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Original Paper

Acta Haematol 2010;124:197–200 DOI: 10.1159/000302203

Molecular Characterization of � -Thalassemia in Adana, Turkey:A Single Center Study

Birol Guvenc   a, c, d Sule Menziletoglu Yildiz   b Ferda Tekinturhan   c Suleyman Dincer   e Inci Akyuzluer   d Secaatin Okten   d Hakan Erkman   d  

a   Department of Hematology, Cukurova University Faculty of Medicine, b   Health Services Vocational School, Cukurova University, c   Hemapheresis, Stem Cell and Cryopreservation Unit, Cukurova University Balcali Hospital, and d   Seyhan Hereditary Blood Disorders Center, Adana , and e   Department of Hematology, Yeditepe University Faculty of Medicine, İstanbul , Turkey

� 3.7 , � 4.2 , -- MED , -- 20.5 , � PA-2 � , � � � anti-3.7 , and � PA-1 � were de-tected. Conclusion: Our results showed that the � -thal mu-tations represent a great heterogeneity and that the - � 3.7 deletion has the highest frequency in Adana.

Copyright © 2010 S. Karger AG, Basel

Introduction

� -Thalassemia ( � -thal), characterized by a decreased or a complete lack of synthesis of a globin chain, is one of the most common inherited disorders of hemoglobin (Hb) synthesis worldwide and commonly found in South-east Asian, Mediterranean and Middle Eastern popula-tions [1, 2] . The � -globin gene cluster is located on chro-mosome 16p13.3 and mutations or deletions affectingeither one or more � -globin genes result in � -thal syn-dromes [3] . The most frequent mutations of � -globin genes are single-gene deletions, and the deletion or inac-tivation of only 1 � -globin gene usually results in insig-nificant hematological findings. The � -thal trait results from the deletion or inactivation of 2 � -globin genes(--/ � � or - � /- � ); however, it exhibits mild microcytic,

Key Words

- � 3.7 deletion � � -Thalassemia � Frequency, � -thalassemia  � Heterogeneity � Molecular properties, � -thalassemia � Mutations

Abstract

Background/Aim: � -Thalassemia ( � -thal) is a widespread genetic disorder throughout the world caused primarily by reduced synthesis of the � -globin chains, and it has been found at a high incidence in Turkey. Our aim in this study was to determine the frequency and molecular properties of � -thal in Adana, Turkey. Material and Methods: A total of 3,000 individuals comprising premarital couples or patients with anemia were screened between 2007 and 2008. Hematolog-ical parameters were analyzed using an automatic cell coun-ter, and to detect the carriers of hemoglobin variants, high-performance liquid chromatography was used. Molecular screening of the � -globin gene was carried out by an Alpha-Globin StripAssay � which is based on multiplex PCR for spe-cific amplification. Results: We have identified 225 cases with � -thal and found that the prevalence of � -thal is 7.5% in this area. In molecular analyses, the � -thal gene mutations

Received: November 30, 2009 Accepted after revision: January 13, 2010 Published online: March 24, 2010

Assoc. Prof. Dr. Birol Guvenc Department of Hematology , Faculty of Medicine, University of Cukurova TR–01330 Balcali, Adana (Turkey) Tel. +90 322 338 6060, ext. 3141, Fax +90 322 338 6938E-Mail bguvenc   @   cu.edu.tr or guvenc.birol   @   gmail.com

© 2010 S. Karger AG, Basel0001–5792/10/1244–0197$26.00/0

Accessible online at:www.karger.com/aha

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Acta Haematol 2010;124:197–200 198

hypochromic anemia with normal or altered Hb A 2 lev-els. When 3 of 4 � -globin genes become inactive, the af-fected individual has only 1 functional � -globin gene pre-senting with Hb H disease, which is characterized by se-vere anemia [4] . The - � 3.7 mutation is very common in the Mediterranean and in Africa.

Adana, located close to the southeastern Mediterra-nean coast, is the fifth most populated city in Turkey, and has been known to be an endemic region for malaria. The Cukurova plain, one of Turkey’s largest centers for agri-cultural production, covers the provinces of Adana, Mer-sin, and Hatay. The major ethnic groups in Adana are the Turks, the Kurds, as well as Arabs, whose ancestors im-migrated from Syria and Egypt centuries ago. There is also a substantial community of migrants from the Bal-kans and Caucasia who have settled in Adana. The great ethnic diversity of this area as well as being a malaria-endemic region means that the frequency of � -thal is high compared to the other parts of Turkey. In addition to that, consanguineous marriages, which contribute to the increased frequency of � -thal, are still very common in Adana.

The molecular techniques mainly used for the identi-fication of deletions/mutations in the � -globin gene in-clude multiplex gap-PCR, the amplification refractory mutation system (ARMS), the restriction fragment length polymorphisms (RFLP) or the reverse dot blot analysis [5–7] . The StripAssay is another molecular technique by which the mutations in human DNA are identified using a strip method in a single procedure. From different sam-ples, such as blood or amniotic fluid, genomic DNA is extracted, and the mutant/wild parts of genes are ampli-fied through multiplex PCR with biotinylated primers. After the PCR products are hybridized on nitrocellulose membrane with allele-specific probes, mutant and wild-type alleles are detected by enzymatic color reaction [8] . The Alpha-Globin StripAssay � (ViennaLab-Labordiag-nostika GmbH, Vienna, Austria) has recently become available and considerably facilitated the � -thal diagnosis in Turkey. In our center, the StripAssay has been used to identify deletions/mutations in the � -globin gene, fol-lowed by a second molecular technique (ARMS or RFLP) to confirm the results.

In this study, we aimed to determine the molecular characterization of the � -thal gene in Adana, Turkey.By using the StripAssay, we detected 2 single-gene dele-tions (- � 3.7 , - � 4.2 ), 3 double-gene deletions (-- MED , -- 20.5 , � � � anti-3.7 ) and 2 point mutations ( � PA-1 � and � PA-2 � ) in the alpha 2 gene.

Materials and Methods

From 2007 to 2008, a total of 3,000 individuals including pre-marital couples and patients presenting with anemia were screened at the Seyhan Hereditary Blood Disorders Center in Adana. Blood samples were collected with EDTA-containing tubes, and com-plete blood counts were performed by a Coulter (Beckman Coul-ter, Brea, Calif., USA) hematological analyzer for all cases. Except in patients diagnosed with iron-deficiency anemia, high-perfor-mance liquid chromatography was utilized to identify the type of Hb for cases with unexplained hypochromic microcytic anemia. For subjects who had an MCV ! 80 fl, MCH ! 25 pg, MCHC ! 30 g/dl and an Hb A 2 level ! 2.5%, molecular diagnostic techniques were used to detect the type of � -thal mutation.

DNAs were isolated from peripheral blood leukocytes accord-ing to the conventional methods. Mutation analysis of the � -glo-bin gene was carried out using an Alpha-Globin StripAssay (cat. No. 4-160), and to verify the results, ARMS or RFLP was used as a second molecular diagnostic method.

Results

According to our results, 225 out of 3,000 subjects were diagnosed with � -thal, meaning that the rate of oc-currence of � -thal is 7.5% in Adana. The PCR-based anal-ysis of 225 blood samples to determine the � -globin gene status demonstrated that the incidence of � -thal, particu-larly the 3.7-kb deletion, was extremely high in Adana. The distributions of mutations were as follows: - � 3.7 / � � (53.33%), -- MED / � � (15.11%), -- 20.5 / � � (6.66%), � PA-2 � / � � (4.0%), � � � anti-3.7 / � � (2.22%), � PA-1 � / � � (1.33%), - � 4.2 / � � (0.44%), - � 3.7 /- � 3.7 (11.55%), - � 3.7 /-- MED (4.0%), - � 3.7 /-- 20.5 (0.88%) and - � 4.2 /- � 4.2 (0.44%) ( table  1 ). These results showed that there was a great heterogeneity of genotype in � -thal in this area.

When some hematological parameters were analyzed in relation to the genotype, the MCV value was found to be the lowest in the - � 3.7 /-- MED , and the highest in the - � 3.7 / � � mutation. The MCH and the MCHC levels were also consistent with this finding. In summary, hemato-logical indices were further reduced as the number of functional genes decreased ( table 2 ).

Discussion

The deletion of a single or double � -globin gene that is located at position 16p13.3 causes � -thal, one of the most common genetic disorders in the world [3] . The preva-lence of � -thal has been found to be 5–15% in the Medi-terranean, 60% in the Middle East, 5–40% in Africa, 15–80% in India, 5–15% in the Far East and 5–80% in New

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Molecular Characterization of � -Thalassemia in Adana

Acta Haematol 2010;124:197–200 199

Zealand, while the incidence has been reported to be ap-proximately 3% in the Cukurova region in southern Tur-key [9] .

To determine the prevalence of � -thal in Adana, we screened a total of 3,000 subjects in a single center and identified 225 cases with � -thal (7.5%). We have noticed that our results were inconsistent with those of Curuk et al. [9] , who reported the frequency of � -thal to be 3% for the Cukurova region. This discrepancy between both studies may be attributed to the smaller sample size used by Curuk et al. and to the fact that the investigation of Curuk et al. included only patients with Hb H, whereas our center has been responsible for carrying out screen-ing programs especially for cases with a risk of thalasse-mia. This must be taken into account when evaluating the findings of the studies. As a result, the high rate of � -thal trait that we noted in our study was considered reason-able.

Moreover, it has been hypothesized that � -thal is widespread in malaria-endemic regions in the world and is considered protective against severe malaria infections [10] . The Cukurova region, particularly Adana, was one of the malaria-endemic areas in Turkey up until a couple of decades ago. Additional and significant reasons con-tributing to the increased rate of � -thal in Adana could be the great ethnic diversity and the high incidence of consanguineous marriages. These 2 factors might also be the main causes of the genotypic heterogeneity in � -thal mutations that we noticed in our study. We have identi-fied 11 different genotypes and our results demonstrated that - � 3.7 / � � and -- MED / � � were the most prevalent geno-types in subjects with � -thal in Adana followed by - � 3.7 /- � 3.7 and -- 20.5 / � � mutations. These data were found to be consistent with those presented in previous studies in Turkey [9, 11] .

In the Mediterranean area, widespread mutations are the - � 3.7 and - � 4.2 single � -globin gene deletions and the -- MED and - � 20.5 double-gene deletions [7, 12] . The most common genotypes in Iraq reported by Al-Allawi et al. [13] were - � 3.7 / � � , -- MED / � � and - � 3.7 /- � 3.7 which were detected in 84.3% of the study group. - � 3.7 single-gene deletion has also been stated as the most frequent muta-tion in Ashkenazi Jews and in Spain [14, 15] . Similarly, Siala et al. [16] from Tunisia and Di Bella et al. [17] from Italy showed that - � 3.7 carriers are widespread in those countries. On the other hand, the SEA and the THAI de-letions that we have not identified in our cohort are com-monly encountered in the Asian populations [5, 18] .

Our ultimate goal in this study was to detect individu-als with � -thal in Adana, and to provide appropriate ge-netic counseling for the affected subjects and their fami-lies. Although prenatal diagnosis is not mandatory for couples with � -thal, if there is a risk of hydrops fetalis, the prenatal approach has been accepted as an efficient way to prevent serious obstetric complications in mothers carrying hydropic fetuses in the late period of gestation [4] . Chorionic villus sampling is the most widely used technique to analyze DNA samples of fetuses in the pre-natal diagnosis in the first trimester of gestation. Never-theless, 1–2% potential risk of missed abortion because of the procedure has been reported. Thus, serial 2-dimen-sional ultrasound, prenatal diagnosis of fetal cells in ma-ternal blood and preimplantation genetic diagnosis have been proposed as options to reduce the risk of miscar-riage [19, 20] .

Since the hematological indices are only slightly al-tered in heterozygous � -thal forms, accurate diagnosis can be provided only by molecular analysis. The relation-

Table 1. Distribution of �-thal mutations

Genotype n %

-�3.7/�� 120 53.33--MED/�� 34 15.11--20.5/�� 15 6.66�PA-2�/�� 9 4.0���anti-3.7/�� 5 2.22�PA-1�/�� 3 1.33-�4.2/�� 1 0.44-�3.7/-�3.7 26 11.55-�3.7/--MED 9 4.0-�3.7/--20.5 2 0.88-�4.2/-�4.2 1 0.44

Total 225 100

Table 2. Some hematological parameters (means 8 SD) of the�-thal genotypes (n = 218)

Genotype n MCV, fl MCH, pg MCHC, g/dl

- �3.7/�� 120 75.5787.55 25.6582.35 32.9581.05--MED/�� 34 63.4583.05 20.2080.90 31.8580.05-�3.7/-�3.7 26 68.8589.15 24.7786.40 30.5182.35--20.5/�� 15 70.0586.05 23.3081.70 33.3580.45-�3 .7/--MED 9 62.1081.15 18.9580.55 31.4080.30�PA-2�/�� 9 75.5082.85 25.8081.90 34.3081.20���anti-3.7/�� 5 71.4088.10 23.4283.95 35.8580.40

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ship between hematological parameters and the geno-types observed in our study group can be seen in table 2 . El-Kalla and Baysal [21] showed that there was a signifi-cant decrease in the MCV value with decreasing numbers of the � -globin genes. In our study, we also noticed a con-sistent correlation between the MCV, MCH and MCHC values and the number of functional � -globin genes. Still, using solely the hematological indices to define the type of genotype in � -thal forms is not reliable.

PCR-based methods, DNA sequencing, gene map-ping, Southern blot analysis, or multiple ligation-depen-dent probe amplification have been frequently used to de-tect common or novel deletions in the � -globin genes. The StripAssay used in this study has been validated in recent studies [8] , and we can suggest that it provides a simple, rapid, and cost-effective molecular diagnostic way to detect � -thal mutations.

It appears that � -thal is very common and still a sig-nificant problem in Adana, and accurate diagnosis is one of the most important steps in the prevention of � -thal traits and for individuals with Hb H disease. The high rate of abnormal Hbs, mainly Hb S, as well as the � -thal trait in Adana results in the increased number of affect-ed newborns. An early diagnosis is, therefore, necessary to implement proper preventive health care measures, which are crucial to ensure quick treatment of potential-ly serious complications, and to improve the awareness of other devastating � -thal and � -thal syndromes in the communities. Prospective and methodological analyses particularly during infancy and pregnancy should be performed to evaluate potential risk factors related to se-vere disease and to formulate future medical interven-tions and treatment policies.

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