Published online February 26, 2007
PEDIATRICS Vol. 119 No. 3 March 2007, pp. e764-e767 (doi:10.1542/peds.2006-1385)

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EXPERIENCE & REASON

Marked Hyperbilirubinemia Associated With the Heme Oxygenase-1 Gene Promoter Microsatellite Polymorphism in a Boy With Autoimmune Hemolytic Anemia

Stephan Immenschuh, MD^a, Ying Shan, MD, MPH^b, Hartmut Kroll, MD^a, Sentot Santoso, PhD^a, Wilhelm Wössmann, MD^c, Gregor Bein, MD^a and Herbert L. Bonkovsky, MD^b

^a Institute of Clinical Immunology and Transfusion Medicine
^c Department of Pediatric Hematology and Oncology, Justus Liebig University, Giessen, Germany
^b Department of Medicine and the Liver-Biliary-Pancreatic Center, University of Connecticut Health Center, Farmington, Connecticut

ABSTRACT

Mild hyperbilirubinemia is a clinical feature of hemolysis. Here we describe a boy with marked elevation of serum bilirubin values (maximum: 70 mg/dL) during an acute episode of autoimmune hemolytic anemia, which returned to within the reference range after clinical improvement. The boy was a homozygous carrier of short alleles of the heme oxygenase-1 (HO-1) gene GT dinucleotide-repeat promoter polymorphism, which is associated with increased activity and inducibility of the heme-degrading enzyme HO-1, which catalyzes the production of bilirubin. In addition, heterozygosity of the uridine 5'-diphosphate-glucuronosyl-transferase 1A1 promoter polymorphism that is linked with Gilbert syndrome was found in this patient. Because bilirubin production plays a critical role during the neonatal period, the HO-1 promoter polymorphism may be an important genetic factor for the clinical outcome of neonatal hyperbilirubinemia.

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Key Words: bilirubin • oxidative stress • genotype-phenotype correlation • genetic testing • kernicterus

Abbreviations: UGT1A1, uridine 5'-diphosphate-glucuronosyl-transferase 1A1 • HO, heme oxygenase • AIHA, autoimmune hemolytic anemia • PCR, polymerase chain reaction

Bilirubin is a double-edged sword that, on the one hand, is a potent antioxidant¹ with clinically relevant protective effects^2,3 but, on the other hand, is potentially toxic.⁴ In neonatal hyperbilirubinemia, high concentrations of bilirubin may cause kernicterus and death.⁵ Serum levels of bilirubin are determined by a fine-tuned balance of production and elimination and a complex interplay of various genetic factors such as the uridine 5'-diphosphate-glucuronosyl-transferase 1A1 (UGT1A1) TATAA element polymorphism, which regulates the steady-state activity of this key enzyme for bilirubin conjugation.⁶ Production of bilirubin is intimately linked to heme oxygenase (HO), which catalyzes the first and rate-controlling step of heme degradation and generates biliverdin that is converted into bilirubin via biliverdin reductase.^7,8 Genetic deficiency of HO-1, the inducible isoform of HO, leads to anemia, hepatomegaly, and lymph node swelling in knock-out mice,⁹ and these clinical features have also been described in a human case of genetic HO-1 deficiency.¹⁰

Upregulation of HO-1 protects against a variety of pathophysiological conditions such as inflammation^11–13 and sickle cell crisis.¹⁴ A promoter polymorphism of the human HO-1 gene, which is correlated with and determines the level of expression and inducibility of the endogenous gene, has been shown to be associated with the susceptibility to develop various diseases.^15,16 Specifically, short alleles of the GT dinucleotide-repeat polymorphism within the promoter of the human HO-1 gene (<25 repeats) cause high levels of HO-1 gene induction in response to oxidative-stress stimuli.¹⁵ In genetic association studies, carriers of short alleles of the HO-1 GT-repeat polymorphism exhibited a lower incidence of smoking-related emphysema, atherosclerosis, and diabetes mellitus.¹⁶ However, little is known regarding the clinical impact of this HO-1 gene polymorphism during hemolysis in which large amounts of heme that are released from erythrocytes may induce HO-1 gene expression.¹⁷ This could be of particular importance in hemolytic conditions such as autoimmune hemolytic anemia (AIHA) and neonatal hyperbilirubinemia.

Here we report a case of marked hyperbilirubinemia during an acute hemolytic episode of AIHA in a boy who is a homozygous carrier for short alleles of the HO-1 gene GT-repeat promoter polymorphism but not for the long TATAA element polymorphism of the UGT1A1 element.

CASE REPORT

A 14-year-old white boy with a history of chronic relapsing autoimmune thrombocytopenia presented with an acute onset of jaundice, pallor, and hepatosplenomegaly. We obtained a blood count, which showed pancytopenia with a hemoglobin level of 3.4 g/dL, white blood cell count of 1.7 x 10⁹/L, and platelet count of 64 x 10⁹/L; the peripheral blood film exhibited spherocytosis and reticulocytosis. Hemolysis was confirmed by additional laboratory findings (initial lactate dehydrogenase: >1007 U/L [reference: <200 U/L]; absent haptoglobin; elevated plasma hemoglobin), and results of direct and indirect Coombs' tests were positive. No laboratory signs of extrahepatic cholestasis (alkaline phosphatase: 45 U/L [reference: <500 U/L]), liver cell necrosis (alanine aminotransferase: 33 U/L [reference: 10–50 U/L]), or impaired liver function (albumin: 4050 mg/dL [reference: 3500–5200 mg/dL]; pseudocholinesterase: 7054 U/L [reference: 5100–12600 U/L]) were detected, and the patient was diagnosed with Evans syndrome (AIHA and thrombocytopenia). The boy needed up to 5 packed red blood cell transfusions daily to maintain a hemoglobin level of 4 to 5 g/dL. The initial serum bilirubin level of 18 mg/dL (16.6 mg/dL unconjugated bilirubin) rose to a peak of 70 mg/dL (18 mg/dL unconjugated bilirubin) after 5 days (Fig 1). Total and unconjugated bilirubin levels were measured with an automated system (Architect; Abbott Diagnostics, Wiesbaden, Germany) with commercially available assays (Total Bilirubin [order No. B8G621] and Direct Bilirubin [order No. B8G631]; Abbott Diagnostics), which are based on the diazo method. Treatment with glucocorticoids and intravenous immunoglobulin slightly slowed hemolysis, but transfusion dependency persisted. Two doses of cyclophosphamide had no effect, and rituximab was given 4 times in weekly intervals. After performance of a splenectomy and treatment with cyclosporine, hemolysis improved and serum bilirubin levels of the patient decreased to reference values (0.7 mg/dL), when hemolysis stopped completely. No obvious neuropsychiatric sequelae of kernicterus were observed during the hospital stay of the patient or after discharge.

View larger version (10K): [in this window] [in a new window]   FIGURE 1 Serum bilirubin values in a patient with an acute episode of AIHA. Bilirubin values were determined over 36 days after admission of the patient. Hemoglobin (Hb) values are shown for a comparison.

 
Genotyping for HO-1 promoter allelic variants was performed with DNA from peripheral blood by polymerase chain reaction (PCR) amplification of the GT repeat containing promoter fragments of the HO-1 gene with forward primer 5'-AGAGCCTGCAGCTTCTCAGA-3' and reverse primer 5'-ACAAAGTCTGGCCATAGGAC-3'. After gel separation, PCR-amplified products were sequenced and the numbers of GT repeats were determined. The patient and his brother were homozygous carriers of short alleles (21 and 22 GT repeats; Fig 2). Similarly, the mother was a homozygous carrier (two 22-GT repeats), whereas the father was a heterozygous carrier of short and long alleles (21 and 30 GT repeats). Genotyping for the long UGT1A1 TATAA element mutation, which leads to Gilbert syndrome, with increased serum bilirubin levels was performed with forward primer 5'-GTCACGTGACACAGTCAAACATT-3' and reverse primer 5'-CTCCACAGCCATGGCGCCTTT-3' that flank the TA-repeat region of the UGT1A1 TATAA element.¹⁸ The patient and other family members were heterozygous for the longer UGT1A1 TATAA-box mutation (data not shown).

View larger version (27K): [in this window] [in a new window]   FIGURE 2 HO-1 promoter allelic variants in the patient and his family. Genomic DNA of the patient and his family was extracted from peripheral blood. Promoter fragments of the HO-1 gene with GT repeats were amplified by PCR with specific primers, and the products were separated on an agarose gel. After extraction of fragments from the gel, the procedure was repeated. Samples were sequenced, and the numbers of GT repeats are indicated by the arrows with the legend below the gel. The patient and his brother were carriers of the 21 and 22 GT repeats, having received one allele from the father (21 GT repeats) and another allele from the mother (22 GT repeats). bp indicates base pairs.

 

DISCUSSION

We have described a boy with an unusually marked elevation of serum bilirubin values during an acute hemolytic episode of AIHA (Fig 1). The patient was homozygous for the short GT dinucleotide-repeat promoter polymorphism of the HO-1 gene, which is the inducible isoform of the heme-degrading enzyme HO (Fig 2). The GT-repeat polymorphism is associated with an increased expression and inducibility of the human HO-1 gene, which in turn increases the production of bilirubin in a coupled enzyme reaction with biliverdin reductase. It is widely accepted that hemolysis leads to mild hyperbilirubinemia, but serum bilirubin concentrations of >6 mg/dL are not commonly encountered in this clinical condition.¹⁹ Although we cannot rule out the possibility that Evans syndrome (AIHA and thrombocytopenia) may cause more severe hemolysis than AIHA alone, this possibility seems unlikely. Other causes of hyperbilirubinemia such as extrahepatic obstruction, liver cell necrosis, or impaired liver function were not observed in this patient. Thus, to our knowledge, this is the first reported case in which massive hyperbilirubinemia is associated with the short HO-1 gene GT dinucleotide-repeat promoter polymorphism. The "cutoff" length for the short allele of the HO-1 GT-repeat polymorphism is <25 GT repeats, as initially defined by Yamada et al¹⁵ and subsequently confirmed in a number of follow-up studies.¹⁶ Another genetic polymorphism that may lead to hyperbilirubinemia (Gilbert syndrome) is associated with the numbers of TA repeats in the TATA box of the UGT1A1 gene.¹⁸ Genotyping of the UGT1A1 TATAA element in this patient ruled out full-blown Gilbert syndrome, but a potential ancillary effect of limited UGT1A1 activity is not excluded, because the patient was heterozygous for this polymorphism (data not shown). Moreover, because the coding region of the UGT1A1 gene has not been sequenced, compound heterozygosity for UGT1A1 coding and promoter sequence variants cannot be ruled out. Thus, in the present case, homozygosity for the short GT-repeat polymorphism of the HO-1 gene is likely to explain the unusually marked elevation of serum bilirubin levels via an increased supply of the HO-1 inducer heme during an acute episode of AIHA.

Bilirubin protects against various diseases that are related to oxidative stress (for a review see ref ³), and these beneficial effects have been attributed to the potent antioxidant function of this compound.^1,4 Because HO is the major producer of endogenous bilirubin, the increased inducibility of HO-1 resulting from the short GT-repeat polymorphism may explain the role of HO-1 as a protective genetic factor in such diseases.¹⁶ By contrast, high serum levels of bilirubin can be toxic in neonatal hyperbilirubinemia, in which condition infants with high levels of unconjugated serum bilirubin may develop and die as a result of kernicterus.⁵ Therefore, the balance of production and elimination of bilirubin is of particular importance during the neonatal transition period, and an increased inducibility of HO-1 because of the short allele of the HO-1 gene promoter polymorphism may significantly contribute to the severity of neonatal hyperbilirubinemia. A recent study on Japanese infants on this issue did not find an association between the severity of neonatal hyperbilirubinemia and the length of the HO-1 gene GT-repeat promoter polymorphism.²⁰ The results of the latter report, however, have to be interpreted with caution, because ethnic background seems to be an important factor for the clinical impact of the HO-1 promoter polymorphism.¹⁶

CONCLUSIONS

Unusually marked hyperbilirubinemia during hemolysis was observed in a patient who was homozygous for short alleles of the HO-1 gene GT dinucleotide-repeat promoter polymorphism. Thus, this HO-1 gene polymorphism may be a prognostic factor for the severity of neonatal hyperbilirubinemia and is worthy of additional study in conditions of bilirubin overproduction.

FOOTNOTES

Accepted Sep 27, 2006.

Address correspondence to Stephan Immenschuh, MD, Institut für Klinische Immunologie und Transfusionsmedizin, Justus-Liebig-Universität Giessen, Langhansstrasse 7, 35392 Giessen, Germany. E-mail: stephan.immenschuh{at}immunologie.med.uni-giessen.de

The authors have indicated they have no financial relationships relevant to this article to disclose.

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