A Pathogenic ANK1 c.5096G>A Mutation Disrupting the Ankyrin-1/β-Spectrin Interaction in Hereditary Spherocytosis.

Hereditary spherocytosis (HS) is a common inherited hemolytic anemia characterized by spherical erythrocytes, splenomegaly, and increased osmotic fragility, with ANK1 mutations being the most frequent cause. We identified a novel ANK1 mutation (c.5096G>A, p.R1699K) in a patient with classic HS phenotypes and a family history of hemolytic anemia. To explore its pathogenicity, we performed a series of hematological and morphological analyses. Compared with wild-type mice, the Ank1 c.5198G>A knock-in mice displayed typical HS features. Hematologically, they showed reduced MCV, MCH, and MCHC. The EMA fluorescence intensity was significantly decreased, and RBCs exhibited increased osmotic fragility. Morphologically, peripheral smears revealed abundant spherocytes, and electron microscopy showed a transformation from normal biconcave discs to spherical erythrocytes. Additionally, the Ank1 c.5198G>A knock-in mice presented with significant splenomegaly. Mechanistically, coimmunoprecipitation analysis indicated a significantly weakened association between ankyrin-1 and β-spectrin in the Ank1 c.5198G>A knock-in mice. This study provides in vivo evidence that the Ank1 R1733K mutation impairs erythrocyte membrane stability and cytoskeletal integrity, recapitulating the HS phenotype in mice. Our findings support the pathogenicity of the ANK1 c.5096G>A mutation and offer a useful model for further investigating the molecular mechanisms of HS.