Systematic analysis of NDUFAF6 in complex I assembly and mitochondrial disease

Abstract Isolated complex I (CI) deficiencies are a major cause of primary mitochondrial disease. Two-thirds of CI deficiencies arise from defects in CI assembly factors (CIAFs) that are not part of the CI holoenzyme. The biochemistry of CIAFs is poorly defined, making their role in CI assembly unclear and confounding interpretation of potential disease-causing genetic variants. To address these challenges, we devised a deep mutational scanning approach to systematically assess the function of thousands of NDUFAF6 genetic variants. Guided by these data, biochemical analyses, and cross-linking mass spectrometry, we discovered that the CIAF NDUFAF6 facilitates incorporation of NDUFS8 into CI and reveal that NDUFS8 overexpression rectifies NDUFAF6 deficiency. Our data further provide experimental support of pathogenicity for seven novel NDUFAF6 variants associated with human pathology and add phenotypic annotation for 5,675 additional variants. Overall, our work defines the function of NDUFAF6 and provides a comprehensive clinical resource for diagnosing NDUFAF6-related diseases.