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Published on: December 7, 2010
Cell-based analysis of CAD variants identifies individuals likely to benefit from uridine therapy
Francisco Del Caño-Ochoa1,2,3, Bobby G Ng4, Malak Abedalthagafi5
1Genome Dynamics and Function Program, Centro de Biología Molecular Severo Ochoa, CSIC-UAM, Madrid, Spain.
Insights
A new cell-based assay accurately identifies pathogenic variants in the CAD gene, crucial for pyrimidine biosynthesis. This helps diagnose 11 individuals with CAD deficiency who may benefit from uridine therapy.
Area of Science:
- Biochemistry
- Genetics
- Metabolic Disorders
Background:
- Autosomal recessive variants in CAD, a key enzyme in pyrimidine de novo biosynthesis, cause a severe inborn metabolic disorder.
- Diagnosis is challenging due to CAD's large size, numerous missense variants, and nonspecific clinical presentation.
- Uridine supplementation is a potential treatment for this disorder.
Purpose of the Study:
- To develop a reliable assay for assessing the pathogenicity of CAD variants.
- To identify individuals with CAD deficiency who could benefit from uridine therapy.
Main Methods:
- Generated a human CAD-knockout cell line using CRISPR/Cas9, dependent on uridine for survival.
- Utilized a complementation assay with recombinant CAD to identify variants inactivating the enzyme.
- Tested 34 variants using the CAD-knockout cell line.
Main Results:
- Identified 16 out of 34 tested variants as deleterious to CAD activity.
- Confirmed 11 individuals with biallelic pathogenic CAD variants and consistent clinical phenotypes.
- Established a CAD deficit in 11 subjects, enabling description of their clinical presentation.
Conclusions:
- A novel cell-based assay effectively determines the pathogenicity of CAD variants.
- The assay identified 11 individuals with CAD deficiency eligible for uridine therapy.
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.
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