A Novel-G814fs Knock-In Mouse Model Reveals Phenotypic Heterogeneity Among TruncatingMutations in X-Linked Alport Syndrome.

Abstract

: X-linked Alport syndrome (XLAS) arises from pathogenic variants in. Truncating variants are generally classified as severe, but whether clinically meaningful heterogeneity exists within this group remains unclear. This study aimed to establish a novelknock-in mouse model based on a clinical variant and to determine whether truncating mutation position influences disease severity.: A de novoframeshift variant, c.2440delG, was identified in a patient with severe early-onset XLAS. A-G814fs knock-in mouse was generated by CRISPR/Cas9 on the C57BL/6J inbred mouse strain background and compared with the establishednonsense model using survival analysis, serial functional measurements, kidney histopathology, transmission electron microscopy, and RNA sequencing.: The-G814fs knock-in mouse was successfully generated and showed loss of glomerular α5(IV) collagen chain expression. Compared with G5X mice, G814fs mice exhibited shorter survival (median 141 vs. 161.5 days,= 0.0004), earlier onset of proteinuria, and more severe kidney functional decline. By 16 weeks, G814fs mice also showed more severe glomerular basement membrane abnormalities and more extensive glomerulosclerosis. RNA sequencing revealed a shared inflammatory gene signature in both models, together with selective upregulation of genes related to the PPAR signaling pathway and fatty acid metabolism in G814fs kidneys.: This study reports a novel de novoframeshift variant and establishes the first-G814fs knock-in mouse model. Direct comparison with the G5X model shows that distinct truncatingmutations can be associated with substantially different disease severity, providing a useful platform for future mechanistic and therapeutic studies in XLAS.