In the last decade, undiagnosed disease programs have emerged to address the significant number of individuals with suspected but undiagnosed rare genetic diseases. In our single-center study, we have launched a pilot program for pediatric patients with undiagnosed diseases in the second-largest university hospital in the Czech Republic. This study was prospectively conducted at the Department of Pediatrics at University Hospital Brno between 2020 and 2023. A total of 58 Czech patients with undiagnosed diseases were enrolled in the study. All children underwent singleton WES with targeted phenotype-driven analysis. We identified 28 variants, including 11 pathogenic, 13 likely pathogenic, and 4 VUS according to ACMG guidelines, as diagnostic of genetic diseases in 25 patients, resulting in an overall diagnostic yield of 43%. Eleven variants were novel and had not been previously reported in any public database. The overall clinical utility (actionability) enabling at least one type of change in the medical care of the patient was 76%, whereas the average number of clinical implications to individual patient care was two. Singleton WES facilitated the diagnostic process in the Czech undiagnosed pediatric population. We believe it is an effective approach to enable appropriate counseling, surveillance, and personalized clinical management.

Center of Precision Medicine Department of Pathology University Hospital Brno Faculty of Medicine Masaryk University Brno Czech Republic

Central European Institute of Technology Masaryk University Brno Czech Republic

Centre of Molecular Biology and Genetics Department of Hematology Oncology and Internal Medicine Masaryk University and University Hospital Brno Brno Czech Republic

Department of Biology Faculty of Medicine Masaryk University Kamenice 5 625 00 Brno Czech Republic

Department of Medical Genetics and Genomics University Hospital Brno Faculty of Medicine Masaryk University Brno Brno Czech Republic

Department of Pediatric Anesthesiology and Intensive Care Medicine University Hospital Brno and Faculty of Medicine Masaryk University Brno Czech Republic

Department of Pediatric Neurology University Hospital Brno Faculty of Medicine Masaryk University Brno Czech Republic

Department of Pediatric Oncology Faculty of Medicine University Hospital Brno Masaryk University Brno Czech Republic

Department of Pediatrics University Hospital Brno Faculty of Medicine Masaryk University Cernopolni 9 613 00 Brno Czech Republic

Department of Pharmacology CZECRIN Masaryk University Faculty of Medicine Brno Czech Republic

Department of Simulation Medicine Faculty of Medicine Masaryk University Brno Czech Republic

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

Institute of Medical Biochemistry and Laboratory Diagnostics 1st Faculty of Medicine Charles University and General University Hospital Prague Prague Czech Republic

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