Beta-thalassemia (β-thal) is an inherited monogenic disease, caused by mutations in the β-globin gene, characterized by the partial (β+) or total (β0) reduction of β-globin chains synthesis.1,2 The defect in the β-globin gene results in ineffective erythropoiesis, which leads to microcytic hypochromic anemia, a decrease in hemoglobin A synthesis, and excess of α-globin chains, the latter being responsible for the disease pathophysiology.3 As a consequence of these alterations, individuals may be clinically classified as β-thal major, resulting from homozygosis of β0 or β+ mutations, or in rare cases due to compound heterozygous (β0/β+), requiring chronic blood transfusions, as well as iron chelation therapy due to iron overload (IO).3 β-thal intermedia patients (β+/β+ or β0/β+), in which part of the homozygotes develops milder forms of the disease, ranging from moderate to severe anemia, requiring or not periodic transfusions.1,3 Finally, β-thal carriers, which are heterozygous (β0/β or β+/β), are usually asymptomatic.1 Thus, it is a complex group of diseases with a quite variable pathophysiology.

The FOXO3 gene encodes a protein that plays a key role in numerous cellular processes by controlling the transcription of several target genes. The FOXO3 activity investigation during erythropoiesis indicated fundamental functions for this protein in several steps, such as the maintenance of the hematopoietic stem cell (HSC) pool, cell cycle progression in the early-stage erythroblasts, erythroid maturation in the late-stage erythroblasts, correct temporal expression of genes responsible for the maturation and enucleation of erythroblasts, being also important in mitochondrial clearance due to autophagy (reviewed in reference4).

The FOXO3 knockout in mice has been described as causing a significant increase in reticulocytes, decreased red blood cell count, terminal erythroid maturation and defective erythrocyte production.5,6 There are also reports indicating that loss of the transcriptional function of the FOXO3 is involved in the pathophysiology of some chronic erythroid disorders, including β-thal.7 The loss of the transcriptional function may lead to an increase in myeloproliferation, immunological deficiencies and anemia due to failure in the transcriptional regulation of the antioxidant enzyme expressions, thus causing impairment in the control of the levels of reactive oxygen species in the HSCs (reviewed in reference4). Thus, the activity of FOXOs and the cellular redox state are intrinsically linked and the FOXO3 role can be accurately summarized as being a central component of resistance to cellular stress.8

Studies, including genome-wide association studies (GWAS), have been conducted relating the single nucleotide polymorphisms (SNPs), belonging to the FOXO3 gene, to human longevity and prevention of life-limiting diseases.9,10 The literature has shown that the polymorphism rs3800231 (35-2764A > G), located in the third intron of this gene, is related to gender-independent longevity9,10 and has never been studied in hematological diseases. As demonstrated, the mechanisms that guarantee the correct transcriptional regulation of the FOXO3 gene are essential for its activity to trigger appropriate cellular responses. Thus, the presence of the aforementioned FOXO3 polymorphism could contribute to alterations in the transcriptional activity of this gene, acting as a modifier of β-thal phenotypic manifestations, according to its great relevance in the erythropoiesis process.

The molecular defects that result in thalassemia are extremely diverse, with more than 350 existent β-thal mutations reported in the IthaGenes database.2 Therefore, the genetic and physiopathological understanding of the basis for disease expression variability is still unclear. The FOXO3 transcription factor is the main active FOXO within HSCs, acting as a key mediator of erythroid terminal maturation, enucleation and regulation of the antioxidant transcriptional response program.4 Recently, it has been demonstrated that the loss of FOXO3 activity is one of the causes of dysregulated erythropoiesis in β-thal cells.7

In a study with β-thal intermedia mice, the process of ineffective erythropoiesis was demonstrated to be occasioned by the inactivation of the FOXO3 that led to oxidative damage in late erythroblasts. This downregulation of the FOXO3 is due to persistent activation of the EPOR-PI3K/AKT/mTOR pathway,18 suggesting that the activation of the FOXO3 could be beneficial in this blood disorder and thus, different FOXO3 activating agents are being tested in vitro and animal models, such as resveratrol and rapamycin. Resveratrol administration increased Hb levels, reduced the reticulocyte count and improved erythrocyte survival,19 while rapamycin enhanced γ-globin mRNA expression, HbF production,20 erythroid cell maturation and β-globin production,18 improving the anemia outcome through FOXO3 activation. Thereby, genetic polymorphism effects on the FOXO3 gene expression have the potential to improve our understanding of β-thal pathophysiology and ineffective erythropoiesis.

The FOXO3 polymorphism rs3800231 reveals an extensive ethnic and geographic variability, showing an interesting association with human longevity but with contradictory results.10,21,22 This SNP presents a global minor allele frequency (MAF) of A = 0.46 and G = 0.53, while European populations present A = 0.32 and G = 0.67. In this study, we observed that the allele frequencies in the control group (A = 0.41 and G = 0.59) approach the global MAF, which is to be expected, due to the great miscegenation that occurred in the country. However, unexpectedly, we observed that the allelic frequencies observed among the β-thalassemic individuals (A = 0.59 and G = 0.41) differ greatly from those observed in the European population. This finding stands out because southeastern Brazil received a large number of immigrants of Mediterranean origin.23 Despite its widespread worldwide distribution, thalassemia has been known for many years as "Mediterranean Anemia" because of its high prevalence in individuals whose ancestors originated from Italy, Greece, and Armenia.2 These novel findings regarding the SNP allele frequencies, never before assessed in a hematological disorder, differ from those expected, given the ethnic relationship between β-thal and Europe. Thus, this polymorphic variant is an intriguing target in the study of this hematological disease, considering the essential function of the FOXO3 gene in erythropoiesis.

The characteristic ineffective erythropoiesis found in β-thalassemic patients triggers a cascade of compensatory mechanisms, resulting in different clinical manifestations.1 In this study, we have shown that the most frequent manifestations in our homozygous study group were IO, splenomegaly, and bone complications, e.g., osteopenia and osteoporosis. Interestingly, different findings in the scientific literature have related, directly or indirectly, these three clinical manifestations with the FOXO3 differential expression, as well as with the SNP rs3800231. In addition, the present study has observed a close relationship between the IO and bone complications, since all individuals with bone complications also presented IO. The IO damages the balance of the osteoblast metabolism and induces apoptosis in vitro, which may be the main reason for bone loss and osteoporosis.24 In addition, researchers demonstrated that the IO significantly threatened the growth of osteoblastic cells and that the FOXO3 binds directly to the DUSP14 gene promoter, whose overexpression significantly suppressed the effect of the IO in the osteoblastic cells, thus indicating that the interaction of the FOXO3 and DUSP14 may be responsible for the cellular defense under stress by the IO in these cells.25

Moreover, regarding bone complications, studies have shown that the FOXO3 is the predominant isoform expressed in bone cells.26 Loss of the transcriptional activity of the FOXO3 by gene silencing leads to an increased apoptosis of osteoblastic cells and a decreased number of osteoblasts and bone formation rate and bone mass.27,28 It is also known that the loss of FOXOs (FOXO1, 3, and 4) function in mice resulted in increased apoptosis of osteoblasts and osteocytes, resulting in an osteoporotic phenotype, characterized by decreased bone mass in the cortical and spongy regions. Thus, the function of the FOXO was considered indispensable to bone mass homeostasis.26 Additionally, FOXO3 levels are elevated during osteoblast differentiation, corresponding to an increased capacity of the human mesenchymal stem cells (hMSCs) to deal with oxidative stress, which is essential for their proper differentiation to osteoblasts.29 In addition, comparisons made between aging-related characteristics with the presence of 15 SNPs of the FOXO3 in the elderly of Danish origin found a significant reduction in the risk of bone fracture for the recessive alleles of 10 of these SNPs, among them the G allele of the SNP rs3800231.30

The FOXO3 protein levels are abundantly high in the spleen.9 In a mice study, those with a FOXO3 deficiency exhibited splenomegaly, increased splenocyte numbers, and extramedullary hematopoiesis, with a higher frequency of erythrocytic and granulocytic lines. In addition, the bone marrow was hypocellular, with a decreased production of mature erythroid cells, these findings being further corroborated by histopathological analysis.17 Therefore, these observations, along with the significant differences in genotypic and allelic frequencies found between control and thalassemic groups, the high frequency of wild homozygotes (AA) among the β-thal homozygotes presenting IO, splenomegaly and bone complications, were very suggestive that the FOXO3 polymorphism could influence the chance of clinical condition development.

We concluded that the FOXO3 polymorphism investigated did not play a significant role as a modifier of the clinical manifestations observed in the β-thal homozygotes studied. However, it is worth mentioning that the small sample size in some subgroups might have influenced our findings. Thus, taking into consideration the studies summarized above, the hypothesis postulated in this study is still plausible, suggesting that the SNP rs3800231, as well as others in the FOXO3 gene, might be involved in the broad spectrum of clinical subphenotypes of the individuals with beta-thalassemia.

F.F.T. and V.S.B.: study concept and design; data analyses and interpretation and manuscript preparation.

D.G.H.S.: data analyses and interpretation and critical review of the manuscript.

J.V.O.: data statistical analysis.

C.R.B.D.: study concept and design.

The authors declare no conflicts of interest.