Sickle cell anemia – Nitric oxide related genetic modifiers of hematological and biochemical parameters

1 Introduction

Sickle cell anemia (SCA) is an autosomal recessive hereditary anemia characterized by the presence of hemoglobin S (Hb S) and is one of the most common human genetic disorders world-wide [20], This disease is caused by a single point mutation in the beta-globin gene, located on chromosome 11, with a corresponding amino acid substitution at the sixth position of the beta-globin chain [10]. Hemolytic anemia and vaso-occlusion are the major features of this disease. Red blood cell rheology and adhesiveness play a key role in the occurrence of vaso-occlusive like events in sickle cell anemia, with stiffness of sickle erythrocytes and topography of cell membrane affecting adhesion to the endothelium [2].

SCA patients present clinical and hematologic variability that cannot be only explained by the single mutation in the beta-globin gene. Others genetic modifiers and environmental effects are important in the clinical phenotype [18]. Nitric oxide (NO), a gas with potent vasodilation, antiadhesive and antioxidative properties, represents a critical molecule for proper endothelial function and maintenance of a patent vascular lumen [7, 17]. A study shows that systemic vascular resistance in SCA is positively correlated with red blood cell deformability, and negatively with nitric oxide metabolites level in SCA children [12]. The endothelial NO synthase (eNOS), an enzyme that generates NO, is expressed in the endothelium, encoded by a gene (eNOS) located on chromosome 7 [19]. A recent study found that red blood cell nitric oxide synthase modulates red blood cell deformability in SCA [14]. SCA patients present endothelial dysfunction due to impaired bioavailability of endogenous nitric oxide caused by scavenging of NO by cell-free plasma hemoglobin and consumption of L-arginine amino acid by cell-free arginase enzyme released from hemolyzed red blood cell [24]. Several polymorphic variants have been described in the eNOS gene [6]. The present study aims to explore the association between hematological and biochemical parameters with genetic variants from eNOS gene, in pediatric SCA patients.

2 Patients and methods

3 Results

Polymorphisms analyzed for the eNOS gene are: the variable number of tandem repeats in intron 4, eNOS 4a/b; a SNP in the promoter region, rs2070744; and another in exon 7, rs1799983. Results from this study show a significant statistical association between some parameters and genetic variants at eNOS gene. An increased reticulocyte count and high LDH levels were associated with both the rs2070744_TT (Table 1 and Fig. 1) and the rs1799983_GG genotypes (Table 2 and Fig. 2) and high levels of neutrophils were associated with the eNOS4a allele at intron 4 VNTR (Table 3 and Fig. 3). Rs2070744_CC was not found in the population. There is no significant difference between the other parameters and genetic variants/ haplotypes at eNOS gene (Table 1, 2, 3 and 4).

4 Discussion

Sickle cell anemia is one of the most common human genetic disorders world-wide and represents a major public health problem. Considering the unusual clinical heterogeneity, it is imperative that the significance of polymorphisms with clinical relevance be delineated.

In this study we analyzed three well-characterized polymorphisms: intron 4 (27 bp VNTR), rs2070744 (–786TC) and rs1799983 (Glu298Asp). Results showed a significant statistical association between commonly measured hemolysis biomarkers and two of the eNOS polymorphisms, an increased reticulocyte count and high LDH levels were associated with both, the rs2070744_TT (Table 1 and Fig. 1) and the rs1799983_GG (Table 2 and Fig. 2) genotypes at eNOS gene. Concerning rs2070744_TT, results are not unexpected since the corresponding T allele in the promoter region was already reported associated with lower levels of gene expression [22]. Our results confirm once more a functional effect of this polymorphism on eNOS gene. Regarding the other polymorphism (rs1799983), its effect is still controversial since the aspartate substitution at 298 does not seem to have a major effect in modulating eNOS activity in vivo, however an association of the T allele with pre-eclampsia [11] and ischemic heart disease [5] was reported strengthening the importance of this polymorphism. Studies with intron 4 VNTR polymorphism revealed an association between the 4a allele with coronary heart disease [16] and renal disease [4, 23]. Another study found that eNOS intron 4 VNTR gene polymorphism is related to endothelial dysfunction and vasculopathy in sickle cell disease [19]. As modulators of the inflammatory response, neutrophils are in high number in SCA patients. In this study higher number of neutrophils were associated with the eNOS4a allele (Table 3 and Fig. 3). NO exerts regulatory effects on neutrophil, with low concentrations being stimulatory [1, 8]. A study showed that levels of expression of two of the alleles (eNOS4a/b) dependents on the promoter SNP rs2070744 [22]. Thus, association of the a allele with higher number of neutrophils may be related with eNOS haplotypes, instead of eNOS genotypes alone. In fact, that has been suggested in other studies [13, 22]. However we did not observe statistically significant differences once analyzing haplotypes, probably because of the low sample size.

Our results reinforce the importance of NO bioactivity in SCA. We presume that NO, and its precursors might be used as therapy to improve the quality of life of SCA patients.