Molecular analysis of abnormal hemoglobins in beta chain in Aegean region of Turkey and first reports of hemoglobin Andrew-Minneapolis and Hb Hinsdale from Turkey

Abstract

Objectives

The Agean is one of the regions in Turkey where thalassemias and abnormal hemoglobins (Hbs) are prevalent. Combined heterozygosity of thalassemia mutations with a variety of structural Hb variants lead to an extremely wide spectrum of clinical and hematological phenotypes which is of importance for prenatal diagnosis.

Methods

One hundred and seventeen patients and carriers diagnosed by hemoglobin electrophoresis (HPLC), at risk for abnormal hemoglobinopathies were screened for mutational analysis of the beta-globin gene. The full coding the 5′ UTR, and the 3′ UTR sequences of beta-globin gene (GenBank accession no. U01317) were amplified and sequenced.

Results

In this study, a total of 118 (12.24%) structural Hb variant alleles were identified in 1341 mutated beta-chain alleles in Medical Genetics Department of Ege University between January 2006 and November 2013.

Discussion

Here, we report the mutation spectrum of abnormal Hbs associated with the beta-globin gene in Aegean region of Turkey.

Conclusion

In the present study, the Hb Hinsdale and Hb Andrew-Minneapolis variants are demonstrated for the first time in the Turkish population.

Keywords:

• Abnormal hemoglobins
• Hb Andrew-Minneapolis
• Hb Hinsdale

Introduction

Thalassemias and abnormal hemoglobins (Hbs) are one of the important health problems in Turkey with a highest prevalence in western and southern coastal regions. The Ministry of Health and National Hemoglobinopathy Council have reported an overall 2.1% prevalence of the beta-thalassemia trait in Turkey. The latest official report revealed that prevalence of beta-thalassemia carriers is as high as 13.1% in the Antalya region while Hb S carrier status is more common in the Cukurova region with a frequency of 10.0%.¹ The prevalence of hemoglobinopathies has been given as 2.2–4.3% for the whole Aegean Region.^2–5 Although there have been a considerable number of studies on beta-thalassemia and alpha-thalassemia, few studies have ever been published concerning abnormal structural variants in Turkey.⁶

Abnormal structural Hbs are the most common hemoglobinopathies after thalassemias and more than 80 abnormal forms of Hb including alpha, beta, and gamma chain variants have been reported in the Turkish population.^7,8

In this study, the mutation spectrum of abnormal Hbs associated with the beta-globin gene in individuals from the Aegean region of Turkey was evaluated. Nine different abnormal Hbs were detected. Among these the Hb Hinsdale and Hb Andrew-Minneapolis were reported for the first time in Turkish population.

Material method

One hundred and seventeen patients and carriers diagnosed by hemoglobin electrophoresis (HPLC), at risk for abnormal hemoglobinopathies, were screened for mutational analysis of the beta-globin gene from January 2006 to November 2013. DNA was extracted from peripheral EDTA anti-coagulated whole blood using the MagNA Pure LC automated system (Roche Applied Science, Manheim, Germany) following the manufacturer's instructions. The full coding the 5′ UTR, and the 3′ UTR sequences of beta-globin gene (GenBank accession no. U01317) were amplified and sequenced. PCR products were purified using the ExoSAP-IT (GE Healthcare, Little Chalfont, UK). The PCR fragments were sequenced by the BigDye terminator V3.1 Cycle Sequencing ready reaction system (Applied Biosystems, CA, USA) according to the manufacturer's instructions. Sequence analysis was performed on an ABI Prism 3100-Avant DNA sequencer (Applied Biosystems).

Results

In this study, a total of 118 (12.24%) structural Hb variants alleles were identified in 1341 mutated beta-chain alleles in unrelated patients and carriers for abnormal Hbs. Nine different abnormal Hb gene mutations were prevalent in the population from the Aegean region of Turkey. These were Hb S, Hb D-Los Angeles, Hb G-Coushatta, Hb O-Arab, Hb E-Saskatoon, Hb Tunis, Hb Knossos, Hb Hinsdale, and Hb Andrew-Minneapolis which were found either in heterozygous, homozygous, or compound heterozygous states. In several cases, abnormal Hbs were in compound heterozygous state with beta-thalassemia. The list of these conditions is given in Table 1.

Table 1. Genotypes of abnormal hemoglobins associated with the beta-globin gene in Aegean region of Turkey

The most common mutation identified was [beta 6 (A3) Glu to Val] (Hb S) with a frequency of 49.1% in the overall mutated alleles of the beta-globin gene. This was followed by three respectively common mutations Hb D-Los Angeles (27.9%), Hb G-Coushatta (10.1%), and Hb O-Arab (8.4%). Allele frequency of the other five mutations detected in patients analyzed was 4.4% in total (Table 2).

Table 2. Allele frequencies of abnormal hemoglobins associated with the beta-globin gene in the Aegean region of Turkey

Discussion

Abnormal Hbs are a group of genetic disorders that results in abnormal structure of one of the globin chains of the Hb molecule. These variants can be found worldwide, and the distribution pattern and frequency vary region to region. A recent study from Turkey suggested that the number of variant Hbs is higher than expected.⁸ Altay⁶ reported 25 abnormal Hbs associated with the beta-globin gene in the Turkish population in 2002. In the following 9 years that number has increased by the addition of new variants named, Hb Pyrogos,⁹ Hb Volga,¹⁰ Hb Tyne,¹¹ Hb Yaizu,¹² Hb D-Ouled Rabah,¹³ Hb Tunis,¹⁴ and Hb Crete,¹⁵ Hb Ernz,¹⁶ Hb Sarrebourg,¹⁷ and Hb İzmir.¹⁸ Seven of the nine Hb variants found in this study have been previously reported in the other regions of Turkey.

Hb Andrew-Minneapolis [beta 144(HC1)Lys→Asn] is a rare, abnormal Hb with an asparagine residue substituted for a lysine at position 144 of the beta-globulin chains that result in the formation of a Hb molecule with high oxygen affinity causing secondary polycythemia.¹⁹ In the human Hb variant Hb Hinsdale [beta 139 (H17)Asn→Lys], lysine is substituted for asparagine at position beta-139 of the beta-globulin chains resulting in the formation of a Hb molecule with low oxygen affinity.²⁰ Our study is important in terms of being the first to report the Hb Hinsdale and Hb Andrew-Minneapolis variants in the Turkish population.

Hb S [Cod 6 (G-T) Glu-Val] results from a single base mutation in the beta-globin gene and is the most common abnormal Hb worldwide.²¹ Hb S occurs in three forms: homozygous Hb S which results in sickle cell disease, heterozygous Hb S trait and with another beta-chain abnormality such as sickle-hemoglobin C disease (Hb SC), sickle beta-plus-thalassemia (Hb S/β+) and sickle beta-zero-thalassemia (Hb S/β0). Hb SC is more common in individuals of African descent and milder than sickle cell disease.²² In the present study, majority of the patients with abnormal Hbs either had sickle cell anemia or sickle cell/beta-thalassemia disease. No Hb SC disease was identified. Fifty-eight cases were found to be associated with Hb S. Of these, 6 were homozygous, 40 were heterozygous carriers, and 12 were compound heterozygous for Hb S and beta-thalassemia. Depending on the severity of beta-thalassemia mutations, the clinical phenotype varies widely in compound heterozygous for Hb S and beta-thalassemia.²³ Therefore, clinical findings of our patients varied widely and they are summarized in Table 2. Epidemiological studies showed that the frequency of Hb S varies from region to region but its overall frequency throughout Turkey is 0.3%,⁶ making it the most prevalent abnormal Hb in Turkey. Therefore as expected, Hb S was the most prevalent abnormal Hb among structural hemoglobinopathies in our study.

Hb D-Los Angeles [Cod 121 (G-C) Glu-Gln] is an abnormal type of Hb with an amino acid substitution of glutamine for glutamic acid at codon 121 of the beta-globin gene is the second most common abnormal Hb after Hb S in Turkey.⁶ Hb D occurs in four forms: heterozygous Hb D trait, Hb D-thalassemia, Hb SD disease, and the rare homozygous Hb D disease which usually presents as mild hemolytic anemia and mild-to-moderate splenomegaly.²⁴ In the present study, 33 cases were found to be associated with Hb D-Los Angeles. Of these, 31 were heterozygous carriers, 1 was Hb SD disease, and 1 was compound heterozygous for Hb D-Los Angeles and IVS 1.110 mutation. Hb S/Hb D-Los Angeles disease produces clinically significant conditions like sickle cell anemia and chronic hemolytic anemia of moderate severity as in our case. Compound heterozygosity of beta-thalassemia is associated with a mild form of the disease. In this study, we identified Hb D-Los Angeles with a frequency of 27.9% in the overall abnormal structural Hbs of the beta-globin gene and it was the second most common allele as expected.

Hb G-Coushatta [Cod 22 (A-C) Glu-Ala] has been identified as a rare Hb variant in other studies of the Turkish population.⁶ We identified 12 Coushatta alleles in our study. Two of them were compound heterozygous for Hb G-Coushatta and IVS1.110 [(G-A)]. The remaining 10 were carriers for this variant. Totally Hb G-Coushatta, which resulted in 10.1% of the mutant alleles, was the third most common abnormal Hb in our region. The fourth most frequent abnormal Hb was the Hb O-Arab [Cod 122 (G-A) Glu-Lys], with an allelic frequency of 8.5%. It is also the fourth most common abnormal Hb in Turkey.⁶ We identified nine Hb O-Arab carriers and one being compound heterozygote for Hb O-Arab and IVS 2.745[C>G].

Hemoglobin E (Hb E) [Cod 26 (G-A) Glu-Lys] has been reported to be the second most common abnormal Hb after Hb S in the world.²⁵ Although the studies from other reports of Turkey states that this mutation produces the third most common abnormal Hb in Turkey, in our study we did not identify any Hb E allele.

A recent epidemiological study from our region revealed Hb S was the most common abnormal Hb with a frequency of 0.33% followed by Hb D-Los Angeles (0.13%), Hb E (0.06%), and Hb C (0.01%), respectively. This study showed that abnormal Hbs in the Aegean Region in Turkey were highly heterogeneous.² A likely explanation for differing result in our study is that the findings of this previous study were depended solely on interpretation of Hb electrophoresis test results performed for the purpose of premarital screening and no specific mutation analysis was performed. It is known that different Hbs have similar electrophoretic mobility causing misdiagnosis.

In conclusion, these findings suggest that abnormal Hbs are common in the Aegean region of Turkey. This study was also the first to be conducted on the mutation spectrum of abnormal Hb mutations in the Aegean region and includes the first reported finding of Hb Andrew-Minneapolis and Hb Hinsdale from Turkey.

Disclaimer statements

Contributors The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Funding None.

Conflicts of interest The authors certify that there is no conflict of interest with any financial organization regarding the material discussed in the manuscript.

Ethics approval This study does not require ethical approval.