Karen L Zimowski1, Teodolinda Petrillo2, Michelle D Ho2, Julie Wechsler1, Jordan E Shields1, Gabriela Denning3, Navdeep Jhita3, Angel A Rivera3, Miguel A Escobar4, Christine L Kempton5, Rodney M Camire2,6, Christopher B Doering1. 1. Aflac Cancer and Blood Disorders Center, Emory University/Children's Healthcare of Atlanta, Atlanta, Georgia, USA. 2. The Children's Hospital of Philadelphia, The Raymond G. Perelman Center for Cellular and Molecular Therapeutics, Philadelphia, Pennsylvania, USA. 3. Expression Therapeutics, Tucker, Georgia, USA. 4. University of Texas Houston Health Science Center, Houston, Texas, USA. 5. Department of Hematology and Medical Oncology, Emory University School of Medicine, Atlanta, Georgia, USA. 6. Division of Hematology, Department of Pediatrics, Perelman School of Medicine, The University of Pennsylvania, Philadelphia, Pennsylvania, USA.
Abstract
BACKGROUND: Elucidating the molecular pathogenesis underlying East Texas bleeding disorder (ET) led to the discovery of alternatively spliced F5 transcripts harboring large deletions within exon 13. These alternatively spliced transcripts produce a shortened form of coagulation factor V (FV) in which a large portion of its B-domain is deleted. These FV isoforms bind tissue factor pathway inhibitor alpha (TFPIα) with high affinity, prolonging its circulatory half-life and enhancing its anticoagulant effects. While two missense pathogenic variants highlighted this alternative splicing event, similar internally deleted FV proteins are found in healthy controls. OBJECTIVE: We identified a novel heterozygous 832 base pair deletion within F5 exon 13, termed F5-Atlanta (F5-ATL), in a patient with severe bleeding. Our objective is to investigate the effect of this deletion on F5 and FV expression. METHODS & RESULTS: Assessment of patient plasma revealed markedly elevated levels of total and free TFPI and a FV isoform similar in size to the FV-short described in ET. Sequencing analyses of cDNA revealed the presence of a transcript alternatively spliced using the ET splice sites, thereby removing the F5-ATL deletion. This alternative splicing pattern was recapitulated by heterologous expression in mammalian cells. CONCLUSIONS: These findings support a mechanistic model consisting of cis-acting regulatory sequences encoded within F5 exon 13 that control alternative splicing at the ET splice sites and thereby regulate circulating FV-short and TFPIα levels.
BACKGROUND: Elucidating the molecular pathogenesis underlying East Texas bleeding disorder (ET) led to the discovery of alternatively spliced F5 transcripts harboring large deletions within exon 13. These alternatively spliced transcripts produce a shortened form of coagulation factor V (FV) in which a large portion of its B-domain is deleted. These FV isoforms bind tissue factor pathway inhibitor alpha (TFPIα) with high affinity, prolonging its circulatory half-life and enhancing its anticoagulant effects. While two missense pathogenic variants highlighted this alternative splicing event, similar internally deleted FV proteins are found in healthy controls. OBJECTIVE: We identified a novel heterozygous 832 base pair deletion within F5 exon 13, termed F5-Atlanta (F5-ATL), in a patient with severe bleeding. Our objective is to investigate the effect of this deletion on F5 and FV expression. METHODS & RESULTS: Assessment of patient plasma revealed markedly elevated levels of total and free TFPI and a FV isoform similar in size to the FV-short described in ET. Sequencing analyses of cDNA revealed the presence of a transcript alternatively spliced using the ET splice sites, thereby removing the F5-ATL deletion. This alternative splicing pattern was recapitulated by heterologous expression in mammalian cells. CONCLUSIONS: These findings support a mechanistic model consisting of cis-acting regulatory sequences encoded within F5 exon 13 that control alternative splicing at the ET splice sites and thereby regulate circulating FV-short and TFPIα levels.
Authors: Julie A Peterson; Sweta Gupta; Nicholas D Martinez; Brandon Hardesty; Susan A Maroney; Alan E Mast Journal: J Thromb Haemost Date: 2021-12-07 Impact factor: 5.824