An Aγ-globin G->A gene polymorphism associated with β⁰39 thalassemia globin gene and high fetal hemoglobin production

A total of 75 β-thalassemia patients were recruited for this study, including 31 β⁰39/β⁰39, 33 β⁰39/β⁺IVSI-110, 9 β⁺IVSI-110/β⁺IVSI-110, one β⁰IVSI-1/β⁺IVSI-6 and one β⁰39/β⁺IVSI-6 patient. The β-thalassemia patients have been recruited at Ferrara Hospital and Rovigo Hospital. The Declaration of Helsinki was followed for the collection of blood samples from β-thalassemia patients; furthermore, specific approvals by the Ethical Committees of Ferrara Hospital and Rovigo Hospital were obtained. All the β-thalassemia patients duly signed the informed consent form before blood sampling.

The genomic DNA from β-thalassemia patients was extracted from 500 μL of whole blood using the QIAamp® DNA Blood Mini Kit (Qiagen, Hilden, Germany) as described in Bianchi et al. [15]. PCR amplification of β-, Aγ- or Gγ-globin genes and DNA sequencing methods used in this study have been previously described by Bianchi et al. [15]. The nucleotide sequences of the PCR primers are reported in Table 1. BMR Genomics (Padua, Italy) performed gene sequencing.

The two-phase liquid culture procedure was employed as previously described [26, 27]. The erythroid differentiation status of ErPCs was verified analyzing transferrin receptor (TrfR) and glycophorin A (GYPA) expression by FACS (fluorescence-activated cell sorting) using the BD FACScan™ system (Becton, Dickinson & Company, Franklin Lakes, NJ, USA) and anti-human CD71 (TrfR) FITC-conjugated antibody (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) and anti-human CD235a (GYPA) antibody PE-conjugated (Miltenyi Biotec GmbH) as described elsewhere [28, 29]. Production of hemoglobins was assessed by high performance liquid chromatography (HPLC) as described elsewhere [11, 28].

The results reported in this paper are usually presented as average ± SD. The one-way ANOVA (ANalyses Of VAriance between groups) software was used for compare statistical differences between groups. The paired t test of the GraphPad Prism Software was used to obtain the p values. Differences were considered statistically significant when p < 0.05 (*) and highly significant when p < 0.01 (**) [28, 29].

In order to verify whether mutations affecting the LYAR-binding site of the Aγ-globin gene are present within our β-thalassemia patient population, sequencing of the Aγ-globin genes was performed using genomic DNA isolated from a total of 75 β-thalassemia patients, including 31 β⁰39/β⁰39, 33 β⁰39/β⁺IVSI-110, 9 β⁺IVSI-110/β⁺IVSI-110, one β⁰IVSI-1/β⁺IVSI-6 and one β⁰39/β⁺IVSI-6 patient. Examples of the sequencing results obtained are shown in Fig. 1a, which indicates that one (G->A) rs368698783 polymorphism was found in position +25 of the Aγ-globin gene, modifying the LYAR-binding sequence from 5′-GGTTAT-3′ to 5′-GATTAT-3′. For this reason, we called this mutation rs368698783 Aγ(+25 G->A) (see its location in Fig. 1b). In the examples reported in Fig. 1a, the representative homozygous (G/G), heterozygous (G/A) and homozygous mutated (A/A) genomic sequences are shown. In addition, as indicated in the representative examples shown in Fig. 1a, the G/G genotype is linked to the XmnI(−/−) haplotype; in contrast the G/A and A/A Aγ(+25) genotypes are linked to XmnI(−/+) and XmnI(+/+) haplotypes, respectively. Figure 1b shows the location of the mutation within the 5’UTR sequence of the Aγ-globin gene and the nucleotide change concerning the 5′-GGTTAT-3′ LYAR binding site proposed by Ju et al. Notably, no other nucleotide variations affecting the LYAR-binding sequence were found in these 75 patients. Moreover, no other mutations were found in the 607 bp and 613 bp sequenced regions of the Aγ-globin and Gγ-globin genes, respectively, with the exception of a 4 bp deletion residing in the promoter region of the Aγ-globin gene (HBG1: g.-225_-222delAGCA) [30], found in three XmnI(−/−), Aγ(+25 G/G) β⁰39/β⁺IVSI-110 patients. In 16/75 patients (21%) this Aγ(+25 G->A) polymorphism was found in the heterozygous (G/A) state, while the Aγ(+25) homozygous (A/A) state was found only in four patients. While we cannot exclude the presence of other mutations in the Aγ-globin genes of sub populations of β-thalassemia patients, we can conclude that the Aγ(+25 G->A) concerning the rs368698783 polymorphism is the most frequent mutation affecting this Aγ-globin gene region within our population, well representative of the Mediterranean area.

Table 2 shows that in all the patients analyzed the Aγ(+25 G->A) rs368698783 polymorphism is strictly linked to the Gγ-XmnI polymorphism. In fact all the 55 Gγ-XmnI(−/−) patients were found to be Aγ(+25 G/G). In addition, all the 16 Gγ-XmnI(−/+) patients were found to be Aγ(+25 G/A) and the four Gγ-XmnI(+/+) patients were found to be Aγ(+25 A/A). This very interesting distribution allows to hypothesize that the XmnI polymorphism, when present in this β-thalassemia patient population, is physically linked to the Aγ(+25 G->A) polymorphism.

The results shown in Fig. 2 show that only one of the 9 β⁺IVSI-110/β⁺IVSI-110 patients was found to be Gγ-XmnI(−/+) and Aγ(+25 G/A) (11.1%). The other patients were Gγ-XmnI(−/−) and Aγ(+25 G/G) (88.9%). No patients exhibited a Gγ-XmnI(+/+) and Aγ(+25 A/A) combination. By sharp contrast, in the β⁰39/β⁺IVSI-110 cohort, the Gγ-XmnI(−/+) and Aγ(+25 G/A) patients were found to be 6 (18.2%), 27 being Gγ-XmnI(−/−) and Aγ(+25 G/G) (81.8%). Also in this case no patients exhibited a Gγ-XmnI(+/+) and Aγ(+25 A/A) combination. Finally, in the β⁰39/β⁰39 cohort the Gγ-XmnI(−/+) and Aγ(+25 G/A) patients were found to be 7 (22.6%), 20 being Gγ-XmnI(−/−) and Aγ(+25 G/G) (64.5%). Unlike the β⁺IVSI-110/β⁺IVSI-110 and the β⁰39/β⁺IVSI-110 cohorts, four β⁰39/β⁰39 patients exhibited a Gγ-XmnI(+/+) and Aγ(+25 A/A) combination (12.9%). These data, when analyzed together with the data shown in Table 2, support the hypothesis that the Gγ-XmnI and Aγ(+25 G->A) polymorphisms might be preferentially linked with the β⁰39 thalassemia mutation. To verify this hypothesis the family trees of all the available families with β-thalassemia patients carrying at least one β⁰39-globin gene were analyzed with respect to β-globin genes and Gγ-XmnI and Aγ(+25 G->A) polymorphisms.

Figure 3a shows the family trees of a β⁰39/β⁺IVSI-6, two β⁰39/β⁰39 (out of three present in our cohort) and 5 β⁰39/β⁺IVSI-110 patients (out of the 6 available). The results obtained firmly demonstrate that in the β⁰39/β⁺IVSI-6 family (Fe89) the Gγ-XmnI and Aγ(+25 G->A) polymorphisms are linked to the β⁰39 gene (Fig. 3a, upper left side of the panel). In the two families with β⁰39/β⁰39 patients (Fig. 3a, upper middle side of the panel), one β⁰39-globin gene (Fe29) and both β⁰39-globin genes (Fe77) are associated with the Gγ-XmnI and Aγ(+25 G->A) polymorphisms. More importantly, when families with β⁰39/β⁺IVSI-110 were considered, one (Fe11) was not informative (the genome of the mother was not available), while in patients Fe31, Fe34, Fe88 and Fe91 the β⁰39 genotype was structurally linked to the Gγ-XmnI and Aγ(+25 G->A) polymorphisms combination. These polymorphisms were not associated with the β⁺IVSI-110 gene, as summarized in Fig. 3b. These data clearly indicate that in the population analyzed the Gγ-XmnI and Aγ(+25 G->A) polymorphisms cosegregate with the β⁰39-globin gene mutation in the majority of the families of compound-heterozygous β⁰-thalassemia patients.

In order to verify possible relationships between the Gγ-XmnI and Aγ(+25 G->A) polymorphisms configuration, 30 available β⁰39/β⁰39 patients were recruited, peripheral blood isolated, erythroid precursor cells (ErPCs) selected and erythropoietin (EPO)-induced as elsewhere reported [27, 28]. After 7 days of EPO treatment, the erythroid differentiation was confirmed by benzidine-staining (looking at hemoglobin production) and FACS analysis of transferrin receptor and glycophorin A expression, as reported elsewhere [28] (data not shown). After demonstration that more than 80% of the cells were benzidine, TrfR and GYPA positive, HPLC analysis was performed to quantify fetal hemoglobin (HbF) production. Figure 4 (panels a and b) shows the representative HPLC profile of two ErPC lysates (arrowed in Fig. 4c), displaying a low (Fig. 4a) and high (Fig. 4b) HbF relative production. All the data obtained are shown in Fig. 4c, in which the ErPCs are stratified with respect to the Gγ-XmnI and Aγ(+25 G->A) polymorphisms configuration. As clearly evident, a significant correlation can be observed between the Gγ-XmnI and Aγ(+25 G->A) polymorphisms configuration and elevated production of HbF. In fact the average HbF production by ErPCs from β⁰39/β⁰39, Gγ-XmnI(+/+) and Aγ(+25 A/A) patients was 66.1 ± 14.1%, a value significantly higher than those found in ErPCs from Gγ-XmnI(−/−) and Aγ(+25 G/G) or Gγ-XmnI(−/+) and Aγ(+25 G/A) patients. This finding suggests that Gγ-XmnI and Aγ(+25 G->A) polymorphisms should be present in both alleles for maximal potentiation of HbF production, even if not “per se” sufficient and probably acting with other “HbF modifiers”, since all the Gγ-XmnI(+/+) and Aγ(+25 A/A) patients did not carry any detectable alteration of the α-globin gene asset. In any case, the cellular system here described might help to dissect genetic control of fetal-hemoglobin persistence and disease phenotypes, especially considering the possibility to access cellular biobanks from β-thalassemia patients stratified with respect to genotype, Gγ-XmnI and Aγ(+25) polymorphisms, enabling cryopreservation and usage of the cryopreserved and thawed cells for molecular biology studies [31].