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INTRODUCTION: Cholestasis is the impairment of normal bile flow causing accumulation of bile salts, lipids, and bilirubin in blood which presents as Jaundice. Jaundice beyond 2 weeks of age is rare in infancy with worldwide incidence of 1 in 2500 live births. Biliary atresia is the most common extra hepatic cause of cholestasis in late neonatal and infancy period. Cholestasis and hyperbilirubinemia cause irreversible brain and liver damage if not diagnosed and treated early. CASE PRESENTATION: A 3-week-old neonate presenting with progressive yellowish discoloration of eyes and skin. Explorative laparotomy found anatomically normal liver and biliary tree, but a lymph node obstructing the common bile duct. DISCUSSION: This case was particularly unique as history of illness and initial investigations were suggestive of biliary atresia. However, the patient had lymph nodes with no history of any triggers to lymphadenopathy. It is a rare case of obstruction of biliary flow in this age group. CONCLUSION: Despite biliary atresia being the commonest cause of obstructive jaundice in infancy, it is important to rule out other causes like lymph nodes obstructing the biliary tree.

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Glucose-6-phosphate dehydrogenase (G6PD) deficiency and variants of the UDP-glucuronosyltransferase 1A1 (UGT1A1) gene are the most common genetic causes of neonatal unconjugated hyperbilirubinemia (NUH). In this review, we searched PubMed for articles on the genetic causes of NUH published before December 31, 2022, and analyzed the data. On the basis of the results, we reached eight conclusions: (1) 37 mutations of the G6PD gene are associated with NUH; (2) the clinical manifestation of G6PD deficiency depends not only on ethnicity but also on the molecular mechanisms underlying the deficiency (and thus its severity); (3) of mutations in the UGT1A1 gene, homozygous c.-53A(TA)6TAA > A(TA)7TAA is the main cause of NUH in Caucasians and Africans, whereas homozygous c.211G > A is the main genetic cause of NUH in East Asians; (4) in Indonesian neonates, homozygous c.-3279T > G is the most common cause of NUH development, and neither c.-53 A(TA)6TAA > A(TA)7TAA nor c.211G > A causes it; (5) in breast-fed East Asian neonates, the TA7 repeat variant of the UGT1A1 gene protects against the development of NUH; (6) G6PD deficiency combined with homozygous c.211G > A variation of the UGT1A1 gene increases the risk of severe NUH; (7) in Pakistani and Caucasian patients with Crigler-Najjar syndrome type 2 (CN-2), point mutations of the UGT1A1 gene are widely distributed and frequently occur with variation at nucleotide -53, whereas in Asian patients with CN-2, compound homozygous variations in the coding region are frequently observed; and (8) records of G6PD deficiency and UGT1A1 variation status for a neonate offer useful pharmacogenomic information that can aid long-term care. These results indicate that timely diagnosis of NUH through molecular tests is crucial and that early initiation of treatment for the neonates and educational programs for their parents improves outcomes.

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OBJECTIVE: To compare the association of unbound bilirubin (UB), total serum bilirubin (TSB), and bilirubin:albumin molar ratio (BAMR) with acute bilirubin encephalopathy (ABE), as assessed by bilirubin-induced neurologic dysfunction (BIND) score, in infants with significant hyperbilirubinemia (TSB ≥20 mg/dL or underwent exchange transfusion). STUDY DESIGN: In this prospective cohort study, infants ≥34 weeks of gestational age with significant hyperbilirubinemia during the first 2 postnatal weeks were eligible, unless they had craniofacial malformations, chromosomal disorders, TORCH (toxoplasmosis, other infections, rubella, cytomegalovirus and herpes simplex) infections, surgery, or a family history of congenital deafness. TSB, serum albumin, and UB were measured at hospital admission using the colorimetric, bromocresol green, and modified peroxidase method, respectively. Infants were evaluated on admission for ABE using a standardized neurologic examination and assigned a BIND score by trained physicians. Infants with a total BIND score of 0 were deemed to not have ABE, whereas those with a score ≥1 were deemed to have ABE. RESULTS: A total of 151 infants were studied, among whom 37 (24.5%) had ABE. Of these, 19 had mild ABE (BIND score 1-3) and 18 had moderate-to-severe ABE (BIND score 4-9). On logistic regression, UB, but not TSB or BAMR, was associated with ABE (aOR 1.64; 95% CI 1.17-2.3). On ordered logistic regression, UB, but not TSB or BAMR, was associated with severity of ABE (aOR 1.76; 95% CI 1.28-2.4). CONCLUSIONS: Our findings of the association between UB and ABE indicate that BIND scoring may be useful for evaluation of ABE in infants ≥34 weeks of gestational age.

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Key Clinical Message: Crigler-Najjar syndrome type 2 should be suspected in any young patient presenting with isolated indirect hyperbilirubinemia where all other common etiologies have been excluded. It is a relatively benign condition that responds to phenobarbitone. Abstract: Crigler-Najjar syndrome (CNS) type 2 is an inborn cause of isolated indirect hyperbilirubinemia characterized by a partial deficiency of the enzyme uridine 5'-diphosphate-glucuronosyltransferase (UGT) responsible for bilirubin conjugation. Typically, this condition is diagnosed based on clinical manifestations, supplemented by enzyme analysis if feasible, and exhibits a significant response to phenobarbitone, known for its enzyme-inducing properties. In this case, we present a young male patient who had experienced recurrent isolated indirect hyperbilirubinemia since early childhood, with negative results in the hemolytic workup. The patient exhibited a UGT1A1 gene defect and demonstrated a highly favorable response to phenobarbitone treatment. The purpose of this report is to raise awareness among physicians about this benign condition and underscore the importance of avoiding unnecessary investigations.

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PURPOSE: To evaluate the difference in cerebral blood flow in neonates with and without extreme unconjugated hyperbilirubinemia. METHODS: Transcranial Doppler parameters of 26 full term newborns with extreme unconjugated hyperbilirubinemia (UCH) were compared to 13 postnatal age and sex matched normal healthy neonates serving as controls. Resistance index (RI), pulsatility index (PI) and peak systolic velocity (PSV) were measured in the middle cerebral, internal carotid and posterior cerebral arteries on both sides by transcranial color Doppler ultrasound. RESULTS: An increase in cerebral blood flow (decreased RI, PI and increased PSV) was observed in the extreme unconjugated hyperbilirubinemia (UCH) group. There was positive correlation between total serum bilirubin level and peak systolic velocity and vice versa with resistivity and pulsatility indices. Eight neonates developed clinical features of acute bilirubin encephalopathy and showed significantly increased peak systolic velocity in the right middle cerebral artery compared to those with normal outcome. Resistivity index and pulsatility index were lower in patients managed by exchange transfusion compared to those managed with phototherapy. CONCLUSION: An increase in cerebral blood flow was observed in neonates with UCH compared to those without hyperbilirubinemia. By assessing the cerebral blood flow velocity, resistivity index (RI), and pulsatility index (PI) of particular intracranial arteries, the transcranial Doppler can identify the at-risk neonates, for development of neurological affliction in extreme unconjugated hyperbilirubinemia.

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Objective: We aimed to investigate the clinical and genetic risk factors associated with neonatal severe unconjugated hyperbilirubinemia. Methods: This was a retrospective, 1:1 matched, case-control study. We included 614 neonates diagnosed with severe unconjugated hyperbilirubinemia (serum total bilirubin level ≥425 µmol/L or serum total bilirubin concentration that met exchange transfusion criteria) from the China Neonatal Genomes Project in Children's Hospital of Fudan University. Clinical exome sequencing data were analyzed using a data analysis pipeline of Children's Hospital of Fudan University. The factors associated with severe unconjugated hyperbilirubinemia were assessed using univariable and multivariable logistic regression analyses. Interaction analyses were examined between clinical and genetic risk factors. Results: ABO/Rh incompatibility hemolysis (odds ratio [OR] 3.36, 95% confidence interval [CI] 2.32-4.86), extravascular hemorrhage (OR 2.95, 95% CI 2.24-3.89), weight loss (OR 5.46, 95% CI 2.88-10.36), exclusive breastmilk feeding (OR 3.56, 95% CI 2.71-4.68), and the homozygous mutant of UGT1A1 211G>A (OR 2.35, 95% CI 1.54-3.59) were all identified as factors significantly associated with severe unconjugated hyperbilirubinemia. The presence of UGT1A1 211G>A mildly increased the risk of severe unconjugated hyperbilirubinemia caused by ABO/Rh incompatibility hemolysis (OR 3.98, 95% CI 2.19-7.23), although the effect is not statistically significant. Conclusion: ABO/Rh incompatibility hemolysis, extravascular hemorrhage, weight loss, exclusive breastmilk feeding, and the homozygous mutant of UGT1A1 211G>A were found to be risk factors for severe unconjugated hyperbilirubinemia. Clinical factors remain the most crucial and preventable determinants in managing severe unconjugated hyperbilirubinemia, with a minimal genetic contribution. The establishment of preconception care practices and the reinforcement of screening for the aforementioned risk factors are essential steps for preventing severe unconjugated hyperbilirubinemia.

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Cyclosporine A (CSA) is an immunosuppressive drug that has improved transplant survival rates. However, its use is often limited because it is thought to be linked to the development of chronic kidney disease after kidney transplants. This study aimed to investigate the protective effects and underlying mechanisms of physiological unconjugated (UC) hyperbilirubinemia mediated by UGT1A1 antisense oligonucleotide in a mouse model of CsA-induced chronic kidney disease, and match these with that of chitosan (CH) as a natural chelator against kidney injury. In the current study, CsA-treated mice were given an intravenous injection of UGT1A1 antisense morpholino oligonucleotide (16 µg/kg) every third day for 14 days. In serum samples, bilirubin, creatinine, and urea were determined. Markers of oxidative stress, antioxidant activities, and mRNA expression of target genes PPAR-α, cFn, eNOS, NF-B, AT1-R, ETA-R, Kim-1, and NGAL were measured in the kidney tissues. Moreover, histopathological examinations were carried out on the kidney tissue. Physiological UC hyperbilirubinemia could be a promising protective strategy against CsA-induced kidney disease in transplant recipients. UGT1A1 antisense oligonucleotide-induced physiological UC hyperbilirubinemia serum significantly protected against CsA-induced kidney dysfunction. UCB acts as a signaling molecule that protects against kidney disease through different mechanisms, including antioxidant, anti-inflammatory, and hormonal action, by activating nuclear hormone receptors (PPAR-α). Moreover, it significantly downregulated mRNA expression of NF-kB, ETA-R, iNOS, AT1-R, cFn, Kim-1, and NGAL in the kidney tissue and alleviated CsA-induced kidney histological changes in CsA-treated mice.

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BACKGROUND: Neonatal hyperbilirubinemia is observed in most newborns, and 5-15% of neonates require phototherapy. Phototherapy is effective but often prolongs hospitalization and has both short-term and potential long-term harms. The aim of this systematic review and meta-analysis was to evaluate the role of ursodeoxycholic acid (UDCA) combined with phototherapy in neonatal hyperbilirubinemia. METHODS: A literature search was conducted on September 1, 2021; 590 studies were screened, and 17 full texts were assessed by two authors. We included randomized controlled trials with or without placebo intervention. Primary outcomes were changes in total bilirubin levels at 24 hours and phototherapy duration. We calculated mean differences with 95% confidence intervals (CI). RESULTS: Six studies with 880 neonates were included. Of these studies, only two used a placebo-controlled double-blinded design. The overall risk of bias was high in one and moderate in four of the included studies. The mean decrease in the total bilirubin level during the first 24 hours was 2.06 mg/dL (95% CI 0.82-3.30; six studies) greater in the UDCA treatment group. The phototherapy duration was 19.7 hours (95% CI 10.4-29.1; five studies) shorter in the UDCA treatment group. CONCLUSIONS: We found low-quality evidence that UDCA as an adjuvant to phototherapy seems to decrease total bilirubin faster and shorten phototherapy duration compared to standard treatment. Further studies are needed to confirm the efficacy, acute and long-term outcomes, and safety before implementing UDCA as an adjuvant to phototherapy in neonatal hyperbilirubinemia.

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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.

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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.

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OBJECTIVE: To investigate the clinical and genetic causes of neonatal unconjugated hyperbilirubinemia. STUDY DESIGN: We included 1412 neonates diagnosed with unconjugated hyperbilirubinemia (total serum bilirubin >95 percentile for age), from the China Neonatal Genomes Project between August 2016 and September 2019, in the current study. Clinical data and targeted panel sequencing data on 2742 genes including known unconjugated hyperbilirubinemia genes were analyzed. RESULTS: Among the 1412 neonates with unconjugated hyperbilirubinemia, 37% had severe unconjugated hyperbilirubinemia, with total serum bilirubin levels that met the recommendations for exchange transfusion. Known clinical causes were identified for 68% of patients. The most common clinical cause in the mild unconjugated hyperbilirubinemia group was infection (17%) and in the severe group was combined factors (21%, with infection combined with extravascular hemorrhage the most common). A genetic variant was observed in 55 participants (4%), including 45 patients with variants in genes associated with unconjugated hyperbilirubinemia and 10 patients with variants that were regarded as additional genetic findings. Among the 45 patients identified with unconjugated hyperbilirubinemia-related variants, the genes were mainly associated with enzyme deficiencies, metabolic/biochemical disorders, and red blood cell membrane defects. G6PD and UGT1A1 variants, were detected in 34 of the 45 patients (76%). CONCLUSIONS: Known clinical causes, which varied with bilirubin levels, were identified in approximately two-thirds of the patients. Genetic findings were identified in 4% of the patients, including in patients with an identified clinical cause, with G6PD and UGT1A1 being the most common genes in which variants were detected.

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The reason for reporting this case is to remind that some microorganisms may cause hemolysis leading to early and severe hyperbilirubinemia by secreting hemolysin in cases; where bilirubin levels cannot be successfully decreased despite effective phototherapy, intravenous immunoglobulin, and even exchange transfusion, or in cases of increased rebound bilirubin (although urinary tract infection is associated with increased conjugated bilirubin fraction and prolonged jaundice). The most common causes of hemolysis are ABO/Rh incompatibility and enzyme deficiencies such as glucose-6-phosphate dehydrogenase (G6PDH), pyruvate kinase (PK), and galactose-1-phosphate uridyltransferase (GALT). Our patient was a male infant, weighing 3,160 g, at 38 + 4 gestational week; he was referred to our unit with total bilirubin level of 14.7 mg/dL recorded at the postnatal 20th hour and was initiated treatment with intensive phototherapy and prepared for exchange transfusion. The G6PD, PK, and GALT enzyme levels studied at the postnatal 96th hour and reducing substances in urine were detected to be normal/negative, whereas complete urinalysis revealed pyuria (7 leukocytes per each high power field). α-hemolysis-producing 105 colony-forming unit/mL Enterobacter cloacae grew on blood agar in the urine culture. As reported in our case, hemolysin-secreting α and ß-hemolytic bacteria can lead to severe and early hemolysis and unconjugated hyperbilirubinemia, as in blood type incompatibility and enzyme deficiencies.

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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.

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A clinical trial using adeno-associated virus serotype 8 (AAV8)-human uridine diphosphate glucuronosyltransferase 1A1 (hUGT1A1) to treat inherited severe unconjugated hyperbilirubinemia (Crigler-Najjar syndrome) is ongoing, but preclinical data suggest that long-term efficacy in children is impaired due to loss of transgene expression upon hepatocyte proliferation in a growing liver. This study aims to determine at what age long-term efficacy can be obtained in the relevant animal model and whether immune modulation allows re-treatment using the same AAV vector. Neonatal, suckling, and juvenile Ugt1a1-deficient rats received a clinically relevant dose of AAV8-hUGT1A1, and serum bilirubin levels and anti-AAV8 neutralizing antibodies (NAbs) in serum were monitored. The possibility of preventing the immune response toward the vector was investigated using a rapamycin-based regimen with daily intraperitoneal (i.p.) injections starting 2 days before and ending 21 days after vector administration. In rats treated at postnatal day 1 (P1) or P14, the correction was (partially) lost after 12 weeks, whereas the correction was stable in rats injected at P28. Combining initial vector administration with the immune-suppressive regimen prevented induction of NAbs in female rats, allowing at least partially effective re-administration. Induction of NAbs upon re-injection could not be prevented, suggesting that this strategy will be ineffective in patients with low levels of preexisting anti-AAV NAbs.

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BACKGROUND: Neonatal jaundice is one of the most common problems in neonates. Effective treatment of jaundice requires therapeutic intervention with high quality phototherapy. Over recent years, several studies reported fiberoptic phototherapy to be less effective than conventional phototherapy in term neonates. Our study aimed to compare the effectiveness of fiberoptic phototherapy with a larger illuminated area and higher irradiance to conventional phototherapy methods. METHODS: This was a randomized controlled trial conducted at the Kilimanjaro Christian Medical Centre (KCMC). A total of 41 term neonates, less than 7 days of age with unconjugated hyperbilirubinemia were randomized. Thirteen (13) neonates were allocated to receive fiberoptic phototherapy, 13 to blue light conventional phototherapy and 15 to white light conventional phototherapy. Effectiveness was assessed by comparing the duration of phototherapy, bilirubin reduction rate and side effects of treatment. The data was analyzed with the independent t-test. RESULTS: The mean overall bilirubin reduction rate was comparable in the fiberoptic phototherapy group (0.74%/h) and the blue light conventional phototherapy group (0.84%/h), with no statistically significant difference (p-value 0.124). However, white light conventional phototherapy had a significantly lower mean overall bilirubin reduction rate (0.29%/h) as compared to fiberoptic phototherapy (p-value < 0.001). The mean treatment duration of phototherapy was 69 h, 68 h and 90 h in the fiberoptic, blue light conventional and white light conventional phototherapy groups respectively. Side effects such as loose stool and skin rash were noted in some participants who received conventional phototherapy. No side effects of treatment were noted in the fiberoptic phototherapy group. CONCLUSION: The effectiveness of fiberoptic PT and blue light conventional PT were comparable in terms of bilirubin reduction rate and treatment duration, whereas fiberoptic phototherapy was more effective than white light conventional PT, with a significantly higher bilirubin reduction rate and shorter treatment duration. Fiberoptic phototherapy may mitigate side effects caused by conventional phototherapy. TRIAL REGISTRATION: The Pan African Clinical Trial Registry, PACTR202004723570110 . Registered 22nd April 2020- Retrospectively registered.

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There is limited literature on Gilbert's syndrome (GS) in children with persistent unconjugated hyperbilirubinemia from Indian subcontinent. All patients (< 18 y of age) with genetically confirmed GS were included, and their profile was analysed. A total of 170 subjects were confirmed as having GS as per genetic analysis (133 with homozygous and 37 with heterozygous status). Majority were diagnosed in the adolescent age group (mean age 13.6 y). The median serum total bilirubin (TB) levels were around 3.3 mg/dl with maximum levels reaching upto 18 mg/dl. Around 15% subjects had an associated condition including hematological or hepatobiliary disease amongst others. GS is an important but under-recognised cause of unexplained unconjugated hyperbilirubinemia in Indian pediatric subjects. It may co-exist with other hematological and hepatobiliary disorders, and complicate the clinical/laboratory picture. Extent of hyperbilirubinemia may fluctuate to levels much higher than what is usually described in current world literature.

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BACKGROUND: Both Gilbert's syndrome (GS) and hereditary spherocytosis (HS) are common genetic disorders. However, comorbidity of GS with HS has always been considered a rare phenomenon, and it can impede accurate diagnoses in the presence of isolated unconjugated hyperbilirubinemia. CASE SUMMARY: In a study on Levitt's carbon monoxide (CO) breath test for the differential diagnosis of isolated hyperbilirubinemia, we found six GS patients with HS in 6 mo. The patients, including five males and one female, aged 25-58 years, were from four families and generally in good health. Their chronic fluctuating jaundice and/or hyperbilirubinemia had been diagnosed as simple constitutional jaundice for 6-30 years. Liver function tests showed isolated unconjugated hyperbilirubinemia with serum total bilirubin ranging from 20.7-75.4 µmol/L. Blood hemoglobin was normal in five cases, and slightly decreased in one (11.5 g/dL). Overt hemolytic signs were absent, while erythrocyte lifespan determined by the newly developed Levitt's CO breath test was significantly short (15-50 d), definitely demonstrating the presence of hemolysis. Given that their unconjugated hyperbilirubinemia compared inappropriately with hemolytic severity, as indicated by the hemoglobin level, further combined genetic tests for both UGT1A1 and hereditary erythrocyte deficiencies were conducted. These tests confirmed, at last, the coexistence of GS with HS. CONCLUSION: Comorbidity of GS and HS might not be uncommon in isolated unconjugated hyperbilirubinemia. While CO breath test would sensitively detect the hemolysis, the discordance between the hyperbilirubinemia and hemoglobin level could strongly indicate the coexistence of GS and HS.

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A large fraction of DNA variants impairs pre-mRNA splicing in human hereditary disorders. Crigler-Najjar syndrome (CNS) is characterized by a severe unconjugated hyperbilirubinemia caused by variants in the UGT1A1 gene. We previously reported one CNS-type II patient with two splice-site variants in trans (c.864+5G>T and c.996+2_996+5del). According to MaxEntScan, both disrupt their corresponding donor sites (c.864+5G>T: 6.99 → 2.28; c.996+2_996+5del: 5.96 → -11.02), so they were selected for subsequent functional tests. Given the unavailability of patient RNA, we constructed an UGT1A1 splicing-reporter minigene with exons 1-4 to characterize the underlying splicing anomaly. The variant c.996+2_996+5del generated two aberrant transcripts, Δ(E2) (exon 2 skipping/64%) and ▼(E2q135) (intron retention of 135-nt/36%), which lead to the loss of 18 conserved amino-acids and the gain of 45 new ones of a critical functional domain, respectively. The c.864+5G>T variant mainly produced the aberrant transcript Δ(E1q141) (141-nt deletion/70.4%) and the full-length isoform (29.6%). Δ(E1q141) would provoke the loss of 47 amino-acids of the N-terminal domain that encodes for substrate specificity. Thus, the three anomalous transcripts are likely to inactivate UGT1A1. Moreover, this patient is also homozygous for the promoter variant A(TA)7TAA that decreases UGT1A1 expression by 70%, so the full-length transcript produced by c.864+5G>T would be even more reduced (<9%), thus supporting the diagnosis of CNS-type II. Therefore, minigenes represent valuable tools for the functional and clinical classifications of genetic variants.