title: A novel 33-Gene targeted resequencing panel provides accurate, clinical-grade diagnosis and improves patient management for rare inherited anaemias.
abstract: Accurate diagnosis of rare inherited anaemias is challenging, requiring a series of complex and expensive laboratory tests. Targeted next-generation-sequencing (NGS) has been used to investigate these disorders, but the selection of genes on individual panels has been narrow and the validation strategies used have fallen short of the standards required for clinical use. Clinical-grade validation of negative results requires the test to distinguish between lack of adequate sequencing reads at the locations of known mutations and a real absence of mutations. To achieve a clinically-reliable diagnostic test and minimize false-negative results we developed an open-source tool (CoverMi) to accurately determine base-coverage and the 'discoverability' of known mutations for every sample. We validated our 33-gene panel using Sanger sequencing and microarray. Our panel demonstrated 100% specificity and 99·7% sensitivity. We then analysed 57 clinical samples: molecular diagnoses were made in 22/57 (38·6%), corresponding to 32 mutations of which 16 were new. In all cases, accurate molecular diagnosis had a positive impact on clinical management. Using a validated NGS-based platform for routine molecular diagnosis of previously undiagnosed congenital anaemias is feasible in a clinical diagnostic setting, improves precise diagnosis and enhances management and counselling of the patient and their family.
authors.affiliation: BRC Blood Theme and BRC/NHS Translational Molecular Diagnostics Centre, John Radcliffe Hospital, Oxford, UK.|Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.
authors.forename: Noémi B A
authors.initials: NB
authors.lastname: Roy
authors.affiliation: BRC Blood Theme and BRC/NHS Translational Molecular Diagnostics Centre, John Radcliffe Hospital, Oxford, UK.
authors.forename: Edward A
authors.initials: EA
authors.lastname: Wilson
authors.affiliation: BRC Blood Theme and BRC/NHS Translational Molecular Diagnostics Centre, John Radcliffe Hospital, Oxford, UK.
authors.forename: Shirley
authors.initials: S
authors.lastname: Henderson
authors.affiliation: Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.
authors.forename: Katherine
authors.initials: K
authors.lastname: Wray
authors.affiliation: Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.
authors.forename: Christian
authors.initials: C
authors.lastname: Babbs
authors.affiliation: Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.
authors.forename: Steven
authors.initials: S
authors.lastname: Okoli
authors.affiliation: Department of Haematology, Oxford University Hospitals NHS Foundation Trust, Churchill Hospital, Oxford, UK.
authors.forename: Wale
authors.initials: W
authors.lastname: Atoyebi
authors.affiliation: Division of Pediatric Hematology/Oncology, Children's Hospital at Erlanger, Chattanooga, TN, USA.
authors.forename: Avery
authors.initials: A
authors.lastname: Mixon
authors.affiliation: Department of Haematology, Cork University Hospital, Cork, Ireland.
authors.forename: Mary R
authors.initials: MR
authors.lastname: Cahill
authors.affiliation: Department of Haematology, The Royal Victoria Infirmary, Newcastle-upon-Tyne, UK.
authors.forename: Peter
authors.initials: P
authors.lastname: Carey
authors.affiliation: Department of Haematology, Salisbury NHS Foundation Trust, Salisbury, UK.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Cullis
authors.affiliation: Department of Haematology, Sydney Children's Hospitals Network, Westmead, Australia.
authors.forename: Julie
authors.initials: J
authors.lastname: Curtin
authors.affiliation: BRC Blood Theme and BRC/NHS Translational Molecular Diagnostics Centre, John Radcliffe Hospital, Oxford, UK.
authors.forename: Helene
authors.initials: H
authors.lastname: Dreau
authors.affiliation: Nuffield Department of Clinical Laboratory Sciences, John Radcliffe Hospital, University of Oxford, Oxford, UK.
authors.forename: David J P
authors.initials: DJ
authors.lastname: Ferguson
authors.affiliation: Department of Paediatric Haematology/Oncology, Royal Hospital for Children, Glasgow, UK.
authors.forename: Brenda
authors.initials: B
authors.lastname: Gibson
authors.affiliation: Paediatric Haematology/Oncology Unit, Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital, Oxford, UK.
authors.forename: Georgina
authors.initials: G
authors.lastname: Hall
authors.affiliation: BRC Blood Theme and BRC/NHS Translational Molecular Diagnostics Centre, John Radcliffe Hospital, Oxford, UK.
authors.forename: Joanne
authors.initials: J
authors.lastname: Mason
authors.affiliation: Department of Paediatric Haematology-Oncology, University Hospital Southampton, Southampton, UK.
authors.forename: Mary
authors.initials: M
authors.lastname: Morgan
authors.affiliation: BRC Blood Theme and BRC/NHS Translational Molecular Diagnostics Centre, John Radcliffe Hospital, Oxford, UK.
authors.forename: Melanie
authors.initials: M
authors.lastname: Proven
authors.affiliation: Paediatric Haematology/Oncology Unit, Oxford University Hospitals NHS Foundation Trust, John Radcliffe Hospital, Oxford, UK.
authors.forename: Amrana
authors.initials: A
authors.lastname: Qureshi
authors.affiliation: Laboratorio Diagnóstico UGC de Hematología Hospital Universitario Reina Sofía, Córdoba, Spain.
authors.forename: Joaquin
authors.initials: J
authors.lastname: Sanchez Garcia
authors.affiliation: Division of Haemato-Oncology, Department of Paediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok, Thailand.
authors.forename: Nongnuch
authors.initials: N
authors.lastname: Sirachainan
authors.affiliation: Department of Haematology, Sydney Children's Hospitals Network, Westmead, Australia.
authors.forename: Juliana
authors.initials: J
authors.lastname: Teo
authors.affiliation: Department of Paediatrics, Skåne University Hospital, Lund, Sweden.
authors.forename: Ulf
authors.initials: U
authors.lastname: Tedgård
authors.affiliation: Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK.
authors.forename: Doug
authors.initials: D
authors.lastname: Higgs
authors.affiliation: NHS Blood and Transplant, NHSBT - John Radcliffe Hospital, Level 2, Oxford, UK.
authors.forename: David
authors.initials: D
authors.lastname: Roberts
authors.affiliation: Molecular Haematology Unit, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, Oxford, UK. Irene.roberts@paediatrics.ox.ac.uk.
authors.forename: Irene
authors.initials: I
authors.lastname: Roberts
authors.affiliation: BRC Blood Theme and BRC/NHS Translational Molecular Diagnostics Centre, John Radcliffe Hospital, Oxford, UK. anna.schuh@oncology.ox.ac.uk.
authors.forename: Anna
authors.initials: A
authors.lastname: Schuh
mesh_terms: D000740:Anemia / Q000175:diagnosis* / Q000235:genetics*; D019295:Computational Biology / Q000379:methods; D019468:Disease Management; D056726:Genetic Association Studies; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing / Q000379:methods / Q000592:standards; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D007223:Infant; D008297:Male; D009154:Mutation; D020641:Polymorphism, Single Nucleotide; D035583:Rare Diseases; D015203:Reproducibility of Results; D057188:Workflow
keywords: congenital dyserythropoietic anaemia; inherited anaemia; molecular genetics; next-generation sequencing; pyruvate kinase deficiency