[UGT1A1 gene mutation spectrum with indirect hyperbilirubinemia in children].

Objective: To explore the relevancy between the uridine diphosphate-glucuronylgly-cosyltransferase 1A1 (UGT1A1) gene mutation and the phenotype of indirect hyperbilirubinemia in children. Methods: Sixteen cases with indirect hyperbilirubinemia who visited the Department of Gastroenterology, Children's Hospital of Nanjing Medical University from July 2013 to November 2019 were retrospectively analyzed and were divided into Gilbert syndrome (GS), Crigler-Najjar syndrome type II (CNS-II), and indirect hyperbilirubinemia groups unexplained by UGT1A1 gene mutations. The differences in gene mutation site information and general clinical data were compared. The association between gene mutation spectrum and bilirubin level was explored by t-test analysis. Results: Ten of the sixteen cases with indirect hyperbilirubinemia had GS, three had CNS-II, and three had indirect hyperbilirubinemia unexplained by UGT1A1 gene mutations. A total of six mutation types were detected, of which c.211G > A accounted for 37.5% (6/16), c.1456T > G accounted for 62.5% (10/16), and TATA accounted for 37.5% (6/16), respectively. Compared with the GS group, the CNS group had early disease onset incidence, high serum total bilirubin (t = 5.539, P < 0.05), and indirect bilirubin (t = 5.312, P < 0.05). However, there was no significant difference in direct bilirubin levels (t = 1.223, P > 0.05) and age of onset (t = 0.3611, P > 0.05) between the two groups. There was no significant correlation between the number of UGT1A1 gene mutations and serum bilirubin levels. Children with c.1456T > G homozygous mutations had the highest serum bilirubin levels. Conclusion: The common pathogenic variants of the UGT1A1 gene sequence are c.1456T > G, c.211G > A, and TATA, indicating that these site mutations are related to the occurrence of indirect hyperbilirubinemia and have important guiding significance for the etiological analysis of indirect hyperbilirubinemia in children.

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.

Health-related quality of life and cognitive function in children with Crigler-Najjar syndrome type 1.

BACKGROUND: The aim of the study was to assess the health-related quality of life (HRQOL) and cognitive function in patients with Crigler-Najjar syndrome (CNS) type I and its impact on their lives. METHODS: Twenty-one patients diagnosed with CNS type I aged 1 month to 18 years in the Paediatric Hepatology Unit of Cairo University Children's Hospital were enrolled in this cross-sectional observational study. The patients' health-related quality of life (HRQOL) was assessed using the World Health Organization Quality Of Life BREF questionnaire (WHOQOL-BREF) and the Short Form 36 Health Survey Questionnaire (SF-36). Cognitive function was assessed using the Stanford-Binet Intelligence Scale: Fifth Edition (SB5). RESULTS: All patients had a history of admission to a neonatal intensive care unit, 17 were managed by phototherapy only and 5 also underwent exchange transfusion. According to the WHOQOL questionnaire, 11 cases (52.4%) had a low QOL score, and 7 of 13 patients had an average score for their total IQ test. Cases with poor compliance to phototherapy had statistically significantly lower QOL scores (p=0.001), while, according to the SF36 survey, cases who received exchange transfusion had statistically significantly higher cognitive function (p=0.03). There was a positive correlation between the neurological effect as a complication of the disease and poor physical QOL. CONCLUSION: Paediatric patients with CNS have significantly lower HRQOL, especially physically, psychologically and environmentally. It is recommended that assessment of HRQOL should be a routine part of follow-up in CNS patients. Patients whose HRQOL is affected receive regular psychiatric counselling, social support and rehabilitation.Abbreviations: CNS: Crigler-Najjar syndrome; HRQOL: health-related quality of life; IQ: intelligence quotient; NICU: neonatal intensive care unit; QOL: quality of life; SB5: Stanford-Binet intelligence scale: 5th edition; SF-36: Short Form 36 Health Survey Questionnaire; UDGT: uridine diphosphate glucuronosyl transferase; UGT1A1: uridine 5'-diphosphate glucuronosyltransferase; WHOQOL-BREF: World Health Organization Quality of Life Brief Version.

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.

Free Bilirubin Induces Neuro-Inflammation in an Induced Pluripotent Stem Cell-Derived Cortical Organoid Model of Crigler-Najjar Syndrome.

Bilirubin-induced neurological damage (BIND), which might progress to kernicterus, occurs as a consequence of defects in the bilirubin conjugation machinery, thus enabling albumin-unbound free bilirubin (BF) to cross the blood-brain barrier and accumulate within. A defect in the UGT1A1 enzyme-encoding gene, which is directly responsible for bilirubin conjugation, can cause Crigler-Najjar syndrome (CNS) and Gilbert's syndrome. We used human-induced pluripotent stem cell (hiPSC)-derived 3D brain organoids to model BIND in vitro and unveil the molecular basis of the detrimental effects of BF in the developing human brain. Healthy and patient-derived iPSCs were differentiated into day-20 brain organoids, and then stimulated with 200 nM BF. Analyses at 24 and 72 h post-treatment point to BF-induced neuro-inflammation in both cell lines. Transcriptome, associated KEGG, and Gene Ontology analyses unveiled the activation of distinct inflammatory pathways, such as cytokine-cytokine receptor interaction, MAPK signaling, and NFκB activation. Furthermore, the mRNA expression and secretome analysis confirmed an upregulation of pro-inflammatory cytokines such as IL-6 and IL-8 upon BF stimulation. This novel study has provided insights into how a human iPSC-derived 3D brain organoid model can serve as a prospective platform for studying the etiology of BIND kernicterus.

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.

The added value of brain MR spectroscopy in children with Crigler-Najjar syndrome type-I: correlation with demographic, neurodevelopmental, and laboratory findings.

OBJECTIVE: The aim of this study is to demonstrate the role of proton magnetic resonance spectroscopy (1H-MRS) in the detection of brain microstructural changes in patients with Crigler-Najjar syndrome type-I (CNs-I), and its correlation with demographic, neurodevelopmental and laboratory findings. METHODS: Prospective study was conducted on 25 children with CNs-I and 25 age and sex-matched children, who served as control. They underwent multivoxel 1H-MRS of basal ganglion at echo time 135-144 ms. N-acetyl aspartate/Creatine (NAA/Cr) and Choline (Ch)/Cr were calculated and correlated with demographic, clinical, and laboratory findings of patients with CNs-I. RESULTS: There was a significant difference in NAA/Cr and Ch/Cr between patients and controls. The cut-off value for NAA/Cr and Ch/Cr used to differentiate patients from controls were 1.8 and 1.2 with an area under the curve (AUC) of 0.91 and 0.84 respectively. There was a significant difference in MRS ratios between patients with neurodevelopmental delay (NDD) and patients without NDD. The cut-off values for NAA/Cr and Ch/Cr used to differentiate between patients with NDD and patients without NDD were 1.47 and 0.99, with AUC of 0.87 and 0.8 respectively. The NAA/Cr and Ch/Cr were well correlated with family history (p = 0.006 and p < 0.001) respectively, consanguinity (p < 0.001 and p = 0.001), neurodevelopmental delay (p = 0.001 and p = 0.004), serum bilirubin level (r = -0.77, p < 0.001), (r = -0.49, p = 0.014), phototherapy (p < 0.001 and p = 0.32), blood transfusion (p < 0.001 and p = 0.001) respectively. CONCLUSION: 1H-MRS can be a useful tool in the detection of neurological changes in patients with CNs-I; NAA/Cr and Ch/Cr parameters are well correlated with demographic, clinical, and laboratory findings. ADVANCES IN KNOWLEDGE: Our study is the first report on using MRS in assessing neurological manifestations in CNs. 1H-MRS can be a useful tool in the detection of neurological changes in patients with CNs-I.

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.

Diffusion Tensor Imaging of Auditory Pathway in Patients With Crigler-Najjar Syndrome Type I: Correlation With Auditory Brainstem Response.

AIM: To evaluate the role of diffusion tensor imaging of the auditory pathway in patients with Crigler Najjar syndrome type I and its relation to auditory brainstem response. METHODS: Prospective study was done including 12 patients with Crigler Najjar syndrome type I and 10 age- and sex-matched controls that underwent diffusion tensor imaging of brain. Mean diffusivity and fractional anisotropy at 4 regions of the brain and brainstem on each side were measured and correlated with the results of auditory brainstem response for patients. RESULTS: There was significantly higher mean diffusivity of cochlear nucleus, superior olivary nucleus, inferior colliculus, and auditory cortex of patients versus controls on both sides for all regions (P = .001). The fractional anisotropy of cochlear nucleus, superior olivary nucleus, inferior colliculus, and auditory cortex of patients versus controls was significantly lower, with P values of, respectively, .001, .001, .003, and .001 on the right side and .001, .001, .003, and .001 on left side, respectively. Also, a negative correlation was found between the maximum bilirubin level and fractional anisotropy of the left superior olivary nucleus and inferior colliculus of both sides. A positive correlation was found between the mean diffusivity and auditory brainstem response wave latency of the right inferior colliculus and left cochlear nucleus. The fractional anisotropy and auditory brainstem response wave latency of the right superior olivary nucleus, left cochlear nucleus, and inferior colliculus of both sides were negatively correlated. CONCLUSION: Diffusion tensor imaging can detect microstructural changes in the auditory pathway in Crigler Najjar syndrome type I that can be correlated with auditory brainstem response.

[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.

Outcome of liver transplantation and prevalence of liver fibrosis in Crigler-Najjar syndrome.

INTRODUCTION: Crigler-Najjar syndrome (CNS) is a rare inherited disorder that is characterized by high levels of non-hemolytic, unconjugated hyperbilirubinemia leading to brain damage and even death. Liver transplantation (LT) can correct the metabolic defect, but there are little data regarding LT in this patient cohort. The liver parenchyma has been considered to be structurally normal in CNS, but there is growing evidence of clinically silent but histologically significant fibrosis in CNS patients. PATIENTS AND METHODS: We included 13 patients in our retrospective study who underwent LT at our center. Patient survival, graft function, and long-term complications were evaluated over a median follow-up period of 10 years (range: 1-16 years). In addition, the prevalence of histologically relevant fibrosis was characterized. RESULTS: The overall survival among our LT patients was 100%. The graft survival was only 61.5%. During the follow-up period, 5 LT patients had to undergo retransplantation. More than 45% of our patients showed histological signs of fibrosis. CONCLUSION: LT remains the only definite therapeutic option for severe CNS but needs to be considered thoroughly regarding the clinical risk-benefit-ratio and impact on quality of life. Furthermore, hepatic parenchymal injury needs to be considered while evaluating future therapeutic options for CNS.

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.