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Case Studies
Three Different Co-existing α-Thalassemia Mutations
and Sickle Cell Disease in a Pregnant Woman
Fakher Rahim, PhD,1 Fateme Hamid, MSc,2 Hamid Galedari, PhD,3
Gholamreza Khamisipour, PhD,4 Bijan Keikhaei, MD,2 Kaveh Jaseb, MD,2 Najmaldin Saki, PhD2*
ABSTRACT
Thalassemia is the most frequent single-gene defect and overwhelmingly
effects prenatal patients in Iran; the condition is caused by a diverse
range of mutations. We discuss an extremely rare combination of
α-thalassemia, caused by a defect on the short arm
of chromosome 16, is the most common inherited
hemolytic anemia worldwide.1 The disease is
characterized by microcytic hypochromic anemia
and is inherited as an autosomal recessive disorder.
Healthy people have 4 α-genes (2 α1 and 2 α2 genes).
Depending on deletions and mutations in 1 or more
genes, patients have different clinical manifestations,
from mild to severe anemia.2 All patients have variable
degrees of anemia (ie, decreased hemoglobin [Hb]),
decreased mean corpuscular hemoglobin (MCH),
DOI: 10.1309/LMKQEVYZFO7MV6RZ
Abbreviations
Hb, hemoglobin; MCH, mean corpuscular hemoglobin; MCV,
mean corpuscular volume; HbA2, hemoglobin A2; HbS, Sickle
hemoglobin; HbAS, hemoglobin AS; HbSS, hemoglobin subserosal;
HbF, fetal hemoglobin; CBC, complete blood cell count; EDTA,
ethylenediamineteraacetic acid; PCR, polymerase chain reaction;
ARMS, amplification refractory mutation system; RBD, RNA-binding
domain; MLPA, multiplex ligation-dependent probe amplification; PND,
prenatal diagnosis; HbH, hemoglobin H; RBCs, red blood cells; RDW, red
blood cell distribution width; HbA1, hemoglobin A1; NA, not applicable
Toxicology Research Center, Ahvaz Jundishapur University of
Medical Sciences, Ahvaz, Iran; 2Research Center of Thalassemia and
Hemoglobinopathy, Ahvaz Jundishapur University of Medical Sciences,
Ahvaz, Iran; 3Department of Genetics, School of Sciences, Shahid
Chamran University, Ahvaz, Iran; 4Allied Health Sciences School,
Bushehr University of Medical Sciences, Bushehr, Iran
1
*To whom correspondence should be addressed:
E-mail: [email protected]
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α-globin gene disease and sickle cell trait. This combination may
explain a mild form of thalassemia that presents with moderate anemia.
Keywords: α-thalassemia, mutation, determinants, sickle cell disorder
decreased mean corpuscular volume (MCV), and a
normal or slightly decreased level of hemoglobin A2
(HbA2).1 α-thalassemia is highly prevalent throughout
Southeast Asia, India, the Middle East, parts of Africa,
and the Mediterranean countries. The most common
mutations and deletions in Iran are -α3.7, -α4.2, --MED,
-HBA2: c.*94A>G, -α2 PA4: A>G, -α2 CD19 [-G], and
-α2 intervening sequence (IVS-II) -5nt.3 Sickle
cell disease is one of the most prevalent
hemoglobinopathies and is characterized by chronic
hemolytic anemia. A point mutation in the β chain
leads to the substitution of glutamic acid for valine,
resulting in the production of an abnormal Sickle
hemoglobin (HbS; α2βS2).4 Its clinical manifestations are
different depending on the state of inheritance and
heterozygocity or homozygocity for this mutation.
Although the heterozygote form (ie, sickle cell trait or,
hemoglobin AS [HbAS]) is generally asymptomatic, in
homozygote form (sickle cell disease, or hemoglobin
subserosal [HbSS] disease) the patient will have sickle
cell crisis (ie, vaso-occlusion, sequestration, and
aplasticity), acute chest syndrome, and increased
susceptibility to infections. It has also been observed5
that pregnant women with sickle cell disease are
at risk of pre-eclampsia, low birth weight, and fetal
loss. Severity of disease in coinheritance of HbS with
β-thalassemia and/or α-thalassemia determinants
depends on the number and type of genes involved.6
In the electrophoresis of HbSS disease, HbA is not
observed; however, HbS is observed at a level of less
of 85%, along with a percentage of HbA2 in the normal
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Case Studies
range and an increased percentage of fetal hemoglobin
(HbF). In patient with HbAS disease, the HbS is
approximately 40%, HbA is approximately 60%, the
HbA2 percentage is normal or slightly increased, and
the HbF percentage is normal.
Because of demographic diversity, different ethnicities,
and migration, we have observed a spectrum of the
co-inheritance of a variety of mutations and deletions.7
This article reports a novel co-inheritance of 3
α-thalassemia determinants (deletion, gene triplication,
and point mutation) in a pregnant woman with sickle
cell disease.
Material and Methods
This patient was referred by hematologists to the
Prenatal diagnosis (PND) center of Shafa Hospital of
the Ahvaz Jundishapur University of Medical Sciences,
Ahvaz, Iran, with suspicion of α-thalassemia and
sickle cell disease. After preliminary testing, results
for complete blood cell count (CBC), hemoglobin
electrophoresis (ie, acetate cellulose), and iron levels
ruled out iron-deficiency anemia, genetic tests were
conducted to identify and confirm the presence of
thalassemia and/or hemoglobinopathies.
Venous blood was drawn from the patient and her
husband and collected in an ethylenediamineteraacetic
acid (EDTA) tube for hematologic parameters
determination (Sysmex K1000 Hematology Analyzer,
Sysmex Corporation, Kobe, Japan) and Hb
electrophoresis (cellulose acetate in alkaline pH of 8.6);
also, an amniotic-fluid sample was withdrawn from
the fetus. Samples for molecular DNA analysis were
extracted using AccuPrep Genomic DNA Extraction Kit
(Bioneer Corporation, Daejeon, South Korea).
α- and β-globin genes were analyzed using a reverse
dot-blot assay as instructed (ViennaLab Diagnostics
Gmbh, Vienna, Austria). Briefly, in this method
the normal and mutant sequences were amplified
using biotinylated primer sets by polymerase chain
reaction (PCR), then hybridized to fixed probes on
a nitrocellulose strip and finally, visualized by an
enzymatic reaction benefiting from biotin-avidin affinity.
Using the blood-analysis kit, 21 deletions, various
point mutations, and an α-gene triplication could be
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identified simultaneously. Separate gap PCRs for the
3 most frequent deletions (3.7, 4.2, and med) were
performed5 to verify the strip assay result. In cases in
which the point mutation seen in the strip assay was
present, α-globin genes were sequenced for further
confirmation. The β-globin gene was primarily studied
by direct sequencing; the amplification refractory
mutation system (ARMS) and linkage analysis were
used as secondary methods.6 After detection of
α-gene triplication by RNA-binding domain (RBD), the
heterozygous status was checked by multiplex ligationdependent probe amplification (MLPA) genetic testing.8
Further, for β-globin gene sequencing, the forward and
reverse strands were both sequenced to confirm cd6
homozygosity. Written Informed consent was obtained
from the patient involved in this study and her husband.
This study was approved by Ahvaz Jundishapur University
of Medical Sciences ethics committee.
Case History
A 24-year-old pregnant woman, at 7 weeks’ gestation,
from Ahvaz, a city in the southwestern region of
Iran, was sent to our hospital for fever, anemia, and
arthralgia. The patient underwent PND assessment
because of her thalassemia and her HbS background.
Via genetic testing, she was found to have a –α3.7
deletion, anti-3.7 gene triplication, and α-gene point
mutation HBA2: c.*94A>G (Figure 1). In β-globin gene
studies, the patient was shown to have the HBB:
c.20A>T, Glu6Val mutation in homozygous form (Figure
2). Finally, the fetus (proband) was found to have a
single (-α3.7) and a double (--MED) deletion on the α
gene, which was concomitant with heterozygous HBB:
c.20A>T, Glu6Val mutation.
Family Study
The hematological data, clinical manifestation, and
result of hemoglobin analysis of the parents are
summarized in Table 1. The patient’s husband was
26 years old and a carrier of α-thalassemia (Table 1).
The mother tested homozygous for HbS (ie, she does
not have sickle cell trait) and appeared to have no
symptoms of sickle cell anemia. Her α-globin mutation
may modify her risk for symptoms of sickle cell disease
but these factors shouldn’t put her at risk for significant
thalassemia symptoms. However, the fetus, at 7 weeks’
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Figure 1
Red Marker Line (top)
α-globin strip assay by reverse
dot-blot results in the study patient,
showing anti-3.7 α-gene triplication,
α2 poly A-1, and -α3.7 gene
deletion.
Control
1
2
3
4
5
6
7
8
9
– 3.7 single gene del
– 4.2 single gene del
– 20.5 kb double gene del
––MED double gene del
––SEA double gene del
–– THAI double gene del
––FIL double gene del
α1 cd 14 [G>A]
α1 cd 59 [G>A} (Hb Adana)
mutant
mutant
mutant
mutant
mutant
mutant
mutant
mutant
mutant
10
11
α1 cd 14
α1 cd 59
wild type
wild type
12
PCR Control A
Green Marker Line (bottom)
Red Marker Line (top)
Control A
13
14
15
16
17
18
19
20
21
22
23
24
anti-3.7 gene triplication
α2 init [T>C]
α2 cd 19 [–G]
α2 IVS 1 –5nt
α2 cd 59 [G>A]
α2 cd 125 [T>C] (Hb Quong Sze)
α2 cd 142 [T>C] (Hb Constant Spring)
α2 cd 142 [T>A] (Hb Icaria)
α2 cd 142 [A>T] (Hb Pakse)
α2 cd 142 [A>C] (Hb Koya Dora)
α2 poly A-1 [AATAAA>AATAAG]
α2 poly A-2 [AATAAA>AATGAA]
mutant
mutant
mutant
mutant
mutant
mutant
mutant
mutant
mutant
mutant
mutant
mutant
25
26
27
28
29
30
31
α2 init cd
α2 cd 19
α2 IVS 1
α2 cd 59
α2 cd 125
α2 cd 142
α2 poly A
wild type
wild type
wild type
wild type
wild type
wild type
wild type
32
PCR Control B
Blue Marker Line (bottom)
10 A/B
Figure 2
210
220
230
G G TG CAC C T G AC T C C T G TG G AG AA GTC T G CC
β-globin sequence analysis. The arrow indicates the homozygous
Sickle hemoglobin (HbS) [A>T] mutation in the study patient.
Table 1. Hematological Data and Genotypes of the Parents and the Proband With HbS and
α-Thalassemia
MCV MCH MCHCRBCs
Subject
Age (fl) (pg)(g/dL)
(µL)
RDW HbA2 HbF HbA1
(%) (%)(%) (%)HbS
α-Genotype
HbS Genotype
Mother 24 year 69.1 19.7 28.5 4.11 × 10621.8 3.4 1.9 NA 94.7 -α 3.7/ α α
cd6 (GAG>GTG)/cd6 (GAG>GTG)
Father
26 y
70.0 22.4 27.6 5.22 × 10623.1 2.5 1.8 96.7 NA α α /--MEDNA
Proband 7 wkNANA NANA
NA NANA NANA-α 3.7/--MED
cd6 (GAG>GTG)/ N
Abbreviation: MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; RBCs, red blood cells;
RDW, red blood cell distribution width; HbA1, hemoglobin A1; HbS, Sickle hemoglobin; NA, not applicable.
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Case Studies
gestation, was at risk for thalassemia symptoms (ie,
hemoglobin H [HbH] disease).
Discussion
Herein, we report the case of a patient with an
infrequent hereditary arrangement of 4 various genetic
anomalies of hemoglobin genes: the patient and
her husband had 3 mutations on the α-gene and a
sickle-cell anomaly on the β-gene, which is a rare
combination.9,10 The co-occurrence of sickle-cell
anemia with α-thalassemia (due to the 3.7 kb deletion)
is extremely common in ethnic African populations.
What makes this case potentially unique is that the
unusual α-globin point mutation and triplication in the
mother is typically seen in patients of Asian ethnic
origin. The mother’s c.*94A>G (a2PA6) mutation is
presumably located on the same chromosome as the
triplication, which had previously been reported11 in
association with an α-globin triplication. The mutation
in the present case, which was comprised of the
combination of c.*94A>G/triplication and the 3.7 kb
deletion, might mean that the patient has a more
significant level of α-thalassemia than most individuals
with reported sickle cell disease and α-thalassemia.
Sickle cell disease is itself quite variable; factors such
as genetic and environmental influences contribute
to this variability. One of the most important genetic
factors is thalassemia.
Individuals with sickle cell trait are heterozygous for
HbS; therefore, they have a 50% chance of transmitting
the mutation to their son or daughter. For children to
develop sickle cell disease, both of their parents almost
always must carry a mutation. The risk of transmitting
α-thalassemia depends on the nature of the mutation.
As can be observed from the data in this report, this
case is a classic example of moderately severe sickle
α-thalassemia disease. Thalassemia is very common
among people of Mediterranean ethnic origin. The
sickle cell gene also exists more commonly in people
of the same ethnic origin. In the United States, 10%
of the population is at risk of sickle cell anemia; in
northwestern Europe, between 2% and 9% are at risk
for hemoglobin disorders. In some Southeast Asian
countries, as much as 40% of the population may carry
significant hemoglobin mutations, resulting in increased
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rates of infants born with thalassemia.12 Hemoglobin
analyses of the parents of such infants have shown that
the fraction of HbS was extremely high, namely, 94.7%
in the mother, whereas the MCV was decreased in both
parents to less than 71 fl.
The complete or partial deletion of both α-genes in
cis deletion results in no α-chain synthesis; hence,
rare deletions that cause α 0-thalassaemia remove the
regulatory region, which lies 40 to 50 kb upstream
of the α-globin gene cluster, leaving the α-genes
intact. Moreover, the coexistence of the anti-3.7 gene
triplication at a point mutation in cis deletion is a rare
entity that is observed in our case individual.
Patients with HbH disease have only a single active
α-globin gene and manifest moderate to severe
anemia. Deletion of all 4 α-globin genes (--/--),
has the most severe manifestation, known as Hb
Bart’s hydrops fetalis syndrome, which is generally
associated with death in utero.2
The consequences of α-thalassemia on sickle-cell
anemia are possibly connected to an essential effect
on mean corpuscular hemoglobin concentration
(MCHC) and polymerization; further, simultaneous
effects on other properties of sickle cells and the
possibility that the MCHC differences may be
secondary to sickling effects cannot be overlooked.
Studies of the phenotypic results of the interaction
of α-thalassemia and sickle-cell anemia will provide
further insight into the causes of clinical diversity in
sickle cell disease.13 Co-existing α-thalassemia and
sickle cell disease significantly improved hematological
parameters and resulted in fewer blood transfusions,14
may cause musculoskeletal pain,15 and decreases the
risk of cerebrovascular disease in children with sickle
cell anemia.16,17 In conclusion, we report an extremely
rare combination of α-globin gene disease and sickle
cell trait. This combination may explain the mild form
of thalassemia disease that manifests with moderate
anemia. LM
Acknowledgments
We wish to thank all our colleagues in Shafa Hospital
Clinical & Specialty Laboratory. Our work has been
supported by grant from Ahvaz Jundishapur University of
Medical Sciences, Ahvaz, Iran.
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e75
Case Studies
Conflict of interest
The authors declare no conflict of interest.
9. Medinger M, Saller E, Harteveld CL, et al. A rare case of
coinheritance of Hemoglobin H disease and sickle cell trait combined
with severe iron deficiency. Hematol Rep. 2011;3(3):e30.
10.Webster BH, Lammi AT. Thalassaemia and other
haemoglobinopathies in general practice. Aust Fam Physician.
1994;23(8):1485-1490.
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