We investigated two male infant patients who were given a diagnosis of progressive mitochondrial encephalomyopathy on the basis of clinical, biochemical, and morphological features. These patients were born from monozygotic twin sisters and unrelated fathers, suggesting an X-linked trait. Fibroblasts from both showed reduction of respiratory chain (RC) cIII and cIV, but not of cI activities. We found a disease-segregating mutation in the X-linked AIFM1 gene, encoding the Apoptosis-Inducing Factor (AIF) mitochondrion-associated 1 precursor that deletes arginine 201 (R201 del). Under normal conditions, mature AIF is a FAD-dependent NADH oxidase of unknown function and is targeted to the mitochondrial intermembrane space (this form is called AIFmit). Upon apoptogenic stimuli, a soluble form (AIFsol) is released by proteolytic cleavage and migrates to the nucleus, where it induces "parthanatos," i.e., caspase-independent fragmentation of chromosomal DNA. In vitro, the AIFR201 del mutation decreases stability of both AIFmit and AIFsol and increases the AIFsol DNA binding affinity, a prerequisite for nuclear apoptosis. In AIFR201 del fibroblasts, staurosporine-induced parthanatos was markedly increased, whereas re-expression of AIFwt induced recovery of RC activities. Numerous TUNEL-positive, caspase 3-negative nuclei were visualized in patient #1's muscle, again indicating markedly increased parthanatos in the AIFR201 del critical tissues. We conclude that AIFR201 del is an unstable mutant variant associated with increased parthanatos-linked cell death. Our data suggest a role for AIF in RC integrity and mtDNA maintenance, at least in some tissues. Interestingly, riboflavin supplementation was associated with prolonged improvement of patient #1's neurological conditions, as well as correction of RC defects in mutant fibroblasts, suggesting that stabilization of the FAD binding in AIFmit is beneficial.
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