title: Design and application of a 23-gene panel by next-generation sequencing for inherited coagulation bleeding disorders.
abstract: BACKGROUND
  Molecular testing of Inherited bleeding coagulation disorders (IBCDs) not only offers confirmation of diagnosis but also aids in genetic counselling, prenatal diagnosis and in certain cases genotype-phenotype correlations are important for predicting the clinical course of the disease and to allow tailor-made follow-up of individuals. Until recently, genotyping has been mainly performed by Sanger sequencing, a technique known to be time consuming and expensive. Currently, next-generation sequencing (NGS) offers a new potential approach that enables the simultaneous investigation of multiple genes at manageable cost.
  
  
  OBJECTIVE
  The aim of this study was to design and to analyse the applicability of a 23-gene NGS panel in the molecular diagnosis of patients with IBCDs.
  
  
  METHODS
  A custom target enrichment library was designed to capture 31 genes known to be associated with IBCDs. Probes were generated for 296 targets to cover 86.3 kb regions (all exons and flanking regions) of these genes. Twenty patients with an IBCDs phenotype were studied using NGS technology.
  
  
  RESULTS
  In all patients, our NGS approach detected causative mutations. Twenty-one pathogenic variants were found; while most of them were missense (18), three deletions were also identified. Six novel mutations affecting F8, FGA, F11, F10 and VWF genes, and 15 previously reported variants were detected. NGS and Sanger sequencing were 100% concordant.
  
  
  CONCLUSIONS
  Our results demonstrate that this approach could be an accurate, reproducible and reliable tool in the rapid genetic diagnosis of IBCDs.
authors.affiliation: Department of Hematology, H. Universitario de Salamanca, IBSAL, Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain.
authors.forename: J M
authors.initials: JM
authors.lastname: Bastida
authors.affiliation: Instituto de Investigación Biomédica de Salamanca, IBMCC, Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC, Salamanca, Spain.
authors.forename: M
authors.initials: M
authors.lastname: Del Rey
authors.affiliation: Department of Hematology and Clinical Oncology, Centro Regional de Hemodonación, H. Universitario Morales Meseguer, IMIB-Arrixaca, Murcia, Spain.
authors.forename: M L
authors.initials: ML
authors.lastname: Lozano
authors.affiliation: Department of Hematology, H. Universitario de Salamanca, IBSAL, Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain.
authors.forename: M E
authors.initials: ME
authors.lastname: Sarasquete
authors.affiliation: Instituto de Investigación Biomédica de Salamanca, IBMCC, Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC, Salamanca, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Benito
authors.affiliation: Department of Hematology, Hospital Universitario Rio Hortega de Valladolid, Valladolid, Spain.
authors.forename: M E
authors.initials: ME
authors.lastname: Fontecha
authors.affiliation: Department of Hematology, Hospital General de Segovia, Segovia, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Fisac
authors.affiliation: Department of Hematology, Hospital Universitario Rio Hortega de Valladolid, Valladolid, Spain.
authors.forename: L J
authors.initials: LJ
authors.lastname: García-Frade
authors.affiliation: Department of Hematology, Complejo Asistencial de Soria, Soria, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Aguilar
authors.affiliation: Department of Hematology, Complejo Asistencial de Avila, Avila, Spain.
authors.forename: M P
authors.initials: MP
authors.lastname: Martínez
authors.affiliation: Department of Hematology, Hospital Virgen del Puerto de Plasencia, Caceres, Spain.
authors.forename: E
authors.initials: E
authors.lastname: Pardal
authors.affiliation: Department of Hematology, Hospital de El Bierzo, Ponferrada, Spain.
authors.forename: C
authors.initials: C
authors.lastname: Aguilera
authors.affiliation: Department of Hematology, Complejo Asistencial de Leon, Leon, Spain.
authors.forename: B
authors.initials: B
authors.lastname: Pérez
authors.affiliation: Department of Hematology, Hospital de Merida, Badajoz, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Ramos
authors.affiliation: Department of Hematology, Hospital de Merida, Badajoz, Spain.
authors.forename: M R
authors.initials: MR
authors.lastname: Cardesa
authors.affiliation: Department of Hematology, Hospital Rio Carrion, Palencia, Spain.
authors.forename: J M
authors.initials: JM
authors.lastname: Martin-Antorán
authors.affiliation: Department of Hematology, Hospital Rio Carrion, Palencia, Spain.
authors.forename: L A
authors.initials: LA
authors.lastname: Silvestre
authors.affiliation: Department of Hematology, Hospital Clinico Universitario de Valladolid, Valladolid, Spain.
authors.forename: M J
authors.initials: MJ
authors.lastname: Cebeira
authors.affiliation: Department of Hematology, Hospital San Pedro de Alcantara, Caceres, Spain.
authors.forename: N
authors.initials: N
authors.lastname: Bermejo
authors.affiliation: Department of Pediatrics, Hospital Universitario de Salamanca, Salamanca, Spain.
authors.forename: S
authors.initials: S
authors.lastname: Riesco
authors.affiliation: Department of Pediatrics, Hospital Universitario de Salamanca, Salamanca, Spain.
authors.forename: M C
authors.initials: MC
authors.lastname: Mendoza
authors.affiliation: Department of Hematology, H. Universitario de Salamanca, IBSAL, Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain.
authors.forename: R
authors.initials: R
authors.lastname: García-Sanz
authors.affiliation: Department of Hematology, H. Universitario de Salamanca, IBSAL, Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain.
authors.forename: M
authors.initials: M
authors.lastname: González-Díaz
authors.affiliation: Department of Hematology, H. Universitario de Salamanca, IBSAL, Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain.|Instituto de Investigación Biomédica de Salamanca, IBMCC, Centro de Investigación del Cáncer, Universidad de Salamanca-CSIC, Salamanca, Spain.
authors.forename: J M
authors.initials: JM
authors.lastname: Hernández-Rivas
authors.affiliation: Department of Hematology, H. Universitario de Salamanca, IBSAL, Instituto de Investigación Biomédica de Salamanca, Salamanca, Spain.
authors.forename: J R
authors.initials: JR
authors.lastname: González-Porras
mesh_terms: D000293:Adolescent; D000328:Adult; D025861:Blood Coagulation Disorders, Inherited / Q000235:genetics* / Q000473:pathology; D002648:Child; D002675:Child, Preschool; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D005260:Female; D016368:Frameshift Mutation; D017353:Gene Deletion; D056726:Genetic Association Studies; D005820:Genetic Testing / Q000379:methods*; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007223:Infant; D008297:Male; D008875:Middle Aged; D020125:Mutation, Missense; D017422:Sequence Analysis, DNA; D055815:Young Adult
chemical_list: D004247:DNA
keywords: fibrinogen disorders; haemophilia; inherited coagulation factor deficiencies; inherited rare bleeding disorders; molecular diagnosis; next-generation sequencing