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

Abstract

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.

Keywords: Aγ-globin gene polymorphism; Fetal hemoglobin; LYAR; β-thalassemia.

Fig. 1

Sequence analysis of selected Aγ- and Gγ-globin genes. a. Right: representative sequence analysis of the Aγ-globin gene surrounding the region involved in LYAR binding site. The +25(G->A) polymorphism is arrowed. This corresponds to the already known rs368698783 polymorphism, which was not analyzed in full detail in the β-thalassemia patient population (including patients carrying β⁺ and β⁰ mutations). In the examples depicted the +25 Aγ-globin gene sequence is G/G (Fe6), G/A (Fe29) and A/A (Fe44). Left: the same genomic DNA has been sequenced at the XmnI site, found to be −/− in Fe6, −/+ in Fe29 and +/+ in Fe44 samples. b. Location of the −158 XmnI Gγ-globin and +25 Aγ-globin gene sequences within the Aγ- and Gγ-globin genes

Fig. 2

Distribution of the −158 XmnI Gγ-globin and +25 Aγ-globin gene polymorphisms within the studied β-thalassemia patients. The studied 73 patients were divided in β⁰39/β⁰39, β⁰39/β⁺IVSI-110 and β⁺IVSI-110/β⁺IVSI-110 subgroups and the −158 XmnI Gγ-globin and +25 Aγ-globin gene polymorphisms determined by direct sequencing

Fig. 3

Genetic trees of the informative β-thalassemia families studied. a The analysis of the transmission of the −158 XmnI Gγ-globin and +25 Aγ-globin gene sequences from the parents to the studied β-thalassemia patients allowed to determine the linkage to the β⁰39 mutation. b, c Most frequent XmnI Gγ-globin and +25 Aγ-globin genes linked to β⁰39 (b) and β⁺IVSI-110 (c) globin genes. The comparative analysis does not include Fe11 because not informative. N.A. = not available

Fig. 4

Relationship between the −158 XmnI Gγ-globin and +25 Aγ-globin gene polymorphisms and the level of fetal hemoglobin (HbF) in erythroid precursor cells from β⁰39/β⁰39 thalassemia patients. a, b. Representative HPLC analyses of the cytoplasms isolated from two ErPCs populations, one exhibiting low levels of HbF (a) and the other exhibiting high HbF levels (b) (arrowed in panel c). c HbF levels in ErPCs from 30 β⁰39/β⁰39 thalassemia patients (stratified on the basis of the 158 XmnI Gγ-globin and +25 Aγ-globin gene polymorphisms)