Beyond genetics: Deciphering the impact of missense variants in CAD deficiency

Author:

del Caño‐Ochoa Francisco1ORCID,Ng Bobby G.2ORCID,Rubio‐del‐Campo Antonio1ORCID,Mahajan Sonal2ORCID,Wilson Matthew P.3ORCID,Vilar Marçal4ORCID,Rymen Daisy5ORCID,Sánchez‐Pintos Paula67ORCID,Kenny Joanna8ORCID,Ley Martos Myriam9ORCID,Campos Teresa10ORCID,Wortmann Saskia B.1112ORCID,Freeze Hudson H.2ORCID,Ramón‐Maiques Santiago113ORCID

Affiliation:

1. Structure of Macromolecular Targets Unit Instituto de Biomedicina de Valencia (IBV), CSIC Valencia Spain

2. Human Genetics Program Sanford Burnham Prebys Medical Discovery Institute La Jolla California USA

3. Laboratory for Molecular Diagnosis, Center for Human Genetics KU Leuven Leuven Belgium

4. Molecular Basis of Neurodegeneration Unit Instituto de Biomedicina de Valencia (IBV), CSIC Valencia Spain

5. Department of Pediatrics, Center for Metabolic Diseases University Hospitals of Leuven Leuven Belgium

6. Unidad de Diagnóstico y Tratamiento de Enfermedades Metabólicas Congénitas, C.S.U.R. de Enfermedades Metabólicas, MetabERN Hospital Clínico Universitario de Santiago de Compostela La Coruña Spain

7. Instituto de Investigación Sanitaria Santiago de Compostela (IDIS) La Coruña Spain

8. Children's Health Ireland at Crumlin Dublin Ireland

9. Pediatric Neurology Unit Hospital Universitario Puerta del Mar Cádiz Spain

10. Reference Center of Inherited Metabolic Diseases of Hospital de São João Porto Portugal

11. University Children's Hospital, Paracelsus Medical University (PMU) Salzburg Austria

12. Amalia Children's Hospital, Radboudumc Nijmegen The Netherlands

13. Group 739, Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER)–Instituto de Salud Carlos III Valencia Spain

Abstract

AbstractCAD is a large, 2225 amino acid multienzymatic protein required for de novo pyrimidine biosynthesis. Pathological CAD variants cause a developmental and epileptic encephalopathy which is highly responsive to uridine supplements. CAD deficiency is difficult to diagnose because symptoms are nonspecific, there is no biomarker, and the protein has over 1000 known variants. To improve diagnosis, we assessed the pathogenicity of 20 unreported missense CAD variants using a growth complementation assay that identified 11 pathogenic variants in seven affected individuals; they would benefit from uridine treatment. We also tested nine variants previously reported as pathogenic and confirmed the damaging effect of seven. However, we reclassified two variants as likely benign based on our assay, which is consistent with their long‐term follow‐up with uridine. We found that several computational methods are unreliable predictors of pathogenic CAD variants, so we extended the functional assay results by studying the impact of pathogenic variants at the protein level. We focused on CAD's dihydroorotase (DHO) domain because it accumulates the largest density of damaging missense changes. The atomic‐resolution structures of eight DHO pathogenic variants, combined with functional and molecular dynamics analyses, provided a comprehensive structural and functional understanding of the activity, stability, and oligomerization of CAD's DHO domain. Combining our functional and protein structural analysis can help refine clinical diagnostic workflow for CAD variants in the genomics era.

Funder

Generalitat Valenciana

Abraham Lincoln Brigade Archives

European Synchrotron Radiation Facility

National Institute of Neurological Disorders and Stroke

Publisher

Wiley

Subject

Genetics (clinical),Genetics

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