Molecular characterization of β-thalassemia intermedia in the West Bank, Palestine

β-Thalassemia is one of the most common autosomal recessive disorders in the world as well as in Palestine. Up to date, more than 200 genetic mutations affecting the β-globin gene and associated with β-thalassemia have been reported [1], and of these mutations, 18 mutations have been reported among β-thalassemia patients from West Bank region of Palestine [2, 3]. β-Thalassemia intermedia (TI) is a heterogeneous group with a severity that is intermediate between the asymptomatic β-thalassemia trait (TT) and the transfusion-dependent β-thalassemia major (TM) [4]. TI and TM show overlap in their clinical symptoms and the differentiation between the two disorders shall enable TI patients to receive the proper clinical management and to avoid disease complications [5].

Despite the advances in the molecular genetics of TI, most cases of TI are still diagnosed based on clinical symptoms [6]. Genetically, TI results from mutations affecting primarily the β-globin gene production but coinheritance of other globin genes such as α- and γ-globin genes are also common. The molecular basis of TI phenotype can be attributed to three mechanisms: (i) inheritance of mild or silent β mutations, (ii) co-inheritance of α-thalassemia with β-thalassemia major, (iii) co-inheritance of determinants that increase γ-chain production [7‐9]. The contribution of each of these mechanisms to the etiology of TI varies in different ethnic groups and their study is indispensable to adjust the management protocols for TI [5, 10, 11].

Few studies have determined the genetic mutations of TM and TT in the West Bank, but none had analyzed the molecular characterization of TI in the West Bank region, Palestine. So, this study aimed to determine the spectrum of genetic mutations associated with β-thalassemia intermedia as well as to investigate the hematological characteristics and assess the clinical management of TI patients in the West Bank region, Palestine.

The study patients represent a young population with 50.9% of them are less than 18 years old. Analysis of the health data of TI patients showed that 64.7% of them had a regular blood transfusion and the median Hb was 8.3 g/dL. The median serum ferritin among the study patients was 1800 ng/mL and only 31 patients received iron chelation therapy. The decision to transfuse and the frequency of transfusion of TI patients in this study were based on their diagnosis as TI and on Hb level. However, the Thalassemia International Federation recommends that Hb level should not be an indicator for initiation of transfusion therapy for TI patients, except in patients with considerably severe anemia (Hb level < 5 g/dL) [15]. Thus, the transfusion frequency is inappropriate, at least in some patients, and should be reviewed case by case to determine the need as well as the frequency of transfusion. In addition, the high serum ferritin level in most patients suggests that their iron chelation therapy is inadequate. Previous studies showed that a better prognosis for survival was associated with a low serum ferritin level [16‐18].

A total of seven different mutations of β-globin gene were detected and comprised null mutations (β⁰) as well as mild mutations (β⁺) allowing reduced synthesis of β-globin chain. The most common allele of β-globin gene encountered was IVS-I-6 (T > C) with a frequency of 76.5%. In consistency with our results, this allele was found to be the most frequent allele among Israeli Arabs with TI (i.e., Palestinians in Israel; (57.7%)) [19], as well as among β-thalassemia patients from West Bank region (28.7%) [2] and Southern region of West Bank (48.5%) [3] (Table 5). While the IVS-I-6 (T > C) allele was the third frequent allele among Palestinian β-thalassemia patients from Gaza strip [20]. The current study included only TI patients whose genotypes are mostly contributed by mild genotypes (β+) of β-globin gene. This, in turn, may partially explain the different frequencies of the IVS-I-6 (T > C) allele among our TI patients compared to the other two studies from the West Bank [2, 3] that used a mixed sample mostly comprised of TM patients. In addition, the IVS-I-6 (T > C) allele is a mild allele and is probably less frequent among TM patients compared to TI patients. In addition, the different frequencies of β-globin gene mutations among our TI patients compared to β-thalassemia patients from Gaza strip [20] reflect different genetic background of the Palestinian tribes in both regions of Palestine. In contrast, analysis of the frequency of IVS-I-6 (T > C) allele among β-thalassemia patients in neighboring Arab countries showed that this allele was ranked as the second, third, fifth or sixth common allele, among other β-globin gene alleles, in Iraqi Arabs [21], Egyptians [22], Lebanese [23], Jordanians [24] and Syrians [25], respectively (Table 5).

Among our TI patients, 10 genotypes of β-globin gene were detected (Table 2). Of these genotypes, the most frequent single genotype was IVS-I-6 (T > C)/IVS-I-6 (T > C) and was encountered in 74.5% of TI patients. The next common genotypes were IVS-I-110/IVS-I-110 and IVS-II-1/IVS-II-1, where each was encountered in 5.88% of TI patients. The remaining seven genotypes were each detected in 2% of TI patients. These results indicate that the major contributing factor for TI among our study population is the inheritance of the mild β-globin gene allele (β+) and specially the IVS-I-6 (T > C) allele.

The second common mutation identified in this study was the Mediterranean IVS-I-110 G > A with an allele frequency of 7.8%. This allele was found to be the most frequent allele among β-thalassemia patients in Gaza strip (33.9%) [20]. In addition, earlier reports from Palestine, found that the IVS-I-110 G > A allele was the second frequent allele among β-thalassemia patients in West Bank (17.1%) [2], and the third frequent allele among β-thalassemia patients from Southern region of West Bank (9.5%) [3] (Table 5). In contrast, analysis of the frequency of IVS-I-110 G > A allele among β-thalassemia patients in neighboring Arab countries showed that this allele was ranked as the first common allele in Egyptians [22], Lebanese [23], Jordanians [24], Greece [26], and Syrians [25], while it is the third frequent allele in Iraqi Arabs [21].

The third frequent mutation was IVS-II-1 G > A with an allele frequency of 6.9%, which is considered as a severe allele (β⁰). Earlier reports from Palestine, reported this IVS II-1 G > A allele as the eighth and fifth frequent allele among β-thalassemia patients from West Bank (2.9%) [2], and from the Southern region of West Bank (4.4%) [3], respectively. However, this allele was not detected in Gaza strip [20]. It is the most common mutation in Iraqi Arabs 41.2% [21], and the second frequent allele in Israeli Arabs [19].

The next two alleles (IVS-I-1 and Codon 37) were found in our study at a lower frequency compared to previous reports from Palestine [2, 3, 20]. A previous study from Gaza Strip reported the IVS-I-1 allele as the most frequent allele and even showed a milder severity compared to the IVS-I-110 [27]. The allele (IVS-II-848 C > A) was reported earlier in West Bank [2] and at very low frequency among Jordanians [24], Syrians [25] and Iraqi Arabs [21].

The allele beta − 101 C > T was found at a low frequency in our study (2%) and this is the first report of this allele in Palestine. This allele was reported at a lower frequency in Syria [25] and Lebanon [23].

The combination of homozygous or compound heterozygous β-thalassemia with α-thalassemia decreases the excess alpha chains, and thus results in a less severe phenotype. α-Thalassemia mutations were seen in five patients only (9.8%) and in association with three β-globin gene genotypes: one β°/β⁺ plus –α^3.7/αα and heterozygous state for the ^Gγ-globin gene XmnI SNP; two β⁺/β⁺ (one with α2-IVSII-5 nt del and the other with -α^3.7/αα); one β°/β° and α2-IVSII-5 nt del and one β⁺/β^wt plus α-triplication (αα/ααα^anti3.7) (Table 3). Similar findings were reported from regional and neighboring Arab countries, where 7.8% (4/51) of Iraqi [4] and 9.8% (5/52) of Iranian TI patients [28] showed coinheritance of α-thalassemia mutations. The TI phenotype of another patient whose genotype was β⁰/β⁰ which is supposed to be severe is probably ameliorated by co-inheritance of at least two genetic modifiers, namely the existence of an α-thalassemia mutation (α^IVSI(−5nt)α/ αα) and the existence of high HbF level (46.8%). In the latter case, the high levels of HbF are probably caused by genetic modifiers influencing HbF production [29, 30]. In the other two patients whose genotype was β⁺/β⁺ and co-inherited an additional α-thalassemia mutation, their Hb level is slightly above the average Hb of the study sample and thus the α-thalassemia mutation has probably slightly influenced their phenotype. Similar findings were reported in two of four members of a Jordanian family whose genotype was β⁺/β⁺ and –α^3.7/αα, they were reported as non-transfusion dependent [31]. One patient was heterozygous for the β-globin gene (β⁺/β^wt), but he co-inherited the α-triplication (αα/ααα^anti3.7), which explains his TI phenotype. Since such alpha triplication increases the globin chain imbalance among β- thalassemia heterozygote and shifts the disease severity from TT toward the TI phenotype [8, 32, 33]. A similar finding in Israel was reported for a TT patient, who showed severe anemia and splenomegaly, although his genotype was β⁺/β^wt but he co-inherited the α-triplication αα/ααα^anti3.7 [34].

Analysis of the Gγ-globin gene XmnI SNP showed that three patients were homozygous and one patient was heterozygous for this SNP, three of them have the β⁰/β⁰ genotype and one has the β⁰/β⁺ (in addition to –α^3.7/ αα) and the effect of this SNP was clearly illustrated by the high levels of HbF in these patients. The role of Gγ-globin gene XmnI SNP in increasing HbF levels and moderation of thalassemia phenotype is widely known and reported in many earlier studies [4, 14, 35]. The XmnI polymorphism is one of three major HbF quantitative trait loci (QTLs) responsible for HbF variation, and it leads to a less severe phenotype by increasing γ-chain production, which helps to neutralize unbounded α-chains [15, 36]. However, in the present study the ^Gγ-globin gene XmnI polymorphism was not detected in any patient with the genotype β⁺/β⁺ or the β⁺/β^wt. The latter findings are consistent with earlier reports that the XmnI SNP is the commonest ameliorating factor in cases with β° mutations but not β⁺ [4, 37]. However, in our study the percentage of TI patients having the XmnI SNP was low, in consistency with an earlier report from Brazil (9.7%) [38] but inconsistent to previous reports from Iraq (56.8%) [4], Iran (51.9%), [28] Pakistan (23%) [14] and China (26.5%) [39].

It is interesting that the three patients with the β⁰/β⁰ (IVS II-1/ IVS II-1) and homozygous for XmnI SNP not only have a high level of HbF but also have Hb levels (mean ± SD = 8.7 ± 0.86) above the average levels of the study patients. A recent study on a cohort of Palestinian β-thalassemia patients from Gaza strip reported a milder phenotype for the homozygous IVS-I-1 allele and an association between this allele and the high expression level of HbF [27]. Ghoti el al. [27] found XmnI SNP in only 7 out of 15 patients’ homozygotes for the IVS-I-1 allele, thus other genetic factors probably contribute to the high levels of HbF in association with null allele, which yet has to be determined. In addition, other studies have also reported an amelioration of the β⁰ allele by inheritance of the XmnI SNP, including reports from Egypt, that showed that patients with the IVS-II-1 allele have relatively higher XmnI SNP frequency (50%) than IVS-I-6 and IVS-I-110 [40], and a report from Southern Iran showed that 87.5% of patients with the IVS-II-1 allele were homozygous for the XmnI SNP [41]. Thus, the low incidence of β° mutations among our study subjects may partially explain the low frequency of XmnI SNP detected in the present study.

The inheritance of the β ⁺ allele has been shown as the primary genetic modifier in TI in different reports [19, 26, 42] including the present study. The co-inheritance of α-thalassemia has been shown to be an important ameliorating factor in TI cases with either β⁰ allele [43] or β⁺ allele [42]. In addition, QTL affecting HbF expression (HBG2, BCL11A and HBS1L-MYB) have been shown to play an important role in ameliorating the thalassemic phenotype [21, 42‐45]. In each of the three latter QTL, different SNPs have been studied and revealed different incidences among TI patients representing different ethnic groups [21, 42‐45]. In a homogenous group of Sardinian β⁰-thalassemic patients, Galenello et al. [43] reported that all this group (50 cases of TI and 75 cases of TM) was negative for the XmnI SNP (HBG2) and their mild thalassemic phenotype could be mostly attributed to the co-inheritance of BCL11A, HBS1L-MYB SNPs as well as to α-thalassemia mutations. However, in most studies heterogenous groups of TI patients (associated with β⁺ and β⁰ alleles) were analyzed and the role of the three QTL were found to be variable [42‐45] confirming the heterogeneity of TI and reflecting the different genetic background of each ethnic group studied.

In the present study, the frequency of β⁺ / β⁺ genotype is 84.3% (Table 2), and that of β⁰/ β⁰ genotype is 7.8% (Table 2). These findings indicate that the mild phenotype of TI in the majority of cases is due to inheritance of the β⁺ allele. While the co-inheritance of XmnI SNP and/or α-thalassemia could explain the mild phenotype of 5 TI cases from 7 cases associated with β⁰ allele. However, the presence of the other QTL (namely BCL11A and HBS1L-MYB) has not been determined yet.

Further studies with a larger number of patients and analysis of other QTL (BCL11A and HBS1L-MYB) may help reveal additional genetic modifiers contributing to the variations in HbF and milder phenotype associated with the β⁰ allele.