Hemoglobin international journal for hemoglobin research

ISSN: 0363-0269 (Print) 1532-432X (Online) Journal homepage: https://www.tandfonline.com/loi/ihem20

The Spectrum of α-Thalassemia Mutations in , West

Reza Alibakhshi, Keivan Moradi, Mozaffar Aznab, Zahra Dastafkan, Susan Tahmasebi, Mahsa Ahmadi & Leila Omidniakan

To cite this article: Reza Alibakhshi, Keivan Moradi, Mozaffar Aznab, Zahra Dastafkan, Susan Tahmasebi, Mahsa Ahmadi & Leila Omidniakan (2020) The Spectrum of α- Thalassemia Mutations in Kurdistan Province, West Iran, Hemoglobin, 44:3, 156-161, DOI: 10.1080/03630269.2020.1768863 To link to this article: https://doi.org/10.1080/03630269.2020.1768863

Published online: 26 Jun 2020.

Submit your article to this journal

Article views: 4

View related articles

View Crossmark data

Full Terms & Conditions of access and use can be found at https://www.tandfonline.com/action/journalInformation?journalCode=ihem20 HEMOGLOBIN 2020, VOL. 44, NO. 3, 156–161 https://doi.org/10.1080/03630269.2020.1768863

ORIGINAL ARTICLE The Spectrum of a-Thalassemia Mutations in Kurdistan Province, West Iran

Reza Alibakhshia, Keivan Moradia, Mozaffar Aznabb, Zahra Dastafkanc, Susan Tahmasebic, Mahsa Ahmadic and Leila Omidniakanc aDepartment of Biochemistry, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran; bDepartment of Hematology Oncology, School of Medicine, Kermanshah University of Medical Sciences, Kermanshah, Iran; cMedical Genetics Laboratory, Kermanshah University of Medical Sciences, Kermanshah, Iran

ABSTRACT ARTICLE HISTORY In order to identify the a-thalassemia (a-thal) mutation spectrum in Kurdistan Province, West Iran, a Received 26 November 2019 total of 217 individuals, including 154 a-thal carriers and 63 normal subjects were investigated in this Revised 16 April 2020 study. Molecular analysis of a1- and a2-globin genes using multiplex gap-polymerase chain reaction Accepted 16 April 2020 (gap-PCR), amplification refractory mutation system (ARMS)-PCR or direct DNA sequencing, showed 11 a –a3.7 KEYWORDS different -globin variants. The (rightward) deletion (NG_000006.1: g.34164_37967del3804) a a polyA2 -Thalassemia ( -thal); (70.32%), polyadenylation signal (polyA2) site (AATAAA>AATGAA) (a a)(HBA2:c.92A>G) (7.74%), 4.2 codon 59 gene; mutation; ; –a (leftward) deletion (6.45%) and codon 59 (or Hb Adana) (G>A) (aa )(HBA1: c.179G>A) West Iran (4.52%) were the most frequent mutations in the present study. In conclusion, the spectrum of a-thal mutations in Kurdistan Province is closest to that in western (Kurdish and Laki popula- tions). In addition, it was revealed that the codon 59 mutation is common in the Kurdish population. On the other hand, despite the same ethnic background of Kurds in Iran and Iraq, the ––MED I double gene deletion and polyA2 point mutation have different distributions in these two populations. Therefore, further studies are needed to identify the cause of these differences.

Introduction diagnosis. Iran is a country located in the Middle East region. As many thalassemia carriers live in this country, the Deficiencies in the production of normal hemoglobin (Hb)- forming globin proteins result in a group of inherited blood National Thalassemia Screening Program has been estab- disorders called thalassemia [1]. a- And and b-thalassemia lished in Iran for more than 20 years [7]. The results of this (a- and b-thal) are due to mutations occurring on the screening program show different mutation spectra of a- HBA1/HBA2 and HBB genes, respectively. The prevalence of and b-globin genes in different geographic regions of Iran. thalassemia is high in Mediterranean, African, Middle In fact, given the presence of different ethnicities, it is not Eastern, Indian and Southeast Asian populations. surprising to see this diversity in Iran [1,8]. ‘ ’ Accordingly, an area called the Thalassemia Belt has Kurdistan Province is located in the western part of Iran. emerged [1,2]. According to the latest census of population and housing in To date, several mutations have been identified in a1- 2016, more than 1.6 million people live in Kurdistan and a2-globin genes and have been recorded in the HbVar database [3](http://globin.bx.psu.edu/hbvar/menu.html). Province. This province includes 10 counties, named More than 95.0% are deletional mutations [4]. Studies con- , Dehgolan, , , , , ducted in different populations have shown that each popu- Divandarreh, , and (https://www. lation has its own mutational spectrum. Thus, in each amar.org.ir). Kurdistan Province has borders to the north, particular population, there are a few mutations with high northeast, east and south with West Azerbaijan and Zanjan, allelic frequencies and a greater number of rare muta- Hamadan and Kermanshah provinces of Iran, respectively. tions [5]. In addition, it shares a border with Iraq in the west. People Given the high prevalence of patients with thalassemia in of Kurdistan Province speak the Kurdish language. To the populations living in the ‘Thalassemia Belt’ region and the best of our knowledge, no study has yet been in performed impact that these diseases may have on the quality of life of patients’ family members, various screening programs have in Kurdistan Province. Therefore, our aim in this study was a been implemented by different governments [6]. One of the to report the spectrum of -globin gene mutations in the main purposes of these screening programs is to identify Kurdish population living in the southern part of Kurdistan carriers and consequently the possibility of prenatal Province (Figure 1).

CONTACT Dr. Reza Alibakhshi [email protected] Department of Biochemistry, School of Medicine, Kermanshah University of Medical Sciences, Parastar Street, Kermanshah, Kermanshah Province, Iran ß 2020 Informa UK Limited, trading as Taylor & Francis Group HEMOGLOBIN 157

Figure 1. The gray area on the map of Kurdistan Province, West Iran, represents the places of residence of the studied patients.

Materials and methods of the entire HBA1/HBA2 genes (50 and 30 regulatory sequences as well as all exons and introns) was performed in During the period from 2009 to 2019, 154 a-thal carriers TM an ABI PRISM 3130 DNA analyzer (Applied Biosystems, from the southern part of Kurdistan Province (the counties Foster City, CA, USA). The data were analyzed using DNA of Sanandaj, Qorveh, Dehgolan, Marivan, Sarvabad and sequencing analysis version 5.2 software. Sequences of pri- Kamyaran) were referred to the Kermanshah Central mers are available upon request. Laboratory (Reference), Kermanshah, Kermanshah Province, Statistical data analysis was carried out using the a Iran, for the investigation of their -globin gene mutations. Statistical Package for the Social Sciences software, version a The -thal carriers were diagnosed based on their blood 25.0 (https://ibm.com/SPSS-Statistics/). The p values of < indices including mean corpuscular volume (MCV) 80.0 fL <0.05 were considered to be significant for all described < and/or mean corpuscular Hb (MCH) 27.0 pg, and Hb A2 tests. The v2 and independent Student t-test were used to < 3.5%, as well as lack of iron deficiency. In addition to compare categorical variables and mean levels of age and a -thal carriers, 63 subjects (siblings of carriers) with normal hematological factors between carriers and normal subjects, blood indices were chosen as the control group. An respectively. In addition, the analysis of variance (ANOVA) informed patient consent form was obtained from all of the and Scheffe post-hoc analysis were performed to determine participants. Approval was obtained from the Ethics the significance of differences between hematological factors Committee of Kermanshah University of Medical Sciences, in normal genotypes with a-thal genotypes identified in Kermanshah, Kermanshah Province, Iran [Ethics Code: our study. IR.KUMS.REC.1398.731]. We used three different molecular methods to identify the a-thal-causing mutations: 1) multiplex gap-polymerase Results –a3.7 chain reaction (gap-PCR) for detection of (rightward) A total of 217 individuals including 154 a-thal carriers and –a4.2 (NG_000006.1: g.34164_37967del3804), (leftward), and 63 normal subjects were investigated to identify a-globin ––MED I (NG_000006.1: g.24664_41064del16401) deletions; gene mutations. In order to evaluate the gender differences 2) amplification refractory mutation system (ARMS)-PCR in the mean levels of hematological factors, an independent a5nta HBA2 þ þ for detection of ( : c.95 2_95 6delTGAGG), Student t-test was performed separately in both patient and polyadenylation signal A2 (polyA2) site (apolyA2a) control groups (Table 1). According to the obtained results, (AATAAA>AATGAA) (HBA2:c.92A>G), polyA1 no statistically significant differences were observed in the apolyA1a A> G HBA2 ( ) (AATAA AATAA ) (T-Saudi) ( : mean of Hb A2, MCV and MCH levels between males and codon 59 c. 94A>G), codon 59 (A>G) (or Hb Adana) (aa ) females (p > 0.05). However, the mean levels of Hb and red codon 19 (HBA1: c.179G>A), and codon 19 (–G) (a a)(HBA2: blood cell count (RBC) were significantly lower in females c.56delG); 3) direct DNA sequencing of the a1- and a2-glo- than those in males (p < 0.0001). On the other hand, as bin genes for the analysis of samples whose mutations were shown in Table 2, there was no statistically significant differ- a not in the list of mutations examined by the gap-PCR and ence in Hb A2 levels between -thal carriers and normal ARMS-PCR methods. The protocols and PCR cycling condi- subjects (p > 0.05). Moreover, MCV, MCH and Hb were sig- tions have been described by us in detail in our previous nificantly lower and RBC was significantly higher in a-thal studies [9,10] as well as by the Tan et al. [11] study. Briefly, carriers than those in normal subjects (p < 0.0001 for MCV, after purification of PCR products using QIAquick (Qiagen MCH and RBC, and p < 0.001 for Hb). GmbH, Hilden, Germany) PCR purification kit and precipi- a-Thalassemia mutations were detected using gap-PCR, tation with ethanol-sodium acetate, direct DNA sequencing ARMS-PCR and direct DNA sequencing of the a1- and a2- 158 R. ALIBAKHSHI ET AL.

Table 1. Gender difference in the mean levels of hematological factors in both patient and control groups. 12 Group Hb A2 (%) MCH (pg) MCV (fL) Hb (g/dL) RBC (10 /L) a-Thal carriers males (n ¼ 70) 2.48 ± 0.41 23.13 ± 2.41 74.98 ± 5.44 14.78 ± 1.41 6.12 ± 0.61 females (n ¼ 84) 2.56 ± 0.34 24.19 ± 1.88 76.35 ± 4.23 12.80 ± 0.98 5.31 ± 0.41 p Valuea 0.221 0.879 0.088 <0.0001 <0.0001 Control subjects males (n ¼ 29 2.60 ± 0.24 28.18 ± 0.97 83.61 ± 2.51 15.75 ± 1.06 5.63 ± 0.39 females (n ¼ 34) 2.58 ± 0.35 27.97 ± 0.82 84.55 ± 2.94 13.52 ± 0.98 4.85 ± 0.38 p Valuea 0.783 0.345 0.186 <0.0001 <0.0001 a-Thal: a-thalassemia; Hb: hemoglobin; MCH: mean corpuscular Hb; MCV: mean corpuscular volume; RBC: red blood cell count. aThe independent Student t-test.

Table 2. Demographics and hematological factors of the individuals participat- It is estimated that the prevalence of b-thal carriers in ing in the present study. Kurdistan Province is higher than its national average (5.9 Characteristics a-Thal Carriers Control Subjects p Value vs. 4.0%) [24]. However, there is no study to determine the Age 25.88 ± 6.62 27.25 ± 7.82 0.225a a b prevalence of -thal carriers in this province, so there has Males 70 (45.5%) 29 (46.0%) 1.000 a Females 84 (54.5%) 34 (54.0%) – been no published spectrum of -globin gene mutations in a a Hb A2 (%) 2.51 ± 0.39 2.59 ± 0.30 0.134 Kurdistan Province. In the present study, 11 different -glo- MCH (pg) 24.16 ± 2.13 28.07 ± 0.89 <0.0001a < a bin gene variants and 20 different genotypes were detected. MCV (fL) 75.73 ± 4.84 84.12 ± 2.77 0.0001 –a3.7 Parameters The deletion, with an allele frequency of 70.32%, was Hb (g/dL) males 14.78 ± 1.41 15.75 ± 1.06 0.001a the most frequent mutation identified in this study. This fre- Hb (g/dL) females 12.80 ± 0.98 13.52 ± 0.98 0.001a 12 < a quency is one of the highest frequencies reported for this RBC (10 /L) males 6.12 ± 0.61 5.63 ± 0.39 0.0001 et al. RBC (1012/L) females 5.31 ± 0.41 4.85 ± 0.38 0.0001a mutation in Iran. Valaei [8] found the average allele –a3.7 a aThe independent samples t-test. frequency of deletion to be 67.10% in -thal carriers bThe v2 test. distributed in different parts of Iran, with a range of 45.60% in the north to 83.80% in the southeast regions. The polyA2 globin genes. A total of 11 different a-globin variants were point mutation (7.74%) and the –a4.2 deletional mutation found. a-Thalassemia deletional mutations including –a3.7 (6.45%) were the second and third most common mutations (70.32%), –a4.2 (6.45%) and ––MED I (1.94%) comprised in this study, respectively. Both of these mutations have high 78.71% of the total mutated alleles. In addition, polyA2, Hb frequencies in the northern region of Iran. In addition, the Adana and polyA1 point mutations were detected with fre- –a4.2 deletional mutation has a relatively high frequency in quencies of 7.74, 4.52 and 3.87%, respectively. Therefore, Ilam Province, West Iran (Table 5). these six mutations accounted for 94.84% of the total An interesting finding in this study was the relatively mutated alleles (Table 3). high frequency of the Hb Adana mutation in Kurdistan From 154 a-thal carriers, the a-thal mutations were Province (4.52%), which is similar to that reported in found in 126 individuals leading to a total of 155 mutated Kermanshah and Lorestan provinces [10,13]. Except for the alleles. No mutations were found in the remaining 28 a-thal western region, this mutation has been rare in other parts of carriers after direct DNA sequencing (detection rate 81.82%) Iran [8]. The Hb Adana mutation was first reported by (Table 4). From 20 different genotypes detected in this Cur€ uk€ et al. [25] in Turkey. According to a recent review study, the heterozygous and homozygous forms of the –a3.7 article by Singh et al. [26], the frequency of this mutation is deletion (aa/–a3.7 and –a3.7/–a3.7, respectively), aa/apolyA2a, low in Turkey, Iran, Iraq and China, and is high in aa/–a4.2 and aa/aacodon 59 were the most prevalent geno- Indonesia and Malaysia. Hb Adana refers to a point muta- types with frequencies of 43.51, 11.04, 6.49, 3.25 and 3.25%, tion in codon 59 of either the HBA2 (in Indonesia and respectively. The frequencies of other genotypes as well as Malaysia) or HBA1 (in Turkey, Iran and Iraq) genes [26]. In the results of post-hoc analysis are shown in Table 4. line with other studies performed in Iran, the Hb Adana mutation identified in this study was on the HBA1 gene. In the present study, the a 5nta mutation was observed with a Discussion low frequency of 1.29%. This finding is in contrast to other Identification of a-globin gene mutations in different parts studies performed in western Iran in which the frequency of of Iran in the framework of the National Thalassemia the mutation was relatively high [9,12,13]. Screening Program has shown that there are at least 42 dif- From 20 different genotypes detected in this study, the ferent mutations in the Iranian population [8]. A number of most common genotypes had a prevalence of 43.51% these mutations including –a3.7, –a4.2, ––MED I, apolyA2a, (aa/–a3.7), 11.04% (–a3.7/–a3.7), 6.49% (aa/apolyA2a), 3.25% apolyA1a, and a 5nta, have been reported throughout Iran. (aa/–a4.2) and 3.25% (aa/aacodon 59). The other identified Some other mutations, including –(a)20.5 (NG_000006.1: genotypes and their frequencies are shown in Table 4. g.15164_37864del22701), aacodon 59, Hb Constant Spring According to the Valaei et al. [8] review article, the genotype (Hb CS or HBA2: c.427T>C) aCSa (HBA2: c.427T>C) and of –a3.7/––MED (with a frequency of 30.90%) is the most acodon 19a (HBA2: c.56delG) have been reported preferen- common genotype related to Hb H disease in the Iranian tially in specific areas of Iran [8–10,12–23]. population. This genotype was detected in a 28-year-old HEMOGLOBIN 159

Table 3. Frequency of a-thalassemia mutations identified in carriers from Kurdistan Province, West Iran. Thalassemia Mutated Frequency Common name Name HGVS Nomenclature type alleles (n) (%) –a3.7 NA NG_000006.1: g.34164_73967del13804 a-thal del 109 70.32 polyA2 (AATAAA>AATGAA) NA HBA2:c. 92A>G a-thal-2 12 7.74 –a4.2 NA NA a-thal del 10 6.45 codon 59 (G>A) Hb Adana HBA1: c.179G>A a1 variant/a1-thal 7 4.52 polyA1 (AATAAA>AATAAG)NA HBA2:c. 94A>G a-thal-2 6 3.87 codon 75 (GAC>CAC) Hb Q-Iran HBA2: c.226G>C) a2 variant 3 1.94 ––MED I NA NG_0000006.1: g.24664_41064del11641 a-thal del 3 1.94 IVS-I-1 (–5 nt) NA HBA2: c.95þ2_95þ6delTGAGG a-thal-2 2 1.29 codon 19 (–G) NA HBA2: c.56delG a-thal 1 0.65 codon 94 (GAC>TAC) Hb Setif HBA2: c.283G>T a2 variant 1 0.65 codon 94 (þ21 bp duplication) Hb SKMC HBA1: c.283_300þ3dup a1 variant/a1-thal 1 0.65 Total 155 100.00 Hb: hemoglobin; HGVS: Human Genome Variation Society; NA: not applicable; polyA2: polyadenylation signal A2 site; polyA1: polyadenylation signal A1 site; MED: Mediterranean deletion; Hb SKMC: Hb Sheikh Khalifa Medical City.

Table 4. Frequency of a-thalassemia genotypes and associated hematological values in carriers from Kurdistan Province, West Iran, compared with nor- mal genotypes.

Gender Hb A2 (%) MCV (fL) MCH (pg) M F Total (%) Mean ± SD p Value Mean ± SD p Value Mean ± SD p Value Normal (n) 19 34 63 (100.00) 2.59 ± 0.36 84.12 ± 2.77 28.07 ± 0.89 aa/–a3.7 27 40 67 (43.51) 2.53 ± 0.36 1.000 77.05 ± 3.79 <0.0001 24.68 ± 1.63 <0.0001 –a3.7/–a3.7 8 9 17 (11.04) 2.44 ± 0.36 0.989 71.24 ± 2.53 <0.0001 21.85 ± 1.36 <0.0001 aa/apolyA2a 6 4 10 (6.49) 2.42 ± 0.45 0.993 78.54 ± 4.06 0.017 25.67 ± 1.82 0.010 aa/–a4.2 2 3 5 (3.25) 2.44 ± 0.15 1.000 77.58 ± 3.00 0.95 24.56 ± 0.74 0.003 aa/aacodon 59 2 3 5 (3.25) 2.88 ± 0.29 0.972 75.44 ± 4.24 0.002 24.56 ± 1.32 0.003 aa/apolyA1a 1 3 4 (2.60) 2.67 ± 0.09 1.000 71.92 ± 1.60 <0.001 22.30 ± 0.58 <0.0001 –a3.7/–a4.2 1 2 3 (1.95) 2.50 ± 0.10 1.000 71.20 ± 2.02 <0.0001 21.43 ± 0.81 <0.0001 aa/––MED 1 1 2 (1.30) 2.05 ± 0.35 0.995 69.15 ± 0.21 <0.001 21.80 ± 0.42 <0.0001 aa/a–5nta 1 1 2 (1.30) 2.90 ± 0.28 0.999 76.20 ± 1.41 0.434 24.35 ± 0.35 0.230 apolyA1a/acodon 94a 0 1 1 (0.65) 1.40 NA 77.00 NA 24.20 NA –a3.7/apolyA2a 0 1 1 (0.65) 2.70 NA 66.10 NA 19.00 NA aa/acodon 19a 1 0 1 (0.65) 3.10 NA 74.0 NA 24.00 NA aQ-Irana/aQ-Irana 1 0 1 (0.65) 1.70 NA 74.90 NA 25.40 NA –a4.2/–a4.2 1 0 1 (0.65) 2.40 NA 69.80 NA 20.50 NA aacodon 59/apolyA2a 1 0 1 (0.65) 2.20 NA 71.00 NA 22.40 NA –a3.7/––MED 1 0 1 (0.65) 1.10 NA 59.00 NA 18.00 NA –a3.7/aacodon 59 1 0 1 (0.65) 2.50 NA 69.00 NA 22.30 NA aa/aaHb SKMC 0 1 1 (0.65) 2.70 NA 70.00 NA 25.00 NA –a3.7/apolyA1a 1 0 1 (0.65) 2.40 NA 61.00 NA 19.00 NA –a3.7/aQ-Irana 0 1 1 (0.65) 1.70 NA 73.00 NA 23.70 NA Unidentified 14 14 28 (18.18) 2.63 ± 0.34 1.000 78.29 ± 4.57 <0.0001 25.34 ± 2.01 <0.0001 MCV: mean corpuscular volume; MCH: mean corpuscular hemoglobin (Hb); SD: standard deviation; polyA2: polyadenylation signal site 2; Hb SKMC: Hb Sheikh Khalifa Medical City; polyA1: polyadenylation signal site 1.

Table 5. The most common mutations in our study compared to those found in different parts of Iran as well as in the Kurdish population from Iraq. (The results are presented as percentages.) Geographic Region [Ref.] –a3.7 apolyA2a –a4.2 aacodon 59 apolyA1a ––MED I a–5ntaacodon 19a W Iran: present study (Kurdish) 70.32 7.74 6.45 4.52 3.87 1.94 1.29 0.65 W Iran: Ilam (Kurdish) [12] 66.23 3.95 8.33 1.75 2.63 0.00 10.09 0.00 W Iran: Kermanshah (Kurdish) [9] 60.90 9.90 3.10 2.50 3.70 3.20 10.60 NR W Iran: Lorestan (Laki) [13] 56.35 9.39 1.66 4.42 1.10 6.08 15.47 NR N Iraq: Dohuk (Kurdish)a [35] 67.92 NR 3.77 1.89 1.89 22.64 NR NR NE Iraq: Sulaimanihyah (Kurdish) [26] 59.60 1.00 1.90 2.90 3.80 23.10 2.90 NR All regions of Iran [8] 67.10 6.78 4.68 0.25 2.37 3.60 6.90 2.60 N Iran [14,15] 45.60; 49.53 15.19; 15.30 6.20; 8.76 NR 1.60 5.48; 6.90 3.70; 5.49 3.04 NW Iran [16] 75.20 rare 5.20 NR rare 4.62 1.70 NR C Iran [17,18] 57.80; 70.80 4.04; 10.20 4.04; 7.00 0.00; 1.20 0.57; 1.70 0.90; 8.09 5.00; 14.40 1.70; 2.30 S Iran [19,20] 68.60; 71.30 3.90; 4.20 3.70; 3.80 NR 3.03; 4.10 1.90; 2.10 6.70; 8.20 1.70; 3.90 SW Iran [21] 67.03 4.58 2.93 rare 3.60 6.30 4.20 5.01 SE Iran [22,23] 76.50; 83.80 rare 2.50; 3.60 NR rare rare 4.20; 16.50 4.06; 5.70 W Iran: West Iran; NR: not reported; N Iraq: North Iraq; NE Iraq: Northeast Iraq; N Iran: North Iran; NW Iran: Northwest Iran; C Iran: Central Iran; S Iran: South Iran; SW Iran: Southwest Iran; SE Iran: Southeast Iran. aThe authors of this study reported ‘genotype frequencies’ in [35]; however, we converted the ‘genotype frequencies’ into ‘allele frequencies’. man with Hb H disease in our study. The analysis of hema- values. Mohammadpour et al. [27] in their study, which tological factors of this patient showed Hb H ¼ 7.0%, Hb included a-thal patients from Kurdistan and Kermanshah Bart’s ¼ 0.6% and significantly decreased MCV and MCH provinces, found the genotype of –a3.7/––MED in two 160 R. ALIBAKHSHI ET AL.

Kurdish Hb H patients. However, these researchers did not further studies are needed to identify the cause of these specify whether the Hb H patients were from Kermanshah differences. Province or from Kurdistan Province. By separately comparing mean levels of hematological Acknowledgments factors between males and females in both patient and con- trol groups, we found that the Hb and RBC values in males The authors are grateful to the patients and their families for consent- were significantly higher than those in females (p < 0.0001). ing to participate in this study. We especially wish to thank all the peo- ple at the Medical Genetics Laboratory, Kermanshah University of The analysis of other factors (Hb A2, MCV and MCH) did Medical Sciences, Kermanshah, Iran for their great kindness and not show any significant differences between males and collaboration. females (p > 0.05) (Table 1). These results are in line with the results of our previous study [12] as well as the studies conducted by Galanello et al. [28], Usman et al. [29] and Disclosure statement Grau et al. [30]. As shown in Table 2, the MCV, MCH and The authors report no conflicts of interest. The authors alone are Hb levels were significantly lower and RBC was significantly responsible for the content and writing of this article. higher in a-thal carriers than those in normal subjects p < p < ( 0.0001 for MCV, MCH and RBC, and 0.001 for Funding Hb). However, in contrast to the studies of Keikhaei et al. [21] and Denic et al. [31], our findings did not show any This study was supported from the Kermanshah University of Medical a Sciences by grant number [#980659], Kermanshah, Iran. significant difference in Hb A2 between -thal carriers and normal subjects (p > 0.05). On the other hand, the genotypes of –a3.7/––MED and –a3.7/apolyA1a were related to the lowest MCV and MCH values in this study, which are consistent References – with the results of previous studies [12,16,32 35]. [1] Mahdieh N, Rabbani B. b Thalassemia in 31,734 cases with The Kurds are an ethnic group living in parts of Iran, HBB gene mutations: pathogenic and structural analysis of the Iraq, Turkey and Syria. They speak the Kurdish language common mutations; Iran as the crossroads of the Middle East. and are the fourth largest ethnicity in the eastern Blood Rev. 2016;30(6):493–508. [2] Birgens H, Ljung R. The thalassaemia syndromes. Scand J Clin Mediterranean region after Arabs, Persians and Turks [9]. – a Lab Invest. 2007;67(1):11 26. According to Table 5, although the -thal mutation spec- [3] Giardine B, Borg J, Viennas E, et al. Updates of the HbVar trum is similar in the Kurdish provinces of Iran and Iraq, database of human hemoglobin variants and thalassemia muta- the frequency of mutations in these regions may differ. For tions. Nucleic Acids Res. 2014;42(Database issue):D1063–D1069 example, acodon 19a is a rare mutation in the Kurdish popu- (http://globin. cse.psu.edu). lation. In addition, aacodon 59, a rare mutation in non- [4] Mahdavi MR, Hojjati MT, Roshan P. A review on thalassemia and related complications. J Manzandaran Univ Med Sci. 2013; Kurdish populations living in the Middle East, is a prevalent 23(103):139–149. mutation in Kurdish provinces. Despite these similarities, [5] Zhang J, Zhu B-S, He J, et al. The spectrum of a- and b-thalas- the frequency of some mutations such as ––MED I double semia mutations in Yunnan Province of Southwestern China. – gene deletion and apolyA2a point mutation in the Kurdish Hemoglobin. 2012;36(5):464 473. [6] Xu X, Zhou Y, Luo G, et al. The prevalence and spectrum of a provinces of Iran is different from that in the Iraqi Kurdish and b thalassaemia in Guangdong Province: implications for provinces. As reported by Al-Allawi et al. [35,36], the fre- the future health burden and population screening. J Clin quency of ––MED I double gene deletion in Duhok and Pathol. 2004;57(5):517–522. Sulaimaniyah provinces (the two Kurdish-speaking provinces [7] Samavat A, Modell B. Iranian national thalassaemia screening – in northern and northeastern Iraq, respectively) is approxi- programme. BMJ. 2004;329(7475):1134 1137. [8] Valaei A, Karimipoor M, Kordafshari A, et al. Molecular basis mately one quarter of the total mutational frequency of a-thalassemia in Iran. Iran Biomed J. 2018;22(1):6–14. observed in these areas. Al-Allawi et al. [35,36] have not [9] Alibakhshi R, Mehrabi M, Omidniakan L, et al. The spectrum provided a reason for the high frequency of this mutation in of a-thalassemia mutations in Kermanshah Province, West Iran. Hemoglobin. 2015;39(6):403–406. their populations. On the other hand, although the fre- et al. apolyA2a [10] Alibakhshi R, Khalegi S, Akramipour R, Molecular analy- quency of the point mutation has been reported sis of a globin genes non deletional mutations in a thalassemia with a range of 3.95–9.90% in the three Kurdish provinces patients in Kermanshah province. Razi J Med Sci. 2014;21(118): of Iran (Kermanshah, Kurdistan and Ilam), it has been rare 13–21. in Duhok and Sulaimaniyah provinces (Table 5). [11] Tan A-C, Quah TC, Low PS, et al. A rapid and reliable 7-dele- a In conclusion, the spectrum of a-thal mutations in tion multiplex polymerase chain reaction assay for -thalasse- mia. Blood. 2001;98(1):250–251. Kurdistan Province is closest to that in the western provin- [12] Moradi K, Aznab M, Azimi A, et al. a-Thalassemia mutations ces of Iran (Kurdish and Laki populations). In addition, it in Ilam Province, West Iran. Hemoglobin. 2020;44(1):1694033. was revealed that the aacodon 59 mutation is common in the Online ahead of print. et al Kurdish population. On the other hand, despite the same [13] Moradi K, Aznab M, Tahmasebi S, . The spectrum of a-thalassemia mutations in the lak population of Iran. ethnic background of Kurds in Iran and Iraq, the ––MED I Hemoglobin. 2019;43(2):107–111. apolyA2a double gene deletion and point mutation have dif- [14] Hashemi Soteh SMB, Karami H, Mousavi SS, et al. a -globin ferent distributions in these two populations. Therefore, gene mutation spectrum in patients with microcytic HEMOGLOBIN 161

hypochromic anemia from Mazandaran Province, Iran. J Clin [26] Singh SA, Sarangi S, Appiah Kubi A, et al. Hb Adana (HBA2 Lab Anal. 2020; 34(1):e23018. or HBA1: c.179G>A) and a thalassemia: genotype-phenotype [15] Eftekhari H, Tamaddoni A, Mahmoudi Nesheli H, et al. A com- correlation. Pediatr Blood Cancer. 2018;65(9):e27220. prehensive molecular investigation of a-thalassemia in an [27] Mohammadpour L, Keshavarzi F, Zeinali S, et al. a Globin gene Iranian Cohort from Different Provinces of North Iran. mutations in a case series of Kurdish Iranian a thalassemia – Hemoglobin. 2017;41(1):32 37. patients. Sci J Kurdistan Uni Med Sci. 2015;20(3):79–85. et al. [16] Derakhshan SM, Khaniani MS, Afkhami F, Molecular [28] Galanello R, Sollaino C, Paglietti E, et al. a Thalassemia carrier a study of deletional and nondeletional mutations on the -globin identification by DNA analysis in the screening for thalassemia. locus in the Azeri population of Northwestern Iran. Am J Hematol. 1998;59(4):273–278. Hemoglobin. 2016;40(5):319–322. [29] Usman K, Syed ZA, Rao AA. Reference range values of haema- [17] Zarbakhsh B, Farshadi E, Ariani Kashani A, et al. Molecular a tological parameters in healthy Pakistani adults. Pak J Physiol. study of -thalassemia mutations in Iranian potential carriers. – Sci J Iran Blood Transfus Organ. 2010;7(2):70–77. 2007;3(1):19 22. [18] Hadavi V, Taromchi AH, Malekpour M, et al. Elucidating the [30] Grau M, Cremer JM, Bloch W. Comparisons of blood parame- spectrum of a-thalassemia mutations in Iran. Haematologica. ters, red blood cell deformability and circulating nitric oxide 2007;92(7):992–993. between males and females considering hormonal contracep- [19] Faramarzi H, Rastegar M, Moghadami M, et al. a-Thalassemia tion: a longitudinal gender study. Front Psychol. 2018;9:1835. et al. mutations in two provinces of Southern Iran: Fars & Kohkeloye [31] Denic S, Agarwal MM, Al Dabbagh B, Hemoglobin A2 and Bouyer Ahmad. Hemoglobin. 2012;36(2):139–143. lowered by iron deficiency and a-thalassemia: should screening [20] Dehbozorgian J, Moghadam M, Daryanoush S, et al. recommendation for b-thalassemia change? ISRN Hematol. Distribution of a-thalassemia mutations in Iranian population. 2013;2013:1–5. Hematology. 2015;20(6):359–362. [32] Tamaddoni A, Hadavi V, Nejad NH, et al. a-Thalassemia muta- [21] Keikhaei B, Slehi-fard P, Shariati G, et al. Genetics of Iranian tion analyses in Mazandaran province, North Iran. North Iran. a -thalassemia patients: a comprehensive original study. Hemoglobin. 2009;33(2):115–123. – Biochem Genet. 2018;56(5):506 521. [33] Onay H, Aykut A, Karaca E, et al. Molecular spectrum of a-glo- a [22] Saleh-Gohari N, Khosravi-Mashizi A. Spectrum of -globin bin gene mutations in the Aegean region of Turkey: first obser- gene mutations in the Kerman Province of Iran. Hemoglobin. vation of three a-globin gene mutations in the Turkish 2010;34(5):451–460. population. Int J Hematol. 2015;102(1):1–6. [23] Miri-Moghaddam E, Nikravesh A, Gasemzadeh N, et al. [34] Bozdogan ST, Yuregir OO, Buyukkurt N, et al. a-thalassemia Spectrum of a-globin gene mutations among premarital Baluch mutations in adana province, southern Turkey: genotype-phe- couples in southeastern Iran. Int J Hematol Oncol Stem Cell Res. 2015;9(3):138–142. notype correlation. Indian J Hematol Blood Transfus. 2015; – [24] Miri M, Tabrizi Namini M, Hadipour Dehshal M, et al. 31(2):223 228. et al Thalassemia in Iran in last twenty years: the carrier rates and [35] Al-Allawi NA, Badi AI, Imanian H, . Molecular characteri- a the births trend. Iran J Blood Cancer. 2013;6(1):11–17. zation of -thalassemia in the Dohuk region of Iraq. [25] C¸ur€ uk€ MA, Dimovski AJ, Baysal E, et al. Hb Adana or Hemoglobin. 2009;33(1):37–44. a ! b a et al 259(E8)Gly Asp 2, a severely unstable 1-globin variant, [36] Al-Allawi NA, Jalal SD, Rasheed NS, . The spectrum of observed in combination with the –(a)20.5 kb a-thal-1 deletion a-thalassemia mutations in the Kurdish population of in two Turkish patients. Am J Hematol. 1993;44(4):270–275. Northeastern Iraq. Hemoglobin. 2013;37(1):56–64.