1 Introduction
Both Crigler-Najjar syndrome type 2 (CNS2; OMIM: 606785) and Gilbert syndrome (GS; OMIM: 143500) are autosomal recessive disorders characterized by hyper unconjugated bilirubinemia. The molecular etiology of these diseases is a mutation in the bilirubin UDP-glucuronosyltransferase family 1, polypeptide A1 (UGT1A1), leading to significantly diminished activity of the UGT1A1 enzyme in hepatocytes and a severe decrease in the ability of the liver to produce conjugated bilirubin [
1]. CNS is exceedingly rare in clinical practice, impacting approximately 0.6 to 1 in 1 million newborns globally [
2]. It is marked by jaundice, evident through yellowing of the skin and sclera, without accompanying symptoms such as pruritus, hepatomegaly, or splenomegaly, and the jaundice may intensify due to physical exercise, alcohol intake, and similar factors. The injection of phenobarbital or other enzyme-inducing drugs can significantly reduce serum bilirubin levels; nevertheless, persistent hyperbilirubinemia increases the risk of gallstone formation and related complications in CNS patients [
3].
Deletions, alterations in intron splicing donor and acceptor sites, missense mutations, exon skipping, insertions, or formation of a stop codon in the
UGT1A1 gene are common mutations in CNS. Most patients with CNS2 have a homozygous or compound heterozygous mutation that reduces enzyme activity to less than 10% of normal, and phenobarbital treatment usually improves this residual enzyme activity [
4]. This study explores a 28-year-old male patient who presented with recurrent right upper abdominal pain and was diagnosed with CNS2 combined with cholecystitis. Genetic testing revealed a rare double homozygous mutation in the
UGT1A1 gene, and his familial pattern of inheritance, clinical features, diagnosis, and treatment were analyzed.
2 Case report
The 28-year-old patient developed paroxysmal right upper abdominal pain one month before presenting to our hospital; the pain could radiate to the waist and back and was accompanied by nausea and vomiting of gastric contents and bile, yellow sclera, and deepening of the urine color, with no fever or diarrhea. He visited the local hospital, where the emergency CT showed gallbladder stones and cholecystitis, and his symptoms improved after relieving spasm and pain therapy. Three days before receiving treatment at our hospital, the patient experienced a recurrence of abdominal pain with symptoms like a month earlier. He did not use any specific medications, was not addicted to alcohol or tobacco, had no history of blood transfusion, and did not undertake any appropriate tests or treatment for his history of persistent jaundice since birth, with yellowing of the sclera and skin increased by tiredness. The patient was examined with a temperature of 36.5 °C, significant yellowing of the skin and bilateral sclera on the body, a pressure ache in the right upper abdomen without rebound pain, no liver or spleen enlargement, and a positive Murphy’s sign. Laboratory tests indicate significantly elevated levels of both conjugated and unconjugated bilirubin, as well as aminotransferase, alongside increased alkaline phosphatase and γ glutamyl transferase. The inflammatory marker C-reactive protein is approximately doubled, while urine conjugated bilirubin is elevated, though urine bile remains normal. Blood tests reveal no hemolysis and coagulation function, as well as various serum antibodies associated with liver disease, showing no abnormalities (Tab.1). Ultrasound revealed normal liver size and morphology, no mass image, and a 75 mm × 28 mm gallbladder with thickened and roughened wall, 7 mm wall thickness, and strong echo (6 mm × 5 mm). The magnetic resonance cholangiopancreatography (MRCP) of the biliary tract (Fig.1 and 1D) revealed a full gallbladder, thickened walls, increased T2WI signal, and round-like patches of short T1 and T2 signals measuring 2.1 cm × 1.6 cm. The common bile duct and walls were also dilated and thickened. Multiple gallbladder stones and cholecystitis were the major diagnosis. For one week, he received cefoperazone sodium and sulbactam sodium for infection and bicyclol tablets for liver protection. His clinical symptoms improved and his transaminases recovered to twice the upper limit of normal, but his bilirubin did not change. To determine the reason for the patient’s persistent jaundice, an ultrasound-guided percutaneous puncture biopsy of the liver parenchyma was conducted. Pathology revealed mild hepatic lobule inflammation accompanied with mild hepatocellular and capillary biliary sludge, slight edema in the confluent region, absence of significant fibrous tissue hyperplasia, and small bile ducts exhibiting sludge and biliary epithelial atrophy (Fig.1 and 1F). Both parents of the patient were not consanguineous, have no history of blood transfusion, and did not have jaundice, liver function abnormalities, or biliary stones. However, biochemical tests showed mild bilirubin elevation (Fig.1 and 1H), suggesting that the patient’s jaundice at birth may have been inherited.
The patient underwent second-generation sequencing (NGS) and was evaluated for candidate genes and mutations utilizing whole-genome sequencing capture technology. We identified two potentially responsible mutation sites: a double homozygous mutation located in the promoter sequence and exon 1 of the
UGT1A1 gene (NM_000463.3). Subsequent Sanger sequencing was conducted to confirm that the primers for the promoter sequence region were F: 5′-GCCTATTAAGAAACCTAATAAAGCTC-3′, R: 5′-GCATGGGACACCACTGGG-3′; primers for exon 1 were F: 5′-CCCAACCCATTCTCCTACG-3′, R: 5′- GTCCCACTCCAATACACACCTG-3′; primers were synthesized by Synbio Technologies Co., Ltd. (Suzhou, China). The first mutation site, c.-41_-40dupTA (rs3064744), inserts two bases TA into the TATA-box region (ATATAA) of the gene
UGT1A1 promoter, the core promoter element around −30 of the transcription start (Fig. S1), resulting in A(TA)
7TAA, a homozygous mutation in the proband and heterozygous in the parents (Fig.2). c.686C>A (rs35350960) in exon 1, causing CCG→CAG, and proline substitution by glutamine, resulting in p. Pro229Gln (Fig.2). The ACMG guidelines [
5] classified c.-41_-40dupTA and c.686C>A as likely pathogenic and pathogenic, respectively. The 3D structures of wild type (WT) and UGT1A1 mutant proteins were predicted using Swiss-model and displayed in Pymol. The p.Pro229Gln mutation changes the protein’s three-dimensional structure, which may affect its hydrogen bonding to the leucine at position 233.
Diagnosis of the patient was favoring CNS2 based on clinical presentation, laboratory tests, and genetic testing, while both of his parents were diagnosed with GS due to a compound heterozygous UGT1A1 mutation. After the patient rejected elective surgery, we added phenobarbital to his treatment. Three months later, his total bilirubin decreased to 78.1 μmol/L, the transaminases were normal, and the cholecystitis had no acute attacks.
3 Discussion
CNS is a rare autosomal recessive disorder marked by unconjugated hyperbilirubinemia, which can be categorized into subtypes according to the residual activity of the UGT1A1 enzyme and its response to phenobarbital. Patients with CNS1 exhibit severe and persistent unconjugated hyperbilirubinemia during the neonatal period, with the majority succumbing to bilirubin encephalopathy within two years of birth [
6]. CNS2 patients retain some UGT1A1 enzyme activity, therefore their disease progresses slowly and has a better prognosis, manifesting as persistent jaundice exacerbated by stress, concurrent illnesses, pregnancy, and medications [
7]. While GS is a modest enzyme deficiency that causes mildly raised bilirubin levels and intermittent jaundice, it seldom causes major health issues [
8]. The three illnesses share a genetic origin but differ in enzyme deficiency severity and clinical result, with total bilirubin levels of 342–855 μmol/L (20–45 mg/dL), 103–342 μmol/L (6–20 mg/dL), and 17–103 μmol/L (1–6 mg/dL), respectively [
9]. In this pedigree, the patient’s bilirubin level was significantly higher than his parents’ and caused gallstones, sludge hepatitis, and cholecystitis, but his parents had no associated complications except for a slightly higher bilirubin level than the upper limit of normal value, so the diagnosis of the patient was favoring CNS2, while his parents were diagnosed with GS.
Upon analyzing the familial inheritance pattern relative to the clinical phenotype, we initially hypothesized that the patient possessed a homozygous mutation. However, the phenotypic presence of both parents led us to suspect that they were not merely heterozygous carriers. Subsequent whole-genome sequencing revealed the unexpected finding of two concurrent homozygous mutations in the
UGT1A1 gene: A(TA)
7TAA and P229Q. Nonetheless, double homozygous mutations are infrequently documented, with the G71R and Y486D double homozygous mutation in CNS2 patients being the most recognized, as reported by Yamamoto in 1998, who discovered that this double homozygous mutation exhibited reduced UGT1A1 enzyme activity compared to the single homozygous mutation [
10]. As the proband, he had a more severe phenotype than his compound heterozygous mutant parents. The
UGT1A1 gene is preceded by a promoter region containing the A(TA)
6TAA repetitive sequence, which promotes transcription initiation of
UGT1A1 [
11]. In China, the mutation site A(TA)
7TAA in this region constituted 47.4% of all GS patients, making it the predominant variant among GS patients and the most prevalent mutation site in CNS2 [
12]. P229Q is infrequently observed as a homozygous mutation and predominantly appears as a compound heterozygous mutation, present in both CNS2 and GS patients [
13]. Among 310 individuals, only one exhibited a P229Q homozygous mutation and was diagnosed with GS [
12]. We suggest that the more severe phenotype in the proband is not attributable to a single A(TA)
7TAA and P229Q homozygous mutation, but rather results from the superimposition of the two homozygous variations.
Individuals with CNS exhibit an elevated risk of gallstones and associated complications [
3]. Excess unconjugated bilirubin easily infiltrates the bile, elevating the concentration of insoluble bilirubin. This surplus bilirubin can interact with calcium ions to produce calcium bilirubin precipitates, which serve as nuclei that progressively enlarge, ultimately resulting in the formation of bilirubin stones [
14]. Early diagnosis is challenged by congenital jaundice, which must be distinguished from obstructive jaundice resulting from cholelithiasis. Jaundice impairs hepatocyte function and triggers a systemic inflammatory response, perhaps resulting in a heightened occurrence of systemic and postoperative complications [
15]. For patients with acute cholecystitis who are scheduled to undergo gallbladder surgery and jaundice, it is difficult to assess the impact of hyperbilirubinemia on the surgery, so due to the high risk of surgery, more conservative treatment is chosen, and after the bilirubin has been reduced to normal, surgery is performed. However, in patients with CNS, the bilirubin cannot return to normal and the patient may undergo recurrent episodes of cholecystitis. Whether the patient wanted elective surgery or not, he should have been actively treated for hyperbilirubinemia to reduce complications. Phenobarbital is the first-line treatment for CNS2 patients and induces UDPGT activity to increase bilirubin clearance by increasing hepatic uptake, storage, and excretion [
4]. The patient had a notable decrease in bilirubin following phenobarbital therapy and did not suffer from more acute cholecystitis episodes; thus, we believe that early bilirubin level reduction (< 103 μmol/L) may mitigate the occurrence of sequelae like cholecystitis, though further studies with longer follow-up are needed to confirm this observation.
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