Automated patch-clamp supports functional PIEZO1 variant classification in red blood cells in hereditary xerocytosis

Hereditary xerocytosis (HX) is a dominantly inherited hemolytic anemia caused by gain-of-function mutations in the mechanosensitive ion channel PIEZO1. While genetic sequencing identifies PIEZO1 variants, many remain classified as variants of uncertain significance (VUS), complicating clinical interpretation. We developed an automated patch-clamp (APC) assay using customized recording chips with microfluidic flow channels generating physiologically relevant shear stress, combined with pharmacological modulation, for PIEZO1 activation. The assay was validated in HEK293 cells overexpressing human PIEZO1, then applied to red blood cells (RBCs) from two patients carrying PIEZO1 variants: R2456H (class 5, pathogenic) and G909V (class 3, VUS). RBCs from the R2456H patient showed 8-fold elevated mechanosensitive currents compared to healthy donors, with sustained activation profiles indicating delayed inactivation. G909V showed currents similar to healthy controls. Comparative flow cytometry showed comparable PIEZO1 surface expression between R2456H and healthy RBCs, indicating altered channel gating rather than increased surface expression. Electrophysiological findings correlated with clinical parameters consistent with RBC dehydration in the R2456H patient. In this proof-of-concept study. APC distinguished a known pathogenic PIEZO1 gain-of-function variant from a VUS with currents comparable to healthy controls. By complementing genetic sequencing with functional evidence, this approach may support diagnostic classification in selected patients with suspected HX.

Keywords: Q3 2026