F5‐Atlanta: A novel mutation in F5 associated with enhanced East Texas splicing and FV‐short production

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.

F5‐Atlanta: Factor V‐short strikes again

“…The interesting paper by Zimowski et al reports on an African‐American patient with severe bleeding symptoms and massively elevated plasma levels of FV‐short and TFPIα (>20‐fold), in the presence of a heterozygous deletion of 832 bp in F5 exon 13 (c.2413_3244del, F5 ‐Atlanta, Figure 1 ). 5 Although the bleeding mechanism is essentially the same as in the East Texas and Amsterdam disorders, this new case is remarkable for its extreme laboratory and clinical phenotypes and unusual genetic mechanism. The F5 ‐Atlanta deletion introduces a frameshift and premature stop codon in exon 13, preventing the expression of full‐length FV (by nonsense‐mediated decay of its mRNA), but not of FV‐short, where the whole deletion is spliced out as part of the FV‐short‐specific intron.…”

“…While the F5 ‐East Texas mutation strengthens the donor splice site of the FV‐short‐‐specific intron 3 and F5 ‐Amsterdam creates an entirely new donor splice site, 4 F5 ‐Atlanta does not affect the donor or acceptor splice sites, but (most probably) alters the regulatory framework of FV‐short splicing. In fact, the massive up‐regulation of FV‐short expression associated with the F5 ‐Atlanta deletion strongly suggests that the deleted region contains one or more splicing silencers that normally suppress FV‐short splicing 5 . Abolition of these negative regulator(s) would cause relatively more F5 pre‐mRNA transcribed from the F5 ‐Atlanta allele to be processed into FV‐short mRNA, increasing the FV‐short/full‐length FV transcript ratio.…”

“… 1 Their physiological relevance is illustrated by the bleeding tendency associated with the genetic up‐regulation of FV‐short, a minor splicing isoform of FV that preferentially binds TFPIα, as in the East Texas 2 , 3 and Amsterdam 4 bleeding disorders. In this issue, Zimowski et al describe a third and more extreme example of this bleeding mechanism, 5 providing new information on the regulation of FV‐short expression.…”

“…In fact, the massive up‐regulation of FV‐short expression associated with the F5 ‐Atlanta deletion strongly suggests that the deleted region contains one or more splicing silencers that normally suppress FV‐short splicing. 5 Abolition of these negative regulator(s) would cause relatively more F5 pre‐mRNA transcribed from the F5 ‐Atlanta allele to be processed into FV‐short mRNA, increasing the FV‐short/full‐length FV transcript ratio. This could also explain the patient’s overall increased FV level, as the biosynthesis and secretion of FV‐short, which lacks most post‐translational modification sites in the B‐domain, is likely to be more efficient than that of full‐length FV, resulting in relatively more secreted protein (as observed in recombinant expression systems).…”

Antisense-Mediated Down-Regulation of Factor V-Short Splicing in a Liver Cell Line Model

“…FV-short was originally identified in a family with FV-short over-expression due to a F5 gene mutation that enhances FV-short splicing, resulting in 10-fold increased TFPIα levels and a bleeding tendency (East Texas bleeding disorder) [22,27]. More recently, two other F5 gene mutations (F5-Amsterdam [28] and F5-Atlanta [29]) up-regulating FV-short splicing and plasma TFPIα levels have been reported in unrelated patients with similar histories of trauma-related bleeding.…”