Blood, Vol. 94 No. 7 (October 1), 1999:
pp. 2259-2262
Coinheritance of Gilbert Syndrome Increases the Risk for Developing
Gallstones in Patients With Hereditary Spherocytosis
By
Emanuele Miraglia del Giudice,
Silverio Perrotta,
Bruno Nobili,
Claudia Specchia,
Giovanna d'Urzo, and
Achille Iolascon
From the Department of Pediatrics, Second University of Naples,
Naples, Italy; the Department of Health Sciences, Biostatistics Unit,
University of Genoa, Italy; and the Department of Pediatrics,
University of Bari, Bari, Italy.
 |
ABSTRACT |
The precocious formation of bilirubinate gallstones is the most
common complication of hereditary spherocytosis (HS), and the
prevention of this problem represents a major impetus for splenectomy
in many patients with compensated hemolysis. Because Gilbert syndrome
has been considered a risk factor for gallstone formation, there are
reasons for postulating that the association of this common inherited
disorder of hepatic bilirubin metabolism with HS could increase
cholelithiasis. To test this hypothesis, 103 children with mild to
moderate HS who, from age 1, have undergone a liver and biliary tree
ultrasonography every year, were retrospectively examined. The 2-bp
(TA) insertion within the promoter of the uridine diphosphate-glucuronosyltransferase gene (UGT1A1), associated with
Gilbert syndrome, was screened. The risk of developing gallstones was
statistically different among the 3 groups of patients: homozygotes for
the normal UGT1A1 allele, heterozygotes, and homozygotes for the allele
with the TA insertion. Fitting a Cox regression model, in
fact, a statistically significant hazard ratio of 2.19 (95% confidence
interval: 1.31 to 3.66) was estimated from one to the next of these
genetic classes. The individual proneness to form gallstones from TA
insertion in the TATA-box of the UGT1A1 promoter should be considered
during the follow-up of patients with HS. Although patients with HS
were the only ones studied, extrapolating these data to patients who
have different forms of inherited (eg, thalassemia, intraerythrocytic
enzymatic deficiency) or acquired (eg, autoimmune hemolytic anemia,
hemolysis from mechanical heart valve replacement) chronic hemolysis
can be warranted.
© 1999 by The American Society of Hematology.
| |
INTRODUCTION |
HEREDITARY SPHEROCYTOSIS (HS) is a common
hemolytic anemia and, taking into account the heterogeneity of
expression, it has a prevalence of approximately 1:2,000 in Europe and
North America. The underlying primary molecular lesion is
heterogeneous; it may affect several membrane proteins including
spectrin, ankyrin, band 3, and, more rarely, protein
4.2.1,2 Most patients do have well-compensated hemolysis.
It is only when complications occur that they are brought to medical
attention. Because the early formation of bilirubinate gallstones is
the most common complication occurring in about half of HS patients,
the prevention of this problem represents a major impetus for
splenectomy in many patients with mild or moderate
hemolysis.3 The pathogenesis of gallstones in HS is related
to the high biliary concentration of monoconjugated bilirubin which, in
turn, may play a pivotal role in gallstone formation by behaving as a
source of unconjugated monohydrogenated bilirubin and as a possible
coprecipitant with calcium.4
Gilbert syndrome, a benign condition of decreased bilirubin conjugation
because of diminished activity of the conjugating enzyme uridine
diphosphate-glucuronyl transferase (UGT1A1), has been associated with
an increased production of monoconjugated bilirubin.5 A
variant promoter for the UGT1A1 gene containing a two-base pair
addition (TA) in the TA(6)TAA element has been recently
described at the homozygous state in patients with Gilbert syndrome.6 The extra nucleotides have been shown to
decrease the expression of the UGT1A1 gene and consequently the
bilirubin conjugation.7 Homozygosity for the promoter
variant is frequently encountered in European and African populations
with a prevalence of 11% to 19%.8 Coinheritance of
Gilbert syndrome has been found to be associated with increased
bilirubin levels in patients with heterozygous 
-thalassemia or
glucose-6-phosphate dehydrogenase (G6PD) deficiency and with increased
incidence of neonatal icterus in G6PD deficiency.9-11
Simultaneous presence of Gilbert syndrome has been described as a
possible cause of HS misdiagnosis.12-14 We
recently showed that newborns with HS, homozygotes for the UGT1A1
allele with the (TA) insertion in the promoter, have enhanced jaundice,
which requires phototherapy.15
The study's aim was to determine whether Gilbert syndrome and HS, if
co-inherited, could act synergistically to increase gallstone formation. Children with HS (103) were subdivided by their UGT1A1 genotype (normal, heterozygotes, and homozygotes for the mutation causing Gilbert syndrome) and retrospectively examined. Regardless of
their erythrocyte membrane alteration, patients with HS, who coinherited Gilbert syndrome, had an almost 5-fold greater tendency to
form gallstones than normal HS patients.
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MATERIALS AND METHODS |
Patients.
One hundred three unsplenectomized children, with mild to moderate HS
(mean age: 9.8 ± 4 years; range, 2 to 14 years), belonging to 93 families, were clinically examined in a retrospective way. Classification of the disease's clinical forms (mild and moderate) was
performed by following well-established criteria.3
Particularly, mild HS is associated with compensated hemolysis without
anemia, whereas hemolysis is incompletely compensated in moderate HS. But, in these patients, hemoglobin levels are constantly higher than 80 to 90 g/L and reticulocytes are usually less than 10%.
To detect gallstones early, these patients have undergone a liver and
biliary tree ultrasonography every year, starting at age 1. It must be
underscored that abdominal ultrasonography represents the most reliable
and readily accessible study to detect gallstones and that it has been
reported to have a success rate as high as 95%.16
Erythrocyte membrane study.
The pattern of red blood cell membrane proteins was evaluated by sodium
dodecyl sulfate-polyacrylamide gel electrophoresis, by using, with some
modifications,17 both the discontinuous buffer system of
Laemmli18 with acrylamide linear gradient from 5% to 15%
and the continuous buffer system of Fairbanks et al19 with
exponential gradient of acrylamide from 3.5% to 17%.
Genetic analysis.
Genomic DNA was extracted from peripheral blood leukocytes by using
standard methods. Screening of the TA insertion in the A(TA)6 TATAA motif within the promoter of the UGT1A1 gene
was performed by polymerase chain reaction with the primers described by Monaghan et al.7 Analysis of the amplified DNA fragments was performed in a 6%, silver stained, polyacrylamide gel.
Statistical analysis.
Continuous data are presented as mean ± SD.
Differences between gaussian variables mean values were
assessed by using the Student t-test; the significance is
defined as P < .05. Rates of developing gallstones were
calculated for each patient category by dividing the number of events
(ie, gallstone formation) by the cumulative number of event-free years
of observation. Assuming that the hazard rate was probably not constant
during the follow-up, the Kaplan-Meier estimates were calculated. The
plots of the complement of Kaplan-Meier estimates for each genotype
were compared by a logrank test.
Finally, a Cox regression model was used to get an
estimate of the hazard ratio (HR) of gallstone formation in function of the time of their development between the 3 classes of patients. A 95% confidence interval (CI) was calculated to measure the
statistical precision of the estimate.
| |
RESULTS |
We found 41 (40%) of the HS patients homozygous for the UGT1A1
wild-type allele (TA(6)/TA(6))
and 39 (37%) heterozygotes
(TA(6)/TA(7)). Twenty-three
(22%) patients were homozygotes for the mutated allele (TA(7)/TA(7)). The frequency
distribution of the 3 different genotypes was similar to the normal
population in Southern Italy.20
The average reticulocyte number was 212 ± 60 (109/L) in
the group of homozygous wild-type allele patients, 220 ± 75 (109/L) in heterozygotes, and 195 ± 55 (109/L) in homozygotes for the mutated allele. The
nonsignificant differences among the 3 groups suggest a similar degree
of hemolysis.
Patients with the UGT1A1
TA(7)/TA(7) genotype showed
higher mean serum bilirubin levels than heterozygotes and normal
patients (4.8 ± 1.9 mg/dL, 3.2 ± 1.6 mg/dL, and
2.4 ± 1.3 mg/dL, respectively). Differences among the 3 groups were
significant (TA(7)/TA(7)
v TA(7)/TA(6):
P < .01; TA(7)/TA(7)
v TA(6)/TA(6): P < .0005 ).
Five of 41 (12%) patients with the UGT1A1
TA(6)/TA(6) genotype developed
gallstones, whereas this complication was discovered in 10 of 39 (26%)
heterozygotes and in 11 of 23 (48%) homozygous patients for the allele
with the TA insertion. Normal, heterozygotes, and homozygotes totaled
428, 384, and 202 for event-free years, and the rates of cholelithiasis
formation were 0.012, 0.026, and 0.055, respectively.
Figure 1 shows the different plots of the
complement of Kaplan-Meier estimates among the 3 genotypes. The
difference between the 3 plots estimated by the logrank test was
statistically significant (P = .003) and represented by every
follow-up time, the cumulative rate difference among the groups. The
coefficient calculated by Cox regression model
(TA(7)/TA(6) v
TA(6)/TA(6) and
TA(7)/TA(7) v
TA(7)/TA(6) ) was 2.19 (95% CI:
1.31-3.66) and represented an estimate of the HR in developing
gallstones. The presence of the mutated UGT1A1 allele, nevertheless,
does not appear to be a major determinant of the age of gallstone
formation. Although gallstone diagnosis occurred at a younger age in
patients with the UGT1A1 TA(7)/TA(7)
genotype, it was not significantly different compared with the normal
and heterozygous children (8.7 ± 4.4 years v 11.2 ± 4.7 years and 10.4 ± 4.6 years, respectively).
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| Fig 1.
Kaplan-Meier probabilities of forming gallstones for 103 patients with HS. Patients are categorized on the basis of their UGT1A1
genotype.
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Combined spectrin and ankyrin reduction was the most frequent
biochemical alteration in the 3 categories of patients, followed by
band 3 deficiency and isolated spectrin reduction
(Table 1). We didn't find statistically
significant differences concerning the tendency to form gallstones when
separating HS patients on the basis of their biochemical phenotype. In
fact, this complication was found in 6 of 23 (26%) patients with
isolated spectrin reduction, in 13 of 46 (28%) patients showing
combined spectrin and ankyrin reduction, and in 7 of 27 (26%) patients
with band 3 reduction.
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Table 1.
Erythrocyte Membrane Protein Deficiencies Found in 103 Patients With HS Subdivided by Their UGT1A1 Genotype
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DISCUSSION |
If and when to splenectomize patients with mild to moderate HS has been
constantly debated. It must be emphasized that splenectomy carries a
surgical risk as well as the risk of postsplenectomy sepsis.21,22 Moreover, it has recently been reported that
after age 40 the rate of myocardial infarction or stroke in HS patients without a spleen is 5.6 times the rate in patients with a spleen and,
consequently, a conservative approach to splenectomy in people with HS
and mild to moderate anemia has been suggested.23 In the
decision regarding splenectomy of HS children with normal or only
slightly low hemoglobin levels and without growth and activity
impairment,24 the likelihood to form gallstones and relative complications may be considered.25-27
We show a statistically significant association between the strongly
associated with Gilbert syndrome UGT1A1
TA(7)/TA(7) genotype and the increased
rate of gallstones in HS children. Furthermore, the relative risk of
gallstones seems to increase in an allele dose-dependent fashion, with
the addition of 1 or 2 mutated UGT1A1 alleles.
These results may be explained by the combined effect of hemolysis from
HS and Gilbert syndrome in producing high levels of bilirubin
monoconjugates. When these interact with bile salts, calcium, or other
biliary components, they could initiate pigment and cholesterol
gallstone development.28-31
The contribution of coinherited Gilbert syndrome to the risk of forming
gallstones should be esteemed during the follow-up of the patient with
spherocytosis. It might be considered also in the decision regarding
splenectomy, although this treatment is debatable in patients with mild
to moderate HS, particularly because new treatments for gallstones
lower the risk of this complication.2
The predisposition to gallstones is greatly increased in HS patients
with Gilbert syndrome but, rather surprisingly, the age of onset for
this complication, although lower compared with the other 2 groups, was
not statistically different. Probably, the number of patients analyzed
and the extension of the follow-up were not large enough to give
statistical importance to this slight difference.
The presence of a wide distribution of membrane protein alterations
(ie, isolated or ankyrin-combined spectrin deficiency and band 3 reduction) in the 3 groups of patients with HS reflects considerable
heterogeneity of the primary molecular disorders. In fact, alterations
of the following genes may, in turn, be involved: SPTB, ANK1, and EPB3.
They map to 14q23-q24.2, 8p11-2, and 17q12-q21, respectively.32 This strongly suggests that the phenomenon
(ie, greater predisposition to form gallstones in HS patients with Gilbert syndrome) is linked to a common phenotype (ie, chronic hemolysis) rather than to a particular molecular defect in HS. For this
reason, although the 103 children investigated belonged to 93 families
(ie, there were 10 couples of siblings), we, performing statistical analysis, didn't cluster families, but chose to treat each
patient as independent.
In patients who coinherited HS and Gilbert syndrome, the formation of
gallstones represents an interesting example of how different genes
(SPTB, ANK1, and EPB3) mutated to produce the same phenotype (HS), in
turn interacting with another mutated gene (UGT1A1 with a [TA]
insertion in the promoter), can produce a multigenic disease.
The association of an increased bilirubin load from chronic hemolysis
and the diminished hepatic conjugation of bilirubin, increases the risk
of gallstones. Consequently, although patients with HS were the only
ones studied, extrapolating these data to patients who have different
forms of inherited (eg, thalassemia, intraerythrocytic enzymatic
deficiency) or acquired (eg, autoimmune hemolytic anemia, hemolysis
from mechanical heart valve replacement) chronic hemolysis can be
warranted.33,34 Although confirmatory studies are still
needed, the assessment of the UGT1A1 genotype might be helpful in the
management of these categories of patients.
Finally, gastroenterologists who study patients with gallbladder
disease, stimulated by these data, might do well to search for the
interaction between Gilbert syndrome and, until now unrecognized, slight hemolysis in some of their patients.
| |
FOOTNOTES |
Submitted February 16, 1999; accepted June 4, 1999.
Supported by a grant from the "Regione Campania: Ricerca Sanitaria
Finalizzata 86/87" and by Telethon Prog. E 645.
The publication costs of this
article were defrayed in part by
page charge payment. This article
must therefore be hereby marked
"advertisement"
in accordance with 18 U.S.C. section
1734 solely to indicate this fact.
Presented in part at the 40th Annual Meeting of the American Society of
Hematology, Miami Beach, FL, December 4-8, 1998 and published in Blood
92:470a, 1998 (abstr, suppl 1) .
Address reprint requests to Emanuele Miraglia del Giudice, MD,
Dipartimento di Pediatria, Seconda Università di
Napoli, Via S.Andrea delle Dame N°4, 80138 Napoli, Italy; e-mail:
nobili{at}unina.it.
| |
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ABSTRACT
INTRODUCTION
