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The challenge of genetically unresolved haemophilia A patients: Interest of the combination of whole F8 gene sequencing and functional assays.
Lassalle, Fanny; Jourdy, Yohann; Jouan, Loubna; Swystun, Laura; Gauthier, Julie; Zawadzki, Christophe; Goudemand, Jenny; Susen, Sophie; Rivard, Georges-Etienne; Lillicrap, David.
Affiliation
  • Lassalle F; CHU Lille, Institut d'Hématologie - Transfusion, Pôle de Biologie Pathologie Génétique, Lille, France.
  • Jourdy Y; Univ Lille, Inserm, U1011 - EGID, Institut Pasteur de Lille, Lille, France.
  • Jouan L; Service d'hématologie biologique, Centre de Biologie et Pathologie Est, Hospices Civils de Lyon, France.
  • Swystun L; EA 4609 Hémostase et Cancer, Université Claude Bernard Lyon 1, Lyon, France.
  • Gauthier J; Integrated Centre for Pediatric Clinical Genomics, CHU Sainte Justine, Montreal, Canada.
  • Zawadzki C; Department of Pathology and Molecular Medicine, Queen's University, Kingston, Canada.
  • Goudemand J; Molecular Diagnostic Laboratory and Division of Medical Genetics, Department of Pediatrics, CHU Sainte Justine, Montreal, Canada.
  • Susen S; CHU Lille, Institut d'Hématologie - Transfusion, Pôle de Biologie Pathologie Génétique, Lille, France.
  • Rivard GE; CHU Lille, Institut d'Hématologie - Transfusion, Pôle de Biologie Pathologie Génétique, Lille, France.
  • Lillicrap D; CHU Lille, Institut d'Hématologie - Transfusion, Pôle de Biologie Pathologie Génétique, Lille, France.
Haemophilia ; 26(6): 1056-1063, 2020 Nov.
Article in En | MEDLINE | ID: mdl-33094873
ABSTRACT

BACKGROUND:

The causative variant remains unidentified in 2%-5% of haemophilia A (HA) patients despite an exhaustive sequencing of the full F8 coding sequence, splice consensus sequences, 5'/3' untranslated regions and copy number variant (CNV) analysis. Next-generation sequencing (NGS) has provided significant improvements for a complete F8 analysis.

AIM:

The aim of this study was to identify and characterize pathogenic non-coding variants in F8 of 15 French and Canadian HA patients genetically unresolved, through the use of NGS, mRNA sequencing and functional confirmation of aberrant splicing.

METHODS:

We sequenced the entire F8 gene using an NGS capture method. We analysed F8 mRNA in order to detect aberrant transcripts. The pathogenic effect of candidate intronic variants was further confirmed using a minigene assay.

RESULTS:

After bioinformatic analysis, 11 deep intronic variants were identified in 13 patients (8 new variants and 3 previously reported). Three variants were confirmed to be likely pathogenic with the presence of an aberrant transcript during mRNA analysis and minigene assay. We also found a small intronic deletion in 6 patients, recently described as causing mild HA.

CONCLUSION:

With this comprehensive work combining NGS and functional assays, we report new deep intronic variants that cause HA through splicing alteration mechanism. Functional analyses are critical to confirm the pathogenic effect of these variants and will be invaluable in the future to study the large number of variants of uncertain significance that may affect splicing that will be found in the human genome.
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Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Factor VIII / Computational Biology / High-Throughput Nucleotide Sequencing / Hemophilia A Type of study: Prognostic_studies Limits: Female / Humans / Male Language: En Journal: Haemophilia Journal subject: HEMATOLOGIA Year: 2020 Type: Article Affiliation country: France

Full text: 1 Collection: 01-internacional Database: MEDLINE Main subject: Factor VIII / Computational Biology / High-Throughput Nucleotide Sequencing / Hemophilia A Type of study: Prognostic_studies Limits: Female / Humans / Male Language: En Journal: Haemophilia Journal subject: HEMATOLOGIA Year: 2020 Type: Article Affiliation country: France