title: Comprehensive massive parallel DNA sequencing strategy for the genetic diagnosis of the neuro-cardio-facio-cutaneous syndromes.
abstract: Variants in 11 genes of the RAS/MAPK signaling pathway have been causally linked to the neuro-cardio-facio-cutaneous syndromes group (NCFCS). Recently, A2ML1 and RIT1 were also associated with these syndromes. Because of the genetic and clinical heterogeneity of NCFCS, it is challenging to define strategies for their molecular diagnosis. The aim of this study was to develop and validate a massive parallel sequencing (MPS)-based strategy for the molecular diagnosis of NCFCS. A multiplex PCR-based strategy for the enrichment of the 13 genes and a variant prioritization pipeline was established. Two sets of genomic DNA samples were studied using the Ion PGM System: (1) training set (n =15) to optimize the strategy and (2) validation set (n = 20) to validate and evaluate the power of the new methodology. Sanger sequencing was performed to confirm all variants and low covered regions. All variants identified by Sanger sequencing were detected with our MPS approach. The methodology resulted in an experimental approach with a specificity of 99.0% and a maximum analytical sensitivity of ≥ 98.2% with a confidence of 99%. Importantly, two patients (out of 20) harbored described disease-causing variants in genes that are not routinely tested (RIT1 and SHOC2). The addition of less frequently altered genes increased in ≈ 10% the diagnostic yield of the strategy currently used. The presented workflow provides a comprehensive genetic screening strategy for patients with NCFCS in a fast and cost-efficient manner. This approach demonstrates the potential of a combined MPS-Sanger sequencing-based strategy as an effective diagnostic tool for heterogeneous diseases.
authors.affiliation: 1] Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal [2] Abel Salazar Institute for the Biomedical Sciences of the University of Porto, Porto, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Justino
authors.affiliation: Department of Genetics, Hospital de Santa Maria, Lisboa, Portugal.
authors.forename: Patrícia
authors.initials: P
authors.lastname: Dias
authors.affiliation: Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
authors.forename: Maria
authors.initials: M
authors.lastname: João Pina
authors.affiliation: Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
authors.forename: Sónia
authors.initials: S
authors.lastname: Sousa
authors.affiliation: Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal.
authors.forename: Luís
authors.initials: L
authors.lastname: Cirnes
authors.affiliation: Department of Genetics, Hospital de Santa Maria, Lisboa, Portugal.
authors.forename: Ana
authors.initials: A
authors.lastname: Berta Sousa
authors.affiliation: 1] Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal [2] Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: José
authors.initials: J
authors.lastname: Carlos Machado
authors.affiliation: 1] Institute of Molecular Pathology and Immunology of the University of Porto, Porto, Portugal [2] Faculty of Medicine, University of Porto, Porto, Portugal.
authors.forename: José Luis
authors.identifier: 0000000171324094
authors.initials: JL
authors.lastname: Costa
mesh_terms: D000015:Abnormalities, Multiple / Q000175:diagnosis* / Q000235:genetics*; D001483:Base Sequence; D004476:Ectodermal Dysplasia / Q000175:diagnosis / Q000235:genetics; D059472:Exome; D019066:Facies; D005183:Failure to Thrive / Q000175:diagnosis / Q000235:genetics; D056726:Genetic Association Studies; D005820:Genetic Testing; D006330:Heart Defects, Congenital / Q000175:diagnosis / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000737:chemistry / Q000235:genetics; D008969:Molecular Sequence Data; D010641:Phenotype; D015203:Reproducibility of Results; D016415:Sequence Alignment
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; C112913:SHOC2 protein, human