Aplicación de la secuenciación masiva de dna al diagnóstico de los defectos congénitos de glicosilación y de glucogenosis
Author
Medrano Rodríguez, CeliaAdvisor
Pérez-Cerdá, CeliaEntity
UAM. Departamento de Biología MolecularDate
2017-03-09Subjects
Genética médica - Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 09-03-2017Esta obra está bajo una licencia de Creative Commons Reconocimiento-NoComercial-SinObraDerivada 4.0 Internacional.
Abstract
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).
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