Distinct Clinical Phenotypes in KIF1A-Associated Neurological Disorders Result from Different Amino Acid Substitutions at the Same Residue in KIF1A

KIF1A is a neuron-specific kinesin motor responsible for intracellular transport along axons. Pathogenic KIF1A mutations cause KIF1A-associated neurological disorders (KAND), a spectrum of severe neurodevelopmental and neurodegenerative conditions. While individual KIF1A mutations have been studied, how different substitutions at the same residue affect motor function and disease progression remains unclear. Here, we systematically examine the molecular and clinical consequences of mutations at three key motor domain residues-R216, R254, and R307-using single-molecule motility assays and genotype-phenotype associations. We find that different substitutions at the same residue produce distinct molecular phenotypes, and that homodimeric mutant motor properties correlate with developmental outcomes. In addition, we present the first analysis of heterodimeric KIF1A motors-mimicking the heterozygous context in patients-and demonstrate that while heterodimers retain substantial motility, their properties are less predictive of clinical severity than homodimers. These results highlight the finely tuned mechanochemical properties of KIF1A and suggest that dysfunctional homodimers may disproportionately drive the diverse clinical phenotypes observed in KAND. By establishing residue-specific genotype-phenotype relationships, this work provides fundamental insights into KAND pathogenesis and informs targeted therapeutic strategies.