“Gilbert’s-like” syndrome as part of a spectrum of persistent unconjugated hyperbilirubinemia in post-chronic hepatitis patients

Gilbert’s syndrome (GS) patients present with remittent unconjugated hyperbilirubinemia. In this study, we investigated the correlation between polymorphisms in the gene encoding UDP-glucuronosyltransferase, UGT1A1, and the development of unconjugated hyperbilirubinemia in clinical GS and post-hepatitis hyperbilirubinemia. Blood samples were collected from 285 patients, including 85 patients who were clinically diagnosed with GS, 70 patients who had indirect hyperbilirubinemia during the recovery period of chronic liver diseases, 109 patients with normal hepatic function and 21 chronic active hepatitis patients. All samples were tested for the presence of the *28/*6 UGT1A1 genotype by pyrosequencing. Compared with the GS-control group, a significant difference in variations of the UGT1A1*28/*6 allele gene was found in GS patients. The post-hepatitis group showed a significant difference in the UGT1A1*28/*6 allele gene frequency distribution relative to that in the hepatitis control group. There were no significant differences between the GS group and post-hepatitis group in the distribution of the UGT1A1*28/*6 allele gene frequency and UGT1A1 diplotypes. UGT1A1*28/*6 gene polymorphisms in patients who had indirect hyperbilirubinemia while recovering from chronic liver diseases presented similar patterns as those seen for GS patients. These findings suggest that a “Gilbert’s-like” syndrome might be part of the spectrum of persistent unconjugated hyperbilirubinemia in post-chronic hepatitis patients.

persistent or intermittent mild unconjugated hyperbilirubinemia can be detected in many patients with chronic persistent hepatitis. For these patients, there is no indication for the treatment for liver inflammation because the liver enzyme indices are usually normal, viral DNA is undetectable, and ultrasound tests often do not indicate liver disease. Routine treatments for jaundice are usually not effective for these patients, and persistent unconjugated hyperbilirubinemia may seriously affect the quality of life for some patients. Such patients are often classified as having so-called "post-hepatitis hyperbilirubinemia". Felsher et al. demonstrated that the mean UGT1A1 activity was significantly lower in 12 patients with chronic persistent hepatitis, and these patients also presented with persistent or intermittent mild unconjugated hyperbilirubinemia compared with healthy individuals 14 . However, the number of cases in this study was small; thus, the possible involvement of UGT1A1 in post-hepatitis hyperbilirubinemia remains unclear.
GS and chronic persistent liver disease are two distinct diseases. UGT1A1 activity has been linked to the development of indirect hyperbilirubinemia in both clinical GS and post-hepatitis hyperbilirubinemia, raising the question of whether "Gilbert's-like" aberrations in bilirubin metabolism are part of the spectrum of hyperbilirubinemia in post-chronic persistent hepatitis and what is the role of gene UGT1A1 polymorphisms in this condition. In this study, we investigated the correlation between UGT1A1 gene polymorphisms and the development of unconjugated hyperbilirubinemia in GS and post-hepatitis hyperbilirubinemia, and provide a new strategy for the possible aetiology, pathogenesis and therapy for "Gilbert's like" syndrome in persistent or intermittent mild unconjugated hyperbilirubinemia in post-chronic liver disease.

Discussion
Our study investigated the characteristics of UGT1A1 polymorphisms in Chinese patients with post-hepatitis hyperbilirubinemia and Gilbert's syndrome (GS). UGT1A1*28/*6 gene polymorphisms are correlated with the development of unconjugated hyperbilirubinemia in both clinical GS and post-hepatitis hyperbilirubinemia.
Patients carrying the UGT1A1*28/*6 gene polymorphism who had indirect hyperbilirubinemia while recovering from chronic liver diseases can present with a pattern similar to that seen for GS patients.
Although UGT1 genetic diversity in the healthy Chinese population has been reported and GS is correlated with UGT1A1*28/*6 gene polymorphisms 7,15-18 , there is limited information concerning the genetic variation of UGT1A1 in Chinese GS patients. The influence of genetic variations and other factors, whether alone or (c) STB/IB levels for different UGT1A1 genotypes indicated significant differences (P = 0.000). The STB level was markedly elevated when individuals were homozygous for UGTA1*28 or *6. STB: serum total bilirubin; IB: indirect bilirubin.
combined, on GS pathology is unclear. Our study found that the gene allele frequency of UGT1A1 *28 was 0.465 and 0.101 in the GS group and control group, respectively (Table 1). Meanwhile, the allele gene frequency of the UGT1A1 *6 mutation was 0.288 and 0.142 in the GS group and control group, respectively (Table 1). Compared with the GS-control group, a significant difference in the variation of the UGT1A1*28/*6 allele gene was found in GS patients (P < 0.05).
During the recovery period, the liver enzyme indices and serum bilirubin levels of hepatitis patients were typically reduced gradually to normal levels. However, similar to GS patients, persistent or intermittent mild unconjugated hyperbilirubinemia can be detected in post-hepatitis patients. The possible pathogenesis of post-hepatitis hyperbilirubinemia remains unclear, and there is limited clinical guidance for clear diagnostic criteria and therapy for these patients, which can result in patient and physician confusion as well as variations in treatment practices. Our results indicated a significant difference in the UGT1A1*28/*6 allele gene frequency distribution in the post-hepatitis hyperbilirubinemia group compared with that in the hepatitis-control group, but there was no significant difference in UGT1A1*28/*6 gene polymorphisms between the GS and post-hepatitis hyperbilirubinemia groups. The gene polymorphisms of UGT1A1*28/*6 in patients who had indirect hyperbilirubinemia during the recovery period from chronic liver diseases presented with a similar pattern to that of GS patients but differed from that for chronic active hepatitis patients.
Although GS patients may experience hyperbilirubinemia, treatment generally is not necessary 19,20 . Pyrosequencing of UGT1A1*28/*6 gene polymorphisms could thus be used as an effective auxiliary method for GS diagnosis after excluding other possible causes of indirect hyperbilirubinemia. Persistent or intermittent unconjugated hyperbilirubinemia could also present in patients during recovery from chronic liver diseases. If these patients carry a UGT1A1 mutation, we would recommend that they not be treated. The pyrosequencing assay for UGT1A1*28/*6 gene polymorphisms has a certain degree of specificity and sensitivity 21 and may provide evidence for the clinical diagnosis and treatment of GS and post-hepatitis hyperbilirubinemia.
In conclusion, UGT1A1*28/*6 gene polymorphisms are correlated with the development of unconjugated hyperbilirubinemia in clinical GS and post-hepatitis hyperbilirubinemia. Clinicians should be aware that "Gilbert's-like" syndrome might be part of a spectrum characterizing persistent unconjugated hyperbilirubinemia in post-chronic hepatitis patients.

Materials and Methods
Subjects. Blood samples were collected from 285 hospitalized patients and outpatients who were treated between August 2012 and February 2015 at Wuhan Union Hospital, Tongji Medical College, Huazhong University of Science and Technology. All the study participants were members of the Han population. Eighty-five patients were clinically diagnosed as having Gilbert's Syndrome (GS; male: n = 67, female: n = 18, average age 35 ± 14 yrs.). The diagnostic criteria for GS were as follows: (1) a history of intermittent jaundice lasting more than six months that worsened with fatigue, alcohol consumption, infection, menstruation or stress; (2) an unconjugated bilirubin and conjugated bilirubin level that was normal or less than 20 percent of total bilirubin, respectively; a serum AST level that was normal, with the exception of other liver diseases and haemolysis; (3) the absence of other diseases that could cause elevated unconjugated bilirubin levels such as hyperthyroidism or drug side effects; (4) the absence of Crigler-Najjar syndrome types I and II. In addition, 109 patients with normal hepatic function served as the GS control group (male: n = 58, female: n = 51, average age 53 ± 12 yrs.). All the control individuals denied a history of jaundice, abnormal liver function and liver disease. The post-hepatitis hyperbilirubinemia group included 70 patients (male: n = 52, female: n = 18, average age: 41 ± 13 yrs.) who had unconjugated hyperbilirubinemia while recovering from chronic hepatic diseases (chronic hepatitis b, viral hepatitis: n = 30, patients with hepatitis b cirrhosis: n = 16, 3 patients with alcoholic liver cirrhosis: n = 3, chronic alcoholic liver disease: n = 6, fatty liver: n = 13, chronic schistosomiasis liver disease: n = 2). The hepatitis-control group included 21 chronic active hepatitis patients (male: n = 19, female: n = 2, average age: 48 ± 12 yrs.). Clinical information was recorded in detail for all four patient groups. The study was approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology. The study methods were carried out in accordance with approved guidelines, and all the subjects provided written informed consent prior to study enrolment.