Crigler-Najjar syndrome: looking to the future does not make us forget the present.

Recently, the safety and efficacy of gene therapy were evaluated in patients with Crigler-Najjar syndrome (CNS). Although it is a promising curative option for CNS, many doubts still persist about its long-term efficacy and safety. Furthermore, there is a risk of overlooking several unresolved problems still present in current clinical practice. This letter is a call for action on crucial open issues that remain nowadays an unmet need in the management of CNS patients.

Synthetic augmentation of bilirubin metabolism in human pluripotent stem cell-derived liver organoids.

UGT1A1 (UDP glucuronosyltransferase family 1 member A1) is the primary enzyme required for bilirubin conjugation, which is essential for preventing hyperbilirubinemia. Animal models lack key human organic anion transporting polypeptides with distinct epigenetic control over bilirubin metabolism, necessitating a human model to interrogate the regulatory mechanism behind UGT1A1 function. Here, we use induced pluripotent stem cells to develop human liver organoids that can emulate conjugation failure phenotype. Bilirubin conjugation assays, chromatin immunoprecipitation, and transcriptome analysis elucidated the role of glucocorticoid antagonism in UGT1A1 activation. This antagonism prevents the binding of transcriptional repressor MECP2 at the expense of NRF2 with associated off-target effects. Therefore, we introduced functional GULO (L-gulonolactone oxidase) in human organoids to augment intracellular ascorbate for NRF2 reactivation. This engineered organoid conjugated more bilirubin and protected against hyperbilirubinemia when transplanted in immunosuppressed Crigler-Najjar syndrome rat model. Collectively, we demonstrate that our organoid system serves as a manipulatable model for interrogating hyperbilirubinemia and potential therapeutic development.

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.).

[A family study of the compound heterozygous mutation of the UGT1A1 gene causing Crigler-Najjar syndrome type II].

Objective: To investigate the family gene features in Crigler-Najjar syndrome (CNS) type II. Methods: The UGT1A1 gene and related bilirubin metabolism genes were comprehensively analysed in a CNS-II family (3 CNS-II, 1 Gilbert syndrome, and 8 normal subjects). The genetics basis of CNS-II were investigated from the perspective of family analysis. Results: In three cases, compound heterozygous mutations at three sites of the UGT1A1 gene (c.-3279T > G, c.211G > A and c.1456T > G) caused CNS-II. Gilbert syndrome and CNS-II were not significantly associated with distribution or diversity loci. Conclusion: The compound heterozygous pathogenic mutations (c.-3279T > G, c.211G > A, and c.1456T > G) at three loci of the UGT1A1 gene may be the feature of the newly discovered CNS-II family genes based on the CNS-II family study.

Novel combined UGT1A1 mutations in Crigler Najjar Syndrome type I.

BACKGROUND: Uridine diphosphate-glucuronosyl transferase 1A1 (UGT1A1), which is the major UGT1 gene product, is located on chromosome 2q37. The expression of UGT1A1 is relatively managed by a polymorphic dinucleotide repeat inside the promoter TATA box consisting of 5-8 copies of a TA repeat. A (TA) 6TAA is considered as the wild type. The A (TA) 7TAA allele has been identified as the most frequent allele in the Caucasian populations while A (TA) 8TAA allele remains the rarest allele worldwide in North Africa, including the Arab populations. METHODS: The spectrum of UGT1A1 genetic mutations in seventeen Tunisian children affected by persistent unconjugated hyperbilirubinemias is represented in addition to their relatives, notably parents, sisters, and brothers. Tunisian children, from 16 unrelated families as well as a 17th family without CN1 affected child, were originated from the West Center of Tunisia. The promoter region and coding exons of the UGT1A1 were PCR amplified, subsequently subjected to Sanger sequencing. RESULTS: The frequencies of genotypes in CN1 patients were as follows (TA) (7/7) (12/17: 70.6%) and (TA) (8/8) (5/17: 29.4%). All patients harbored the c.1070A>G mutation of exon 3 (UGT1A1*16) in the homozygous state. Among relatives of our patients (n = 16), who were all heterozygotes for UGT1A1*16, 13/16 (81.25%) had a heterozygous state for UGT1A1∗1/UGT1A1∗28 or (TA) (6/7) and, 18.75% (3/16) were heterozygous for UGT1A1∗28/UGT1A1∗37 or (TA) (7/8) of the promoter polymorphisms. CONCLUSION: UGT1A1*16 accompanied with UGT1A1*28 or UGT1A1*37 had a specific geographic and ethnic distribution for CN pathogenesis in this Tunisian cohort.

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.

Paternal uniparental disomy of chromosome 2 resulting in a concurrent presentation of Crigler-Najjar syndrome type I and long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency.

This is the first report of the concurrent development of long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and Crigler-Najjar syndrome type 1 (CNs1) inherited via uniparental disomy of chromosome 2, which are both autosomal recessive pathologies. Through an expanded newborn metabolic panel, a male infant was identified as having an acylcarnitine pattern typical for LCHADD, later confirmed to be caused by a well-characterized pathogenic variant in the HADHA gene located at 2p23. Prolonged non-hematologic jaundice requiring repetitive phototherapy prompted further genetic analysis, leading to the identification of another genetic abnormality consistent with CNs1, which was caused by a novel pathogenic variant in the UGT1A1 gene located at 2q37. The two identified point mutations in chromosome 2 were homozygous and present on separate arms, which indicated potential uniparental disomy. Microarray analysis of the genetic material from the patient and his parents confirmed paternal isodisomy of chromosome 2. Further studies are needed to identify other possible pathogenic variants located on the same defective chromosome, evaluate the combined effect of the two metabolic abnormalities, and plan the best possible treatment and care.

The relationship between UGT1A1 gene & various diseases and prevention strategies.

UDP-glucuronyltransferase 1A1 (UGT1A1) is a member of the Phase II metabolic enzyme family and the only enzyme that can metabolize detoxified bilirubin. Inactivation and very low activity of UGT1A1 in the liver can be fatal or lead to lifelong Gilbert's syndrome (GS) and Crigler-Najjar syndrome (CN). To date, more than one hundred UGT1A1 polymorphisms have been discovered. Although most UGT1A1 polymorphisms are not fatal, which diseases might be associated with low activity UGT1A1 or UGT1A1 polymorphisms? This scientific topic has been studied for more than a hundred years, there are still many uncertainties. Herein, this article will summarize all the possibilities of UGT1A1 gene-related diseases, including GS and CN, neurological disease, hepatobiliary disease, metabolic difficulties, gallstone, cardiovascular disease, Crohn's disease (CD) obesity, diabetes, myelosuppression, leukemia, tumorigenesis, etc., and provide guidance for researchers to conduct in-depth study on UGT1A1 gene-related diseases. In addition, this article not only summarizes the prevention strategies of UGT1A1 gene-related diseases, but also puts forward some insights for sharing.

[Liver histologic changes in children with type 1 of Crigler-Najjar syndrome]. / Gistologicheskie izmeneniya pecheni u detei s sindromom Kriglera-Nadzhara 1 tipa.

Background. Crigler-Najjar syndrome (CNS) is a rare genetic disorder found in less than 1 per 1.000.000 births. It happens as a result of an error in UGT1A1 enzyme which can cause high unconjugated bilirubin levels. OBJECTIVE: To describe liver histology changes in patients who have undergone liver transplantation. METHODS: This retrospective cross-sectional study was performed to evaluate the liver pathologies of patients with type 1 of Crigler-Najjar syndrome (CNS1). We analyzed medical records and liver histologic specimens of 53 children who were transplanted in Namazi Hospital Organ Transplant Center affiliated with Shiraz University of Medical Sciences between 2009 and 2019. We studied the tissue of the explanted liver, which was replaced by transplants. Most of the patients were less than 2 years old, with an average age of 1.7 years. The collected data were analyzed using SPSS 22 software. RESULTS: The prevalent pathology found in the liver of these patients was periportal fibrosis (96.2%). Cholestasis was the second common finding (94.3%) followed by pericentral fibrosis (86.7%) and ductal reaction (22.6%). A significant correlation was only present between phototherapy time and ductal reaction grade. CONCLUSION: Our results indicated a high prevalence of fibrosis of different grades among CNS 1 patients which bolds the necessity of histologic examination before considering treatments such as gene therapy or hepatocyte transplantation.

Novel mutations in Uridyl-diphosphate-glucuronosyl-transferase 1A1 (UGT1A1) gene in Tunisian patients with unconjugated hyperbilirubinemia.

INTRODUCTION: Unconjugated hyperbilirubinemia (UCB) is a feature of Gilbert's syndrome (GS) and Crigler-Najjar's syndrome (CNS), which are two hereditary defects in bilirubin metabolism. Both syndromes are linked to mutations in the UGT1A1 gene, which cause either the decrease or the absence of the UGT1A1 enzymatic activity. Here, we investigated the molecular basis of the UGT1A1 gene in Tunisian patients presenting with unconjugated hyperbilirubinemia. METHODS: Twenty-four patients with UCB were investigated. The screening protocol for hemoglobinopathies, enzymopathies, and membrane defects was executed in all patients. Afterward, the molecular analysis of the entire UGT1A1 gene was performed by DNA Sanger sequencing. Several bioinformatic tools were used to explore the effects of novel mutations. RESULTS: Fifteen different UGT1A1 variations were identified, among which four are described here for the first time. In exon 5, the c.1412C > G; p.(Ala471Gly) and c.1589C > T; p.(Ser530Phe) mutations were detected in patients presenting with CNS type I and GS, respectively. In the 3'UTR region of UGT1A1, the c.*90C > T mutation was detected in 3 patients with CNS type I. In the same region, the c.*388C > T defect was found in a GS patient. A deleterious and damaging effect on the UGT1A1 protein were predicted for both exonic mutations. Furthermore, novel microRNAs were identified as targetting the mutated sequences for the 3'UTR mutations. CONCLUSION: Our study provides novel data on UCB among Tunisians. Furthermore, we report four novel mutations associated with both GS and CNS. The identification of these mutations increases the spectrum of the UGT1A1 mutations and contributes to an understanding of the molecular abnormalities associated with unconjugated hyperbilirubinemia.

[Analysis of mutation site characteristics of Gilbert syndrome and Crigler--Najjar syndrome in relation to uridine diphosphate glucuronosyltransferase A1 gene].

Objective: To investigate the mutation characteristics and clinical relevance of Gilbert syndrome (GS) and Crigler-Najjar syndrome (CNS) in relation to uridine diphosphate glucuronosyltransferase A1 (UGT1A1) gene. Methods: The characteristics of UGT1A1 gene mutation and their clinical relevance were analyzed by searching PubMed and Human Gene Mutation Databases. Results: A total of 163 mutation sites were found in the UGT1A1 gene since November 16, 2018. The following patterns existed at the above sites: (1) the numbers of gene mutations occurring between different exons of UGT1A1 was related to GS or CNS phenotypes, and were positively correlated with the length of the exon; (2) nonsense point mutations was mainly occurred in type I of CNS; (3) GS, Crigler-Najjar syndrome type II compound heterozygous mutation sites had a certain combination and distribution, among which - 3279t > G mutation was found in all four GS complex heterozygous compositions; (4) UGT1A1 gene mutation sites reported in Asia had marked aggregation in c.211-c.558. Conclusion: UGT1A1 gene mutation characteristics and clinical relevance varies with different mutation sites, reporting areas and populations. This study has reference value for basic research and clinical diagnosis and treatment of GS and CNS.

A case report of a novel 22 bp duplication within exon 1 of the UGT1A1 in a Sudanese infant with Crigler-Najjar syndrome type I.

BACKGROUND: Crigler Najjar type 1 is a rare autosomal recessive condition caused by the absence of UDPGT enzyme due to mutations in the UGT1A1 gene. This enzyme is responsible for elimination of unconjugated bilirubin from the body by glucuronidation. Affected individuals are at risk for kernicterus and require lifelong phototherapy. Liver transplant is the only definitive treatment. CASE PRESENTATION: Here we report a case of a 6 month old Sudanese female infant with CN1 whose molecular analysis revealed a novel homozygous 22 base pair duplication (c.55_76dup) in the coding exon 1 of the UGT1A1 gene. This 22 bp duplication causes a frame shift leading to a premature stop codon. She underwent a successful liver transplant at 7 months of age and is doing well at 1 year follow-up. CONCLUSION: This study shows that molecular diagnosis helps in precise diagnosis of CN1 and in prognosis, prompt medical intervention and appropriate therapy. This particular 22 bp duplication within the coding region of UGT1A1 can be a founder mutation in the Sudanese population.

Crigler-Najjar Syndrome Type 1: Pathophysiology, Natural History, and Therapeutic Frontier.

BACKGROUND AND AIMS: We describe the pathophysiology, treatment, and outcome of Crigler-Najjar type 1 syndrome (CN1) in 28 UGT1A1 c.222C>A homozygotes followed for 520 aggregate patient-years. APPROACH AND RESULTS: Unbound ("free") bilirubin (Bf ) was measured in patient sera to characterize the binding of unconjugated bilirubin (BT ) to albumin (A) and validate their molar concentration ratio (BT /A) as an index of neurological risk. Two custom phototherapy systems were constructed from affordable materials to provide high irradiance in the outpatient setting; light dose was titrated to keep BT /A at least 30% below intravascular BT binding capacity (i.e., BT /A = 1.0). Categorical clinical outcomes were ascertained by chart review, and a measure (Lf ) was used to quantify liver fibrosis. Unbound bilirubin had a nonlinear relationship to BT (R2  = 0.71) and BT /A (R2  = 0.76), and Bf as a percentage of BT correlated inversely to the bilirubin-albumin equilibrium association binding constant (R2  = 0.69), which varied 10-fold among individuals. In newborns with CN1, unconjugated bilirubin increased 4.3 ± 1.1 mg/dL per day. Four (14%) neonates developed kernicterus between days 14 and 45 postnatal days of life; peak BT  ≥ 30 mg/dL and BT /A ≥ 1.0 mol:mol were equally predictive of perinatal brain injury (sensitivity 100%, specificity 93.3%, positive predictive value 88.0%), and starting phototherapy after age 13 days increased this risk 3.5-fold. Consistent phototherapy with 33-103 µW/cm2 •nm for 9.2 ± 1.1 hours/day kept BT and BT /A within safe limits throughout childhood, but BT increased 0.46 mg/dL per year to reach dangerous concentrations by 18 years of age. Liver transplantation (n = 17) normalized BT and eliminated phototherapy dependence. Liver explants showed fibrosis ranging from mild to severe. CONCLUSION: Seven decades after its discovery, CN1 remains a morbid and potentially fatal disorder.

Disease burden of Crigler-Najjar syndrome: Systematic review and future perspectives.

BACKGROUND AND AIM: Crigler-Najjar syndrome (CNS) results from biallelic mutations of UGT1A1 causing partial or total loss of uridine 5'-diphosphate glucuronyltransferase activity leading to unconjugated hyperbilirubinemia and its attendant risk for irreversible neurological injury (kernicterus). CNS is exceedingly rare and has been only partially characterized through relatively small studies, each comprising between two and 57 patients. METHODS: A systematic literature review was conducted to consolidate data on the patient, caregiver, and societal burden of CNS. RESULTS: Twenty-eight articles on clinical aspects of CNS were identified, but no published data on its humanistic or economic burden were found. In patients with complete UGT1A1 deficiency (type 1 CNS [CNS-I]), unconjugated bilirubin levels increase 3-6 mg/dL/day during the newborn period and reach neurologically dangerous levels between 5 and 14 days of age. Phototherapy is the mainstay of treatment but poses significant challenges to patients and their families. Despite consistent phototherapy, patients with CNS-I have worsening hyperbilirubinemia with advancing age. Liver transplantation is the only definitive therapy for CNS-I and is increasingly associated with excellent long-term survival but also incurs high costs, medical and surgical morbidities, and risks of immunosuppression. CONCLUSIONS: Crigler-Najjar syndrome is associated with a substantial burden, even with existing standards of care. The development of novel disease-modifying therapies has the potential to reduce disease burden and improve the lives of CNS patients and their families.

Prevalence and Relevance of Pre-Existing Anti-Adeno-Associated Virus Immunity in the Context of Gene Therapy for Crigler-Najjar Syndrome.

Adeno-associated virus (AAV) vector-mediated gene therapy is currently evaluated as a potential treatment for Crigler-Najjar syndrome (CN) (NCT03466463). Pre-existing immunity to AAV is known to hinder gene transfer efficacy, restricting enrollment of seropositive subjects in ongoing clinical trials. We assessed the prevalence of anti-AAV serotype 8 (AAV8) neutralizing antibodies (NAbs) in subjects affected by CN and investigated the impact of low NAb titers (<1:5) on liver gene transfer efficacy in an in vivo passive immunization model. A total of 49 subjects with a confirmed molecular diagnosis of CN were included in an international multicenter study (NCT02302690). Pre-existing NAbs against AAV8 were detected in 30.6% (15/49) of screened patients and, in the majority of positive cases, cross-reactivity to AAV2 and AAV5 was detected. To investigate the impact of low NAbs on AAV vector-mediated liver transduction efficiency, adult wild-type C57BL/6 mice were passively immunized with pooled human donor-derived immunoglobulins to achieve titers of up to 1:3.16. After immunization, animals were injected with different AAV8 vector preparations. Hepatic vector gene copy number was unaffected by low anti-AAV8 NAb titers when column-purified AAV vector batches containing both full and empty capsids were used. In summary, although pre-existing anti-AAV8 immunity can be found in about a third of subjects affected by CN, low anti-AAV8 NAb titers are less likely to affect liver transduction efficiency when using AAV vector preparations manufactured to contain both full and empty capsids. These findings have implications for the design of liver gene transfer clinical trials and for the definition of inclusion criteria related to seropositivity of potential participants.

A novel deletion with two pathogenic variants of UGT1A1 causing Crigler-Najjar syndrome in two unrelated Chinese.

Two Chinese female infants from two unrelated families were diagnosed with Crigler-Najjar syndromes-I (CNS-I) and CNS-II respectively. The CNS-I patient had Serum Total Bilirubin Concentration (STBC) peaked at 26.1 mg/dL. She was not responsive to Phenobarbital and received liver transplantation at 2-year-old. The CNS-II patient's STBC fluctuated between 10.2 mg/dL and 17.4 mg/dL and had a milder phenotype. Sequencing of Uridine Diphosphate Glucuronosyl Transferase 1A1 (UGT1A1) revealed the CNS-I patient carried a heterozygous pathogenic variant in c.392 T > C (p.Leu131Pro) and the CNS-II patient carried a heterozygous pathogenic variant in c.1456 T > G (p.Tyr486Asp). Furthermore, a novel deletion spanning exons 2-4 of UGT1A1 were detected in both patients. We studied two family members' genotyping results of UGT1A1 to clarify the inheritance of this microdeletion. To our knowledge, this is probably the first time showing 2 CNS cases both carrying compound heterozygous variations of a known pathogenic variant and a novel microdeletion.

A novel UGT1A1 gene mutation causing severe unconjugated hyperbilirubinemia: a case report.

BACKGROUND: Crigler-Najjar syndrome (CNs) presents as unconjugated hyperbilirubinemia, as a result of UGT1A1 deficiency, and can be categorized in a severe (type I) and mild (type II) phenotype. CNs type II patients usually benefit from phenobarbital treatment that induces residual UGT1A1 activity. CASE PRESENTATION: Here we present a CNs type II patient that is not responsive to phenobarbital treatment, which can be explained by two heterozygous mutations in the UGT1A1 gene. A 3 nucleotide insertion in the HNF-1α binding site in the proximal promoter previously reported in a Crigler-Najjar patient on one allele and a novel two nucleotide deletion in exon 1, resulting in a frameshift and a premature stop codon. CONCLUSION: In newly diagnosed CNs patients with unconjugated bilirubin levels consistent with CNs type II but that are unresponsive to phenobarbital treatment, disruption of the HNF-1α binding site in the proximal promoter should be considered as a probable cause. Upon confirming a mutation in the HNF-1α site, phenobarbital treatment should be stopped or at least be reconsidered because of its sedative effects and its teratogenic properties.