title: Detection of new pathogenic mutations in patients with congenital haemolytic anaemia using next-generation sequencing.
abstract: BACKGROUND
  Congenital haemolytic anaemia (CHA) refers to a group of genetically heterogeneous disorders, mainly caused by changes in genes encoding globin chains, cytoskeletal proteins and red cell enzymes, in which accurate diagnosis can be challenging with conventional techniques.
  
  
  METHODS
  To set-up a comprehensive assay for detecting mutations that could improve aetiological diagnosis, we designed a custom panel for sequencing coding regions from 40 genes known to be involved in the pathogenesis of CHA, using the Ion Torrent™(Thermo Fisher Scientific, S.L. Waltham, MA, USA) Personal Genome Machine (PGM) Sequencer. A control group of 16 samples with previously known mutations and a test group of 10 patients with unknown mutations were included for assay validation and application, respectively.
  
  
  RESULTS
  In the test group, we identified pathogenic mutations in all cases: four patients had novel mutations in genes related to membrane defects (SPTB, ANK1, SLC4A1 and EPB41), four were homozygous or compound heterozygous for mutations in genes related to enzyme deficiencies (GPI, TPI1 and GSS), one had a mutation in the HBB gene and another presented a homozygous mutation in the ADAMTS13 gene.
  
  
  CONCLUSIONS
  Ion PGM sequencing with our custom panel is a highly efficient way to detect mutations causing haemolytic anaemia, including new variations. It is a high-throughput detection method that is ready for application in clinical laboratories.
authors.affiliation: BioCruces Health Research Institute, Barakaldo, Spain.
authors.forename: R
authors.identifier: http://orcid.org/0000-0002-1240-1958
authors.initials: R
authors.lastname: Del Orbe Barreto
authors.affiliation: BioCruces Health Research Institute, Barakaldo, Spain.|Department of Haematology, Cruces University Hospital, Barakaldo, Spain.
authors.forename: B
authors.initials: B
authors.lastname: Arrizabalaga
authors.affiliation: BioCruces Health Research Institute, Barakaldo, Spain.
authors.forename: A B
authors.initials: AB
authors.lastname: De la Hoz
authors.affiliation: BioCruces Health Research Institute, Barakaldo, Spain.|Department of Genetics, Physical Anthropology and Animal Physiology, Faculty of Medicine and Odontology, University of the Basque Country (UPV/EHU), Bilbao, Spain.
authors.forename: Á
authors.initials: Á
authors.lastname: García-Orad
authors.affiliation: BioCruces Health Research Institute, Barakaldo, Spain.|Molecular Genetics Laboratory, Genetics Service, Cruces University Hospital, Barakaldo, Spain.
authors.forename: M I
authors.initials: MI
authors.lastname: Tejada
authors.affiliation: BioCruces Health Research Institute, Barakaldo, Spain.|Department of Haematology, Cruces University Hospital, Barakaldo, Spain.
authors.forename: J C
authors.initials: JC
authors.lastname: Garcia-Ruiz
authors.affiliation: Serviço de Hematologia Clínica, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal.
authors.forename: T
authors.initials: T
authors.lastname: Fidalgo
authors.affiliation: Serviço de Hematologia Clínica, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal.
authors.forename: C
authors.initials: C
authors.lastname: Bento
authors.affiliation: Research Centre for Anthropology and Health (CIAS), Department of Life Sciences, University of Coimbra, Coimbra, Portugal.
authors.forename: L
authors.initials: L
authors.lastname: Manco
authors.affiliation: Serviço de Hematologia Clínica, Centro Hospitalar e Universitário de Coimbra, Coimbra, Portugal.
authors.forename: M L
authors.initials: ML
authors.lastname: Ribeiro
mesh_terms: D000745:Anemia, Hemolytic, Congenital / Q000175:diagnosis / Q000235:genetics*; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D006720:Homozygote; D006801:Humans; D009154:Mutation; D017422:Sequence Analysis, DNA / Q000295:instrumentation*
keywords: Congenital anaemia; haemolysis; molecular diagnosis; mutations; next-generation sequencing