Rotor Syndrome: Glucuronidated Bile Acidemia From Defective Reuptake by Hepatocytes.

Organic anion transporting polypeptide (OATP) 1B1 (gene, solute carrier organic anion transporter family member 1B1 [SLCO1B1]) and OATP1B3 (SLCO1B3) serve as transporters for hepatic uptake of important endogenous substances and several commonly prescribed drugs. Inactivation of both proteins together causes Rotor syndrome. How this OATP1B1/1B3 defect disturbs bile acid (BA) metabolism is largely unknown. In this study, we performed detailed BA analysis in 3 patients with genetically diagnosed Rotor syndrome. We found that BAs glucuronidated at the C-3 position (BA-3G) accounted for 50% or more of total BAs in these patients. In contrast but similarly to healthy controls, only trace amounts of BA-3G were detected in patients with constitutional indocyanine green excretory defect (OATP1B3 deficiency) or sodium-taurocholate cotransporting polypeptide (NTCP; gene, solute carrier family 10 member 1 [SLC10A1]) deficiency. Therefore, substantial amounts of BA-3G are synthesized in hepatocytes. The cycling pathway of BA-3G, consisting of excretion from upstream hepatocytes and uptake by downstream hepatocytes by OATP1B1/1B3 may exist to reduce the burden on upstream hepatocytes. Conclusion: Detailed BA analysis revealed glucuronidated bile acidemia in patients with Rotor syndrome. Further exploration of the physiologic role of glucuronidated BAs is necessary.

Low efficacy of recombinant SV40 in Ugt1a1-/- mice with severe inherited hyperbilirubinemia.

In contrast to AAV, Simian Virus 40 (rSV40) not inducing neutralizing antibodies (NAbs) allowing re-treatment seems a promising vector for neonatal treatment of inherited liver disorders. Several studies have reported efficacy of rSV40 in animal models for inherited liver diseases. In all studies the ubiquitous endogenous early promoter controlled transgene expression establishing expression in all transduced tissues. Restricting this expression to the target tissues reduces the risk of immune response to the therapeutic gene. In this study a liver specific rSV40 vector was generated by inserting a hepatocyte specific promoter. This increased the specificity of the expression of hUGT1A1 in vitro. However, in vivo the efficacy of rSV40 appeared too low to demonstrate tissue specificity while increasing the vector dose was not possible because of toxicity. In contrast to earlier studies, neutralizing antibodies were induced. Overall, the lack of a platform to produce high titered and pure rSV40 particles and the induction of NAbs, renders it a poor candidate for in vivo gene therapy.

Analysis of uridine diphosphate glucuronosyl transferase 1A1 gene mutations in neonates with unconjugated hyperbilirubinemia.

This study was carried out to analyze uridine diphosphate (UDP)-glucuronosyltransferase 1A1 (UGT1A1) gene mutations in neonates with unconjugated hyperbilirubinemia, from two different ethnic groups. Polymerase chain reaction and gene sequencing were used to analyze the differences in genotypes and allele frequencies of different gene mutations among the ethnic groups; this was followed by checking their correlation with the serum bilirubin level and the occurrence of unconjugated hyperbilirubinemia in neonates. Our results reveal that the UGT1A1 mutant genotype, 211G>A, is distributed differently in the case vs control groups, as well as in the Zhuang vs Han ethnic groups. Moreover, this difference is statistically significant (P < 0.05); the total serum bilirubin (TSB) and unconjugated bilirubin (UCB) levels in patients carrying the single homozygous mutation, 211G>A, were markedly higher than that in patients without the mutation (P < 0.05). Furthermore, the TSB and UCB levels were significantly different between patients carrying single or compound 211G>A heterozygous mutation, (TA)6/7, and 1941C>G/2042C>G heterozygous mutation, and patients without mutation (P > 0.05). Our findings suggest that the 211G>A mutation in the first exon may be a risk factor for unconjugated hyperbilirubinemia in Zhuang and Han neonates. The serum bilirubin levels seem to be affected by the homozygosity or heterozygosity of the UGT1A1 gene mutation; 211G>A homozygous mutation is an important factor that causes a rise in bilirubin in neonates with unconjugated hyperbilirubinemia.

Clinical features of three girls with mosaic genome-wide paternal uniparental isodisomy.

Here we describe three subjects with mosaic genome-wide paternal uniparental isodisomy (GWpUPD) each of whom presented initially with overgrowth, hemihyperplasia (HH), and hyperinsulinism (HI). Due to the severity of findings and the presence of additional features, SNP array testing was performed, which demonstrated mosaic GWpUPD. Comparing these individuals to 10 other live-born subjects reported in the literature, the predominant phenotype is that of pUPD11 and notable for a very high incidence of tumor development. Our subjects developed non-metastatic tumors of the adrenal gland, kidney, and/or liver. All three subjects had pancreatic hyperplasia resulting in HI. Notably, our subjects to date display minimal features of other diseases associated with paternal UPD loci. Both children who survived the neonatal period have displayed near-normal cognitive development, likely due to a favorable tissue distribution of the mosaicism. To understand the range of UPD mosaicism levels, we studied multiple tissues using SNP array analysis and detected levels of 5-95%, roughly correlating with the extent of tissue involvement. Given the rapidity of tumor growth and the difficulty distinguishing malignant and benign tumors in these GWpUPD subjects, we have utilized increased frequency of ultrasound (US) and alpha-fetoprotein (AFP) screening in the first years of life. Because of a later age of onset of additional tumors, continued tumor surveillance into adolescence may need to be considered in these rare patients.

Liver repopulation with bone marrow derived cells improves the metabolic disorder in the Gunn rat.

BACKGROUND: Reversible ischaemia/reperfusion (I/R) liver injury has been used to induce engraftment and hepatic parenchymal differentiation of exogenous beta2-microglubulin(-)/Thy1(+) bone marrow derived cells. AIM: To test the ability of this method of hepatic parenchymal repopulation, theoretically applicable to clinical practice, to correct the metabolic disorder in a rat model of congenital hyperbilirubinaemia. METHODS AND RESULTS: Analysis by confocal laser microscopy of fluorescence labelled cells and by immunohistochemistry for beta2-microglubulin, 72 hours after intraportal delivery, showed engraftment of infused cells in liver parenchyma of rats with I/R, but not in control animals with non-injured liver. Transplantation of bone marrow derived cells obtained from GFP-transgenic rats into Lewis rats resulted in the presence of up to 20% of GFP positive hepatocytes in I/R liver lobes after one month. The repopulation rate was proportional to the number of transplanted cells. Infusion of GFP negative bone marrow derived cells into GFP positive transgenic rats resulted in the appearance of GFP negative hepatocytes, suggesting that the main mechanism underlying parenchymal repopulation was differentiation rather than cell fusion. Transplantation of wild type bone marrow derived cells into hyperbilirubinaemic Gunn rats with deficient bilirubin conjugation after I/R damage resulted in 30% decrease in serum bilirubin, the appearance of bilirubin conjugates in bile, and the expression of normal UDP-glucuronyltransferase enzyme evaluated by polymerase chain reaction. CONCLUSIONS: I/R injury induced hepatic parenchymal engraftment and differentiation into hepatocyte-like cells of bone marrow derived cells. Transplantation of bone marrow derived cells from non-affected animals resulted in the partial correction of hyperbilirubinaemia in the Gunn rat.

Dual hereditary jaundice: simultaneous occurrence of mutations causing Gilbert's and Dubin-Johnson syndrome.

BACKGROUND & AIMS: Dubin-Johnson syndrome is recessively inherited, conjugated hyperbilirubinemia induced by mutations in the ABCC2/MRP2 gene encoding the canalicular transporter for conjugated bilirubin. Gilbert's syndrome is recessively inherited, unconjugated hyperbilirubinemia caused by decreased conjugation rate of bilirubin associated mostly with homozygous A(TA) 7 TAA variant of the TATAA-box in the UGT1A1 gene promoter. Our aim was to establish the molecular diagnosis in a 3-year-old male with atypical, intermittent, predominantly unconjugated, hyperbilirubinemia. METHODS: 99m Tc-HIDA cholescintigraphy was used for imaging the biliary tree. Expression of ABCC2/MRP2 protein in hepatocytes was investigated immunohistochemically. UGT1A1 and ABCC2/MRP2 genes were sequenced from genomic DNA, and the mutations were verified by fragment analysis, sequencing the cloned exons, and restriction fragment length polymorphism. RESULTS: Cholescintigraphy revealed delayed visualization of the gallbladder. A brown granular lipopigment differing from melanin-like pigment reported in Dubin-Johnson syndrome was present in hepatocytes, but, otherwise, liver histology was normal. ABCC2/MRP2 protein was not detected on the canalicular membrane of hepatocytes, and 2 novel mutations were found in the ABCC2/MRP2 gene: a heterozygous in-frame insertion-deletion mutation 1256insCT/delAAACAGTGAACCTGATG in exon 10 inherited from the father and a heterozygous deletion 4292delCA in exon 30 inherited from the mother. In addition, the patient was homozygous for -3279T>G and A(TA) 7 TAA mutations in the UGT1A1 gene promoter. CONCLUSIONS: Our patient represents a case of digenic mixed hyperbilirubinemia-a distinct type of constitutive jaundice resulting from coinherited defects in ABCC2/MRP2 and UGT1A1 genes.

Umbilical vein and placental vessels from newborns with hereditary haemorrhagic telangiectasia type 1 genotype are normal despite reduced expression of endoglin.

Hereditary haemorrhagic telangiectasia, HHT, is an autosomal dominant disorder that affects approximately 1 in 8000 people. HHT1 is associated with mutations in the ENG (Endoglin) gene and with haploinsufficiency. The disorder is characterized by focally dilated vessels, which can lead to arteriovenous malformations and serious complications even in young children. In the current study, umbilical cord and placenta samples from newborns with ENG mutations were analyzed to estimate the level of corresponding protein and look for potential vascular dysplasia. We confirmed, using metabolic labelling and flow cytometry, that endoglin levels were significantly reduced to median values of 47 per cent (range 32-56 per cent) and 58 per cent (46-90 per cent), respectively, in human umbilical vein endothelial cells derived from newborns with ENG mutations (HHT1 group; n=18) relative to samples from newborns shown not to have the familial mutation (non-HHT group). We also quantified the relative expression of endoglin by estimating the endoglin/PECAM-1 staining ratio in tissue sections. We observed significantly lower values in the HHT1 group, compared to the non-HHT group for the umbilical vein (n=9; median 0.6 vs 0.9; ranges 0.2-1.0 and 0.5-1.5) and for placental stem villus vessels (n=9 and 10; median 0.42 vs 0.93; ranges 0.24-0.58 and 0.56-1.18). No differences in the estimated umbilical vein cross-sectional area and in the proportion of vessels present in placental villi were observed in sections from the HHT1 group relative to the non-HHT group. Thus, blood vessels from HHT1 individuals are maintained intact in the umbilical vein and placenta during pregnancy and delivery, despite a significant reduction in endoglin expression.

A new rat mutant with chronic conjugated hyperbilirubinemia and renal glomerular lesions.

A new mutant strain of inbred Sprague Dawley rats with autosomal recessive hyperbilirubinuria, were studied by biochemical, histologic, and ultrastructural methods. The plasma bilirubin concentration in the homozygote was significantly higher than that of the heterozygote, and about 80% of the bilirubin was conjugated. Plasma BSP and ICG clearance were both severely delayed in the homozygote. Plasma BSP elimination kinetics suggested that the pathophysiologic defect was not hepatic uptake or storage but rather in secretion into bile. Histopathology of the liver demonstrated brown pigment in the hepatocytes that appeared to be lipofuscin. The electron microscopic features of the hepatic pigment resembled those of the Dubin-Johnson syndrome. Homozygote histopathology also revealed glomerular lesions with mesangial expansion and proliferation in the kidneys. Immunohistologic studies disclosed mesangial granular deposition of IgG, IgA, and to a lesser degree, IgM and C3. These renal changes resembled those of IgA nephropathy. The spontaneous hyperbilirubinuric rat (EHBR) may be a useful animal model for studying constitutive conjugated hyperbilirubinemia, bilirubin metabolism, cholestasis, and glomerulonephropathy subsequent to hepatic dysfunction.

[Ultrasonic examination of the epigastrium in persons with Gilbert's syndrome]. / Ultrazvukové vysetrení nadbrisku u osob s Gilbertovým syndromem.

In 29 subjects with Gilbert's syndrome during ultrasonographic examination of the epigastrium in real time a significantly larger anteroposterior dimension of the right liver lobe (by cca 11%] was found. In the other investigated dimensions no significant deviations were found, not even a significantly higher echogeneity of the liver parenchyma. In two instances multiple cholecystolithiasis was detected. Sonography, a non-invasive method with a high yield, is an important link in the examination programme of subjects with Gilbert's syndrome.

[Manifestation of Gilbert syndrome (Meulengracht disease) following orthotopic liver transplantation: a rare cause of postoperative hyperbilirubinemia]. / Manifestation eines Gilbert-Syndroms (Morbus Meulengracht) nach orthotoper Lebertransplantation: Seltene Ursache einer postoperativen Hyperbilirubinämie.

In a 33 years old woman an orthotopic liver transplantation was performed because of an endstage posthepatic liver cirrhosis. Postoperatively the unconjugated bilirubin levels remained elevated although liver enzyme values were within normal range. Because hemolysis, rejection, infection or biliary obstruction could be excluded we suspected a Gilbert's Syndrome and were able to confirm this diagnosis by low caloric intake and nicotinic acid test. This case report therefore describes the first time the transplantation of a clinically harmless metabolic disorder but inborn error of metabolism by liver grafting into the recipient.

Pericanalicular microfilaments of hepatocytes in patients with familial non-hemolytic hyperbilirubinemia.

We observed pericanalicular webs (PCW) of liver cells in cases with familial non-hemolytic hyperbilirubinemia using electron microscopy. The area and width of PCW were determined by morphometric methods as a way of quantitating this feature. The mean PCW width was 0.175 +/- 0.003 micron (mean +/- SE) in Dubin-Johnson syndrome and 0.184 +/- 0.005 micron in Rotor's syndrome. In both of these syndromes PCW width was significantly larger than that in Gilbert's syndrome (0.124 +/- 0.003 micron) (p less than 0.01). The mean PCW area was 0.585 +/- 0.017 micron 2 in Dubin-Johnson syndrome and 0.582 +/- 0.030 micron 2 in Rotor's syndrome. Values in these two syndromes were significantly larger than that in Gilbert's syndrome (0.382 +/- 0.014 micron 2) (p less than 0.01). Widths and areas of PCW in these three syndromes were not significantly different between central, intermediate, and peripheral zones of the hepatic lobules. There was a positive correlation between serum direct bilirubin levels and widths or areas of PCW in these syndromes. These results suggested that disturbances of bile flow caused by the dysfunction of pericanalicular microfilaments are partly involved in the pathogenesis of Dubin-Johnson syndrome and Rotor's syndrome.

Astrocyte response to perinatal liver disease, hyperammonemia, and hyperbilirubinemia: an immunohistochemical study.

Astrocytic reaction to perinatal brain damage, which is caused by hyperammonemia, liver disease, hyperbilirubinemia, and a few other conditions, was studied using immunohistochemical methods for the demonstration of glial fibrillary acidic protein (GFAP). We found no increase in GFAP expression in those areas where Alzheimer II astrocytes usually proliferate. Diffuse astrocytic proliferation in the white matter and focal reaction in gray matter, which we ascribe to complicating factors, the foremost of which is anoxia, was found in many of the cases.

Histochemical observations of lipids in the cerebellum of young Gunn rats.

Cerebella of 20-day-old homozygous (jj) and heterozygous (j+) Gunn rats were examined histochemically for the presence of lipids. The j+ rat cerebellum showed no lipid granules, whereas many granules were recognized in the hypoplastic jj rat cerebellum. The degree of appearance of lipid granules differed considerably among the lobes--a high content in the declive and tuber but none in the nodulus. Two kinds of cells showed a strong acid phosphatase activity and contained many lipid granules. It is suggested that the accumulation of lipids, probably cholesteryl esters, is closely related to the enhanced activity of the lysosomal system in the hypoplastic jj rat cerebellum severely injured by bilirubin.