Gilbert or Crigler-Najjar syndrome? Neonatal severe unconjugated hyperbilirubinemia with P364L UGT1A1 homozygosity.

BACKGROUND: Several 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 PRESENTATION: Two 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. CONCLUSION: UGT1A1 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.

UGT1A1 dysfunction increases liver burden and aggravates hepatocyte damage caused by long-term bilirubin metabolism disorder.

UGT1A1 is the only enzyme that can metabolize bilirubin, and its encoding gene is frequently mutated. UGT1A1*6 (G71R) is a common mutant in Asia which leads to the decrease of UGT1A1 activity and abnormal bilirubin metabolism. However, it is not clear whether low UGT1A1 activity-induced bilirubin metabolism disorder increases hepatocyte fragility. ugt1a+/- mice were used to simulate the UGT1A1*6 (G71R) population. Under the same CCl4 induction condition, ugt1a+/- mice showed severer liver damage and fibrosis, indicating that ugt1a1 dysfunction increased liver burden and aggravated hepatocyte damage. In the animal experiment with a continuous intraperitoneal injection of bilirubin, the ugt1a+/- mice livers had more serious unconjugated bilirubin accumulation. The accumulated bilirubin leads to hyperphosphorylation of IκB-α, Ikk-ß, and p65 and a significant increase of inflammatory factor. The α-SMA and Collagen I proteins markedly up-regulated in the ugt1a+/- mice livers. Immunofluorescence and confocal microscopy showed that hepatic stellate cells and Kupffer cells were activated in ugt1a+/- mice. Comprehensive results show that there was a crosstalk relationship between low UGT1A1 activity-bilirubin-liver damage. Furthermore, cell experiments confirmed that unconjugated bilirubin activated the NF-κB pathway and induced DNA damage in hepatocytes, leading to the significant increase of inflammatory factors. UGT1A1 knockdown in hepatocytes aggravated the toxicity of unconjugated bilirubin. Conversely, overexpression of UGT1A1 had a protective effect on hepatocytes. Finally, Schisandrin B, an active ingredient with hepatoprotective effects, extracted from a traditional Chinese medicinal herb, which could protect the liver from bilirubin metabolism disorders caused by ugt1a1 deficiency by downregulating p65 phosphorylation, inhibiting Kupffer cells, reducing inflammation levels. Our data clarified the mechanism of liver vulnerability caused by cross-talk between low UGT1A1 activity bilirubin, and provided a reference for individualized prevention of liver fragility in Gilbert's syndrome.

Bilirubin Decreases Macrophage Cholesterol Efflux and ATP-Binding Cassette Transporter A1 Protein Expression.

BACKGROUND: Mild but chronically elevated circulating unconjugated bilirubin is associated with reduced total and low-density lipoprotein cholesterol concentration, which is associated with reduced cardiovascular disease risk. We aimed to investigate whether unconjugated bilirubin influences macrophage cholesterol efflux, as a potential mechanism for the altered circulating lipoprotein concentrations observed in hyperbilirubinemic individuals. METHODS AND RESULTS: Cholesterol efflux from THP-1 macrophages was assessed using plasma obtained from normo- and hyperbilirubinemic (Gilbert syndrome) humans (n=60 per group) or (heterozygote/homozygote Gunn) rats (n=20 per group) as an acceptor. Hyperbilirubinemic plasma from patients with Gilbert syndrome and Gunn rats induced significantly reduced cholesterol efflux compared with normobilirubinemic plasma. Unconjugated bilirubin (3-17.1 µmol/L) exogenously added to plasma- or apolipoprotein A1-supplemented media also decreased macrophage cholesterol efflux in a concentration- and time-dependent manner. We also showed reduced protein expression of the ATP-binding cassette transporter A1 (ABCA1), a transmembrane cholesterol transporter involved in apolipoprotein A1-mediated cholesterol efflux, in THP-1 macrophages treated with unconjugated bilirubin and in peripheral blood mononuclear cells obtained from hyperbilirubinemic individuals. Furthermore, we demonstrated that bilirubin accelerates the degradation rate of the ABCA1 protein in THP-1 macrophages. CONCLUSIONS: Cholesterol efflux from THP-1 macrophages is decreased in the presence of plasma obtained from humans and rats with mild hyperbilirubinemia. A direct effect of unconjugated bilirubin on cholesterol efflux was demonstrated and is associated with decreased ABCA1 protein expression. These data improve our knowledge concerning bilirubin's impact on cholesterol transport and represent an important advancement in our understanding of bilirubin's role in cardiovascular disease.

Crigler-Najjar syndrome type I in a Turkish newborn caused by a novel mutation and Gilbert type genetic defect.

Crigler-Najjar syndrome (CNS), caused by deficiency of bilirubin uridine diphosphate glucuronosyltransferase (UGT) 1A1, is a rare and autosomal recessive inherited disorder characterized by severe unconjugated nonhemolytic hyperbilirubinemia since birth. We present a girl with CNS type I caused by a novel mutation and Gilbert type genetic defect. Gilbert's Syndrome (GS) and CNS type I both involve abnormalities in bilirubin conjugation secondary to deficiency of bilirubin UGT. The combined defects even in benign genetic forms were shown to cause more serious clinical disease. The patient has been treated with daily home-based phototherapy for more than nine months and considered as a candidate for liver transplantation.

Neonatal hyperbilirubinemia and the bilirubin uridine diphosphate-glucuronosyltransferase gene: the common -3263T > G mutation of phenobarbital response enhancer module is not associated with the neonatal hyperbilirubinemia in Japanese.

BACKGROUND: Neonatal hyperbilirubinemia is frequent and severe in Japanese newborns. Previously, it has been reported that half of the Japanese neonates with severe hyperbilirubinemia carried the 211G > A (p.G71R) mutation of the bilirubin uridine diphosphate-glucuronosyltransferase (UGT1A1) gene causing Gilbert syndrome. Recently, it was reported that the -3263T > G mutation in the phenobarbital response enhancer module in UGT1A1 was associated with the majority of cases of Gilbert syndrome. The gene frequency of the -3263T > G mutation was determined and the relation with neonatal hyperbilirubinemia in Japanese was studied. METHODS: UGT1A1 in 119 neonates born at Yamagata University Hospital, Yamagata, Japan, and 26 subjects who had undergone phototherapy due to severe hyperbilirubinemia at four other hospitals were studied. The gene frequency of -3263T > G mutation in Japanese, Korean, Chinese and German healthy adult controls was also determined. Hyperbilirubinemia was assessed with a Jaundice Meter and UGT1A1 was analyzed by sequence determination or restriction enzyme method. RESULTS: The gene frequency of the -3263T > G mutation was 0.26 in Japanese subjects and was similar to the prevalence in Korean, Chinese and German populations. However, there was no significant increase in the gene frequency of the mutation in the neonates who required phototherapy for hyperbilirubinemia compared to that in the neonates without severe hyperbilirubinemia. In addition, neonates with or without the mutation did not show a significant change in the level of bilirubin and the mutation also did not show a synergic effect with the 211G > A mutation on the level of bilirubin. CONCLUSION: The -3263T > G mutation is not likely to be associated with the neonatal hyperbilirubinemia in Japanese.

Clinical and laboratory manifestations of congenital dyserythropoietic anemia type I in young adults.

OBJECTIVES: Congenital dyserythropoietic anemia (CDA) type I is a rare autosomal recessive macrocytic anemia whose natural history is not well documented. The aim of the present study was to evaluate the clinical picture of the disease in young adults. METHODS: The study sample consisted of 17 patients of mean age 11.9 +/- 5.4 yr (range 18-33 yr) and one older patient (age 44 yr), all Israeli Bedouins. The degree of anemia was evaluated as well as the extent of development of gallstones and iron overload. In each subject we determined the hemochromatosis gene mutations and the uridine dyphosphate-glucoronosyltransferase (UGT-1A) gene polymorphism associated with Gilbert's syndrome. RESULTS: The patients were found to have moderate anemia, with the women displaying lower mean hemoglobin levels than the men (8.2 +/- 0.9 g dL(-1) vs. 10 +/- 1.3 g dL(-1); P=0.0059). The majority of patients (59%) had received at least one blood transfusion, with the women having a significantly higher transfusion requirement. Although delayed puberty was noted, final height and weight were within normal limits, and eight patients had progeny. Biliary stones were found in three of 16 patients, two of whom were homozygous for UGT-1A gene polymorphism. None of the patients carried the common hemochromatosis gene mutation, although serum ferritin levels were moderately elevated (788 +/- 332 ng mL(-1)). CONCLUSIONS: CDA type I in young adults is characterized by moderate macrocytic anemia, more severe in women, and a tendency to cholelithiasis and secondary progressive iron overload. We suggest that iron overload in this patient population should be monitored and chelation therapy initiated when indicated to prevent organ damage

Association of neonatal hyperbilirubinemia with bilirubin UDP-glucuronosyltransferase polymorphism.

OBJECTIVE: The incidence of nonphysiologic neonatal hyperbilirubinemia is twice as high in East Asians as in whites. We studied whether the condition was associated with mutations in the gene for bilirubin uridine 5'-diphosphate-glucuronosyltransferase (UGT1A1), a key enzyme of bilirubin catabolism. DESIGN: We analyzed the UGT1A1 gene in 25 Japanese neonates who had nonphysiologic hyperbilirubinemia (serum bilirubin >257 micromol/L) with no obvious cause. They had all received phototherapy. The background control population consisted of 50 Japanese neonates whose transcutaneous jaundice index was monitored during the first week of life. We detected mutations by direct sequencing of polymerase chain reaction-amplified fragments of the gene. RESULTS: We found a polymorphism for UGT1A1 in exon 1; a G-->A transition at nucleotide 211 caused arginine to replace glycine at position 71 of corresponding protein product (G71R). The frequency of the mutated allele in the hyperbilirubinemic group (0.34) was significantly higher (chi2 = 5.56) than in the control group (0.16). In the control group the peak transcutaneous jaundice index of the carriers of G71R was significantly higher than it was in the normal infants. CONCLUSIONS: The missense mutation causing G71R is the first reported polymorphism for UGT1A1, and the mutation is a risk factor for nonphysiologic neonatal hyperbilirubinemia. The high incidence of hyperbilirubinemia in the Japanese may be attributable to the high frequency of this missense mutation.

Neonatal hyperbilirubinemia and a common mutation of the bilirubin uridine diphosphate-glucuronosyltransferase gene in Japanese.

Neonatal hyperbilirubinemia, which is prevalent among Asian peoples, has been considered as a physiological phenomenon, and its metabolic basis has not been clearly explained. Gilbert syndrome is a common inherited disease of unconjugated hyperbilirubinemia due to decreased bilirubin uridine diphosphate-glucuronosyltransferase (B-UGT), and its role in neonatal jaundice has recently been considered. We have previously reported that the Gly71Arg mutation of the B-UGT gene associated with Gilbert syndrome is prevalent in Japanese, Korean, and Chinese populations and was more frequently detected in neonates with severe hyperbilirubinemia than in control subjects. We have studied 159 Japanese full-term neonates, evaluating the relationship between the B-UGT genotype and the severity of jaundice, as assessed with a transcutaneous bilirubinometer. The gene frequency of the Gly71Arg mutation in these neonates was 0.19, and neonates carrying the Gly71Arg mutation had significantly increased bilirubin levels on days 2-4, manifested in a gene dose-dependent manner. The frequency of the Gly71Arg mutation was 0.47 in the neonates who required phototherapy (i.e., those with more severe hyperbilirubinemia), significantly higher than 0.16 in the neonates who did not require the therapy. The gene frequency of the TA repeat promoter polymorphism, the (TA)7 mutation, was 0.07, and neonates carrying this mutation did not have an increase in bilirubin. These results suggested that the Gly71Arg mutation contributes to the high incidence of neonatal hyperbilirubinemia in Japanese.

Familial unconjugated hyperbilirubinemia syndromes.

Our understanding of the biochemical defects underlying the hepatic forms of congenital, unconjugated hyperbilirubinemias has been greatly enhanced over the past decade. This is mostly due to the availability of pure, labeled bilirubin, the appropriate kinetic analyses, and a better understanding of the mechanisms underlying bilirubin conjugation. Although it is quite obvious that the defect underlying Gilbert's and Crigler-Najjar syndromes is deficient glucuronidation, the molecular explanation may eventually be found in altered composition of the microsomal lipids rather than in a protein defect of glucuronyl transferase. The recognition that Gilbert's syndrome is a quite heterogeneous entity will allow a better understanding of the mode of inheritance of this disorder; its relationship to Crigler-Najjar type II disease also awaits further definition. It is hoped that definition of the molecular defect in Crigler-Najjar type I will lead to better therapeutic modalities, but this remains to be seen.