Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy. Genet Med 2020 Oct;22(10):1598-1605
Date
05/29/2020Pubmed ID
32461667Pubmed Central ID
PMC7521996DOI
10.1038/s41436-020-0833-2Scopus ID
2-s2.0-85085495821 (requires institutional sign-in at Scopus site) 19 CitationsAbstract
PURPOSE: Pathogenic autosomal recessive variants in CAD, 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 of CAD with over 1000 missense variants and the nonspecific clinical presentation. We aimed to develop a reliable and discerning assay to assess the pathogenicity of CAD variants and to select affected individuals that might benefit from uridine therapy.
METHODS: Using CRISPR/Cas9, we generated a human CAD-knockout cell line that requires uridine supplements for survival. Transient transfection of the knockout cells with recombinant CAD restores 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 in CAD and a phenotype consistent with a CAD deficit. We used the CAD-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 of CAD variants, identifying 11 CAD-deficient individuals who could benefit from uridine therapy.
Author List
Del Caño-Ochoa F, Ng BG, Abedalthagafi M, Almannai M, Cohn RD, Costain G, Elpeleg O, Houlden H, Karimiani EG, Liu P, Manzini MC, Maroofian R, Muriello M, Al-Otaibi A, Patel H, Shimon E, Sutton VR, Toosi MB, Wolfe LA, Rosenfeld JA, Freeze HH, Ramón-Maiques SAuthor
Michael Muriello MD Assistant Professor in the Pediatrics department at Medical College of WisconsinMESH terms used to index this publication - Major topics in bold
Aspartate CarbamoyltransferaseCarbamoyl-Phosphate Synthase (Glutamine-Hydrolyzing)
Cell Line
Dihydroorotase
Humans
Uridine
