Thrombospondin-1 Gene Polymorphism Is Associated with Estimated Pulmonary Artery Pressure in Patients with Sickle Cell Anemia

Thrombospondin-1 (TSP1) is a multifunctional glycoprotein present in platelet α-granules that contains domains for adhesive proteins, enzymes, and cell receptors. It is released by activated platelets and is increased in the plasma of patients with sickle cell anemia (SCA). TSP1 is known to mediate adherence of sickle reticulocytes to endothelial cells. Furthermore, TSP1 also inhibits the NO signaling pathway through its binding to the CD47 cell receptor on endothelial cells and platelets. Thus, we hypothesized that genetic variants in TSP1 may contribute to the marked phenotypic variation seen in SCA.

We tested whether genetic variants of the TSP1 gene are associated with biomarkers of SCA severity and specific complications in a cohort of 452 patients with homozygous HbSS disease from the multicenter walk-PHaSST (Treatment of Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy) trial. Genetic data from 406 patients were included in the analysis. Patients ranged in age from 12 to 70 years, and 51% were females. 307 patients were enrolled at various sites in the United States, while 99 patients were enrolled at Hammersmith Hospital in the UK. A total of 10 SNPs were selected, including haplotype tagged SNPs, coding variants, and SNPs potentially regulating gene expression and alternative splicing. DNA isolated from peripheral blood was used for genotyping.

We found that the TSP1 SNPs rs1478604 and rs1478605 were significantly positively correlated with tricuspid regurgitation velocity (TRV) >2.5 m/sec, an echocardiographic marker of pulmonary hypertension associated with increased mortality, under a recessive model (r=0.10, p=0.0288 and r=0.13, p=0.0047 respectively). TRV was also analyzed as a categorical variable using regression analysis, and subjects were grouped based on the following clinically relevant cut-off points, 2.5 m/sec, 2.7 m/sec, and 3 m/sec. Univariate regression analyses showed TRV>2.5 m/sec was independently associated with the recessive genotypes of rs1478604 and rs1478605 (p=0.069 and 0.017 respectively). As previously published, we confirmed the association of TRV with age, gender, NT-proBNP and creatinine, and we adjusted for these variables in multivariate regression analyses for the association of TRV and each SNP. The results showed that the two SNPs were significantly associated with TRV>2.5 m/sec (rs1478604, OR 2.54, 95% CI (1, 6.44), p<0.05 and rs1478605, OR 4.20, 95% CI (1.41, 12.49), p<0.01) even after controlling for these variables.

The results suggest that genetic variations in the TSP1 gene are associated with variations in estimated pulmonary artery pressure in patients with SCA. Both rs1478604 and rs1478605 are localized to the 5' untranslated region. Bioinformatic analysis of these SNPs for putative transcription factor binding sites demonstrated that the two SNPs are associated with both gain and loss of transcription factor binding compared to the wild type. Therefore, both SNPs are potentially functional in TSP1 regulation. These data also suggest possible translational implications in light of earlier pre-clinical reports that showed that mice lacking TSP1 were protected from hypoxia-mediate pulmonary and right ventricular remodeling and showed less alterations in PA pressure.

In populations without SCA, these alleles have been independently linked to risk of malaria, corneal immune function, and gastric cancer. TSP1 activity has also been linked to fibrotic renal disease in a rat model and to IRI-mediated AKI in a murine model. The mechanism implicated in some of these cases involves disordered TSP1 regulation of transforming growth factor beta (TGF-beta), whose superfamily of receptors is dysregulated in the pathogenesis of pulmonary hypertension and kidney disease.

Our findings support a hypothetical model of TSP1 involvement in the pathophysiology of SCA. Efforts to seek validation of the genetic findings from this study in an independent SCA cohort are currently underway.