Distribution of alpha-thalassemia mutations in Iranian population

Abstract

Background

Alpha-thalassemia as one of the most common monogenetic disorders is widely spread over the Mediterranean, Southeast Asian, and Middle Eastern populations, including Iran. Although beta-thalassemia is much more common than alpha-thalassemia, alpha-thalassemia is still one of the main health problems in Iran with different mutation frequencies in various ethnic groups. So the evaluation of alpha-thalassemia mutations could be helpful to detect carriers as well as prevention strategy in Iranian population.

Objectives

The aim of this study was to investigate the spectrum and frequencies of alpha-globin mutations in different ethnic groups of southern Iran.

Materials and methods

Common alpha-globin mutations were evaluated in 4010 Iranian population using a reverse dot blot for all point mutations and gap-polymerase chain reaction.

Results

Out of all individuals, 3993 were distinguished as carriers of alpha-thalassemia mutations. Thirteen types of alpha-thalassemia mutations were discovered. Allele of α^3.7 mutation was the most prevalent (43.84%) followed by the α^IVS1/−5NT allele with the prevalence of 4.91%. The less frequent alleles were Hb ICARIA and α^codon16 with the prevalence of 0.04 and 0.01%, respectively.

Conclusion

Our findings are essential for carrier screening, genetic counseling, and prenatal diagnosis in order to decrease the prevalence of α-thalassemia in Iran which is one of the goals of the national screening program.

Keywords:

• Alpha-globin
• Genetic screening
• Thalassemia

Introduction

Hemoglobin A is a polypeptide that consists of two alpha-globin chains and two non-alpha-globin chains, also four oxygen-binding heme lies in this polypeptide. Any structural defect leads to failure of binding oxygen to hemoglobin called hemoglobinopathy.¹ Alpha-thalassemia (alpha-thalassemia) is one of the most common monogenic disorders that is known by decrease or absence of alpha-globin gene expression. Most of the alpha-thalassemia mutations are a deletion of one (α+) or both alpha-globin genes (α0). Point mutations in alpha-globin genes that are called non-deletion alpha-thalassemia (αα^T or α^Tα) are also seen with less frequency.^2,3

Alpha-globin gene cluster is located on 16p13.3. There are seven different genes including HBA1 and HBA2 in this cluster. HBA1 and HBA2 are identical in most of their sequences and few differences have been shown in 5′-untranslated region (UTR) and introns but they are significantly different in 3′-UTR; however, both of these genes encode an identical peptide with 141 amino acids. Expression of HBA2 is significantly higher than HBA1, so failure of HBA2 is more effective. Mutation rate in HBA2 is, also, higher than that of HBA1. Today, 60 mutations have been reported in HBA2 associated with alpha-thalassemia compared with 34 mutations in HBA1.^4–6

Alpha-thalassemia is more prevalent in countries in which malaria is endemic.⁷ Iran as one of these countries has a high prevalence of alpha-thalassemia.⁸ Because of the variety of ethnic groups in Iran, determination of frequency of mutations in alpha-globin genes is required. Therefore, we decided to evaluate the frequency of alpha-globin genes mutations in each ethnic group of southern Iran.

Materials and methods

This is a cohort study which was conducted in Shiraz University of Medical Sciences from 2000 to 2013 in southern Iran. Four thousand and ten individuals with microcytic hypochromic anemia from seven different ethnic groups (Fars, Bakhtiari, Balouch, Arab, Kord, Turk, and Lor) participated in this study. They were referred for premarital screening to the thalassemia research center of Shiraz University of Medical Sciences, in southern Iran.

All subjects had a mean cell volume (MCV) of <80 fL, mean cell Hb (MCH) of <27 pg, and normal electrophoretic pattern and serum ferritin level. Analysis of red blood cell indices and Hb analysis was performed by the standard methods. Hb variants of samples were determined by high-performance liquid chromatography (Bio-Rad Laboratories, Hercules, CA, USA). DNA was extracted from peripheral blood cells using the QIAamp DNA Blood Mini Kit from QIAGEN. Alpha-thalassemia genotypes were identified by molecular methods including the reverse dot blot (reverse alpha-globin strip assay, ViennaLab, Vienna, Austria) for surveying point mutations, and gap-PCR for other mutations (the primer pair sequences used for PCR are shown in Table 1).

Table 1. The primer pair sets used for gap PCR

This study was approved by the medical ethics committee of Shiraz University and a consent form was taken from all participants.

The ANOVA test was used to determine correlation between genotypes and Hb levels. A Chi-square test was used to determine the relationship between genotype groups and different ethnicity. P-value <0.05 was considered significant.

Results

Out of the 4010 participants, 3993 subjects were carriers of alpha-thalassemia mutations confirmed by molecular methods. Table 2 provides the mean of different hematological parameters in these subjects. The remaining participants (0.42%) showed no mutations in alpha-thalassemia genes.

Table 2. The mean of different hematological parameters in these subjects

Our results showed 13 different mutations among 3993 samples. The –α^3.7 deletion was the most frequent mutated allele (43.84%), followed by α^IVS1/−5nt mutation (4.91%). Hb Constant Spring (Hb CS, codon 142, TAA>CAA(alpha2)) was the third prevalent mutated allele found in our subjects (2.78%). The allele frequencies of mutations are presented in Table 3 with details.

Table 3. Allele frequencies of 13 mutations found in 3993 individuals

We also found hetrozygotes of beta-thalassemia mutation compounds with alpha-thalassemia mutation in 352 patients. Our results showed –α^3.7, α^−5nt, Hb CS, α^codon19, α^4.2, Poly A2, and ααα^anti3.7could be accompanied with beta-thalassemia mutations (Table 3). We, additionally, calculated Hb, MCV, and MCH for each genotype which are listed in Table 4. Because of the small number of samples with particular genotypes, we pooled genotypes which are believed to have the same effect at the hematological level.⁹ The pooled genotypes which we believe to cause the same phenotype are categorized in eight groups: (1) carriers of a single non-deletion alpha-thal mutation, (2) genotypes with a single allele of α+-thal, (3) homozygotes or compound heterozygotes for two alpha+-thalassemia deletion allele, (4) carriers of α0-thalassemia, (5) single homozygotes for a non-deletional alpha-thalassemia, (6) carriers of a poly A1 mutation, (7) compound heterozygotes for a deletion and a non-deletion α+-thalassemia allele, and (8) compounds of an alpha-thalassemia mutation and beta-thalassemia mutation.

Table 4. Hematological parameters in different genotype pools

Analyses of the correlation between pooled genotypes and Hb levels revealed that different genotypes cause different amounts of Hb levels (P = 0.00). Group 7 was completely different from the other groups by considering the Hb levels (P = 0.00), but other groups showed some similarities with each other (Table 5). We also considered the distribution of different mutation groups in different ethnicity groups (Table 6). According to the statistical analysis, there is no significant relationship between group of mutations and ethnicity in Iranian population (P > 0.05).

Table 5. ANOVA post hoc test significance of correlation between different pooled genotypes and Hb levels

Table 6. Distribution of genotype groups in different ethnicity

Discussion

Iranian population is a mixture of different ethnic groups, so clarifying alpha-thal mutations in different regions is necessary for better understanding and prevention of alpha-thalassemia. There have been several studies on this topic in different parts of Iran.^8–11 It has been established that the most common mutation in Iranian population is −α^3.7.^9,10,12–14 In this study, alpha-thalassemia mutations in southern [AQ1] Iran were evaluated and 13 different mutations were found in this area. Like other regions, α^3.7 was defined as the most common allele in our study. This is also comparable with allele distribution studies in neighboring countries.¹⁵ The second and the third mutated alleles in our study were α^IVS1/−5NT and Constant Spring, respectively. This finding is in disagreement with a previous study on the south province of Iran which announced α^codon19 as the second common mutation.¹⁶ Neishabury et al.³ announced α^4.2 as the second most common mutation in Iranian population, but the present results reveal this mutation as having the sixth frequency. To the best of our knowledge, no study regarding the prevalence of genotypes based on ethnicity in Iranian population has been carried out.

The remarkable prevalence of combined alpha and beta-globin mutations in Iran provides a considerable variety of phenotypes due to the coincidence of alpha- and beta-thal mutations.¹⁷ We discovered 352 individuals who had both mutations together at the same time. As it is shown in Table 4, when two genes mutations occur at the same time, Group 8, the amount of HBA2 is increased. This result is compatible with the fact that absence or reduction in beta-globin gene expression increases the amount of delta-globin.¹⁸

The pooled genotype correlations with Hb levels demonstrate that the Hb level in Group 7 is significantly less than other groups and has no correlation with them (Tables 4 and 5). This is expected because Group 7 consists of two mutations in a gene at the same time, so it has to have a higher effect on phenotype.

Conclusion

In Iran, with its high prevalence of alpha-thalassemia carriers, the results of our study could be valuable in the diagnosis of thalassemia carriers for running premarital screening programs. Since 1991, a national premarital screening program has been initiated for the prevention of thalassemia; however, it is still one of the main health problems in Iran. Finding geographical distribution of alpha- and beta-thalassemia allelic frequencies is crucial along with beta-thalassemia carriers in order to have a better diagnosis and management.

Acknowledgements

This study owes a debt of gratitude to the patients and their families for their kind cooperation. Also, We thank Sheryl Nikpoor for editing and improving the use of English in the manuscript.

Disclaimer statements

Contributors JD, SD, and JI contributed to data collection. MM contributed to drafting the manuscript and doing laboratory analysis. SH performed the statistical analysis. AA and AS have done laboratory analysis. MK contributed to the study design, concept, and editing the manuscript.

Funding None.

Conflicts of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of this article.

Ethics approval This study was approved by medical ethics committee of Shiraz University of Medical Sciences.