title: Application of a molecular diagnostic algorithm for haemophilia A and B using next-generation sequencing of entire F8, F9 and VWF genes.
abstract: Currently, molecular diagnosis of haemophilia A and B (HA and HB) highlights the excess risk-inhibitor development associated with specific mutations, and enables carrier testing of female relatives and prenatal or preimplantation genetic diagnosis. Molecular testing for HA also helps distinguish it from von Willebrand disease (VWD). Next-generation sequencing (NGS) allows simultaneous investigation of several complete genes, even though they may span very extensive regions. This study aimed to evaluate the usefulness of a molecular algorithm employing an NGS approach for sequencing the complete F8, F9 and VWF genes. The proposed algorithm includes the detection of inversions of introns 1 and 22, an NGS custom panel (the entire F8, F9 and VWF genes), and multiplex ligation-dependent probe amplification (MLPA) analysis. A total of 102 samples (97 FVIII- and FIX-deficient patients, and five female carriers) were studied. IVS-22 screening identified 11 out of 20 severe HA patients and one female carrier. IVS-1 analysis did not reveal any alterations. The NGS approach gave positive results in 88 cases, allowing the differential diagnosis of mild/moderate HA and VWD in eight cases. MLPA confirmed one large exon deletion. Only one case did have no pathogenic variants. The proposed algorithm had an overall success rate of 99 %. In conclusion, our evaluation demonstrates that this algorithm can reliably identify pathogenic variants and diagnose patients with HA, HB or VWD.
authors.affiliation: José María Bastida Bermejo, Unidad de Hemostasia y Trombosis, Servicio de Hematología, Hospital Universitario de Salamanca-IBSAL, Paseo de San Vicente 58-182, 37007 Salamanca, Spain, E-mail: jmbastida@saludcastillayleon.es.
authors.forename: Jose Maria
authors.initials: JM
authors.lastname: Bastida
authors.forename: Jose Ramon
authors.initials: JR
authors.lastname: González-Porras
authors.forename: Cristina
authors.initials: C
authors.lastname: Jiménez
authors.forename: Rocio
authors.initials: R
authors.lastname: Benito
authors.forename: Gonzalo R
authors.initials: GR
authors.lastname: Ordoñez
authors.forename: Maria Teresa
authors.initials: MT
authors.lastname: Álvarez-Román
authors.forename: M Elena
authors.initials: ME
authors.lastname: Fontecha
authors.forename: Kamila
authors.initials: K
authors.lastname: Janusz
authors.forename: David
authors.initials: D
authors.lastname: Castillo
authors.forename: Rosa María
authors.initials: RM
authors.lastname: Fisac
authors.forename: Luis Javier
authors.initials: LJ
authors.lastname: García-Frade
authors.forename: Carlos
authors.initials: C
authors.lastname: Aguilar
authors.forename: María Paz
authors.initials: MP
authors.lastname: Martínez
authors.forename: Nuria
authors.initials: N
authors.lastname: Bermejo
authors.forename: Sonia
authors.initials: S
authors.lastname: Herrero
authors.forename: Ana
authors.initials: A
authors.lastname: Balanzategui
authors.forename: Jose Manuel
authors.initials: JM
authors.lastname: Martin-Antorán
authors.forename: Rafael
authors.initials: R
authors.lastname: Ramos
authors.forename: Maria Jose
authors.initials: MJ
authors.lastname: Cebeiro
authors.forename: Emilia
authors.initials: E
authors.lastname: Pardal
authors.forename: Carmen
authors.initials: C
authors.lastname: Aguilera
authors.forename: Belen
authors.initials: B
authors.lastname: Pérez-Gutierrez
authors.forename: Manuel
authors.initials: M
authors.lastname: Prieto
authors.forename: Susana
authors.initials: S
authors.lastname: Riesco
authors.forename: Maria Carmen
authors.initials: MC
authors.lastname: Mendoza
authors.forename: Ana
authors.initials: A
authors.lastname: Benito
authors.forename: Ana
authors.initials: A
authors.lastname: Hortal Benito-Sendin
authors.forename: Víctor
authors.initials: V
authors.lastname: Jiménez-Yuste
authors.forename: Jesus Maria
authors.initials: JM
authors.lastname: Hernández-Rivas
authors.forename: Ramon
authors.initials: R
authors.lastname: García-Sanz
authors.forename: Marcos
authors.initials: M
authors.lastname: González-Díaz
authors.forename: Maria Eugenia
authors.initials: ME
authors.lastname: Sarasquete
mesh_terms: D000465:Algorithms; D004252:DNA Mutational Analysis; D003937:Diagnosis, Differential; D005164:Factor IX / Q000235:genetics*; D005169:Factor VIII / Q000235:genetics*; D005260:Female; D005819:Genetic Markers; D020022:Genetic Predisposition to Disease; D006467:Hemophilia A / Q000097:blood / Q000175:diagnosis* / Q000235:genetics*; D002836:Hemophilia B / Q000097:blood / Q000175:diagnosis* / Q000235:genetics*; D006579:Heterozygote; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008297:Male; D025202:Molecular Diagnostic Techniques; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D010641:Phenotype; D011237:Predictive Value of Tests; D015203:Reproducibility of Results; D014841:von Willebrand Factor / Q000235:genetics*
chemical_list: D005819:Genetic Markers; D014841:von Willebrand Factor; C078147:F8 protein, human; D005169:Factor VIII; D005164:Factor IX
keywords: Haemophilia A; haemophilia B; molecular diagnosis; next-generation sequencing; von Willebrand disease