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Blood Cells, Molecules, and Diseases 45 (2010) 29–32
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Blood Cells, Molecules, and Diseases
j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / y b c m d
Natural history of Southeast Asian Ovalocytosis during the first 3 years of life
Vichai Laosombat a,⁎, Vip Viprakasit d, Supaporn Dissaneevate b, Roengsak Leetanaporn c,
Thirachit Chotsampancharoen a, Malai Wongchanchailert a, Sudarat Kodchawan a,
Warangkana Thongnoppakun d, Sarapee Duangchu a
a
Division of Pediatric Hematology & Oncology, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkla 90110, Thailand
Division of Neonatology, Department of Pediatrics, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkla 90110, Thailand
Department of Obstetrics and Gynecology, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkla 90110, Thailand
d
Division of Pediatric Hematology & Oncology, Department of Pediatrics, Faculty of Medicine, Siriraj Hospital, Mahidol University, Bangkoknoi, Bangkok 10700, Thailand
b
c
a r t i c l e
i n f o
Article history:
Submitted 29 March 2010
Available online 24 April 2010
(Communicated by P. Gallagher, M.D.,
29 March 2010)
Keywords:
Anemia
Neonatal hyperbilirubinemia
Southeast Asian Ovalocytosis (SAO)
AE-1 gene
a b s t r a c t
Southeast Asian Ovalocytosis (SAO), the most common red cell membrane disorder found in the Far-East and
Pacific rim, appears to be innocuous in man since it has been identified mostly in non-anemic healthy
individuals. To further substantiate our previous observation that this condition might be symptomatic
particularly in the neonatal period, we studied 1567 newborns from Southern Thailand where SAO is
prevalent. Thirty-one babies (1: 50 with allele frequency of 0.01) have been identified with SAO and
confirmed molecularly to carry a single defective AE-1 (band 3) allele. These babies had significant anemia at
birth due to hemolysis with 51.6% of them developing neonatal hyperbilirubinemia. Co-inheritance of
common UGT1A1 variants in such cases was not associated with their degree of jaundice. Interestingly,
hematology data of these SAO babies became “normalized” in the first 3 years of life without further
evidence of on-going and/or even “compensated” hemolysis.
Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved.
Introduction
Patients and methods
Southeast Asian Ovalocytosis (SAO) is an autosomal form of
hereditary elliptocytosis which is widespread in Southeast Asia [1]
and the South Pacific [2]. This results from the nine-amino-acid
deletion of residues 400-408 due to 27 bp deletion of the band 3 (AE1
or SLC4A1, Anion-Exchanger 1 gene) [3]. AE1 is the major transmembrane protein of the red blood cell and is important in
maintaining both its internal milieu by passive anion transport, and
its shape through attachment via ankyrin to spectrin in the
cycloskeleton [4,5]. The majority of individuals with heterozygosity
for SAO are asymptomatic with no clinically detectable hemolysis [6]
with few exceptions [7]. The homozygous SAO deletion is thought to
be lethal in utero in the embryonic state [1]. Previously, we proposed
that SAO might play a role in anemia and hyperbilirubinemia in
neonates; however, other possible causes of anemia could not be
completely excluded due to the retrospective nature of the studies
[8,9]. Therefore, we prospectively analyzed and followed 31 Thai SAO
infants from birth to 3 years of age to demonstrate the natural history
of this common condition.
We conducted a prospective study between 1 February 2006 and
31 January 2007. This study was approved by the Institute Ethics
Committee. Five milliliters of cord blood of the newborns whose
parents or guardians provided informed consent were analyzed for
CBC, reticulocyte number, blood group and G6PD screening test using
standard techniques [10]. The diagnosis of SAO in the newborns was
made based on criteria previously reported [8]. We collected their
clinical data including gestational age, birth weight, placental weight,
age at detection of pallor, age at detection of jaundice, the highest
microbilirubin level and the duration of treatment by phototherapy.
After birth, other causes of neonatal hemolysis in SAO neonates
including ABO incompatibility, thalassemia and blood loss from fetomaternal hemorrhage (FMH) were excluded [11,12]. Each SAO baby
underwent a full physical examination at the ages of 1, 2, 4, 6, 10, 12,
18, 24, 30 and 36 months to measure weight, height, the size of the
liver and spleen together with CBC and reticulocyte count. Five known
variants of the AE1 gene: Codon 400–408 deletion (SAO 27 bpdeletion), Glycine 701 to Aspartic acid mutation (G701D), Alanine 858
to Aspartic acid mutation (A858D), Serine 773 to Proline mutation
(S773P) and Glutamine 759 to Histidine mutation (Q759H), were
determined as described [9,13]. In addition, three common uridine
diphosphate glucuronosyltransferase 1A1 (UGT1A1) polymorphisms;
TATA box (A(TA)7TAA) mutation (TA)7 at the promoter [14], Glycine
⁎ Corresponding author. Division of Pediatric Hematology & Oncology, Department
of Pediatrics, Faculty of Medicine, Prince of Songkla University, Hat Yai, Songkla 90110,
Thailand. Fax: 66 74 429618.
E-mail addresses: [email protected], [email protected] (V. Laosombat).
1079-9796/$ – see front matter. Crown Copyright © 2010 Published by Elsevier Inc. All rights reserved.
doi:10.1016/j.bcmd.2010.03.010
30
V. Laosombat et al. / Blood Cells, Molecules, and Diseases 45 (2010) 29–32
71 to Arginine mutation (G71R) [15] and Phenylalanine 83 to Leucine
mutation (F83L) [16] were molecularly identified.
Data analysis
Statistical analyses were conducted with STATA 7 software.
Student's t-test was used to compare continuous data and chi squared
test and Fisher exact test, where appropriate, to compare categorical
data.
Results
From 1567 specimens of cord blood studied, we detected typical
SAO morphology in 31 neonates indicating a prevalence of SAO in our
population of 1:50 (allele frequency of 0.01). Four of them were
associated with other conditions: two with Hb E trait, one with
premature baby and one with FMH. Demographic data of 1,536 nonSAO newborns compared with SAO neonates are shown in Table 1.
The SAO group had a significantly higher percentage of pallor at birth;
74.2% compared to 7.6% in the non-SAO group, and jaundice 67.7%
compared to 12% in the non-SAO group (Table 1). SAO was not
detected in neonates of Chinese descent. SAO neonates had a higher
percentage of blood group A than that those found in non-SAO
neonates. Hematology data after the first 24 h were compared
between SAO and 802 non-SAO babies whose data were available
and revealed Hb, Hct and RBC values to be significantly lower in
neonates with SAO than in controls, in contrast to RDW and
reticulocyte values, that were significantly higher. These findings
together with greater jaundice suggest that significant hemolysis
Table 1
Clinical features of the newborn SAO and non-SAO babies.a
Characteristics
NB-SAOb (n = 31)
NB-non-SAOc (n = 1536)
P-value
Sex, M:F
Gestation age (weeks)
Birth weight (g)
Placenta weight (g)
Pallor
Jaundice
Highest MBd (mg%)
MB N 15 mg%
MB N 20 mg%
Phototherapy
Ethnicity
Thai
Muslim
Chinese
Blood group:
O
A
B
AB
16:15
38.4 ± 1.5
2,968 ± 460
613 ± 158
23 (74.2%)
21 (67.7%)
15.2 ± 3.4
14 (45.2%)
4 (12.9%)
21 (67.7%)
785:751
38.7 ± 2.1
3,098 ± 481
627 ± 113
116 (7.6%)
185 (12.0%)
12.3 ±3.1
127 (17.2%)
8 (0.5%)
188 (12.2%)
0.955
0.343
0.136
0.545
b 0.001
b 0.001
b 0.001
b 0.001
b 0.001
b 0.001
26 (83.9%)
5 (16.1%)
0
1377 (89.6%)
128 (8.3%)
15 (1.0%)
0.298
0.123
0.580
9 (29.0%)
13 (41.9%)
8 (25.8%)
1 (3.2%)
578
364
474
116
0.322
0.019
0.540
0.363
Hematology after birth
Agef (days)
Hb (g/L)
Hematocrit (%)
RBC (x 1012/L)
MCV (fL)
MCH (pg)
MCHC (g/dL)
RDW (%)
Reticulocyte (%)
Reticulocyte (x 1010/L)
NB-SAOb (n = 31)
1.97 ± 0.98
141.3 ± 15.3
41.1 ± 4.49
4.03 ± 0.53
102.7 ± 5.17
35.4 ± 2.99
34.4 ± 2.06
17.5 ± 1.67
12.22 ± 3.72
48.75 ± 14.32
NB-non-SAOe (n = 802)
2.6 ± 1.0
164.0 ± 23.5
49.4 ± 6.33
4.78 ± 0.75
100.9 ± 6.75
34.3 ± 2.80
33.9 ± 1.56
15.1 ± 1.94
8.49 ± 4.31
39.48 ± 19.00
a
(37.6%)
(23.7%)
(30.9%)
(7.6%)
b 0.001
b 0.001
b 0.001
0.14
0.029
0.130
b 0.001
b 0.001
0.010
The data are presented as mean ± SD or (percent).
Newborn with SAO.
Newborn with non-SAO.
d
MB = microbilirubin.
e
Those with available hematology data.
f
Age = age at determination (days), P b 0.05 is considered as a statistically significant
difference.
b
c
occurred in SAO neonates resulting in neonatal anemia. Our molecular
analysis showed that all SAO cases carried only causative AE-1
mutation. Co-inheritance of common UGT1A1 variants in these SAO
babies did not seem to aggravate the degree of jaundice; the highest
bilirubin in individuals with wild type (n = 10) and UGT1A1 heterozygotes (14 of TA6/TA7, 2 of G71R and 1 of F83L) were 14.68 ± 3.17
and 14.61 ± 2.88 mg%, respectively. Only one case with homozygous
for G71R variants had the highest bilirubin level (22.93 mg%) among
the SAO babies. During three years follow-up, these SAO babies were
unremarkable with normal growth and development. None has been
found to have hepatosplenomegaly, hemolytic or anemic crisis during
the first 3 years of life. Their Hb Hct and RBC values appeared to
decline to the nadir at 2 months of age, compatible with normal
physiological anemia of childhood (Fig. 1). Interestingly, these anemia
parameters gradually increased afterwards to the normal range.
However, this normalization did not seem to be a result of
compensated hemolysis since the reticulocyte values remained
constant throughout (Fig. 1).
Discussion
In Southeast Asian countries, allele frequency of SAO among
populations in Malaysia, Indonesia and Papua New Guinea were 0.01
[17], 0.01–0.09[18,19], and 0.01–0.15[19], respectively. Our result of
0.01 in Southern Thailand was similar to that of Malaysia which is our
neighboring country on the mainland. It is widely thought that SAO is
an asymptomatic trait without significant hemolysis. A large cohort in
Papua New Guinean children has shown that children with SAO
(n = 16) had a hematological profile similar to that of their
community controls (n = 225) [20]. Infection with malaria appeared
to have a greater effect in children with SAO as their Hb and RBC were
lower than those of controls. However, there was no direct evidence of
apparent hemolysis in such cases and this higher degree of malaria
anemia might result from a selective loss of infested ovalocytes in SAO
subjects. Only a few symptomatic cases with SAO have been reported
with chronic hemolysis with increased reticulocytes; however it
remains unclear whether the band 3 deletion was the only causative
mutation in such cases [7]. Recently, several studies mainly derived
from populations in our region have shown that compound
heterozygous of SAO with other AE-1 mutations; G701D, ΔV850,
A858D, S773P and Q759H could result in hemolytic anemia even
though all reported cases also developed autosomal recessive distal
renal tubular acidosis (dRTA) and some might have had different red
cell morphology [21]. However, stomatocytic ovalocytes remained
present in some interactions (SAO 27 bp-deletion/A858D and SAO
27 bp-deletion/ΔV850). Therefore, it is important to determine
whether neonatal anemia found in this study is not caused by
interaction of SAO with other AE-1 mutants, one of which (G701D)
has previously been identified in our population [13]. Indeed, all SAO
babies carried only the SAO 27 bp-deletion and our 3 yr-follow-up
demonstrated that they all had normal growth with normal acid-base
balance (data not shown) making the possibility of dRTA, and hence
co-inheritance of another AE-1 mutant(s), unlikely.
By excluding other possible cause of anemia at birth, we further
confirmed that SAO might be “symptomatic” by causing “hemolysis”
at birth. Why prenatal and neonatal conditions have set this “rigid”
red cell to be on the verge of overt hemolysis remains to be elucidated,
but it is likely that the pathophysiology might involve inelasticity by
conformational or deformational change of band 3 and an inability of
the deleted band 3 to transport anions [22]. Interestingly, an adaptive
process in SAO babies has developed during their first 3 years of life
since there was no further appearance of hemolysis. Their hematology
showed normal pattern of “physiologic anemia of childhood” and
complete return to the baseline as in controls at the same period. This
warrants further study to explore what physical and biochemical
properties of these ovalocytes might be additionally deranged at birth
V. Laosombat et al. / Blood Cells, Molecules, and Diseases 45 (2010) 29–32
31
Fig. 1. Hematologic data (mean and 95% CI) of patients with SAO at each period of time during the first 3 years of life.
compared to those abnormalities which have been described later in
their life [21].
Acknowledgments
We thank our patients and their parents or guardians for their
participation in this study. This study was supported by the Thai
Government budget grant to V. L. and the Thailand Research Fund
(TRF) and BIOTEC, Thailand to V.V. Thanks to Mr. Edward B. McNeil
(Epidemiology Unit, Faculty of Medicine, Prince of Songkla University,
Songkla, Thailand) for graphic presentation. Thanks to Dr. Alan F
Geater (Epidemiology Unit, Faculty of Medicine, Prince of Songkla
University, Songkla, Thailand) for editing the manuscript.
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