Pulmonary arterial hypertension is a severe condition causing significant morbidity and mortality in patients with scleroderma. By exome sequencing we previously identified rare deleterious variants in the ASB2 gene as genetic modifiers to clinic progression of scleroderma-associated pulmonary arterial hypertension (SSc-PAH). The ASB2 gene encodes a member of the ankyrin repeat and SOCS box-containing (ASB) protein family; and the encoded protein is a subunit of the E3 ubiquitin ligase complex that mediates the degradation of actin-binding proteins. We conducted functional studies to confirm that ASB2 gene may represent a putative genetic risk factor for SSc-PAH.
We quantified ATP8B4 mRNA expression in peripheral blood of SSc-PAH patients (n=23) and SSc controls (n=8) utilizing qRT-PCR. Expression levels of genes were calculated using the 2−ΔΔCt method, with GAPDH as endogenous control. Correlation between levels of ATP8B4 gene expression and clinical measurements of SSc-PAH severity was assessed by Pearson correlation using the SPSS Statistical Package. We further validated ASB2 expression in primary human pulmonary arterial smooth muscle cells (HPASMCs), the target tissue for ASB2 localization, in response to hypoxia challenge for 48 hours.
We observed significantly decreased expression of ASB2 gene in the blood of SSc-PAH patients compared to the SSc group (p<0.05). Moreover, expression levels of ASB2 were inversely correlated with pulmonary vascular resistance (r=0.676, p=0.006), a key determinant of PAH prognosis among SSc-PAH patients. We further validated ASB2 expression in HPASMCs. Levels of ASB2 gene expression were decreased 3.06-fold after exposure of the cells to 4% O2 for 48 hours (p=0.009), similar trend was observed in protein immunoblot analyses of ASB2, showing a significant reduction of 46% in response to hypoxia (p=0.01).
Our findings suggest ASB2 is involved in the pathogenesis of SSc-PAH representing a novel risk factor and therapeutic target for SSc-PAH patients.