Background and ObjectivesCoenzyme Q₁₀(CoQ₁₀)–deficient cerebellar ataxia can be due to pathogenic variants in genes encoding for CoQ₁₀biosynthetic proteins or associated with defects in protein unrelated to its biosynthesis. Diagnosis is crucial because patients may respond favorably to CoQ₁₀supplementation. The aim of this study was to identify through whole-exome sequencing (WES) the pathogenic variants, and assess CoQ₁₀levels, in fibroblasts from patients with undiagnosed cerebellar ataxia referred to investigate CoQ₁₀deficiency.MethodsWES was performed on genomic DNA extracted from 16 patients. Sequencing data were filtered using a virtual panel of genes associated with CoQ₁₀deficiency and/or cerebellar ataxia. CoQ₁₀levels were measured by high-performance liquid chromatography in 14 patient-derived fibroblasts.ResultsA definite genetic etiology was identified in 8 samples of 16 (diagnostic yield = 50%). The identified genetic causes were pathogenic variants of the genesCOQ8A(ADCK3) (n = 3 samples),ATP1A3(n = 2),PLA2G6(n = 1),SPG7(n = 1), andMFSD8(n = 1). Five novel mutations were found (COQ8An = 3,PLA2G6n = 1, andMFSD8n = 1). CoQ₁₀levels were significantly decreased in 3/14 fibroblast samples (21.4%), 1 carrying compound heterozygousCOQ8Apathogenic variants, 1 harboring a homozygous pathogenicSPG7variant, and 1 with an unknown molecular defect.DiscussionThis work confirms the importance ofCOQ8Agene mutations as a frequent genetic cause of cerebellar ataxia and CoQ₁₀deficiency and suggestsSPG7mutations as a novel cause of secondary CoQ₁₀deficiency.