Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy

Purpose Pathogenic autosomal recessive variants inCAD, encoding the multienzymatic protein initiating pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder treatable with a dietary supplement of uridine. This condition is difficult to diagnose given the large size ofCADwith over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity ofCADvariants and to select affected individuals that might benefit from uridine therapy. Methods Using CRISPR/Cas9, we generated a humanCAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinantCADrestores growth in absence of uridine. This system determines missense variants that inactivate CAD and do not rescue the growth phenotype. Results We identified 25 individuals with biallelic variants inCADand a phenotype consistent with a CAD deficit. We used theCAD-knockout complementation assay to test a total of 34 variants, identifying 16 as deleterious for CAD activity. Combination of these pathogenic variants confirmed 11 subjects with a CAD deficit, for whom we describe the clinical phenotype. Conclusions We designed a cell-based assay to test the pathogenicity ofCADvariants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy.