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Identification of the mutation IVS 1–5 (G>C) of the β-hemoglobin gene (Hbβ) by
RDBH in patients with β-thalassemia in Azerbaijan

Article  in  Cytology and Genetics · May 2015

DOI: 10.3103/S0095452715030020

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ISSN 00954527, Cytology and Genetics, 2015, Vol. 49, No. 3, pp. 178–182. © Allerton Press, Inc., 2015.
Original Russian Text © G.A. Akbarova, 2015, published in Tsitologiya i Genetika, 2015, Vol. 49, No. 3, pp. 46–50.

Identification of the Mutation IVS 15 (G>C) of the βHemoglobin

Gene (Hbβ) by RDBH in Patients with βThalassemia in Azerbaijan
G. A. Akbarova
Baku State University, Baku, Zahid Khalil 23, AZ1148 Azerbaijan
email: gunay.akbarova@bsu.az
Received May 28, 2014

Abstract—The hematological and molecular genetic analysis of samples taken from patients with suspected
βthalassemia were made by Reverse DotBlot Hybridization StripAssay (RDBH StripAssay). A complete
blood count (HB, MCH, MCV, MCHC, RBC, Hct, HbA2, HbF) was run, serum iron and ferritin concen
trations were monitored, and molecular analysis by RDBH was carried out. Two patients who were the het
erozygous carriers of the mutation IVS 15 (G>C) got the diagnosis of β+thalassemia minor. The mutation
IVS 15 (G>C) in the compound with mutations IVS 1110 (G>A) or IVS 16 (T>C) determines the devel
opment of βthalassemia intermedia. RDBH is an easy and economical method for molecular diagnostics of
βthalassemia, especially in cases if the analysis of hematological parameters gives dubious results.

Keywords: βthalassemia, IVS 15 (G>C), screening, compounds, Reverse Hybridization StripAssay, the
Republic of Azerbaijan
DOI: 10.3103/S0095452715030020

INTRODUCTION years, such modern techniques of molecular biology as

The gene of βhemoglobin is a small (1.6 kb) and
single gene, as well as similar loci of the gene complex
(ε, γG and γA; pseudogene ψβ and gene δ) with a length
of ~60 kb; it is situated in 11p15 [1]. The coding
sequence of βhemoglobin gene begins at the 51st base
of the first exon with a start codon ATG, continues
through the second exon, and ends at the 130th base of
the third one with a stop codon TAA [2].
The gene products of βcluster together with α
cluster located on the distal end of the short arm of
chromosome 16 (30 kb) form three types of hemoglo
bin: HbA, HbA2, and HbF.
βthalassemia is a group of hemoglobin disorders
inherited in an autosomal recessive fashion and caused
by reduced (β+) or absent (β0) synthesis of beta chains
of hemoglobin. As a result, an excess of alpha chains
affects the process of erythropoiesis and decreases the
lifespan of erythrocytes [3, 4]. Presently, more than
200 mutations of the betaglobin gene are known [2].
In the population of Azerbaijan, βthalassemia is of
special interest. Its frequency is highest among all
inherited diseases and reaches 15–20% of heterozy
gous carriership in some regions [5]. Approximately
200 newborns with homozygous βthalassemia are
born here every year. Their quantity also increases
because of intermarriages, which are traditionally
widespread in Azerbaijan [6].
The use of sensitive and accurate diagnostic tech
niques is of key importance in high quality screening,
including the diagnostics of βthalassemia. In recent
Reverse DotBlot Hybridization StripAssay (RDBH)
and direct sequencing have become indispensable in
diagnostics of this disease. The RDBH method is
rapid, highly sensitive, costeffective, and nontoxic. It
allows for testing up to 48 samples during 4 hours and
detecting more than 20 mutations of βthalassemia
(on every Strip).
The mutation IVS 15 (G>C), which is rather rare
in the populations of Azerbaijan, was found in earlier
studies [5, 7]. This mutation is more typical of East
Asian and IndoAsian populations and populations of
UAE. The mutation IVS 15 (G>C) is caused by sub
stitution (transversion) G>C in the 147th nucleotide
in the 5th position of the first intron which leads to
wrong splicing of the first exon with 5’ end [8]. This
mutation is most often associated with other types of
mutations and abnormal type hemoglobins.
The purpose of this work was to perform hemato
logical and molecular genetic analysis (RDBH) of
samples taken from patients with suspected βthalas
semia from the town of Shirvan (Saatlinskii region), to
confirm diagnoses made before and to identify the
types of mutations in cases when diagnosis is con
firmed.
MATERIALS AND METHODS
The subjects of this study were nine patients of both
sexes (five female and four male) aged from 1.5 to
29 years with previously diagnosed anemia of chronic

178
 IDENTIFICATION OF THE MUTATION IVS 15 (G>C)
Table 1. Hematological parameters of the patients with βthalassemia minor (mean values)
IVS16(T>C)
Laboratory parameters IVS15(G>C)/wt
RBC, 106/µL 5.02
Hb, g/dL 12.0
Hct, % 32.3
MCV, fL 68.0
MCH, pL 21.8
MCHC, g/dL 34.5
HbA2, % 5.12
HbF, % 1.5
Serum iron, ng/µL 1.4
Serum ferritin, ng/µL 72.0
disease and thalassemia intermedia. The blood sam
ples of the subjects were collected in test tubes with
EDTA. Red blood cells count (RBC), the level of
hemoglobin (Hb), hematocrit (Hct), and red blood
cell indices—mean corpuscular volume (MCV),
mean corpuscular hemoglobin (MCH) and mean cor
puscular hemoglobin concentration (MCHC)—were
detected with the use of a Sysmex KX21N2 blood
analyzer (Japan). Moreover, hemoglobin fractions
were studied by highresolution liquid chromatogra
phy (HPLC, BioRad). The levels of serum iron and
ferritin were determined by enzyme immunoassays
(AxSym Abbot). For the extraction of DNA from
blood, we used Life Technologies: PureLink Genomic
DNA Mini Kit (United States) according to the manual.
The DNA concentration was determined by Nano
Drop 2000/2000 s Spectrophotometer, λ = 260/230 nm,
and λ = 260/280 nm (Thermo Fisher Sci., United
States).
Multiplex PCRamplification was carried out with
the use of Nuclear Laser Medicine Kits (Italy) and
consisted of two steps: (1) preparation—15 μL of
Amplification Mix, 5 μL of Taq DNApolimerase, and
5 μL of DNA; (2) PCRdenaturation—94°C/2 min;
annealing: 94°C/15 s–58°C/30 s–72°C/45 s (35 cycles);
final elongation—72°C/3 min (Thermal Cycler verity
2720, Life Technologies, United States).
Reverse hybridization consisting of three stages—
hybridization, washing, and chromatography—was
carried out with the use of Nuclear Laser Medicine
Kits (Italy) (cod. AC006: 23 mutations of βthalas
semia) and Autolipa 48 Instrument (Innogenetics,
Belgium) according to the manual.
RESULTS
In previous genetic studies of the population, we
found signs of mild forms of thalassemia [5]. The
results of more detailed studies confirmed our suspi
cions: the analysis of hematological parameters of
CYTOLOGY AND GENETICS Vol. 49 No. 3
179
IVS1110(G>A)
Normal values, wt/wt
wt
4.97 5.8 4.5–6.0
12.3 11.2 12–18
31.1 35.0 Women: 37–47, men: 42–52
70.4 73.5 82–98
23.0 22.7 27–32
35.0 36.5 33–37
6.01 4.9 <3%
2.2 1.9 <1%
1.6 1.2 Women: 0.4–1.6, men: 0.5–1.7
73.1 78.4 Women: 6–81, men: 30–250
patients and their nearest relatives showed that six
patients had thalassemia minor and three patients had
thalassemia intermedia. Further observation showed
that the patients with thalassemia minor had no clini
cal signs or just slight skin pallor caused by anemia and
no history of blood transfusion. The blood count of
these patients detected anemia with normal concen
tration of erythrocytes; lower values of MCV, MCH,
and MCHC; higher level of HbA2; and almost normal
level of HbF. In three patients, the level of HbA2 was
lower, but effective erythropoiesis and the presence of
some adult erythrocytes were observed, which corre
sponds with the clinical picture of thalassemia inter
media. None of these patients had any distinct clinical
signs of thalassemia or history of transfusion. The
mean values of hematological parameters in patients
with βthalassemia minor and intermedia are pre
sented in Tables 1 and 2.
The molecular genetic analysis of the betaglobin
gene with the use of RDBH StripAssay showed that the
patients with no or few clinical signs of thalassemia are
heterozygous carriers of the mutations IVS 15
(G>C), IVS 16 (T>C) and IVS 1110 (G>A). Three
patients with more distinct signs were found to be
compounds—IVS 15 (G>C)/IVS 16 (T>C) and
IVS 15 (G>C)/IVS 1110 (G>A) (Fig. 1). The par
ents of compounds were asymptomatic carriers of
βthalassemia.
The hematological parameters of a 25yearold
mother of two childrencompounds corresponded
with values typical of the carriers of β+thalassemia
(MCV: 67.0↓, MCH: 21.2↓, HbA2: 5.12↑, HbF:1.8↑,
RBC: 5.63). The father of compounds (29 years) was a
carrier of the mutation IVS 1110 (G>A) without clin
ical signs (MCV: 73.5↓, MCH: 22.7↓, HbA2: 4.9↑,
HbF: 1.9↑, RBC: 5.8). His sister (22 years) was also a
heterozygous carrier of this mutation.
In another family, both parents were heterozygous
carriers of different types of βglobin mutations: the
2015
180 AKBAROVA

Table 2. Hematological parameters of the patients with βthalassemia intermedia (mean values)
Laboratory parameters IVS15(G>C)/IVS16(T>C) IVS15(G>C)/IVS1110(G>A) Normal values, wt/wt
RBC, 106/µL 3.2 3.93 4.5–6.0
Hb, g/dL 6.8 7.3 12–18
Hct, % 24.2 25.3 Women: 37–47, men: 42–52
MCV, fL 62.0 67.6 82–98
MCH, pL 20.8 21.9 27–32
MCHC, g/dL 34.9 35.9 33–37
HbA , % 5.0 4.0 <3%
HbF, % 12.2 10.1 <1%
Serum iron, ng/µL 5.5 6.15 Women: 0.4–1.6, men: 0.5–1.7
Serum ferritin, ng/µL 102.9 207.65 Women: 6–81, men: 30–250

mother (28 years) had genotype IVS 16 (T>C)/wt and
hematological parameters typical of the carriers of
β+thalassemia (MCV: 70.4↓, MCH: 23.0↓, HbA2:
6.01↑, HbF: 2.2↑, RBC: 4.97); the father (28 years)
had genotype IVS 15 (G>C)/wt (MCV: 68.0↓, MCH:
21.0↓, HbA2: 5.1↑, HbF: 1.9↑, RBC: 5.11); moreover,
his brother (24 years) was a heterozygous carrier of the
mutation IVS 15 (G>C).
In three patients, hematological parameters dif
fered significantly from those presented in Table 2. For
example, the parameters of a 4yearold girl were typ
ical of βthalassemia intermedia (MCV: 67.2↓, MCH:
21.7↓, HbA2: 3.8↑, HbF: 8.9↑, RBC: 3.76↓, Hct:
23.1↓, Iron↑). Her 1.5yearold brother had similar
parameters (MCV: 68↓, MCH: 22↓, HbA2: 4.2↑,
HbF: 11.2↑, RBC: 4.1↓, Hct: 27.5↓, Iron↑). These
two patients are the carriers of genotype IVS 15
(G>C)/IVS 1110 (G>A) (Fig. 2).
Another girl aged 10 years with genotype IVS 15
(G>C)/IVS 16 (T>C) also had hematological param
eters typical of βthalassemia intermedia (MCV:
62.0↓, MCH: 20.8↓, HbA2: 5.0↑, HbF: 12.2↑, RBC:
3.2↓, Hct: 24.2↓, Iron↑) (Fig. 3).
DISCUSSION
The results of our studies showed that a number of
diagnoses of anemia previously set in district hospitals
were not confirmed. For example, increased level of
reticulocytes (265 × 109; normal values are 75–170 ×
109) and slight anemia (Hb: 11.5↓) in a 25yearold
mother of two childrencompounds who was a het
erozygous carrier of βthalassemia minor IVS 15
(G>C)/wt, as we discovered later, were treated by iron
preparations, which certainly did not have the neces
sary effect.

– Red Marker Line (Top) – Red Marker Line (Top)

sequence sequence
– Control alteration – Control alteration
– – mutant – 101 C>T – – mutant – 101 C>T
– – mutant – 92 C>T – – mutant – 92 C>T
– – mutant – 87 C>G – – mutant – 87 C>G
– – mutant – 30 T>A – – mutant – 30 T>A
– – mutant codon 5 –CT – – mutant codon 5 –CT
– – mutant codon 6 G>A (HbC) – – mutant codon 6 G>A (HbC)
– – mutant codon 6 A>T (HbS) – – mutant codon 6 A>T (HbS)
– – mutant codon 6 –A – – mutant codon 6 –A
– – mutant codon 8 –AA – – mutant codon 8 –AA
– – mutant codon 8/9 +G – – mutant codon 8/9 +G
– – mutant codon 30 G>C – – mutant codon 30 G>C
– – mutant IVS 1.1 G>A – – mutant IVS 1.1 G>A
– – mutant IVS 1.2 T>A – – mutant IVS 1.2 T>A
IVS 1.5 [G→C] – – mutant IVS 1.5 G>C IVS 1–5 [G→C] – – mutant IVS 1.5 G>C
– – mutant IVS 1.6 T>C – – mutant IVS 1.6 T>C
– – mutant IVS 1.110 G>A IVS 1–110 [G→A] – – mutant IVS 1.110 G>A
– – mutant IVS 1.116 T>G – – mutant IVS 1.116 T>G
– – mutant IVS 1.25 25 bp del – – mutant IVS 1.25 25 bp del
– – mutant codon 39 C>T – – mutant codon 39 C>T
– – mutant codon 44 –C – – mutant codon 44 –C
– – mutant IVS 2.1 G>A – – mutant IVS 2.1 G>A
– – mutant IVS 2.745 C>G – – mutant IVS 2.745 C>G
– – mutant IVS 2.844 C>G – – mutant IVS 2.844 C>G
– – wild tipe – 101 to –87 – – wild tipe – 101 to –87
– – wild tipe – 30 – – wild tipe – 30
– – wild tipe – codon 5 to 9 – – wild tipe – codon 5 to 9
– – wild tipe – codon 30 to IVS 1.6 – – wild tipe – codon 30 to IVS 1.6
– – wild tipe – IVS 1.110 and 1–25 – – wild tipe – IVS 1.110 and 1–25
– – wild tipe – codon 39 – – wild tipe – codon 39
– – wild tipe – codon 44 – – wild tipe – codon 44
– – wild tipe – IVS 2.1 – – wild tipe – IVS 2.1
– – wild tipe – IVS 2.745 – – wild tipe – IVS 2.745
– – wild tipe – IVS 2.844 – – wild tipe – IVS 2.844
– Green Marker Line/bottom) – Green Marker Line/bottom)

Fig. 1. RDBH StripAssay: samples of patients with geno Fig. 2. RDBH StripAssay: samples of children with geno
type IVS 15 (G>C)/wt. type IVS 15 (G>C)/IVS 1110 (G>A).

CYTOLOGY AND GENETICS Vol. 49 No. 3 2015

 IDENTIFICATION OF THE MUTATION IVS 15 (G>C) 181

– Red Marker Line (Top)

sequence
– Control alteration
– – mutant – 101 C>T
– – mutant – 92 C>T
– – mutant – 87 C>G
– – mutant – 30 T>A
– – mutant codon 5 –CT
– – mutant codon 6 G>A (HbC)
– – mutant codon 6 A>T (HbS)
– – mutant codon 6 –A
– – mutant codon 8 –AA
– – mutant codon 8/9 +G
– – mutant codon 30 G>C
– – mutant IVS 1.1 G>A
– – mutant IVS 1.2 T>A
IVS 1.5 [G→C] – – mutant IVS 1.5 G>C
IVS 1.6 [T→C] – – mutant IVS 1.6 T>C
– – mutant IVS 1.110 G>A
– – mutant IVS 1.116 T>G
– – mutant IVS 1–25 25 bp del
– – mutant codon 39 C>T
– – mutant codon 44 –C
– – mutant IVS 2.1 G>A
– – mutant IVS 2.745 C>G
– – mutant IVS 2.844 C>G
– – wild tipe – 101 to –87
– – wild tipe – 30
– – wild tipe – codon 5 to 9
– – wild tipe – codon 30 to IVS 1.6
– – wild tipe – IVS 1.110 and 1–25
– – wild tipe – codon 39
– – wild tipe – codon 44
– – wild tipe – IVS 2.1
– – wild tipe – IVS 2.745
– – wild tipe – IVS 2.844
– Green Marker Line/bottom)

Fig. 3. RDBH StripAssay: samples of children with genotype IVS 15 (G>C)/IVS 16 (T>C).

In another family, a 28year old man and his
24yearold brother were found to have βthalassemia
minor and genotype IVS 15 (G>C)/wt. It is worth
noting that only the father of a girlcompound had
slight sallowness of skin, which is caused by accumu
lation of bilirubin in blood because of enhanced eryth
ropoiesis.
The level of reticulocytes was increased because of
early death of erythrocaryocytes caused by disorders in
the synthesis of betachains of globin and accumula
tion of alphachains. The ineffective erythropoiesis in
one of the patients (MCV = 67.0↓ fL) resulted in
impaired production of erythrocytes and hemolysis,
though MCHC was normal (35.2 g/dL). In male
patients from another family, MCV was 69.2 and 68.0 fL
and MCHC was normal (34.2 ans 35.6 g/dL). In their
childrencompounds, the decreased level of hemoglo
bin (6.8↓ and 7.3↓ g/dL) resulted, first of all, from
decreased erythrocyte volume, since mean concentra
tion of hemoglobin (MCHC) was normal (34.9 and
35.9 g/dL).
The blood concentration of iron in compounds was
rather high, though there had been no blood transfusions:
serum iron concentration was 5.5 and 6.15 ng/μL and
serum ferritin concentration was 102.9 and 207.65 ng/μL.
Such concentrations result from enhanced absorption
of iron caused by the inhibition of hepsidin—a liver
hormone, the main regulator of iron metabolism,
which inhibits the absorbtion of iron and its reutiliza
tion in macrophages.
In children with thalassemia intermedia, the
increased rate of erythropoiesis seems to cause the
inhibition of hepsidin and enhanced iron absorption.
In βthalassemia, GDF15 produced by bone marrow
can inhibit the activity of hepsidin in cases of iron
overload by changing the rate of expression of hepsidin
gene (HAMP) [9–11]. The excess of iron normally
leads to an increase in the rate of hepsidin, which
inhibits its absorption. However, this does not occur in
patients with thalassemia intermedia, which causes
increased level of iron in compounds.
All examined children had slight arrhythmia
caused by the effect of increased iron level on cardio
vascular system; they also had enlarged liver because of
intense extramedullary hemopoiesis.
Thus, the hematological and molecular genetic
analysis of asymptomatic carriers confirmed the pres
ence of βthalassemia in these patients. The differen
tial blood count aimed at distinction of thalassemia
minor from anemia of chronic disease (which was the
previous diagnosis of examined carriers) has to include
the concentrations of serum ion, serum ferritin, HbA2
and HbF.
CONCLUSIONS
RDBH is an easy and economical method for
molecular diagnostics of βthalassemia, especially in
cases if the analysis of hematological parameters gives
dubious results. In studied families, the parents were of
reproductive age and planed to have more children;
therefore, prenatal diagnostics by RDBH was recom
mended, since this method requires minimal amount
of DNA and provides immediate results, which is
important because therapeutic abortion should be
considered in cases of fetal disease.
ACKNOWLEDGMENTS
This work was supported by the Foundation for
Scientific Development under the President of the
Republic of Azerbaijan, project no. EIFMob22013
4(10)13/06/3.

CYTOLOGY AND GENETICS Vol. 49 No. 3 2015

182 AKBAROVA
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Translated by Ya. Atmanskikh
CYTOLOGY AND GENETICS Vol. 49 No. 3 2015