Clinical and molecular characterization of a novel pathogenic AIFM1 E336K mutation connecting mitochondrial dysfunction and neurodegeneration
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Mutations in the AIFM1, which encodes the apoptosis-inducing factor (AIF), are associated with a broad spectrum of neurometabolic disorders, yet their pathogenic mechanisms remains incompletely defined. In this work, we identified and comprehensively characterized a novel hemizygous AIFM1 mutation, c1006G > A (E336K), in male patients with a progressive childhood onset hereditary axonal sensorimotor polyneuropathy inherited in an X-linked recessive pattern, accompanied by sensorineural hearing loss but without cognitive impairment. Their clinical phenotype was consistent with Charcot-Marie-Tooth disease type 4 (CMTX4). Patient-derived fibroblasts exhibited reduced AIF protein stability despite preserved mRNA levels, impaired growth under OXPHOS-dependent conditions, decreased basal respiration, and altered assembly of mitochondrial respiratory supercomplexes. These defects were accompanied by reduced CHCHD4 abundance and decreased mitochondrial mass. Biochemical analyses of the purified E336K protein revealed compromised FAD retention, decreased thermal stability, impaired NADH affinity, destabilization of the charge-transfer complex required for AIF:CHCHD4 interaction, and a shift in coenzyme preference toward NADPH. Structurally, the substitution of Glu336 with Lys remodels the electrostatic environment of the NADH-binding cleft, thereby impairing redox function and weakening CHCHD4 binding. Despite these defects, the E336K mutation preserved DNA binding, nuclease activity, and binding to nuclear partners, although parthanatos induction was attenuated in patient fibroblasts. Collectively, these molecular alterations converge on disrupted mitochondrial bioenergetics and dynamics, providing a direct mechanistic link to the patient’s neurodegenerative course. These findings advance our understanding of AIFM1-related mitochondrial disorders and establish a framework for future precision molecular therapies.
