Abstract
Coagulation factor V (FV) is procofactor essential to normal hemostasis. Recently, a point mutation in exon 13 of the F5 gene (FV-East Texas; FV-ET), which encodes the B-domain, has been shown to result in an inherited moderate bleeding diathesis termed East Texas Bleeding Disorder. The mutation (F5 c. A2350G, according to the latest annotated reference sequence for the F5 transcript, NM_000130.4, originally reported as A2440G) greatly enhances alternative splicing at an otherwise weak splice site, which leads to the production of a new FV isoform, FV-short. FV-short, via an exposed acidic region, binds with high affinity to tissue factor pathway inhibitor alpha (TFPIα) and is thought to stabilize the inhibitor in blood thereby increasing its concentration via changes in clearance mechanisms. High levels of TFPIα as well as other regulatory mechanisms contribute to inhibition of thrombin generation leading to a clinical bleeding phenotype. Another family with a different point mutation (FV-Amsterdam, F5 c.C2588G) downstream from FV-ET was recently identified and found to have similar clinical and laboratory characteristics. A 43 year-old male with a history of recurrent intramuscular hematomas underwent laboratory investigation to identify the etiology of his bleeding tendency. Laboratory testing revealed a prolonged PT and aPTT that failed to correct after 50:50 mixing with normal human plasma (NHP) and normal activity levels of factors II, V, VII, VIII, IX, X, XI, XII, and XIII. A lupus anticoagulant was not detected. A thrombin generation assay revealed a prolonged lag time versus control plasma (20 min vs. 6 min). These findings were similar to FV-ET, and further studies revealed that the concentration of total TFPI and TFPIα were elevated in the patient. Surprisingly however, the levels of TFPI were much higher compared to those measured in FV-ET. Relative to NHP, total TFPI and TFPIα were increased 11-and 22-fold, respectively for the patient compared to 4- and 5-fold for FV-ET (Table 1). Remarkably, a point mutation in exon 13 was not identified. Rather, analysis performed on genomic DNA identified a heterozygous 832 base pair (bp) deletion within exon 13 (c.2413-3244del). This deletion is predicted to result in the omission of 277 amino acids preceding a frameshift and translation of 12 non-native amino acids followed by a stop codon.While this alteration should result in a functionally null allele, causing the patient to have 50% FV levels, the clinical and biochemical findings do not align with this genomic data. Western blotting of the patient's plasma using anti-FV heavy or light chain antibodies revealed the presence of full length (FL)-FV (330 kDa) protein as well as a strong band at 250 kDa representing FV-short. Using recombinant FV-short as a control, quantitative western blotting revealed the patient has very high levels of FV-short (50 nM) compared to FV-ET plasma (5-7 nM; Table 1). To reconcile these data with the genetic information, we performed RT-PCR analysis of blood cell RNA. Several different FV transcripts were identified including a putative FL-FV transcript, a transcript containing the 832 bp frameshift deletion, and a FV-short transcript identical to that from FV-ET. The latter finding was unexpected as the 832 bp deletion is located 3' to the FV-ET donor splice site. We hypothesize that encoded within this novel 832 bp deletion is a sequence that functions as a cis-acting element which regulates FV-short production under normal physiologic conditions. FV-short is constituently expressed to a minor degree in unaffected individuals (~0.2-0.4 nM). Removal of this 832 bp sequence through genetic deletion is predicted to facilitate upregulation of FV-short biosynthesis via enhanced splicing at the FV-ET donor/acceptor splice sites. The identification of the FV-Atlanta deletion and the FV-ET and FV-Amsterdam point mutations highlight a complicated mechanism by which FV-short levels are modulated by alternative splicing. Since FV-short has a major influence on TFPIα levels, these collective findings are clinically relevant and may represent an additional level of regulation existing in the initiation and propagation of coagulation.
Zimowski: National Hemophilia Foundation/Shire: Other: Clinical Fellowship Award Recipient. Denning: Expression Therapeutics: Employment. Jhita: Expression Therapeutics: Employment. Rivera: Expression Therapeutics: Employment. Escobar: Bayer: Honoraria, Membership on an entity's Board of Directors or advisory committees; Shire: Honoraria, Membership on an entity's Board of Directors or advisory committees; CSL Behring: Honoraria, Membership on an entity's Board of Directors or advisory committees; Genentech: Honoraria, Membership on an entity's Board of Directors or advisory committees; NovoNordisk: Honoraria, Membership on an entity's Board of Directors or advisory committees. Kempton: Genentech: Membership on an entity's Board of Directors or advisory committees. Camire: Bayer: Consultancy, Research Funding; Pfizer: Consultancy, Patents & Royalties, Research Funding; Novo Nordisk: Research Funding; Spark Therapeutics: Membership on an entity's Board of Directors or advisory committees.
