Pediatr Blood Cancer 2010;54:758–760

BRIEF REPORTNeonatal Cholestasis and Glucose-6-P-Dehydrogenase Deficiency

Uwe Kordes, MD,1* Andrea Richter, MD,2 Rene Santer, MD,3 Hansjorg Schafer, MD,4 Dominique Singer, MD,5

Josef Sonntag, MD,6 Ulrike Steuerwald, MD,7 Reinhard Schneppenheim, MD,1 and Gritta Janka, MD1

INTRODUCTION

Neonatal indirect hyperbilirubinemia is an emergency requiring

close diagnostic and often prompt therapeutic attention, but it is

uncommon for the pediatric hematologist to become involved.

Neonatal cholestasis indicates hepatobiliary dysfunction, it is much

rarer, it does not usually evolve from the former nor is it usually

related to erythrocytic disorders. Thus it may be particularly

rewarding to reconcile both with a single hematological diagnosis.

CLINICAL DATA

A preterm male infant was delivered in gestational week 35þ 2

by cesarean section because of fetal distress to a 26-year-old gravida

II para II of Russian ancestry. Last trimester gestational diabetes

was well controlled with insulin. Body weight was 2,030 g (<10

percentile), length 43 cm (<10 percentile), head circumference

32.5 cm (<50 percentile), APGAR 9/10/10, pH 7.34. Blood groups

were A Rhesus positive, ccD.EE, Kell negative, direct Coombs test

negative (neonate) and A Rhesus positive, ccD.EE, Kell negative,

antibody screening test negative (mother). Hemolytic anemia was

detected at 2 hr (Table I).

Phototherapy was started, it was stopped subsequently

when severe cholestasis developed. Peak serum total bilirubin was

24 mg/dl and peak conjugated bilirubin was 17 mg/dl on day 5, non-

conjugated bilirubine remained below 12.6 mg/dl (Table I). Except

for a single initial blood glucose level of 40 mg/dl the infant was

euglycemic and normocalcemic.

The infant displayed no irritability, hyperexcitability, had a

normal pitched cry, no apnea-bradycardia, mild muscular hypo-

tonia, mild hepato- and no splenomegaly, a right-sided inguinal

hernia, no hematoma. Complexion was gray-brown. Erythrocyte

transfusions, ursodeoxycholic acid, and vitamin E were given.

Serology and/or PCR excluded active hepatitis A, B, C, HIV-1/-

2, CMV, EBV, HSV, VZV, measles, mumps, rubella, parvovirus

B19, adenovirus, toxoplasmosis, syphilis. Blood cultures were

sterile, CRP and IL-6 repeatedly within normal limits. Blood levels

were normal for a1-antitrypsine, chitotriosidase, acyl-carnitine,

galactose-1-phosphate uridylyltransferase, TSH. Genomic ana-

lysis of JAG1 did not detect any known mutation of Alagille’s

syndrome. Analysis of the UGT1A1 promoter detected a wild-type

(TA)6TAA(6/6) excluding the European type Gilbert–Meulen-

gracht syndrome.

On ultrasound the liver reflex appeared slightly enlarged and

irregular with non-distended bile ducts and a rudimentary gall

bladder. An explorative laparatomy and herniotomy was performed,

intra-operative retrograde cholangiography demonstrated normal

anatomy of intra-hepatic bile duct and ductus communis with

normal bile flow.

Liver biopsy showed a normal lobular structure and bile

ducts, extra-medullary hematopoiesis, few lymphocytic infiltrates,

swollen hepatocytic cytoplasm, few necrosis and giant cells,

moderate siderosis and marked intralobular and cytoplasmatic bile

retention (Fig. 1). Cerebral ultrasound on days 1 and 22 detected two

small periventricular cysts, no evidence for hemorrhage, no basal

ganglia abnormalities, and regular cerebral blood flow.

The G6PD activity on a semiquantitative fluorescent spot

test obtained from the Guthrie card was <5%, in a repeat test

3 months after the fourth erythrocyte transfusion G6PD activity was

7.1 mmol substrate consumption/1011 erythrocytes/min (reference

30.6� 4.5).

The infant was discharged at 4 weeks with 2,595 g weight on

folic acid supplementation. Subsequent baseline hemoglobin

stabilized at 90 g/L, reticulocytes at 25%, liver enzymes and

bilirubin normalized. At 9 months a viral infection triggered a non-

cholestatic hemolytic crisis requiring a transfusion (hemoglobin

52 g/L, LDH 1,327 U/L). On follow-up he has no findings of overt

or subtle bilirubin-induced neurological dysfunction and is

We report a Caucasian neonate with chronic non-spherocytichemolytic anemia due to a class I G6PD deficiency. A novelmutation missense mutation in exon eight of the G6PD gene wasdetected (c.827C>T p.Pro276Leu). Bilirubin peaked on day 5 at24 mg/dl with a conjugated bilirubin of 17 mg/dl. Jaundice resolved

within 4 weeks. A detailed work-up failed to reveal other specificfactors contributing to cholestasis. Severe hemolytic disease of thenewborn may cause cholestasis even in the absence of associatedprimary hepato-biliary disease. Pediatr Blood Cancer 2010;54:758–760. � 2010 Wiley-Liss, Inc.

Key words: cholestasis; glucose-6-P-dehydrogenase deficiency; inspissated bile syndrome; kernicterus; neonatal jaundice

� 2010 Wiley-Liss, Inc.DOI 10.1002/pbc.22390Published online 5 January 2010 in Wiley InterScience(www.interscience.wiley.com)

——————1Department of Pediatric Hematology and Oncology, University

Medical Center Eppendorf, Hamburg, Germany; 2Department of

Pediatric Gastroenterology, University Medical Center Eppendorf,

Hamburg, Germany; 3Department of Pediatric Metabolic Disease,

University Medical Center Eppendorf, Hamburg, Germany;4Department of Pathology, University Medical Center Eppendorf,

Hamburg, Germany; 5Department of Neonatology and Pediatric

Intensive Care Unit, University Medical Center Eppendorf,

Hamburg, Germany; 6Kinderklinik, Stadtisches Klinikum Luneburg,

Luneburg, Germany; 7Screening Laboratory Metabscreen, Hannover,

Germany

Conflict of interest: nothing to report.

*Correspondence to: Uwe Kordes, Department of Pediatric

Hematology and Oncology, University Medical Center Eppendorf,

Martinistr 52, 20246 Hamburg, Germany. E-mail: kordes@uke.de

Received 13 October 2009; Accepted 29 October 2009

reaching normal developmental milestones. Audiological testing

shows normal distortion product and transient evoked otoacoustic

emissions indicating normal hair cell function, assessment of

auditory nerve and brainstem function with evoked potentials is

pending.

Genomic sequencing of exons 2–13 of the G6PD gene detected

hemizygosity for a novel missense mutation in exon 8 (c.827C>T

p.Pro276Leu), termed variant Hamburg. Maternal leukocytes were

heterozygous for the same point mutation, indicating germline

transmission, maternal erythrocytic G6PD activity was low

normal with 27.4 mmol substrate consumption/1011 erythrocytes/

min, excluding marked non-random X-chromosome inactivation.

DISCUSSION

This case represents a rare sporadic WHO class I G6PD

deficiency with<10% enzyme activity associated with chronic non-

spherocytic hemolytic anemia. About 400 variants of the human

G6PD enzyme and more than 140 mostly missense mutations

leading to amino acid substitutions affecting enzyme stability have

been described. Class I defects result from more than 60 known

sporadic independent missense mutations which cluster in exon

10 encoding the dimerization domain [1]. There is a minor cluster in

exon 8, thus variant Pro276Ser as well as nearby variants Wexham

(Ser278Phe) and La Jolla (Ser278Pro) also cause class I G6PD

deficiency similar to variant Hamburg.

Interestingly, carboxyhemoglobin measurements have shown

that hemolysis is not the main determinant of neonatal hyper-

bilirubinemia in Mediterranean G6PD deficiency [2]. Decreased

hepatocyte enzyme activity for UGT1A1, G6PD along with

prematurity and maternal diabetes can all contribute to an increased

bilirubin production-conjugation index.

In the present case, however, there was significant ongoing

hemolysis at birth causing hyperbilirubinemia, likely triggered by

prenatal oxidative stress. Progression to frank cholestasis was

unexpected.

Several reports mention cholestasis occurring in neonatal

hemolysis (see references quoted in Ref. [3]), and the inspissated

bile syndrome has been suggested as a possible underlying

mechanism. Inspissated bile may cause obstruction and dilatation

of the biliary ducts requiring pharmacological choleresis with

ursodeoxycholic acid or even surgical drainage in selected cases.

Notably, ductular dilatation was absent in our case. Bile salt

changes and hyperbilirubinbilia in cystic fibrosis may now be a more

common cause for inspissated bile syndrome rather than hemolytic

disease of the newborn. In the era before maternal antibody

screening and rhesus prophylaxis, inspissated bile syndrome may

have been more common in Coombs positive hemolytic disease of

the newborn [4,5].

The complex pharmacogenomics of bilirubin metabolism

involves a family of organic anion transport proteins located at

the hepatocytic basolateral membrane modulating total and

even conjugated bilirubin levels [6] as well as UDP glucuronyl-

transferase and ATP-binding cassette transporter at the canalicular

Pediatr Blood Cancer DOI 10.1002/pbc

Fig. 1. HE 10�: liver biopsy, overview (A). HE 20�: multinucleated

giant cell, cholestatic droplets in liver cell cytoplasm, and bile duct (B).

HE 20�: extramedullary hematopoiesis including eosinophils around

liver vein (C). Prussian blue, 20�: hemosiderin deposits around liver

vein (D). [Color figure can be viewed in the online issue, which is

available at www.interscience.wiley.com.]

TABLE I. Laboratory Data

Hour 2 Day 5 Day 15 Day 31 Day 60

Hemoglobin (g/L) 83a 85b 71a 76 80

Reticulocyte count (%) 19 28 30 10

Leukocytes (/nl) 60c 15 12.9 12

Platelets (/nl) 138 113 443 250

Total bilirubin (mg/dl) 9 24 9.5 3.1 0.8

Conjugated bilirubin (mg/dl) 1.7 17 7.3 2.3

AST (U/L) 34 127 115 32

ALT (U/L) 18 104 104 29

LDH (U/L) 2,179 1,000 247 215 195

Total bile acid (mmol/L) 30 36

Lioprotein X (g/L) 0.56 0.16

g-GT (U/L) 70 41 23 194 64

aPretransfusion, differential without spherocytes, schistocytes, or bite cells; bPosttransfusion; cIncluding

normoblasts.

Neonatal Cholestasis in G6PD Deficiency 759

membrane. Disturbances of the latter usually have a more severe or

progressive cholestatic phenotype (Dubin Johnson syndrome,

progressive familial intrahepatic cholestasis types 1–3, benign

recurrent intrahepatic cholestasis). It is conceivable that partial

insufficiency of any of these factors might precipitate cholestasis

at times of hemolytic stress. Intrauterine growth retardation would

be a transient risk factor for such an insufficiency not present at

the time of the second non-cholestatic hemolytic episode in our

case.

The possibility that neonatal hepatitis was a contributing factor

was considered. Many differential diagnoses previously labeled

idiopathic neonatal hepatitis [7] were excluded. Delayed rise of

transaminases (Table I) and negative microbiological findings

argue against an intrauterine infection or hypoxic liver damage.

Multinucleated giant cells, single cell necrosis, and lymphocytic

infiltrates may occur in livers stressed with extramedullary

hematopoiesis in hemolytic disease of the newborn [4]. Suboptimal

maternal gylcemic control, not present in this case, may lead to

adverse fetal outcome in infants of diabetic mothers including an

increased risk of unconjugated hyperbilirubinemia, probably as a

result of polycythemia and increased hemolysis of glycosylated

erythrocytes.

Should screening for G6PD deficiency be performed in

presumed low-risk or non-endemic populations? While this is a

report of a rare sporadic class I G6PD deficiency, it is also a reminder

that intermarriage and migration are reasons to reconsider exclusion

of G6PD deficiency from routine screening in northern countries

[1]. G6PD deficiency and early discharge policy put infants at risk

for bilirubin-induced neuropathies. Acute hemolytic crisis may

require interventional transfusions to avert renal failure. Rapid and

easy screening may be performed with the fluorescent spot test.

Hemizygosity for c.827C>T p.Pro276Leu of G6PD

causes chronic non-spherocytic hemolytic anemia. Self-limited

cholestatic liver disease may occur when bilirubin production in

hemolytic disease of the newborn exceeds the capacity for neonatal

choleresis.

REFERENCES

1. Cappellini MD, Fiorelli G. Glucose-6-phosphate dehydrogenase

deficiency. Lancet 2008;371:64–74.

2. Kaplan M, Vreman HJ, Hammerman C, et al. Contribution of

haemolysis to jaundice in Sephardic Jewish glucose-6-phosphate

dehydrogenase deficient neonates. Br J Haematol 1996;93:822–

827.

3. McAdams RM, Dotzler SA, Winter LW, et al. Severe hemolytic

disease of the newborn from anti-e. J Perinatol 2008;28:230–

232.

4. Dunn P. Obstructive jaundice and haemolytic disease of the newborn.

Arch Dis Child 1963;38:54–61.

5. Sivan Y, Merlob P, Nutman J, et al. Direct hyperbilirubinemia

complicating ABO hemolytic disease of the newborn. Clin Pediatr

(Phila) 1983;22:537–538.

6. Sanna S, Busonero F, Maschio A, et al. Common variants in the

SLCO1B3 locus are associated with bilirubin levels and uncon-

jugated hyperbilirubinemia. Hum Mol Genet 2009;18:2711–2718.

7. Balistreri WF, Bezerra JA. Whatever happened to ‘‘neonatal

hepatitis’’? Clin Liver Dis 2006;10:27–53, v.

Pediatr Blood Cancer DOI 10.1002/pbc

760 Kordes et al.