Up to now, genetic analysis of human inherited diseases is based on gene-by-gene Sanger sequencing. In locus-heterogeneous disorders, genes are sequenced according to their population frequency after performing time-consuming cellular and/or biochemical assays in an attempt to reduce to a single or a small group of candidate genes to be analyzed. In order to improve the genetic diagnosis of congenital disorders of glycosylation (CDG) and glycogen storage diseases (GSD), two locus-heterogeneous disorders affecting the glycosylation pathways or the enzymes involved in the glycogen metabolism respectively, we applied the cutting-edge DNA next-generation sequencing (NGS) technology. We have evaluated three different NGS-based tests to analyze 39 CDG and 47 GSD suspected patients. First, we captured the exome of targeted genes (TES) previously described to be associated to CDG or GSD. Diagnosis rate of the customized CDG panel was low probably due to the limited number of genes captured of this emerging disorder. In order to improve the diagnosis rate of both diseases, we captured the whole exome (whole-exome sequencing, WES) or the exome of genes associated with human genetic diseases as annotated in OMIM database (clinical exome sequencing, CES). Both tests were used to broaden the number of analyzable genes beyond those already described, allowing the detection of mutations in non-CDG or GSD-associated genes causing symptoms that might overlap with them. Besides, using WES we identified mutations in a new gene not associated to any disease so far. Additionally, NGS showed higher sensibility than Sanger sequencing since it avoided allele dropout in two samples and allowed the detection of a copy number variation in ALG1. Moreover, it is noteworthy the accuracy and sensibility of the technology in the detection of variants located in genes with high sequence-homology, such as ALG1. Overall, we reached a genetic diagnosis of approximately 50% of the patients analyzed. In CDG, we identified 23 variants, 12 of them novel, in 13 previously described CDG-genes. Furthermore, we described a new CDG-gene not associated to any human pathology so far (CCDC115). Comprehensive functional studies performed on the patient-derived fibroblasts indicated that the protein codified by CCDC115 is probably involved in the Golgi homeostasis, impairing protein glycosylation. In GSD we identified 23 changes, 10 of them novel, in 6 already-described genes. Besides, we identified an overall of six patients bearing mutations in genes not associated to the initially suspected disease. The accurate genetic diagnosis allowed in five patients to undergo an accurate tailored treatment improving their outcome. Summing up, our results highlight the usefulness of NGS to be applied to the diagnosis of two locus-heterogeneous disorders. It is noteworthy that an accurate genetic diagnosis is needed to provide a genetic counseling, to prescribe tailored treatments and to provide insights to guide research towards new therapies based on the mechanism of actions of the mutations identified (precision medicine).

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 09-03-2017