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Hemoglobin, 2014; 38(3): 161–164! 2014 Informa Healthcare USA, Inc. DOI: 10.3109/03630269.2014.893531

ORIGINAL ARTICLE

a-Globin Gene Mutations in Isfahan Province, Iran

Arezo Karamzade1, Hadi Mirzapour1, Majid Hoseinzade2, Sara Asadi2, Tahere Gholamrezapour2, Parvaneh Tavakoli2,and Mansoor Selebi2,3,4

1Department of Genetics and Molecular Biology, School of Medicine, Isfahan University of Medical Sciences, Isfahan, Iran2Genetics Laboratory, Alzahra University Hospital, Isfahan University of Medical Sciences, Isfahan, Iran3Pediatric Inherited Disease Research Centre, Isfahan, Iran4Medical Genetics Centre of GENOME, Isfahan, Iran

Abstract

a-Thalassemia (a-thal) encompasses a spectrum of mutations including deletion and pointmutations on the a-globin chains that is characterized by a reduction or complete absence ofa-globin genes. Most of the a-thal cases are deletions involving one (a+) or both (a0) a-globingenes, although point mutations (aTa or aaT) are found as well. In this study, 314 individualswith low hematological values, normal Hb A2 who were not affected with b-thal or irondeficiency, were investigated for the presence of a-thal mutations. The most common deletionwas �a3.7 (rightward) with a frequency of 70.7%, followed by a�5 nt (–TGAGG) (8.7%), �a4.2

(leftward) (4.7%), the polyadenylation signal (polyA2) site (AATAAA4AATGAA) (4.2%), �(a)20.5

(3.8%), Hb Constant Spring [Hb CS, a142, Stop!Gln; HBA2: c.427T4C] (2.9%), polyA1(AATAAA4AATAAG) and acodon 19 (GCG4GC–, a2) (16%), and – –MED (0.9%). The results of thisstudy may be valuable for designing a plan for carrier screening, premarital genetic counseling,prenatal diagnosis (PND) and reducing excessive health care costs to an affordable level inIsfahan Province, Iran.

Keywords

a-Thalassemia (a-thal), Iran, Isfahan Province,mutations

History

Received 20 July 2013Revised 25 November 2013Accepted 4 December 2013Published online 13 May 2014

Introduction

a-Thalassemia (a-thal) is one of the most common monogenic

disorders in the world and is more prevalent throughout the

Mediterranean countries, the Middle East, Southeast Asia and

Africa (1). a-Globin genes are located on chromosome 16 and

flaws in these genes lead to reduction or complete absence of

a-globin gene expression. The majority of a-thal mutations

are deletions involving one (a+) or both (a0) a-globin genes,

although point mutations (aTa or aaT) are found as well (2).

Reduction in a-globin chain synthesis, excess of g- and

b-globins due to reduction of a-globin leads to Hb Bart’s (g4)

and Hb H (b4) in fetuses and adults, respectively (3). There is

not any significant clinical feature in deletion of one copy of

the a gene but deletion of two a genes results in microcytic

hypochromic anemia, while Hb A2 is normal. Because of the

vast mutation spectrum in different populations and regions,

identification of a-globin gene defects can be helpful in

improved screening, prenatal diagnosis (PND) and patient

care.

Iran is one of the countries with a high prevalence of

thalassemia in a region called the ‘‘thalassemia belt.’’ The

spectrum of a-thal mutations has been studied in many

different region of Iran and previous studies showed that

�a3.7 (rightward deletion), – –MED and �a4.2 (leftward

deletion) are the most common mutations in Iranian patients

(4,5). However from Isfahan Province, located in the central

region of Iran with a population of more than 4.8 million

(Figure 1), no related studies have been reported. Due to its

location, Isfahan Province has a heterogeneous population

that encompasses different ethnic group such as Fars,

Bakhtiari Lurs, Armenians, Qashqais and Persian Jews. In

this study, we investigated the a-thal mutation spectrum

among potential carriers who were referred to the Genetics

Laboratory at the Alzahra University Hospital, Isfahan City,

Isfahan Province, Iran, which is the main university hospital

of Isfahan and one of the largest in Iran.

Materials and methods

Three hundred fourteen individuals (161 males and 153

females, age range 18–35 years old) from Isfahan Province,

referrals from the Genetics Laboratory, Alzahra University

Hospital during the past 2 years (2010–2012), were enrolled

in this study. Ethnic groups of the participants consisted of

unrelated Fars, Bakhtiari Lurs, Armenians and Qashqais

individuals who all resided in the province. All subjects had

microcytic hypochromic anemia with a mean cell volume

(MCV) of580.0 fL and mean cell Hb (MCH) of527.0 pg and

normal Hb A2 levels. Patients with confirmed b-thalassemia

Address correspondence to Dr. Mansoor Salehi, Genetics Laboratory,Alzahra University Hospital, Isfahan University of Medical Sciences,Hezar Jarib Street, Isfahan 81744-174, Iran. Tel: +98-311-792-2486;Fax: +98-311-668-8697. E-mail: m_salehi@med.mui.ac.ir

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(b-thal) or iron deficiency anemia were excluded from the

study. Most of the subjects were from different cities of the

province rather than the central area of Isfahan City.

After informed consent was obtained, a blood sample

(10 mL) was drawn from each patient and genomic DNA was

extracted using the high salt extraction method as described

by Miller et al. (6). The multiplex polymerase chain

reaction (PCR) protocol was used as a primary screening test

for common deletional mutations (�a3.7, �a4.2, �(a)20.5

and – –MED). Primers and PCR conditions were as described

by Chong et al. (7). This was followed by reverse hybridization

using the a-globin StripAssay (ViennnaLab Diagnostics,

Vienna, Austria) to detect the following mutations that

were not covered by PCR: codon 14 (G4A) (a1), codon

19 (–G) (a2), IVS-I,�5 nt (–TGAGG) (a2), codon 142 (T4C)

(a2), codon 59 (G4A) (a2), polyadenylation signal

(polyA1) site (AATAAA4AATAAG) (a2) and polyA2

(AATAAA4AATGAA) (a2). DNA sequencing was per-

formed on samples where no mutations were identified.

Ethics approval was obtained from the Ethics Committee of

the Isfahan University of Medical Sciences. The data were

statistically analyzed using the Statistical Package for Social

Sciences; version 21 software (SPSS Inc, Chicago, IL, USA).

Results

Different mutations were found in 287 out of 314 participants

(88.0%) and no mutations in the remaining 27 individuals

(Table 1). Of the possible genotypes, �a3.7/aa was by far the

most common genotype (60.5%) followed by the a�5 nt/aagenotype (8.5%). The �a3.7 allele was the most common

mutation (70.7%) found in 308 a-globin genes overall. The

second and third most frequent a-globin gene defects were

a�5 nt (8.7%) and �a4.2 (4.8%).The remaining mutations were

polyA2 (4.2%), �(a)20.5 (3.8%), Hb Constant Spring [Hb CS,

a142, Stop!Gln; HBA2: c.427T4C] (2.9%), polyA1 (1.6%),

acodon 19 (1.6%), – –MED (0.9%) and acodon 14 (0.003%). DNA

sequencing was performed on 27 subjects without mutation,

but no a-globin mutation was detected.

Discussion

Iran is located in a region with a high prevalence of

thalassemia and national premarital screening was started in

1991 (8). During this program, good progress was achieved in

the knowledge of a- and b-thal molecular defects. Studies on

defects of the a-globin genes were initiated parallel to b-thal

based on studies in different regions of the country and the

most common a-globin gene mutations have been identified.

Although, a-thal mutations are frequent in Iran and Isfahan

Province is one of the largest provinces of Iran (approximately

7.0% of the population live in this province), but the spectrum

of a-thal mutations have never before been investigated. As

expected, the �a3.7 deletion (70.7%) was the most prevalent

a-thal mutation in Isfahan and this result is in agreement with

other parts of the country (9–12) and in neighboring countries

as well (13). Prevalence of the �a3.7 deletion is the same as in

the neighboring provinces of Fars and Kohgiluyeh Boyer

Figure 1. Map of Iran showing the provinces discussed in this study.

162 A. Karamzade et al. Hemoglobin, 2014; 38(3): 161–164

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Ahmad (71.7%), in Kerman Province it is significantly higher

(83.8%), while in Gilan and Mazandaran, two northern

provinces, are lower still than others with 42.5 and 44.9%,

respectively. This high prevalence of the �a3.7 deletion may

be due to the high consanguinity level in Iranian marriages.

After the common �a3.7 deletion, the �5 nt deletion

(8.7%) was the second most frequent defect on the a-globin

chain, this being in agreement with a study from the

neighboring provinces of Fars, Kohgiluyeh Boyer Ahmad

(7%) and Kerman (5.7%). Regarding double gene deletions,

�(a)20.5 (3.8%) was more prevalent than the – –MED mutation

(0.9%) in Isfahan Province, while in northern provinces, the

– –MED mutation occurred more frequently than the �(a)20.5

mutation. Table 2 shows the frequency of mutations in

different region of Iran including Kerman, Gilan,

Mazandaran, Fars and Kohgiluyeh Boyer Ahmad provinces

(9–12). All enrolled patients in those studies had microcytic

hypochromic anemia and all criteria were the same as in the

studies that were mentioned earlier (MCV580.0 fL, MCH of

527.0 pg, normal Hb A2 levels and no b-thal or iron

deficiency anemia) (9–12).

Twenty-seven (8.5%) individuals were found not to carry

any of the common mutations involved in this study and the

low hematological values of these subjects might have been

caused by other types of defects not included in our study.

The numbers for Kerman, Gilan, Mazandaran, Fars and

Kohgiluyeh Boyer Ahmad provinces were 4.2, 8.7, 11.0 and

1.25%, respectively. In these cases, better analysis could be

achieved by multiple ligation-dependent probe amplification

(MLPA) for rare and novel deletional mutations. As

microcytic hypochromic anemia could be a result of b-thal

and iron deficiency as well as a-thal, patients with

confirmed b-thal trait and iron deficiency were not included

in this study.

a-Thalassemia as a socio-health problem imposes a high

financial cost in at-risk populations annually. Prevention of

hydrops fetalis and Hb H disease is the most effective way

to diminish excessive mental and economical burden on the

patient’s family members and government. As many indi-

viduals are carriers of both a- and b-thal, elevated Hb A2

level in b-thal may lead to misdiagnosis during PND and

will result in missed a-thal cases. Therefore, after ruling out

b-thal traits, molecular analysis for a-globin mutations is

essential for at-risk couples. It seems that in countries

where the rate of consanguineous marriages is high,

screening for a-thal is necessary for prevention of the

burden of hydrops fetalis and Hb H disease in this at-risk

population. Knowing the incidence of each involved muta-

tion in a specific region will be useful for planning to

design carrier screening, premarital genetic counseling,

PND and reducing excessive health care costs to an

affordable level for Isfahan Province, Iran.

Table 1. a-Thalassemia Mutations and Mean [± SD (standard deviation)] Hematological Values.

Genotype n % Hb (g/dL) MCH (pg) MCV (fL) Hb A2 (%) Hb F (%)

�a3.7/aa 190 60.5 13.8 ± 1.5 24.8 ± 1.6 76.6 ± 3.8 2.4 ± 0.3 0.5 ± 0.1a�5 nta / aa 27 8.5 13.7 ± 1.5 24.7 ± 1.1 77.4 ± 2.3 2.4 ± 0.2 0.5 ± 0.2apolyA2a/aa 12 3.8 13.5 ± 1.6 24.2 ± 1.0 76.1 ± 2.1 2.3 ± 0.5 0.7 ± 0.2�a3.7/�a3.7 9 2.8 12.2 ± 1.0 23.0 ± 1.6 73.4 ± 2.3 2.8 ± 0.6 0.6 ± 0.2�(a)20.5/aa 8 2.5 13.3 ± 1.2 20.7 ± 1.0 66.2 ± 3.8 2.6 ± 0.8 0.5 ± 0.1aCSa/aa 7 2.2 12.8 ± 2.3 23.8 ± 2.9 73.5 ± 6.0 2.7 ± 0.3 0.6 ± 0.3�a4.2/aa 7 2.2 14.6 ± 2.0 26.3 ± 1.5 76.0 ± 0.3 2.6 ± 0.3 0.5 ± 0.1acodon 19a/aa 5 1.5 15.1 ± 1.5 24.7 ± 0.6 77.3 ± 2.4 2.7 ± 0.2 0.5 ± 0.1apolyA1a/aa 5 1.5 12.5 ± 1.9 22.0 ± 2.5 73.0 ± 1.5 2.3 ± 0.9 0.3 ± 0.2�a3.7/�a4.2 4 1.2 14.3 ± 2.2 24.0 ± 2.9 76.0 ± 4.3 2.3 ± 0.4 0.5 ± 0.2�a3.7/�(a)20.5 4 1.2 11.0 ± 0.7 17.5 ± 1.3 61.0 ± 3.0 1.9 ± 0.9 0.6 ± 0.1– –MED/aa 3 0.9 12.6 ± 2.0 21.5 ± 0.8 68.0 ± 1.1 2.2 ± 0.1 0.6 ± 0.2�a4.2/�a4.2 2 0.6 13.7 ± 1.3 23.0 ± 1.0 76.5 ± 3.0 2.4 ± 0.9 0.6 ± 0.1�a3.7/aCSa 2 0.6 12.7 ± 3.0 21.6 ± 0.2 67.3 ± 4.0 1.7 ± 3.0 0.6 ± 0.1acodon 14a/aa 1 0.3 15.3 26.1 79.0 2.2 0.6�a3.7/apolyA2a 1 0.3 12.0 21.9 68.2 1.8 0.6No mutation 27 8.5 14.1 ± 1.6 24.3 ± 2.0 75.5 ± 3.4 2.3 ± 0.4 0.5 ± 0.1Total 314 100.0

Table 2. Allele Frequency of a-Thalassemia Mutations in Different Regions of Iran.

Provinces Isfahan (%) Kerman (%) Gilan (%) Mazandaran (%)Fars and Kohgiluyeh

Boyer Ahmad (%)

�a3.7 70.7 83.8 42.5 44.9 71.9a�5 nt (–TGAGG) (a2) 8.7 5.7 8.8 6.5 7.0�a4.2 4.8 3.7 4.4 9.1 3.6apolyA2 (AATAAA4AATGAA) 4.2 5.0 12.4 18.2 3.9�(a)20.5 3.8 0.0 1.8 2.1 0.5Hb CS (HBA2: c.427 T4C) (a2) 2.9 1.3 10.6 3.3 3.7apolyA1 (AATAAA4AATAAG) 1.6 5.0 3.5 1.4 4.2acodon 19 (GCG4GC–) (a2) 1.6 0.0 0.9 4.0 1.7– –MED 0.9 0.3 8.8 4.3 1.9References this study 9 10 11 12

DOI: 10.3109/03630269.2014.893531 �-Globin Gene Mutations in Isfahan Province, Iran 163

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Declaration of interest

The authors report no conflicts of interest. The

authors alone are responsible for the content and writing of

this article.

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