Refined classification and phenotype-driven analysis of PIEZO1 variants in hereditary red blood cell and iron disorders.

Interpreting genetic variants in complex genes such as PIEZO1 remains challenging because of marked allelic heterogeneity, relative tolerance to missense variation, and overlapping clinical phenotypes. Gain-of-function variants in PIEZO1 cause dehydrated hereditary stomatocytosis (DHS1, or hereditary xerocytosis), a pleiotropic syndrome characterized by anemia of variable severity and iron overload. In this study, we provided and applied an integrative framework combining the American College of Medical Genetics and Genomics guidelines, quantitative in silico predictions, structural domain annotation, and detailed patient phenotyping to classify 2565 PIEZO1 variants. A Bayesian scoring system with weighted evidence and a composite predictive score enabled reclassification of nearly 1000 variants of uncertain significance and highlighted nonrandom clustering of pathogenic variants within functionally constrained domains, particularly the anchor, inner helix, and C-terminal domains. Genotype-phenotype correlation analysis in 176 in-house DHS cases identified 3 phenotypic clusters, ranging from classical DHS1 with severe hemolysis and iron overload to atypical or subclinical presentations, reflecting domain-specific pathogenic mechanisms. Overall, this reclassification of PIEZO1 variants, integrated with genotype-phenotype correlation analysis, improves diagnostic precision, supports genotype-guided patient management, and underscores the value of integrating structural and clinical data into interpretation of rare genetic variants.