Endothelin-1 but not Endothelial Nitric Oxide Synthase Gene Polymorphism is Associated with Sickle Cell Disease in Africa

Abstract

Sickle cell disease shows marked variability in severity and pathophysiology among individuals, probably linked to differential expression of various adhesion molecules. In this study, we investigated the differential distribution, genomic diversity and haplotype frequency of endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) polymorphisms, recently implicated as important in modification of disease severity. One hundred and forty five sickle cell disease patients (HbSS) and 244 adult and pediatric controls, without sickle cell disease (HbAA), were recruited from Mali. Genotypic analysis of the functionally significant eNOS variants (T786C, G894T and intron 4) and endothelin-1 (G5665T) was carried out with a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Our results show that the wild type alleles are the most frequent for all eNOS variants between cases and controls. Allelic and genotypic frequencies of eNOS polymorphic groups are not significantly different between cases and controls (P > 0.05). In addition, there is no association between eNOS variants and sickle cell disease, contrary to published reports. On the other hand, we report that endothelin-1 (G5665T) mutant variant had the lowest allelic frequency, and is significantly associated with sickle cell disease in Africa (P < 0.05). Similarly, haplotype frequencies were the same between cases and controls, except for the haplotype combining all mutant variants (T, C, 4a; P = 0.01). eNOS polymorphic variants are less frequent, with no significance with sickle cell disease in Africa. On the other hand, endothelin-1 is associated with sickle cell disease, and has the capacity to redefine pathophysiology and possibly serve as modulator of disease phenotype.

Keywords: endothelial nitric oxide synthase; endothelin-1; pathophysiology; polymorphisms; sickle cell disease.

Figure 1

Agarose gel electrophoresis showing variants of the intron 4 (27-bp TR) polymorphism of the endothelial nitric oxide synthase gene. Ladder: 100 bp ladder, where the 500 bp band stains most intensely, was used as a molecular weight marker for all gels.

Figure 2

Agarose gel electrophoresis showing variants of the G894T (rs1799983) polymorphism of the endothelial nitric oxide synthase gene. PCR products were digested in 2U of BanII restriction endonuclease. bp base pairs; Ladder: 100 bp ladder, where the 500 bp band stains most intensely, was used as a molecular weight marker.

Figure 3

Agarose gel electrophoresis showing variants of the T786C (rs2070744) polymorphism of the endothelial nitric oxide synthase gene. PCR products were digested in 2U of MspI restriction endonuclease. bp base pairs; Ladder: 100 bp ladder, where the 500 bp band stains most intensely, was used as a molecular weight marker.