Alpha‐globin gene triplication and its effect in beta‐thalassemia carrier, sickle cell trait, and healthy individual

Abstract The genotype and phenotype correlation between coinheritance of heterozygous beta‐thalassemia with the alpha‐globin triplication is unclear. In this study we have investigated and reviewed alpha triplication frequency in beta‐thalassemia carriers, sickle cell trait, and healthy individuals and its effect on hematological and phenotypical changes. In this study, 4005 beta‐thalassemia carriers, 455 sickle cell trait, and 2000 healthy individuals were included. Molecular characterization of beta and alpha‐thalassemia was performed. The frequencies of alpha‐globin triplication in beta‐thalassemia carriers, sickle cell trait, and healthy individuals were 67 (1.67%), 4 (0.88%), and 18 (0.9%), respectively. In total, the frequency of alpha‐triplications is approximately 89 (1.39%) in Khuzestan province, South of Iran population. We have compared the average hematological parameters of beta‐thalassemia carriers, sickle cell trait, and healthy individuals with and without alpha gene triplication. This mutation did not show any significant effect on the change of blood indices, neither in healthy individuals nor in sickle cell trait and beta‐thalassemia carriers. Therefore, there is no need to take more notice of anti 3.7 mutation in beta‐thalassemia carriers is opposed with some studies reported that the presence of excess alpha‐globin genes in beta‐thalassemia carriers can lead to the phenotype of beta‐thalassemia intermedia. Therefore, not every individual with triplicated alpha globin coinherited with beta‐thalassemia trait will have a significantly lower Hb than normal, and it is highly likely that none of them will need transfusion.

and 7 g/dl, transfusion-independent, clinical symptoms similar to β-thalassemia intermedia), and mild (hemoglobin levels between 9 and 12 g/dl, transfusion-independent, usually do not develop clinically significant problems) clinical forms [2]. Alpha triplication is, in fact, an increase in alpha genes that occurs on one of the chromosomes.
Alpha triplication mechanism is an unequal crossover during the recombination of α1 and α2 hemoglobin, which is the establishment of ααα anti3.7 triplicated allele. The clinical and hematological picture of beta-thalassemia heterozygotes with a triplicate α-globin gene arrangement is variable, ranging from an asymptomatic presentation to a mild to moderate thalassemia intermedia phenotype [3][4][5][6]. This study aims to investigate the frequency of alpha triplication mutations in beta-thalassemia carriers and healthy individuals, and its effects on the red blood cell indices in the south of Iran.

Ethical statement
This study was approved by the Ethics Review Committee of Pasteur Institute of Iran. Informed consent was signed and obtained from all participants following a detailed description of the purpose of the study. All methods were carried out in accordance with relevant guidelines and regulations.

Study subjects
The peripheral blood was taken from those referred to Narges Genet-

Data analysis
The results were examined using t-test, two tails and were compared on the p-value < 0.05 significance level.

Hematological parameters
The average hematological parameters of every studied genotype with alpha gene triplication in three groups were compared with the same mutations with the normal of the alpha-globin gene. Alpha gene triplication did not show any significant role in the changing of blood indices, in healthy individuals, sickle cell trait, and beta-thalassemia carriers. All beta-thalassemia heterozygotes with triplicated α-globin genes, were clinically asymptomatic, and none of them needed a blood transfusion. The data are summarized in Table 1. In this study the level of total hemoglobin in thalassemia intermedia is considered between 6 and 9 g/dl; hence, most of the beta-thalassemia carriers with 19 different mutations associated with alpha triplication were higher than 9 g/dl of hemoglobin. We have just had four beta-thalassemia carriers with alpha triplication (4 of 66, 6%) lower than 9 g/dl of hemoglobin (one female CD36/37-T/wt, 7.9 g/dl, one male IVSII-I (G-A)/ wt, 8.6 g/dl, one female CD82-83(-G)/ wt, 8.5 g/dl, and one female CD8(-AA)/ wt, 8.6 g/dl) which none of them received blood transfusion.

DISCUSSION
There are different studies which showed the frequency of the alphaglobin gene triplication in healthy individuals and thalassemia patients. Our study 4000 0.0045 0.9 The frequency of the alpha-globin gene triplication is varied, and it is dependent on the prevalence of thalassemia disease and some selection mechanisms such as endemic malaria in the studied countries [3,10].
The highest carrier frequency of the anti 3.7 mutation was observed in 4.0% in healthy individuals of Indian, and lowest carrier frequency was reported in Malay population with 0.59%. In this study from the 2000 healthy individuals, 18 had alpha triplication mutation, it can be stated that the frequency of this allele in healthy people in Khuzestan province, particularly in Ahwaz city, equals 0.9% which is lower than previous Iranian study and more than the other countries ( Table 2). The  In this study, we also investigated the average hematological parameters of every studied genotype with alpha gene triplication in comparison with the same mutations with the normal of the alpha-globin gene as well. We did not find any significant role in the changing of blood indices, only a marginal difference in beta-thalassemia carriers if any (Table 1). We have just had four beta-thalassemia carriers with alpha triplication lower than 9 g/dl of hemoglobin (one female CD36/37-T/wt, The general idea about the additional α gene is that the extra α gene aggravates the mild phenotype of the β-thal carrier to the thalassemia intermedia of mild severity, but in most studies, the genotype and phenotype correlation between coinheritance of heterozygous β-thal and the α-globin triplication remains unclear [11][12][13][14].
Previous studies showed that HbA2 and fetal hemoglobin lev- Although we expected that extra chains of α globin gene eliminated by proteolysis did not have a significant effect or very limited effect in phenotype and hematological parameters.
In this study, we also evaluated previous data reported similar genotypes of our studies from patients with triplicated α-globin genes and heterozygous β-thalassemia for Hb (g/dl), transfusion-dependent, and splenectomy. Of 37 affected persons who coinheritance of the βthalassemia heterozygotes (IVSII-I (G-A)/ wt) with triplicated alphaglobin genes, 18 (48.6%) were transfusion-dependent, four underwent splenectomy, and four had Hb less than 9 g/dl. In comparison with previously reported mutations, the results of previous Iranian studies [14,19] reported from the same centers were different significantly. If we exclude these studies [14,19], the results are changed fundamentally, where no one of individuals coinheritance of the β-thalassemia heterozygotes (IVSII-I (G-A)/ wt) with triplicated alpha-globin genes was transfusion-dependent, no one underwent splenectomy, and just one had Hb less than 9 g/dl. It is suggested that the results of these two articles [14,19] necessary to be re-evaluated by a hematologistoncologist and verification of hematology analyzers (automated blood cell counters) are required.
In addition, of 59 β-thalassemia heterozygote (CD39 (C > T)/ wt) with triplicated alpha-globin genes, 11 (18.64%) were transfusiondependent, five (8.5%) underwent splenectomy, and 13 (22.0%) had Hb less than 9 g/dl. Due to comparing reported mutations in the previous studies, it can be recommended that the mutation of codon 39 needs to be taken into consideration and can have a clinical effect (Table 4).
If we omit these studies [14,19], the results are altered. The total numbers of studies persons are reduced to 177. Of 177 affected persons, 15 (8.47%) were transfusion-dependent, seven (3.95%) underwent splenectomy, and 28 (15.82%) had Hb less than 9 g/dl where most of them coinheritance of the β-thalassemia heterozygotes (CD39 (C > T)/ wt) with triplicated alpha-globin genes. The data are summarized in Table 4.
In conclusion, the genotype of triplicated α-globin gene and heterozygosity for β-thalassemia mutation is not necessary to be considered as a cause of β-thalassemia intermedia in our locality. Therefore, it is not essential to offer a prenatal diagnosis test to families and couples carrying an α-globin gene triplication and a heterozygosity for β-thalassemia. Therefore, not every individual with triplicated alpha globin coinherited with beta-thalassemia trait will have a significantly lower Hb than normal, and it is highly likely that none of them will need transfusion. Due to previous studies, it can be recommended that the mutation of codon 39 needs to be taken into consideration.