Genetic Modifiers in β-Thalassemia Intermedia: A Study on 102 Iraqi Arab Patients

Introduction

Beta-thalassemias (β-thal) are autosomal recessive inherited disorders with clinical phenotypes ranging from the severe transfusion-dependent β-thalassemia major (TM) to the asymptomatic β-thalassemia minor. Between these two extremes lies β-thalassemia intermedia (TI), a less severe condition than TM, but more severe than β-thal minor (Camaschella and Cappellini, 1995). A variety of genetic mechanisms are responsible for the latter phenotype, including inheritance of mild or silent β-thalassemia alleles, coinheritance of α-thalassemia, and inheritance of determinants that are associated with increased γ-chain production (Taher et al., 2013). Several genes have been identified that are involved in modified γ-chain production. The resulting increase of hemoglobin F (HbF) levels ameliorates the phenotype by reducing the α:β imbalance and ineffective erythropoiesis. The three major quantitative trait loci (QTLs) are the XmnI -158 C>T promoter polymorphism in the HBG2 gene, the BCL11A gene located on chromosome 2, and the HBS1L-MYB intergenic region on chromosome 6 (Thein et al., 2009; Taher et al., 2013). The BCL11A gene encodes a zinc finger transcription factor that is a critical modulator of hemoglobin switching and γ-gene silencing, and appears to do so by binding to the locus controlling region as well as the intergenic region within the β-gene cluster and not to the γ-gene promoter (Wilber et al., 2011; Xu et al., 2013). The MYB, on the other hand, is a proto-oncogene that encodes for a c-MYB transcription factor playing an essential role in erythroid differentiation and has been shown to modulate HbF levels in healthy as well as those with hemoglobinopathies (Jiang et al., 2006; Menzel et al., 2014). These two QTLs appear to be directly regulated by another key transcription factor, the Kruppell -like factor 1 (KLF 1), and they cooperate with DNA methyltransferase 1 to achieve fetal to adult hemoglobin switch (Tallack and Perkins, 2013; Roosjen et al., 2014). The relative contributions of major QTLs to HbF regulation seem to vary among different populations (Fanis et al., 2014).

There are a limited number of studies on the molecular basis of β-TI from the Middle East [including Iraq] (Al-Allawi et al., 2014) and on the contributions of single-nucleotide polymorphisms (SNPs) in the three major QTLs to its milder phenotype; thus, the current study was initiated to address, in particular, the latter issue through studying a cohort of registered TI patients at a large thalassemia center in Iraq's capital, Baghdad, a city that is widely believed to be representative to a great extent of its Arab population.

Materials and Methods

Results

Discussion

The current study identified the β-thalassemia mutation spectrum among Iraqi Arab TI patients which comprised Mediterranean, Asian-Indian, Turkish, Egyptian, Kurdish, and Saudi Arabian mutations. All these mutations have been reported by earlier reports on β-thalassemia from Iraq, except for -101 (C>T) and codon 26 (G>A) [Hb E] mutations (Al-Allawi et al., 2006, 2013, 2014; Jalal et al., 2010). The promoter sequence mutation -101 (C>T) is considered the most common among silent β-thalassemia mutations in the Mediterranean populations and usually results in a clinical phenotype of nontransfusion-dependent thalassemia if it interacts with a severe β-thalassemia mutation (Maragoudaki et al., 1999). In the current study, the patient in question was compound heterozygous for -101 (C>T) and codon 8 (-AA) with a mild phenotype presenting for the first time at the age of 28 years. Hemoglobin E, on the other hand, is one of the most common hemoglobinopathies in the Indian subcontinent and SE Asia, and the mutation, in addition to being a structural variant, also creates a cryptic splice site leading to a behavior similar to mild β-thal (Olivieri et al., 2008). Hb E/β-thal is usually associated with a TI phenotype as it is the case in the two patients in the current study who were compound heterozygous for Hb E with codon 44 and IVS-II-1, respectively.

The three most common mutations identified in our enrolled β-TI patients were IVS-II-1, IVS-I-6, and IVS-I-110. An earlier study on the spectrum of β-thal mutations in Baghdad among obligate carriers (majority being parents of patients with thal major) revealed that IVS-1-110 and IVS-II-1 mutations were the most frequent, while IVS-I-6 constituted <4% of mutations (Al-Allawi et al., 2013). The higher contribution of IVS-I-6 in the current study is expected since the latter is a mild β⁺ mutation and thus it is more likely to be associated with a TI phenotype in the homozygous and compound heterozygous state. A similar situation has also been reported among Cypriot TI patients (Verma et al., 2007). When looking at our results in the context of surrounding countries, reports from Iran documented IVS-II-1 as the most frequent mutation among their TI patients (Banan et al., 2013), while the mild IVS-1-6 was the most common among Iraqi Kurds, Lebanese, and Italian TI patients (Camaschella et al., 1995; Qatanani et al., 2000; Al-Allawi et al., 2014; Shamoon et al., 2015), and IVS-1-6 and IVS-1-110 were the most common among Cypriot TI patients (Verma et al., 2007). The mutation spectrum of our TI patients seemed to be lying between the reported spectrum of TI in Iran to the East and that reported in Mediterranean countries to the West (Table 1). This finding is consistent with the geographic location of Iraq and its role throughout its long history as a link between the East and the West.

In the majority of studies, the most important contributor to TI was the inheritance of mild β-thalassemia alleles (Camaschella et al., 1995; Qatanani et al., 2000; Verma et al., 2007; Al-Allawi et al., 2014; Shamoon et al., 2015). Our study showed that in 51% of TI patients one or both β-thal alleles were β⁺ (β⁺/β⁰, β⁺/β⁺). It further showed that 46.1% of TI patients were homozygous or compound heterozygous for the severe β⁰ alleles (β⁰/β⁰). Several genetic modulators have been implicated in ameliorating the phenotype in patients with such severe mutations. Previous studies investigated the modifying role of various SNPs in the three major QTLs by comparing patients with TM and TI. In the present study, we have chosen an alternative approach for investigating the effect of the selected SNPs by comparing TI patients with β⁰/β⁰ and β⁺/β⁺ genotypes. In our setting, the XmnI (rs7482144) polymorphism turned out to be the most significant genetic modifier followed by the BCL11A rs10189857 SNP. The rs1427407 in BCL11A, the two SNPs in HBS1L-MYB, and the α-thal status were not found to play a significant role in phenotypic presentation of our cohort (Table 2).

Our data corroborate the findings of many previous studies that among SNPs in the three major QTLs, the XmnI polymorphism has the strongest effect on modifying disease severity of β-thalassemia (Nguyen et al., 2010; Baden et al., 2011; Danjou et al., 2012; Banan et al., 2013). The contributions of SNPs in the other two QTLs have been subject to controversy. While studies on Sardinian patients with β⁰ thalassemias revealed a significant contribution of several SNPs in BCL11A and HBL1S-MYB to the TI phenotype (Galanello et al., 2009; Danjou et al., 2012), Nguyen et al. (2010) disputed the role of these two QTLs in their French TI patients, particularly in the presence of the XmnI polymorphism. The latter authors suggested that the XmnI effect on HbF production, which is potentiated by highly ineffective erythropoiesis of TI, could mask or inactivate the biological expression of the BCL11A and HBS1L-MYB genes. This explanation may also apply to our results.

One limitation of the current study is that it is a cross-sectional study and because of the fact that a good number of our patients were on regular transfusions at the time of enrolment, it was not possible to get HbF levels except in around 60% of patients. The latter would definitely limit the ability to link the SNPs studied to HbF levels. This limitation is not unique to the current study, but is shared by several previously published reports tackling the same issue (Weatherall, 2012); however, the observation of the current study linking some QTL SNPs with a milder phenotype of TI is significant and warrants further studies in Iraqi TI patients with known HbF levels.

In conclusion, the current report, which is the first on Iraqi Arab TI patients, revealed a relatively different mutation spectrum compared to an earlier study on Iraqi Kurds. Moreover, it showed that the main contributors to the less severe TI phenotype were the inheritance of mild β-thal determinants, XmnI polymorphism and, to a lesser extent, the rs10189857 SNP in the BCL11A gene. Further studies, including patients with TM as well as genome-wide association analysis, may be more informative and may uncover other QTLs in this population.