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UGT1A1-related Bilirubin Encephalopathy/Kernicterus in Adults

Background and Aims Bilirubin encephalopathy/kernicterus is very rare in adults. This study is aimed to investigate the clinical manifestations and genetic features of two patients with UGT1A1-related kernicterus. Methods Sanger sequencing analysis was performed to identify UGT1A1 gene mutations in the patients and their families. Bioinformatics analysis was used to predict the potential functional effects of novel missense mutations. Clinical manifestations and biochemical parameters were collected and analyzed. Results Two patients with Crigler-Najjar syndrome type II (CNS2) developed kernicterus in adulthood. Sanger sequencing identified a compound heterozygous mutation in the UGT1A1 gene in patient 1, which was inherited from his mother (G71R) and his father (c.-3279T>G; S191F). Patient 2 carried three heterozygous mutations, namely G71R, R209W and M391K; among which, the M391K mutation has not been reported before. Multiple prediction software showed that the M391K mutation was pathogenic. Symptoms were relieved in the two patients after phenobarbital and artificial liver support treatment. Patient 1 also underwent liver transplantation. Conclusions Adults with CNS2 are at risk for kernicterus. Phenobarbital treatment is beneficial for maintaining bilirubin levels and preventing kernicterus.


Introduction
Bilirubin encephalopathy/kernicterus is an uncommon disabling neurologic disease caused by the toxicity of unconjugated bilirubin (UCB) to the basal ganglia and various brain stem nuclei. 1 Neonatal jaundice is quite common, affecting 60-80% of newborns, as a whole. 2 However, severe hyperbilirubinemia (>20 mg/dL), which may potentially lead to kernicterus and neurodevelopmental complications, is very rare, accounting for less than 2% of newborns. 3 The incidence of kernicterus is about 0.2 to 2.7 cases per 100,000 live births. 1 Common risk factors may include preterm delivery, hemolytic disease [glucose-6-phosphate dehydrogenase deficiency and ABO hemolysis], perinatal infection and exclusive breastfeeding. 1,4 For adults, elevated UCB caused by hemolytic disease and inherited non-hemolytic unconjugated hyperbilirubinemia (bilirubin glucuronidation defects) is relatively common. Bilirubin glucuronidation is regulated by the uridine diphosphate glucuronosyl transferase 1A1 (UGT1A1) enzyme. It is encoded by the UGT1A1 gene, which is located on chromosome 2 (2q37), and covers a promoter, enhancers, and five exons. According to the severity of UGT1A1 enzyme deficiency, inherited unconjugated hyperbilirubinemia can be classified into Crigler-Najjar syndrome type I (CNS1), Crigler-Najjar syndrome type II (CNS2), and Gilbert syndrome (GS). 5,6 CNS1 is the most severe form, determined by a complete lack of bilirubin glucuronidation, and patients exhibit a toxic level of hyperbilirubinemia (≥340 µmol/L) shortly after birth. 7 CNS1 patients usually suffer from bilirubin encephalopathy, and are prone to death within the first 2 years of their lives. 8,9 At present, orthotopic liver transplantation is the only radical treatment. 10,11 CNS2 is characterized with not very high bilirubin (from 103 to <340 µmol/L) 12 and the bilirubin glucuronidation is less than 10% of normal level but not completely eliminated. 13 Although the phenotype of CNS2 is less severe, patients with CNS2 remain vulnerable to brain injury throughout life, especially in the setting of concurrent diseases, after injury, or during surgery. 14 Poddar et al. 15 reported a case of kernicterus in a CNS2 child due to a dramatic increase in UCB caused by hemolysis. GS is a mild hyperbilirubinemia (from normal level up to 80-100 µmol/L) that occurs in 5-10% of the population, 12 with approximately 70% reduction in bilirubin glucuronidation. 16 GS is considered as a benign condition without neurological damage and treatment requirement.
At present, there are few reports of kernicterus in adults.

Subjects and sample collection
The patients and all family members received careful clinical examinations and laboratory assessments by experienced physicians in Beijing You'an Hospital, Capital Medical University. Fasting blood samples were collected from all participants; clinical manifestations and biochemical parameters were collected and analyzed. This study was approved by the Ethics Committee of Beijing You'an Hospital, Capital Medical University, and a written informed consent form was obtained from all participants.

DNA extraction and screening for the mutations in UGT1A1
Genomic DNA was extracted from whole blood using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany), according to the manufacturer's protocol. The promoter, all five exons, exon-intron boundaries, and a region in the distal promoter (the phenobarbital response enhancer module, PBREM) of UGT1A1 were amplified by PCR technology, then purified through agarose gel electrophoresis and sequenced using a 3730XL sequencer (Applied Biosystems Inc., Foster City, CA, USA). Finally, Sanger sequencing data were compared and analyzed by SeqMan software (DNASTAR, Madison, WI, USA).

Patient 1
The patient was a 32-year-old man with a 30-year history of jaundice. In May 2020, he lost his appetite after drinking (about 500 mL beer, 20 g ethanol). He took Chinese medicine (unknown pharmaceutical ingredients) for a week, but the symptoms were not alleviated. Then, he developed dizziness, headache, and mild neuropsychological disorder. Liver function results were abnormal, with decreased albumin (23.8 g/L) and increased aspartate aminotransferase (commonly referred to as AST; 148U/L), alanine aminotransferase (commonly referred to as ALT; 54 U/L), total bilirubin (TB, 411.8 µmol/L), indirect bilirubin (266.3 µmol/L) and direct bilirubin (145.5 µmol/L). Blood routine test showed a high proportion of neutrophils (81.5%) and a decrease in hemoglobin (109 g/L). His prothrombin time activity was 42% and the Coombs test was negative. Blood ammonia (16 µg/dL), fasting plasma lipids, autoantibodies profile, anti-neutrophil cytoplasmic antibody, and immunoglobulins (Igs) including IgG, IgA, IgM, and IgE were normal. The viral hepatitis markers were negative. Computed tomography (referred to as CT) and magnetic resonance imaging showed hepatosplenomegaly (Supplementary Fig.  1). Brain CT scans were normal ( Supplementary Fig. 1). The patient was diagnosed with acute-on-chronic liver failure (ACLF), CNS2 and kernicterus, and received oral phenobarbital, albumin infusion, and anti-infection therapy.
One week after admission, the patient suddenly manifested hematemesis and showed restlessness, then lost consciousness. TB, indirect bilirubin, and blood ammonia levels increased to 532.3 µmol/L, 379.1 µmol/L, and 123 µg/dL, respectively. Gastroscopy revealed cardiac mucosal laceration syndrome (Mallory-Weiss syndrome). The patient was treated with hemostasis, sedation, intramuscular injection of phenobarbital, and artificial liver support treatment of double plasma molecular adsorption system combined with plasma exchange (DPMAS) and plasma exchange. Later, the patient's consciousness gradually recovered and he underwent liver transplantation. Histopathology of the removed liver showed massive and submassive hepatocyte necrosis, cholestasis, and steatosis ( Fig. 1). The patient is now in a relatively stable state. Table 1 and Fig. 2 show his blood test results.
Sanger sequencing of the UGT1A1 gene identified a compound heterozygous mutation in this patient, which was in- Interestingly, the patient's elder sister also showed jaundice since childhood. Her TB was about 400 µmol/L. She was diagnosed with CNS2 but had never developed kernicterus. In addition, the patient's father had mild unconjugated hyperbilirubinemia (TB of 32.1 µmol/L, indirect biliru-bin of 23.2 µmol/L) and was diagnosed with GS. They were both also found to carry the same compound heterozygous mutation as the patient (Fig. 3).

Patient 2
The patient was a 35-year-old man. He had been suffer- Abbreviations: %N, proportion of neutrophils; ALB, albumin; ALP, alkaline phosphatase; Amon, blood ammonia; BAMR, Bilirubin-albumin molar ratio; DB, direct bilirubin; GGT, γ-glutamyl transferase; HGB, hemoglobin; IB, indirect bilirubin; PLT, platelets; TBA, total serum bile acid; WBC, white blood cell.  3). The M391K mutation has not been reported before. Seven software programs were used to predict the pathogenicity of the mutation. As shown in Supplementary Table 1, all software programs showed that the M391K mutation was pathogenic or damaging. The PhastCons score of the mutation was 1, and the corresponding PhyloP value was 2.307, suggesting the high conservation of this amino acid.
The patient was treated with antibiotics, sedation, intramuscular injection of phenobarbital, and artificial liver support treatment with DPMAS and plasma exchange. The patient's neurological status gradually returned to normal. Table 2 and Fig. 4 show the results of his blood investigations.

Discussion
High concentrations of UCB can cause nervous system damage, known as bilirubin encephalopathy or kernicterus. In general, kernicterus is found in infants and young children, especially those who are premature and/or have hemolysis. 1,17 On one hand, the blood-brain barrier of newborns and children is immature. On the other hand, the albumin/ bilirubin-binding capacity and tissue-binding capacity vary significantly among newborns, and these values are particularly low for premature babies. 18 Kernicterus in adults is a rare condition. To date, there are only two published cases. 19,20 In one case, the disorder was associated with liver failure. 19 The other case was an adult with CNS2, who developed kernicterus after laparoscopic surgery. 20 This report describes two new cases of adult kernicterus associated with UGT1A1 mutations (CNS2).
CNS2 is usually caused by missense mutations in the UGT1A1 gene, which reduces enzyme activity but does not eliminate it. 21,22 Most patients with CNS2 have homozygous missense mutations or compound heterozygous mutations. 23 This explains the milder phenotype and inducibility of the residual enzyme activity by phenobarbital administration. Variants c.211G>A (p.G71R) and c.1456T>G (p.Y486D) are the most frequently reported mutation sites in Asian CNS2 patients. [24][25][26] In our patients, UGT1A1 sequencing analysis was performed. Patient 1 is a compound heterozygote with mutations c.-3279T>G, G71R, and S191F. His father and sister share the same UGT1A1 genotype as him, and were diagnosed with GS and CNS2, respectively. But none of them suffered from kernicterus. The above evidence suggests that the same genotype may result in different phenotypes and clinical manifestations. A possible explanation is that a multifactorial etiology including hormonal, environmental, and genetic factors contributes to the development of inherited diseases. Besides, liver histology suggested massive and submassive hepatocytes necrosis and cholestasis, consistent with ACLF and CNS2, respectively. 27,28 Histopathology also showed steatosis, but the related mechanism is not clear. Before the onset of ACLF, Patient 1 drank about 20 g ethanol (500 mL beer) and then took traditional Chinese medicine for 1 week. Considering the relatively small intake of ethanol and the complex composition of Chinese medicine (although the composition is unknown), we speculated that Chinese medicine was the main cause of his acute liver injury, and eventually lead to his ACLF on the basis of Crigler-Najjar syndrome.
Patient 2 is heterozygous for the mutations G71R, R209W and M391K. The mutations G71R, S191F, and R209W are located in exon 1 of the UGT1A1 gene, and have been shown to be associated with CNS2 (moderate hyperbilirubinemia). 29 The mutation c.-3279T>G is located in the phenobarbital response enhancer module, which is related to GS (mild hyperbilirubinemia). 30 The M391K mutation has not been reported yet. All prediction software indicated that the mutation is pathogenic. PhastCons and PhyloP showed a high degree of amino acid conservation, suggesting that the mutation has a great impact on amino acids and the protein. According to the variant classification criteria of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology, 31 the novel M391K mutation is pathogenic to CNS2 in this patient. The effect on the UGT1A1 enzyme's activity needs to be further verified through cell experimentation. In summary, the mutation site, number, and genotype of UGT1A1 are related to bilirubin level.
The pathogenesis of kernicterus has not been fully elu-  cidated but the main reason is excessive bilirubin production (i.e. hemolysis) and/or insufficient liver glucuronidation, leading to higher levels of free unbound bilirubin. The potential mechanism of UCB neurotoxicity is shown in Fig.  5. 18 In our two patients, CNS2 reduced bilirubin glucuronidation. Infection, fever and liver injury cause a decrease in serum albumin levels. Therefore, free unbound bilirubin increased. In infants, a bilirubin-albumin molar ratio (referred to as BAMR) value >0.8 is considered dangerous because bilirubin/albumin binding is unpredictable at these levels. 17 However, for adults, the BAMR value predicting kernicterus is still unclear. In our two patients, bilirubin concentrations increased to 616 µmol/L and 547 µmol/L, respectively, with BAMR value >0.8 in both. Then, kernicterus occurred. Therefore, for patients with CNS2, it is important to avoid particularly high bilirubin levels and maintain normal serum albumin levels. Infection, liver damage, and hemolytic disease should also be avoided. It should be noted that CNS2 patients respond to phenobarbital. To prevent severe hyperbilirubine- There is also decreased dendritic and axonal arborization. A similar pattern is observed in oligodendrocytes with increased apoptosis, impairment of the redox state (oxidative stress), and reduced synthesis of myelin. Microglia react to toxic injury associated with bilirubin by increased release of proinflammatory cytokines and metalloproteinase activity as cells manifest a phagocytic phenotype. A similar proinflammatory pattern is observed in astrocytes, with enhanced release of glutamate and apoptosis. At the same time, cells may reduce the intracellular concentration of bilirubin either by extruding the pigment through the ATP-binding cassette transporters or by increasing the formation of the less toxic products through bilirubin oxidation products (BOXes) and/or cytochrome P-450 enzymes (1a1 and 1a2, in particular). These responses are protective, whereas all others result in cell damage; this suggests that once the intracellular concentration of bilirubin exceeds a toxic threshold (still to be defined), the polymorphic metabolic cascade leading to neurotoxicity ensues. (From Watchko JF, Tiribelli C. Bilirubin-induced neurologic damage-mechanism and management approaches. N Engl J Med 2013; 369: 2025). Abbreviations: cPARP, cleaved poly (adenosine diphosphate-ribose) polymerase; TER, transcellular resistance. mia and kernicterus, phenobarbital therapy should be adhered to. If the patient develops kernicterus, treatments that eliminate the cause, or those such as albumin supplementation and artificial liver support treatment, are effective. Due to severe necrosis and insufficient regeneration of hepatocytes in patient 1, the bilirubin level still slightly fluctuated in the case of artificial liver support treatment. At the same time, although the patient recovered consciousness after albumin supplementation and artificial liver support treatment, increased bilirubin due to CNS2 may lead to recurrence of kernicterus. At present, liver transplantation is the only treatment option that completely replaces UGT1A1 function and normalizes serum bilirubin levels.
In conclusion, although very rare, adults with CNS2 are at risk of kernicterus. Phenobarbital administration helps maintain bilirubin levels and prevent kernicterus.