cmoat, a homolog of the human multidrug resistance gene (hMRP1), was isolated and shown to be expressed in the canalicular membrane of hepatocytes. In the TR− rat, a single-nucleotide deletion in this gene resulted in a reduced messenger RNA level and absence of the protein. It is likely that this mutation accounts for the TR− phenotype.

Discrimination between Crigler-Najjar type I and II by expression of mutant bilirubin uridine diphosphate-glucuronosyltransferase.

Crigler-Najjar (CN) disease is classified into two subtypes, type I and II. The molecular basis for the difference between these types is not well understood. Several mutations in the bilirubin UDP-glucuronosyl-transferase (B-UGT) gene of six CN type I and two CN type II patients were identified. Recombinant cDNAs containing these mutations were expressed in COS cells. B-UGT activity was measured using HPLC and the amount of expressed protein was quantitated using a sandwich ELISA. This enabled us to determine the specific activities of the expressed enzymes. All type I patients examined had mutations in the B-UGT1 gene that lead to completely inactive enzymes. The mutations in the B-UGT1 gene of patients with CN type II only partially inactivated the enzyme. At saturating concentrations of bilirubin (75 microM) CN type II patient A had 4.4 +/- 2% residual activity and CN type II patient B had 38 +/- 2% residual activity. Kinetic constants for the glucuronidation of bilirubin were determined. The affinities for bilirubin of B-UGT1 expressed in COS cells and B-UGT from human liver microsomes were similar with Km of 5.1 +/- 0.9 microM and 7.9 +/- 5.3 microM, respectively. B-UGT1 from patient B had a tenfold decreased affinity for bilirubin, Km = 56 +/- 23 microM. At physiological concentrations of bilirubin both type II patients will have a strongly reduced conjugation capacity, whereas type I patients have no B-UGT activity. We conclude that CN type I is caused by a complete absence of functional B-UGT and that in CN type II B-UGT activity is reduced.

UGT1A1*28 Allele and Coronary Heart Disease: The Rotterdam Study

Oxidative reactions, such as lipid oxidation and the formation of oxygen radicals, are involved in the pathophysiology of arteriosclerosis and coronary heart disease (CHD) (1). Bilirubin, the metabolic waste product of heme degradation, is an endogenous antioxidant and could thus have a protective effect against CHD (2). In vitro, conjugated and unconjugated bilirubin are scavengers of peroxyl radicals and are able to protect human LDL against peroxidation (3)(4). In vivo, increased serum bilirubin was shown to produce an enhanced antioxidant status of serum (5). In addition, an association between low serum bilirubin and the risk of CHD has been observed in several studies (6)(7)(8). A recent family heart study inferred the existence of a major gene for high bilirubin concentrations that may protect against CHD (9). A likely candidate gene responsible for high serum bilirubin is the gene encoding the hepatic enzyme bilirubin UDP-glucuronosyltransferase (UGT1A1). Glucuronidation is an essential step in the biliary excretion of bilirubin, and decreased UGT1A1 activity leads to increased serum concentrations of unconjugated bilirubin (10). A common etiology of decreased UGT1A1 activity is the insertion of a TA in the TATAA box in the promoter region of the UGT1A1 gene (11). Transcription of the UGT1A1 gene is fivefold lower in individuals with this allele, designated UGT1A1*28 . Individuals homozygous for the UGT1A1*28 allele (genotype 7/7) have mildly increased serum bilirubin compared with heterozygotes (genotype 6/7) and homozygous wild-type individuals (genotype 6/6) (12). In Caucasian populations, the frequency of individuals homozygous for this allele is 10–16% (11)(12). This is close to the 12% for the inferred major described in the family study (9 …

Anion exchanger 2 is essential for spermiogenesis in mice

Na+-independent anion exchangers (AE) mediate electroneutral exchange of Cl- for \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \setlength{\oddsidemargin}{-69pt} \begin{document} \begin{equation*}{\mathrm{HCO}}_{3}^{-}\end{equation*}\end{document} ions across cell membranes, being involved in intracellular pH and cell volume regulation and in transepithelial hydroionic fluxes. Bicarbonate activation of adenylyl cyclase is known to be necessary for sperm motility and sperm capacitation, and a few studies have suggested a possible role of AE carriers in reproduction. Among the four AE genes identified in mammals thus far, only Ae2 (Slc4a2) has been determined to be expressed in the male reproductive system, especially in developing spermatozoa and in epididymal epithelium. Most AE genes drive alternative transcription, which in mouse Ae2 results in several Ae2 isoforms. Here, we generated mice carrying a targeted disruption of Ae2 that prevents the expression of the three AE2 isoforms (Ae2a, Ae2b1, and Ae2b2) normally found in mouse testes. Male Ae2-/- mice (but not female Ae2-/- mice) are infertile. Histopathological analysis of Ae2-/- testes shows an interruption of spermiogenesis, with only a few late spermatids and a complete absence of spermatozoa in the seminiferous tubules. The number of apoptotic bodies is increased in the seminiferous tubules and in the epididymis, which also shows squamous metaplasia of the epididymal epithelium. Our findings reveal an essential role of Ae2 in mouse spermiogenesis and stress the recently postulated involvement of bicarbonate in germ-cell differentiation through the bicarbonate-sensitive soluble-adenylyl-cyclase pathway.

Splice-Site Mutations: A Novel Genetic Mechanism of Crigler-Najjar Syndrome Type 1

Crigler-Najjar syndrome type 1 (CN-1) is a recessively inherited, potentially lethal disorder characterized by severe unconjugated hyperbilirubinemia resulting from deficiency of the hepatic enzyme bilirubin-UDP-glucuronosyltransferase. In all CN-1 patients studied, structural mutations in one of the five exons of the gene (UGT1A1) encoding the uridinediphosphoglucuronate glucuronosyltransferase (UGT) isoform bilirubin-UGT1 were implicated in the absence or inactivation of the enzyme. We report two patients in whom CN-1 is caused, instead, by mutations in the noncoding intronic region of the UGT1A1 gene. One patient (A) was homozygous for a G-->C mutation at the splice-donor site in the intron, between exon 1 and exon 2. The other patient (B) was heterozygous for an A-->G shift at the splice-acceptor site in intron 3, and in the second allele a premature translation-termination codon in exon 1 was identified. Bilirubin-UGT1 mRNA is difficult to obtain, since it is expressed in the liver only. To determine the effects of these splice-junction mutations, we amplified genomic DNA of the relevant splice junctions. The amplicons were expressed in COS-7 cells, and the expressed mRNAs were analyzed. In both cases, splice-site mutations led to the use of cryptic splice sites, with consequent deletions in the processed mRNA. This is the first report of intronic mutations causing CN-1 and of the determination of the consequences of these mutations on mRNA structure, by ex vivo expression.

Redistribution of canalicular organic anion transport activity in isolated and cultured rat hepatocytes

The hepatocanalicular transport of a large number of organic anions, such as bilirubin glucuronides and glutathione conjugates in the rat, is mediated by an adenosine triphosphate (ATP)‐dependent transport system, which is termed canalicular multispecific organic anion transporter (cMOAT). This system is mainly defined by its deficiency in mutant TR− rats. We have previously reported that in cultured hepatocytes the fluorescent organic anion glutathione‐bimane (GS‐B) accumulates in intracellular vesicles and that this transport is mediated by cMOAT. We now show that this intracellular accumulation of fluorescent organic anion is largely absent in freshly isolated hepatocytes but appears when cells are incubated in suspension at 37°C or cultured for periods of 1 to 24 hours. The appearance of intracellular cMOAT activity coincides with the disappearance of 70% of cMOAT activity from the plasma membrane as measured by the transport activity of the cells for the organic anion dinitrophenyl‐glutathione (GS‐DNP). Both the appearance of intracellular cMOAT and the disappearance of transport activity from the plasma membrane were completely inhibited at temperatures below 20°C. Residual cMOAT activity in 24‐hour cultured hepatocytes could be further diminished by incubation of the cells with 1 μmol/L monensin or 10 mmol/L methylamine. We conclude that after disruption of the cell polarity by collagenase isolation of the hepatocytes, remnants of apical membrane containing cMOAT are rapidly endocytosed when the cells are kept at 37°C. Evidence suggests that at least part of the transporters may recycle back to the plasma membrane after endocytosis. These observations may be relevant for the understanding of regulation of canalicular transport.

Crigler-Najjar Syndrome in the Netherlands: Identification of four novel UGT1A1 alleles, genotype-phenotype correlation, and functional analysis of 10 missense mutants

Crigler‐Najjar syndrome (CN), caused by deficiency of UGT isoform 1A1 (UGT1A1), is characterized by severe unconjugated hyperbilirubinemia. In this study we have analyzed 19 CN patients diagnosed in The Netherlands (18) and in Belgium (1), and have identified 14 different UGT1A1 mutations, four of which are novel. Two mutations were present in several unrelated patients, suggesting the presence of two founder effects in The Netherlands. In addition, we show linkage of the UGT1A1∗︁28 promoter polymorphism (rs5719145insTA) to three structural mutations. Functional studies of partial active UGT1A1 mutants are limited. Therefore, we performed in vitro studies to determine the functional activity of seven missense mutants identified in this study and of three reported previously. In addition to bilirubin, we also determined their activity toward eight other UGT1A1 substrates. We demonstrate that five mutants have residual activity that, depending on the substrate, varies from not detectable to 94% of wild‐type UGT1A1 activity. The identification of four novel pathogenic mutations and the analysis of residual activity of 10 UGT1A1 missense mutants are useful for clinical diagnosis, and provides new insights in enzyme activity, whereas the identification of two founder mutations will speed up genetic counseling for newly identified CN patients in The Netherlands. Hum Mutat 30:1–8, 2009.

Persistent unconjugated hyperbilirubinemia after liver transplantation due to an abnormal bilirubin UDP-glucuronosyltransferase gene promotor sequence in the donor

BACKGROUND/AIMS Gilberts syndrome is genetically characterized by an extra TA element in the TATAA-box of the promotor region upstream of the bilirubin UDP-glucuronosyltransferase (UGT1A) coding region (Bosma et al. N Engl J Med 1995; 333: 1171-5). Persistent unconjugated hyperbilirubinemia is occasionally observed in liver transplant recipients with an otherwise normal liver function. We postulate that these patients could have received a liver from a donor with the Gilberts syndrome genotype. Therefore, we investigated the UGT1A-gene TATAA-box in DNA from liver graft donors of jaundiced and non-jaundiced recipients. METHODS DNA was obtained from stored donor lymphocytes and the number of TA elements in the TATAA-box of the UGT1A-gene promotor region was analyzed by polymerase chain-reaction. RESULTS We observed two liver transplant recipients with persistent unconjugated hyperbilirubinemia. They received liver grafts from donors who were homozygous for an abnormal A(TA)7TAA-box in the UGT1A-gene. Four of 10 non-jaundiced recipients received livers from donors who were homozygous for the normal A(TA)6TAA-box and six received livers from donors who were heterozygous with a normal A(TA)6TAA-box on one allele and a prolonged A(TA)7TAA-box on the other allele. CONCLUSIONS This study shows that liver graft recipients with persistent unconjugated hyperbilirubinemia may have received a liver from a donor with an abnormal TATAA-box in the bilirubin UGT1A-gene promotor region.

778. A Novel Genetically Modified Adenovirus Vector Shows Enhanced Infectivity towards Ex Vivo Pancreatic Cancer

Top of pageAbstract Conditionally replicating adenovirus (CRAd) represents a potential therapeutic option for pancreatic cancer (PC). Clinical trials have shown safety and specificity of such adenovirus (Ad) vectors. However, transduction of tumor cells is limited by the low expression of the primary Adenovirus Receptor (CAR) on PC. To overcome this problem, Ad vectors were developed with peptides in the fiber that should allow targeting to PC. A peptide ligand for the Ephrin A2 receptor was incorporated and this vector (Ad-YSA) showed an increase of tumor cell transduction in vitro. Ad-RGD served as a positive control. Since animal models have proven to be of little value in predicting the outcome of oncolytic virotherapy, we used the tissue slice system to explore modified Ad behavior in human tissue. Fresh tissue from normal pancreas and pancreatic adenocarcinoma was obtained from surgical specimens (Whipple procedure). Slices, generated with the Krumdieck tissue slicer, were cultured in medium supplemented with growth factors in a 5/95% CO2/O2 environment. The slices were infected with 1e8 to 5e8 viral particles (wildtype and fiber modified virus harboring the green fluorescent reporter protein (GFP) gene under control of a CMV promoter (Ad.CMV.GFP). As an internal control, to correct for slice size, viability and CAR expression, an equivalent amount of wildtype Ad.CMV.dsRED per slice was added. Three days later reporter protein production was measured by fluorimetry. Morphologic features were determined by HE-staining. These experiments were performed both with primary pancreatic tissue and with tumor tissue. According to morphology, pancreas and tumor tissue could be kept alive for at least three days. Normal pancreas did not show increased transduction efficacy with Ad-RGD or Ad-YSA. Compared to wildtype Ad, Ad-RGD and Ad-YSA revealed a 1.5 to 8.6 and 1.8 to 7.6-fold increase in transduction of pancreatic cancer, respectively (n=7). We observed persistent interpatient variability of infection profiles, which may be related to differential receptor expression. We currently examine CAR, integrin and EphA2 receptor expression in the tissue slices. Our results demonstrate that targeting to the ephrin A2 receptor can augment pancreatic cancer transduction. The tissue slicer technology appears to be of great value to study host-virus interactions. (See Figure)