Identification of New Deep Intronic Variants Causing Hemophilia A by Whole F8 Gene Sequencing, mRNA Analysis and Functional Confirmation, in “Variant-Negative” French and Canadian Patients
ISTH Academy. Lassalle F. Jul 9, 2019; 264604; PB1414 Topic: Hemophilia - Clinical
Fanny Lassalle
Fanny Lassalle
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Identification of New Deep Intronic Variants Causing Hemophilia A by Whole F8 Gene Sequencing, mRNA Analysis and Functional Confirmation, in 'Variant-Negative' French and Canadian Patients

F. Lassalle1, L. Jouan2, Y. Jourdy3, L. Swystun4, J. Gauthier5, C. Zawadzki1, J. Goudemand1, S. Susen1, G.-E. Rivard6, D. Lillicrap4
1Lille University Hospital, Department of Hematology and Transfusion, Lille, France, 2Sainte-Justine University Hospital, Integrated Centre for Pediatric Clinical Genomics, Montreal, Canada, 3Hospices Civils de Lyon, Department of Hematology, Lyon, France, 4Queen's University, Department of Pathology and Molecular Medicine, Kingston, Canada, 5Sainte-Justine University Hospital, Department of Pediatrics, Montreal, Canada, 6Sainte-Justine University Hospital, Department of Hematology and Oncology, Montreal, Canada

Main Topic: Hemophilia and Bleeding (including Transfusion)
Category: Hemophilia - Clinical

Background: The causative variant remains unidentified in 2-5% of hemophilia A (HA) patients despite an exhaustive sequencing of the full F8 coding sequence. Next-generation sequencing (NGS) has provided significant improvements for a complete F8 gene analysis. Although rare, the pathogenic role of deep intronic variants has been documented, in particular relating to their consequences on splicing.
Aims: We aimed to characterize intronic variants in the F8 gene of French and Canadian HA patients with no pathogenic variant identified in the F8 coding region, using NGS, mRNA sequencing and functional confirmation.
Methods: The entire F8 locus (195kb) of 19 patients was sequenced using an NGS capture-method. When available, the F8 mRNA extracted from leukocytes was analyzed in order to detect aberrant transcripts. The pathogenic effect of candidate intronic variants was confirmed by minigene splicing reporter assay.
Results: We confirmed the pathogenic effect of 3 of 14 deep intronic variants that remained after NGS analysis. Two of the variants (intron 13: c.2113+1153G>C; intron 14: c.5220-8563A>G) are associated with an aberrant intronic insertion in the patient's cDNA, leading to the generation of a premature stop codon, thus a truncated protein, and one variant (intron 18: c.5999-27A>G) with an exon skipping event. Both variants were confirmed with mRNA sequencing and minigene analysis. No impact on splicing was found with the minigene assay for 2 candidate pathogenic variants despite good splicing scores in the in-silico analysis with Alamut® Visual. Eight variants remained of uncertain significance.
Conclusions: We report new deep intronic variants responsible for HA by altering the splicing process. Functional analyses are essential to confirm the pathogenic effect of these variants. Confirmatory studies such as these will be invaluable to study the large number of variants of uncertain significance that will be found in the human genome. This study evaluated an approach to those variants that might alter splicing.

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