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

Background Increase of the expression of γ-globin gene and high production of fetal hemoglobin (HbF) in β-thalassemia patients is widely accepted as associated with a milder or even asymptomatic disease. The search for HbF-associated polymorphisms (such as the XmnI, BCL11A and MYB polymorphisms) has recently gained great attention, in order to stratify β-thalassemia patients with respect to expectancy of the first transfusion, need for annual intake of blood, response to HbF inducers (the most studied of which is hydroxyurea). Methods Aγ-globin gene sequencing was performed on 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. Results The results show that the rs368698783 polymorphism is present in β-thalassemia patients in the 5’UTR sequence (+25) of the Aγ-globin gene, known to affect the LYAR (human homologue of mouse Ly-1 antibody reactive clone) binding site 5′-GGTTAT-3′. This Aγ(+25 G->A) polymorphism is associated with the Gγ-globin-XmnI polymorphism and both are linked with the β039-globin gene, but not with the β+IVSI-110-globin gene. In agreement with the expectation that this mutation alters the LYAR binding activity, we found that the Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms are associated with high HbF in erythroid precursor cells isolated from β039/β039 thalassemia patients. Conclusions As a potential explanation of our findings, we hypothesize that in β-thalassemia the Gγ-globin-XmnI/Aγ-globin-(G->A) genotype is frequently under genetic linkage with β0-thalassemia mutations, but not with the β+-thalassemia mutation here studied (i.e. β+IVSI-110) and that this genetic combination has been selected within the population of β0-thalassemia patients, due to functional association with high HbF. Here we describe the characterization of the rs368698783 (+25 G->A) polymorphism of the Aγ-globin gene associated in β039 thalassemia patients with high HbF in erythroid precursor cells.


Background
The β-thalassemias are relevant hereditary hematological diseases caused by nearly 300 mutations of the β-globin gene, leading to low or absent production of adult βglobin and excess of α-globin content in erythroid cells, causing ineffective erythropoiesis and low or absent production of adult hemoglobin (HbA) [1][2][3][4][5]. Increase of the expression of γ-globin genes and high production of fetal hemoglobin (HbF) in β-thalassemia patients is widely accepted as associated with a milder or even asymptomatic disease [6][7][8]. In several cases, high HbF expressing β-thalassemia patients do not need transfusion regimen and, consequently, chelation therapy [6][7][8]. This well recognized finding has prompted researchers to develop efficient HbF inducers for treating β-thalassemia patients expressing low levels of HbF [9][10][11][12][13][14]. On the other hand, the search for HbF-associated polymorphisms (such as the XmnI, BCL11A and MYB polymorphisms) [15][16][17][18][19] has recently gained great attention, in order to stratify β-thalassemia patients with respect to expectancy of the first transfusion, need for annual intake of blood, response to HbF inducers (the most studied of which is hydroxyurea) [20][21][22].
In consideration of the fact that several HbF-related polymorphisms probably act in synergy, the interest in finding novel HbF-related genetic biomarkers has remained high. This field of investigation, in addition to a clear interest in diagnostics and prognostics, might bring novel therapeutic options, in the case the polymorphism(s) is (are) associated with novel therapeutic markers. This field of research has identified several direct or indirect transcriptional repressors of γ-globin gene expression such as BCL11A, KLF1, MYB, Oct-1 [16][17][18][19].
In a recent paper Ju et al. [23] identified a putative novel nuclear protein repressor of γ-globin gene transcription, LYAR (human homologue of mouse Ly-1 antibody reactive clone). The LYAR DNA-binding motif (GGTTAT) was identified by performing CASTing (cyclic amplification and selection of targets) experiments [23]. Results of EMSA (electrophoretic mobility shift assay) and ChIP (chromatin immunoprecipitation) assays confirmed that LYAR binds a DNA region corresponding to the 5′-untranslated region of the Aγ-globin gene. Ju et al. formally demonstrated that LYAR is a strong repressor of human fetal globin gene expression in both K562 cells and primary human adult erythroid progenitor cells. Interestingly, LYAR was found to directly interact also with the methyltransferase PRMT5 which triggers the histone H4 Arg3 symmetric dimethylation (H4R3me2s) mark. Altogether, these data indicate that LYAR acts as a novel transcription factor that binds the γ-globin gene, and is essential for silencing the γglobin gene [23].
The objective of this study was to investigate the presence of genetic variants in β-thalassemia patients potentially affecting the LYAR binding site and the possible association with the most common HbF-associated polymorphism, the XmnI polymorphism [18,24,25]. To this aim we focused our attention on β-thalassemic patients from the north-west Mediterranean area, in particular those carrying the β 0 39 and β + IVSI-110 thalassemia mutations, allowing to compare β 0 -and β + -genotypes. The genomic DNA from these patients was studied by full sequencing of both the Gγ-and Aγ-globin genes.
Genomic DNA extraction, polymerase chain reaction (PCR) and DNA sequencing 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.

Statistical analysis
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].

Results
Presence of the rs368698783 (G->A) Aγ-globin gene polymorphism in β-thalassemia patients 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 β 0 39/β 0 39, 33 β 0 39/β + IVSI-110, 9 β + IVSI-110/β + IVSI-110, one β 0 IVSI-1/β + IVSI-6 and one β 0 39/ 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 LYARbinding 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) β 0 39/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 βthalassemia 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.
The Aγ(+25 G->A) polymorphism is in complete linkage disequilibrium with the XmnI polymorphism 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.

Discussion
Clinical observations have shown that increased levels of fetal hemoglobin (HbF) can ameliorate the severity of the disorders of β-hemoglobin, including β-thalassemia [7]. High HbF levels are associated with transcriptional effects on the γ-globin genes, which are associated with the biological activity of several transcription repressors, including MYB, BCL11A, Oct-1, KLF1 and others [16][17][18][19][31][32][33]. A recent paper has pointed out the attention on a new putative repressor of the γ-globin gene, LYAR (human homologue of mouse Ly-1 antibody reactive clone), recognizing the Aγ-globin gene sequence 5′-GGTTAT-3′. Interestingly, several alterations within this consensus sequence for LYAR are associated with a decrease binding efficiency [23]. At present, no extensive analysis of this sequence has been reported in β-thalassemia patients; no attempts have been made to verify a possible association with the major HbF associated polymorphism, the Gγ-globin-XmnI; finally, no extensive analysis has been reported on possible linkage with β 0 -and β + -globin gene mutations.
The major results of this paper are the following: (a) a G->A mutation at the level of the rs368698783 polymorphism is present in β-thalassemia patients in the 5'UTR sequence (+25) of the Aγ-globin gene, affecting the LYAR binding site 5′-GGTTAT-3′ sequence (Fig. 1); (b) no other mutations of the LYAR binding site were found; (c) this Aγ(+25 G->A) polymorphism is in complete linkage disequilibrium with a promoter variant of the Gγ-globin-gene (the XmnI polymorphism, rs7482144, C->T); (d) the Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms are linked with the β 0 39-globin gene, but not with the β + IVSI-110-globin gene (Figs. 2 and 3). Further genetic analysis in different βthalassemia patient population is necessary (a) to extend this specific finding to other β 0 -thalassemia mutations and (b) to verify the link of Aγ(+25 G->A) and Gγglobin-XmnI (C->T) polymorphisms with the β 0 39-globin gene in a statistically more significant number of patients.

Conclusions
It is interesting to note that the Aγ(+25 G->A) rs368698783 polymorphism is expected to deeply alter the LYAR binding activity, thereby activating the Aγ-globin gene [23]. One possibility, which deserves to be verified in further studies, is that rs368698783, rather than the XmnI polymorphism, could be the physiologically (and even clinically) active variant in hemoglobinopathy patients carrying haplotypes including the XmnI(+) allele.
In respect to this point, our last conclusion is that the Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms might be associated with high HbF in erythroid precursor cells isolated from the β 0 39/β 0 39 thalassemia patients (Fig. 4), in agreement with several studies suggesting the association between XmnI polymorphism and high HbF production [18,24,34,35].
On the other hand, as a potential explanation of our findings, we hypothesize that in β-thalassemia the Gγglobin-XmnI/Aγ-globin-(G->A) genotype is frequently under genetic linkage with β 0 -thalassemia mutations, but not with the β + -thalassemia mutation here studied (i.e. β + IVSI-110). One hypothesis is the very interesting possibility that this genetic combination has been selected within the population of the β 0 -thalassemia patients, due to its functional association with high HbF. , and Telethon (contract GGP10124). This research activity has been also supported by Associazione Veneta per la Lotta alla Talassemia (AVLT), Rovigo.

Availability of data and materials
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.
Authors' contributions RG, GB and NB conceived and designed the experiments. GB performed DNA sequencing and was responsible for the design and interpretation of the relative data; CZ performed the analysis of Aγ(+25 G->A) and Gγ-globin-XmnI polymorphisms; NB and LCC performed the cultures of erythroid precursor cells and the FACS analyses; MRG was responsible of the patient's recruitment at Ferrara Hospital; FC was responsible of the patient's recruitment at Rovigo Hospital; GM performed all the PCR reactions and the relative characterization by agarose gel electrophoresis; MB extracted genomic DNA; IL and AF performed the HPLC analysis; AF performed the statistical analyses; RG, GB, NB and AF wrote the paper. All authors read and approved the final version of the manuscript.

Ethics approval and consent to participate
The collection and processing of the human biological samples for this research were carried out by the Ethics Committee of Ferrara District, number 06/2013 (approved on June 20, 2013), and by CESC (Ethics Committee of Rovigo and Verona Districts), number 36056 (approved on August 5, 2014). The study complies with the Declaration of Helsinki, the principles of Good Clinical Practice and all further applicable regulations. All samples of peripheral blood have been obtained after written documentation of informed consent from patient or legal representative. Copies of the consents have been collected for archiving by the "Day Hospital Talassemici", Divisione Pediatrica of Hospital S. Anna, Ferrara, Italy and by the Department of Transfusional Medicine -ULSS 18, Rovigo, Italy.

Consent for publication
All the subjects involved in the present study gave their consent to publish the data obtained.