Yanyu Zhang,Xing Cui,Jie Xu

1 Department of Traditional Chinese Medicine,Shandong University of Traditional Chinese Medicine,Jinan,China.

2Department of Hematology,Affiliated Hospital of Shandong University of Traditional Chinese Medicine,Jinan,China.

Abstract Objective: To reduce the potential risk in aplastic anemia patients complicated with Gilbert syndrome,and find an effective treatment for the unconjugated hyperbilirubinemia.Material and Methods: The mutation of UGT1A1 gene was identified first via sequencing in patients with Gilbert syndrome complicated by aplastic anemia.Before the treatment for aplastic anemia,bilirubin and phenobarbitone tests were conducted.Patients were then treated for their primary disease and given ursodeoxycholic acid (UDCA) either with or without phenobarbitone.Results: The clinical practice of UDCA,which can alleviate increased bilirubin levels,did not affect the key treatments for aplastic anemia.Conclusions: These results indicate that Gilbert syndrome should be addressed when treating aplastic anemia.Furthermore,abnormal bilirubin levels can be controlled effectively by the UDCA treatment.

Keywords: Gilbert syndrome,Unconjugated hyperbilirubinemia,Ursodeoxycholic acid,Aplastic anemia

Background

Any patient with aplastic anemia especially severe aplastic anemia (AA) should be quickly and fully evaluated as a guide to management.Immunosuppressive therapy (IST) composed of antithymocyte globulin (ATG) in combination with Cyclosporin A (CsA) is the primary and effective treatment for AA,except the hematopoietic stem cell transplant[1].However,As the major treatment,immunosuppressive drugs can cause abnormal bilirubin or transaminase level.Previous studies demonstrated that cyclosporine therapy can induce unconjugated hyperbilirubinemia by inhibiting the uptake and the conjugation of bilirubin or competing with bilirubin for albumin-binding[2-4].The mild symptom of the unconjugated hyperbilirubinemia caused by the immunosuppressive drugs can be eliminated through the dose reduction or medication discontinuing[3],whereas there are other potential causes of the indirect hyperbilirubinemia,including Gilbert syndrome(GS),which is a hereditary unconjugated hyperbilirubinemia caused by defects in the UGT1A1 gene which can induce congenital metabolic disorders[5-7].The diagnosis of GS should be well distinguished from the CsA-induced hyperbilirubinemia in AA patients,which can avoid the unnecessary interference of the therapeutic schedule for AA such as dose reduction or medication discontinuation[8].Therefore,it is crucial to diagnose GS correctly and treat the high bilirubin levels in time,especially for patients who require immunosuppressive treatment.

Case Reports

Case 1

A 37-year-old man with weakness and bleeding presented to our hospital.He was diagnosed with severe aplastic anemia via routine blood testing,a CD55/CD59 test and a bone marrow examination that included bone marrow cytology,biopsy,immunophenotyping,and chromosome analysis.As he was being prepared for treatment with antithymocyte globulin combined with CsA,a physical examination revealed jaundice,and a blood chemistry analysis indicated elevated levels of unconjugated hyperbilirubinemia (total bilirubin 75.3μmol/l and unconjugated bilirubin 55.7μmol/l).There was no evidence of hemolytic disease (negative direct Coombs test) or elevated liver enzymes.His mother had a history of jaundice,and his urine was strongly positive for urobilinogen.Nothing of interest was found in an abdominal computed tomography (CT) scan.The patient was then given a phenobarbitone test (50 mg p.o.per day for 5 days) and a genetic analysis of UGT1A1 for a definitive diagnosis,as shown in Table 1 and Figure 1.The phenobarbitone test was positive,and the patient was found to be a UGT1A1.80:-364C ＞ T,UGT1A1.112:-1352A ＞ C heterozygote.These results confirmed the presence of Gilbert syndrome complicated by severe aplastic anemia.We used ursodeoxycholic acid (UDCA; 250 mg p.o.bid) to control the patient’s bilirubin level while administering ATG and Cyclosporin A.After UDCA treatment was initiated,the total bilirubin and unconjugated bilirubin concentrations were approximately 43.8μmol/l and 37.6μmol/l,respectively (Figure 2).After the patient completed his ATG treatment,UDCA (250 mg p.o.qd or bid) and CsA were used to control his bilirubin and aplastic anemia levels,respectively.

Figure 1.Mutations of the UGT1A1 gene among case-study patients.A: The c.211G ＞ A mutation; B:The c.1112C ＞ T mutation,which is equivocal for this disease; C: The UGT1A1*28 mutation,which changes(TA)6TAA to (TA)7TAA.

Table 1.Mutations of the UGT1A1 gene among case-study patients

Case 2

A 38-year-old woman presented to our hospital with weakness,high leukocyte count,bleeding,and splenomegaly.She was diagnosed with chronic aplastic anemia.Before she was treated with CsA,a physical examination revealed jaundice,and a blood chemistry analysis found elevated unconjugated hyperbilirubinemia (total bilirubin 87.6μmol/l and unconjugated bilirubin 64.6μmol/l),as shown in Figure 2.There was no evidence of hemolytic disease(negative direct Coombs test) or elevated liver enzymes.Her parents had no history of jaundice,and nothing abnormal was found in an abdominal CT scan.Urobilinogen was strongly positive in her urine.The patient was then given a phenobarbitone test (50 mg p.o.per day for 5 days) and a genetic analysis of UGT1A1(Table 1) to allow for a definitive diagnosis.The phenobarbitone test was positive,and the patient was found to be a UGT1A1*6,c.211G ＞ A heterozygote.Therefore,this patient was confirmed to have Gilbert syndrome complicated with CAA.Cyclosporin A was used combined with UDCA (250 mg p.o.qd or bid).After 6 months treatment,the patient’s hemoglobin level increased by 20% to 82g/l,total bilirubin and unconjugated bilirubin concentrations were approximately 35μmol/l and 30μmol/l,respectively.

Figure 2.The bilirubin concentration(μmol/l) of 3 patients.

Case 3

A 35-year-old man presented to our hospital with weakness,bleeding,and jaundice.He was diagnosed with chronic aplastic anemia at another hospital via routine blood test and bone marrow examination that included bone marrow cytology,biopsy,immunophenotyping,and chromosome testing.The patient had been treated with Cyclosporin A.After the patient was admitted to our ward,he was found to have elevated unconjugated hyperbilirubinemia (total bilirubin of 230.6μmol/l and unconjugated bilirubin of 215μmol/l).There was no evidence of hemolytic disease (negative direct Coombs test) or elevated liver enzymes.His father had a history of jaundice.This patient was then given a phenobarbitone test (50 mg p.o.per day for 5 days),and a definitive diagnosis was made using genetic analysis of UGT1A1.The phenobarbitone test was positive,and the patient was found to be a UGT1A1*6,c.211G ＞ A,UGT1A1*28 heterozygote,as shown in Table 1.The patient was confirmed to have Gilbert syndrome.Because the patient had high bilirubin levels,phenobarbitone (25 mg p.o.per day) combined with UDCA was used to control the bilirubin levels.After 1 month of treatment,his total bilirubin level decreased to 71.6μmol/l.He was then treated with CsA (100 mg p.o.bid) combined with phenobarbitone (12.5 mg p.o.per day) and UDCA (250 mg p.o.tid).

Discussion

The incidence rate of GS is approximately 10% in Western countries.Intermittent jaundice is the most important clinical feature of GS,with some patients showing varying degrees of jaundice (slight or moderate) and others not presenting with jaundice at all.The level of unconjugated bilirubin is frequently lower than 70 μmol/l.There are more than 130 different mutations that have been detected on the UGT1A1 gene.The most common mutation is UGT1A1*28,which is only present in Caucasians and decreases the activity of bilirubin glucuronic acid transferase.The c.211G ＞ A mutation,which was found in Patient 1,is only present in Asians.In Patient 3,2 different site mutations were found,which increased the unconjugated bilirubin levels and,consequently,the severity of his jaundice.

Additionally,the immunosuppressive drugs for AA,represented by CsA,can also lead to the unconjugated hyperbilirubinemia.However,the pathogenesis of immunesuppressants-induced hyperbilirubinemia is different from that of GS.Prior studies indicated that drug-induced hyperbilirubinemia is related to the inhibition of OATP1B1 or OATP1B3,but not UGT1A1,implying that the impeding of bilirubin uptake plays an important role in the pathogenesis of drug-mediated high-level unconjugated bilirubin[2].By contrast,the mutation of UGT1A1 is a hallmark of GS,which contribute to the disorder of bilirubin conjugation and excretion[9].Either the hyperbilirubinemia induced by GS or immunesuppressants pose the potential risk in AA patients,which bring about adverse effects on the life quality,the treatment on the primary disease and the outcome of AA patients.On the one hand,the ignorance and wrong diagnosis for GS in AA patients restrict the dosage and continuity of immunesuppressants,which are the pivotal therapy for AA.On the other hand,prior study indicated that the occurrence of GS with UGT1A1 mutation might influence the drug metabolism[10],implying that the characteristic of GS is not conducive to the AA treatment.

Therefore,correct diagnosis and effective treatment for GS that complicated with AA is critical for the control of the primary disease and the development of the life quality of the patients.Herein,we reported that Ursodeoxycholic acid (UDCA) is an effective drug,which alleviated the symptom of GS in the clinic significantly.UDCA originates from the Latin noun ursus,meaning bear,because bear bile contains this substance.UDCA is one of the secondary bile acids and is used to treat patients with primary biliary cholangitis[11] and cystic fibrosis[12].It has also been administered to newborn infants with impaired bile flow[13].Some studies have found that UDCA can reduce the levels of direct bilirubin[11] and unconjugated bilirubin[14,15]and may even prevent neuronal oxidative damage and cell death by reducing the levels of unconjugated bilirubin[16].Our results suggest that UDCA can reduce the bilirubin concentration in these patients.For patients with higher levels of bilirubin (for example,higher than 100μmol/l),however,UDCA is not as effective,and phenobarbitone should be added to the treatment,especially in patients requiring urgent treatment.

There are some reports of Gilbert syndrome combined with hemolytic anemia,such as glucose-6-phosphate dehydrogenase deficiency[17],pyrimidine-5'-nucleotidase Campinas[18] and hereditary spherocytosis[19] .However,there is no report discussing how to control high bilirubin levels when patients must receive immunosuppressant therapies.In this paper,we explored how to control abnormal bilirubin levels to allow treatment of aplastic anemia.

Conclusion

Gilbert syndrome can be controlled when treating aplastic anemia to reduce the risk caused by abnormal bilirubin or transaminase level.Abnormal bilirubin levels can be inhibited by UDCA treatment combined with or without phenobarbitone.

Acknowledgments

Xing Cui collected the clinical cases,analyzed the data,and wrote the manuscript; Yanyu Zhang constructed the pictures.Jie Xu assisted the manuscript writing.All authors have read and approved the manuscript,and ensured that these are the cases.