Thrombospondin-1 Polymorphisms Are Associated with Chronic Kidney Disease in Sickle Cell Anemia

Background: Sickle cell anemia (SCA) is characterized by the "sickling" of red blood cells (RBCs) and red cell rigidity, as well as increased cellular adhesiveness of blood cells and endothelium, leading to vasoocclusion. Thrombospondin-1 protein, secreted during platelet activation, suppresses the activity of nitric oxide synthase and limits downstream effects of nitric oxide at soluble guanylate cyclase, promoting vasculopathy. It is increased in the plasma of SCA patients both at steady state and during acute vasoocclusive episodes, and it mediates sickle cell adhesion in vitro. Thrombospondin gene (THBS1) single nucleotide polymorphisms (SNPs) have been associated with chronic kidney disease in the general population.

Methods: We evaluated the genetic association of two THBS1 SNPs (rs1478605 and rs1478604) by Taqman SNP genotyping with pre-designed primer and probes in a test cohort from the walk-PHaSST study and a validation cohort from the Outcome Modifying Genes in SCD (OMG-SCD) study. Statistical analyses were done utilizing STATA 2014 and SAS v9.2 software. We calculated site-specific allele frequencies using genotype counting and tested for departures from Hardy-Weinberg equilibrium using goodness of fit statistics. Creatinine was log-transformed to meet the assumption of normality. Correlation and regression techniques were used to analyze serum creatinine and its association with each THBS1 SNP. Models were controlled for gender and age at the time of enrollment.

Results: In a test cohort of 414 patients with sickle cell anemia (mean age 35.77 ± 12.37 years, 51.6% female, 45.7% on hydroxyurea, 13.5% on chronic transfusions), we found that the THBS1 SNP rs1478605 was significantly negatively associated with serum creatinine (p=0.007). In an additive genetic model, each addition of the minor allele, G, was associated with a reduction in serum creatinine (p=0.009). This is supported by the finding that carriers of the AA genotype had higher creatinine levels than participants with the GG genotype (0.98 ± 1.31 vs. 0.79 ± 0.35 mg/dL). The same finding was noted with THBS1 SNP rs1478604 (CC: 0.98 ± 1.31 versus TT: 0.83 ± 0.39 mg/dL, p=0.026). To validate these initial findings, we evaluated the association of these two specific SNPs with serum creatinine in a second independent validation cohort of 538 sickle cell anemia patients (mean age 34.21 ± 12.15 years, 55.4% female, 39.3% on hydroxyurea, 6.5% on chronic transfusions). The minor allele (T or G) frequencies of these two SNPs are 0.2919 and 0.3019, respectively, comparable to the test cohort. This independent analysis confirmed a significant negative association of serum creatinine with THBS1 SNPs rs1478605 and rs1478604 in an additive genetic model (p=0.0006 and p=0.0008, respectively). Each addition of the minor allele, G or T in the respective SNPs, was associated with a reduction in serum creatinine, confirming that the minor allele is the protective allele in this model.

Conclusion: Polymorphisms in the thrombospondin-1 gene in adolescents and adults with SCA are associated with chronic kidney disease as indicated by serum creatinine level. We hypothesize that the high risk SNPs confer higher expression of thrombospondin protein, which is known to mediate adhesion of CD36+ sickle reticulocytes to endothelial cells, and to promote vasculopathy through CD47 by suppressing production and downstream signaling of nitric oxide, and promoting NADPH oxidase-driven reactive oxygen species formation. Further investigation of the mechanism of thrombospondin-1 dysregulation in sickle cell nephropathy may produce generalizable information regarding chronic kidney disease in both the sickle cell and general population.

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