Defective nitric oxide metabolism in sickle cell disease

S ickle cell anemia is often called “the first molecular disease,” because it was the first human inherited disease shown to be linked to a single amino acid mutation caused by a single nucleotide substitution in a known gene. This single amino acid mutation causes sickle hemoglobin to polymerize into rigid fibers and rods that renders erythrocytes prone to severe oxidative stress, hemolysis and adhesion. These pathobiological features give rise to intermittent pain, and acute and chronic organ injury. A particularly impressive feature of this monogenic is the extensive spectrum of phenotypic variability of clinical manifestations among different individual patients. Patients have different levels of anemia, and this has prognostic significance. Patients with sickle cell anemia with less severe hemolytic anemia have more frequent vaso-occlusive pain crisis, acute chest syndrome, and osteonecrosis; those with more severe hemolytic anemia have more frequent leg ulcers, priapism, strokes, nephropathy and pulmonary hypertension [1]. The level of hemolytic anemia is driven primarily by variants in two other genes, the alpha and gamma globin genes, which also affect the clinical phenotype. Besides these two well-characterized genes, there is preliminary evidence that several others can promote phenotypic variation in sickle cell anemia [2], although none of them has been fully independently validated. Interestingly, several of these candidate genetic modifiers of sickle cell anemia involve gene products known to affect bioavailability of nitric oxide such as Klotho, while others are involved in vascular regulation (P-selectin, vascular cell adhesionmolecule-1, beta adrenergic receptor, and the gene superfamily that includes transforming growth factor beta, bone morphogenetic protein and their cognate receptors) [2]. Nitric oxide orchestrates a nearly single-minded program of vascular biology: its presence promotes forward blood flow and its deficiency activates a default program of vasoconstriction and hemostasis. Research groups headed by Kaul and Gladwin independently each provided evidence of decreased nitric oxide bioavailability in human sickle cell disease or mouse models [3,4]. In particular, Gladwin’s group developed a well-supported model that nitric oxide is consumed by plasma cell-free hemoglobin released during intravascular hemolysis [4]. Data from these and many other investigators, formulated upon decades of sickle cell clinical epidemiology data, hinted at a role for nitric oxide bioavailability in the phenotypic variability of sickle cell anemia [1]. The contribution of nitric oxide bioavailability to phenotypic variation has been particularly fascinating in sickle cell disease, drawing intense interest and controversy. In this issue of Pediatric Blood and Cancer, Tantawy and coworkers present genomic research results from an Egyptian cohort [5] that address the gene encoding the enzyme that produces nitric oxide in endothelial cells, endothelial nitric oxide synthase (eNOS), also known as NOS3. One eNOS variant in particular, the intron 4 VNTR variant, has been associated weakly or strongly with a huge number of vasculopathy-linked complications outside of sickle cell disease [6]. In sickle cell disease, the eNOS gene intron 4 VNTR appears to be a home run. Although it must be cautioned that so far the findings are limited to a single, small cohort of 51 children with sickle cell anemia, the authors find that the eNOS 4a allele involving a 27 nucleotide tandem repeat is significantly overrepresented in patients with specific vasculopathic complications of sickle cell disease. These particular complications involve biomarkers of pulmonary hypertension, cerebrovascular disease, and nephropathy, a complex or subphenotype of findings already documented to cluster together by epidemiological data, and to be each associated with particularly severe hemolytic anemia in patients with sickle cell disease. This association of an eNOS allele believed to confer diminished NO production appears to augment existing models of pathophysiology of pulmonary hypertension, cerebrovascular disease, and nephropathy involving low NO bioavailability due primarily to scavenging of NO and its precursor arginine by cell-free plasma hemoglobin and arginase released during intravascular hemolysis [1]. More surprisingly, the Egyptian children with SCD who have this genetic marker of low NO production also have biomarker indicators of less robust fetal hemoglobin response to hydroxyurea therapy, with more severe hemolytic anemia, more iron overload and more hemostatic activation [5]. Again, we should be cautious in interpretation of a single small genomics study, but this pattern of their findings is impressively coherent with a host of published data described below. The regulation of fetal hemoglobin has been variably linked to cyclic GMP pathways, which is the major signal transduction pathway downstream of NO. In fact, hydroxyurea can be an NO donor, and it has been proposed that this property contributes to the activity of hydroxyurea in the induction of fetal hemoglobin