Gene Therapy in Patients with the Crigler-Najjar Syndrome.

BACKGROUND: Patients with the Crigler-Najjar syndrome lack the enzyme uridine diphosphoglucuronate glucuronosyltransferase 1A1 (UGT1A1), the absence of which leads to severe unconjugated hyperbilirubinemia that can cause irreversible neurologic injury and death. Prolonged, daily phototherapy partially controls the jaundice, but the only definitive cure is liver transplantation. METHODS: We report the results of the dose-escalation portion of a phase 1-2 study evaluating the safety and efficacy of a single intravenous infusion of an adeno-associated virus serotype 8 vector encoding UGT1A1 in patients with the Crigler-Najjar syndrome that was being treated with phototherapy. Five patients received a single infusion of the gene construct (GNT0003): two received 2×1012 vector genomes (vg) per kilogram of body weight, and three received 5×1012 vg per kilogram. The primary end points were measures of safety and efficacy; efficacy was defined as a serum bilirubin level of 300 µmol per liter or lower measured at 17 weeks, 1 week after discontinuation of phototherapy. RESULTS: No serious adverse events were reported. The most common adverse events were headache and alterations in liver-enzyme levels. Alanine aminotransferase increased to levels above the upper limit of the normal range in four patients, a finding potentially related to an immune response against the infused vector; these patients were treated with a course of glucocorticoids. By week 16, serum bilirubin levels in patients who received the lower dose of GNT0003 exceeded 300 µmol per liter. The patients who received the higher dose had bilirubin levels below 300 µmol per liter in the absence of phototherapy at the end of follow-up (mean [±SD] baseline bilirubin level, 351±56 µmol per liter; mean level at the final follow-up visit [week 78 in two patients and week 80 in the other], 149±33 µmol per liter). CONCLUSIONS: No serious adverse events were reported in patients treated with the gene-therapy vector GNT0003 in this small study. Patients who received the higher dose had a decrease in bilirubin levels and were not receiving phototherapy at least 78 weeks after vector administration. (Funded by Genethon and others; ClinicalTrials.gov number, NCT03466463.).

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.

The Effects of Selenium Supplementation on Gene Expression Related to Insulin and Lipid Metabolism, and Pregnancy Outcomes in Patients with Gestational Diabetes Mellitus: a Randomized, Double-Blind, Placebo-Controlled Trial.

This study was performed to evaluate the effects of selenium supplementation on gene expression related to insulin and lipid metabolism, and pregnancy outcomes in patients with gestational diabetes mellitus (GDM). The current randomized, double-blind, placebo-controlled clinical trial was conducted in 36 patients with GDM. Participants were randomly divided into two groups to intake either 200 µg/day selenium supplements as selenium yeast or placebo (n = 18 each group) for 6 weeks. Selenium supplementation upregulated peroxisome proliferator-activated receptor gamma (P = 0.03) and glucose transporter 1 (GLUT-1) (P = 0.01) in lymphocytes of subjects with GDM compared with the placebo. Selenium supplementation did not affect gene expression of low-density lipoprotein receptor (LDLR) and lipoprotein(a) [Lp(a)]. Supplementation with selenium had a significant decrease in incidence of newborns' hyperbilirubinemia (5.6% vs. 33.3%, P = 0.03) and newborns' hospitalization (5.6% vs. 33.3%, P = 0.03) compared with the placebo. Overall, we found that selenium supplementation for 6 weeks among patients with GDM significantly increased PPAR-γ and GLUT-1 expression, but did not affect gene expression of LDLR and LP(a). It also reduced incidence of newborns' hyperbilirubinemia and newborns' hospitalization. Clinical trial registration number: http://www.irct.ir: IRCT20170513033941N35.

Prolonged Hyperbilirubinemia in a Neonate with a Novel Mutation in the UDP-glucuronosyltransferase 1A1 Gene.

The total bilirubin value of a male infant was 385 µmol/l on day 5. Liver function test results were normal and there was no evidence of sepsis and no hemolysis reaction. Phototherapy was administered and on day 8 the patient's total bilirubin level was 255 µmol/l. Intermittent episodes of hyperbilirubinemia occurred without phototherapy, with the total bilirubin level reaching 335 µmol/l on day 19. A 3-day regimen of phenobarbital was administered and on day 24 his total bilirubin level was 180 µmol/l. The patient was discharged. At the age of 2 months, the total bilirubin value was 27 µmol/l. His direct bilirubin value was <15% of total bilirubin in every determination. A family study of the UDP-glucuronosyltransferase(UGT)1A1 gene showed that the infant carries a homozygous mutation at nucleotide -3279 plus compound heterozygous mutations at nucleotides 782 and 1091. The mutation at nucleotide 782 is a novel finding. Gilbert's syndrome was diagnosed.

Hepatotoxicity from anabolic androgenic steroids marketed as dietary supplements: contribution from ATP8B1/ABCB11 mutations?

BACKGROUND: Though possession of androgenic anabolic steroids (AAS) is illegal, non-prescription use of AAS persists. METHODS: We describe two Caucasian males (aged 25 and 45 years) with cholestatic hepatitis following ingestion of the dietary supplement Mass-Drol ('Celtic Dragon') containing the AAS 2α-17α-dimethyl-etiocholan-3-one,17ß-ol. RESULTS: Despite substantial hyperbilirubinaemia peak gamma-glutamyl transferase (GGT) remained normal. Besides 'bland' intralobular cholestasis, liver biopsy in both found deficiency of canalicular expression of ectoenzymes as seen in ATP8B1 disease. In the older patient, bile salt export pump marking (encoded by ABCB11) was focally diminished. We hypothesized that AAS had either induced inhibition of normal ATP8B1/ABCB11 expression or triggered initial episodes of benign recurrent intrahepatic cholestasis (BRIC) type 1/or 2. On sequencing, ATP8B1 was normal in both patients although the younger was heterozygous for the c.2093G>A mutation in ABCB11, a polymorphism previously encountered in drug-induced liver injury. CONCLUSION: AAS marketed as dietary supplements continue to cause hepatotoxicity in the UK; underlying mechanisms may include unmasking of genetic cholestatic syndromes.

Sustained reduction of hyperbilirubinemia in Gunn rats after adeno-associated virus-mediated gene transfer of bilirubin UDP-glucuronosyltransferase isozyme 1A1 to skeletal muscle.

Crigler-Najjar syndrome is an autosomal recessive disorder with severe unconjugated hyperbilirubinemia due to deficiency of bilirubin UDP-glucuronosyltransferase isozyme 1A1 (UGT1A1) encoded by the UGT1A1 gene. Current therapy relies on phototherapy to prevent life-threatening elevations of serum bilirubin levels, but liver transplantation is the only permanent treatment. Muscle-directed gene therapy has several advantages, including easy and safe access through simple intramuscular injections, and has been investigated in human clinical trials. In this study, we have investigated the efficacy of adeno-associated viral (AAV) vector-mediated muscle-directed gene therapy in the preclinical animal model of Crigler-Najjar syndrome, that is the Gunn rat. Serotype 1 AAV vector expressing rat UGT1A1 under the control of muscle-specific creatine kinase promoter was injected at a dose of 3×10(12) genome copies/kg into the muscles of Gunn rats and resulted in expression of UGT1A1 protein and functionally active enzyme in injected muscles. AAV-injected Gunn rats showed an approximately 50% reduction in serum bilirubin levels as compared with saline-treated controls, and this reduction was sustained for at least 1 year postinjection. Increased excretion of alkali-labile metabolites of bilirubin in bile and urine was detected in AAV-injected animals. High-performance liquid chromatography analysis of bile from AAV-injected Gunn rats showed a metabolite with retention time close to that of bilirubin diglucuronide. Taken together, these data show that clinically relevant and sustained reduction of serum bilirubin levels can be achieved by simple and safe intramuscular injections in Gunn rats. AAV-mediated muscle directed gene therapy has potential for the treatment of patients with Crigler-Najjar syndrome type 1.

A novel mutation in the MC2R gene causing familial glucocorticoid deficiency type 1.

Familial glucocorticoid deficiency (FGD) or hereditary unresponsiveness to adrenocorticotropin (ACTH) is an autosomal recessive disorder characterized by isolated glucocorticoid deficiency associated with normal mineralocorticoid secretion. Mutations in genes encoding either ACTH receptor or melanocortin 2 receptor accessory protein are responsible for the disease in about 50% of cases, named FGD type 1 and type 2, respectively. Patients may present with hyperpigmentation, recurrent infections, failure to thrive, hypoglycemic seizures, and coma in infancy or early childhood. Here we report the case of a 17-day-old newborn diagnosed with FGD type 1 who presented with hyperbilirubinemia and hyperpigmentation, a sign which was erroneously assumed to be due to prolonged phototherapy by the referring physician. Hormone analysis showed low cortisol and high ACTH levels with normal serum electrolytes and renin-aldosterone axis. Genetic analysis revealed a novel homozygous melanocortin 2 receptor mutation p.Leu225Arg in the patient. The healthy parents were heterozygous for the mutation.

Severe hyperbilirubinaemia in a Chinese girl with type I Crigler-Najjar syndrome: first case ever reported in Mainland China.

Jaundice is common in ethnic Chinese infants, but to our knowledge Crigler-Najjar syndrome (CN syndrome) type I has never been reported in China. A Chinese girl with severe jaundice was recently diagnosed to have CN syndrome type I by analyzing the bilirubin-uridinediphospho (UDP)-glucuronosyltransferase gene (UGT1A1). The patient was homozygous for a nonsense mutation that replaced glutamine (CAG, amino acid 239) with stop codon (TAG) at nucleotide number 715 (715C-->T) in exon 1. No mutation was found in exons 2-5. Her parents were heterozygous for the same mutant. The patient had an average bilirubin level of 300-500 mumol/L and a peak of 701 mumol/L. Daily phototherapy for 15 h was required to keep the bilirubin levels within 280-320 mumol/L. The unconjugated hyperbilirubinaemia apparently resulted from homozygous nonsense mutation of UGT1A1, which could completely abolish the UGT activity towards bilirubin (hepatic glucuronidation) and result in CN syndrome type I. Identification of the genetic defect is very useful for gene therapy, especially for DNA/RNA chimera therapy, and can be used as an antenatal screening test to identify the affected offsprings.

Coinheritance of two alpha-spectrin gene defects in a recessive spherocytosis family.

We studied a recessive hereditary spherocytosis (HS) family from Norway in which all four children had haemolytic spherocytosis while spectrin (Sp) deficiency was detected in the proband. Molecular analysis demonstrated that all affected children had inherited the low expression alpha-Sp allele LEPRA (Low Expressed PRAgue) from the father. Haplotyping with a polymorphic dinucleotide repeat for the alpha-Sp gene (alphaVNTR) located in the 3' untranslated region of mRNA showed that all recessive children had inherited the same maternal alpha-spectrin allele. The paternal Sp-alphaLEPRA allele was found in cis of the polymorphic alpha-Sp Bughill allele (alphaBH) characterized by the A970D point mutation in the Sp alpha-chain. This mutation was identified on two-dimensional electrophoresis of Sp tryptic digests as an acidic shift of the alphaII tryptic domains (spots alphaIIa). Analyses of the relative expression of the paternal alpha-Sp Bughill polymorphism in the proband showed that the product of the maternal alpha-Sp gene is almost completely absent from the mature erythrocyte membrane. Comparative analysis between alphaVNTR PCR-amplified from genomic DNA and from cDNA showed that the maternal low expression alpha-Sp allele is associated with a decreased amount of mRNA. Results from molecular and biochemical studies showed that all the affected children of this family are compound heterozygous for two different low expression alpha-Sp alleles: an uncharacterized defective alpha-Sp allele on the maternal side and an alphaLEPRA allele tagged by the alphaIIa polymorphism on the paternal side.

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.