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Researchers Identify How Variant Acts Through Intermediaries to Lead to Bleeding Disorder

NEW YORK (GenomeWeb News) – Acting through a number of steps, a mutation in the coagulation-related F5 gene leads to a bleeding disorder, according to researchers from the University of Texas Health Sciences Center and Lund University in Sweden.

As the investigators reported in the Journal of Clinical Investigation today, that mutation leads the F5 gene to be alternatively spliced, resulting in a truncated protein. That short protein, in turn, associates with another protein involved in coagulation, keeping it in the bloodstream and leading to a bleeding disorder that has been plaguing multiple generations of an east Texas family.

In 2001, a study of this family noted that despite their symptoms of a bleeding disorder — such as bruising and hemorrhaging, among others — family members' coagulation factors tests came back with normal results, though they did have prolonged prothrombin time or activated partial thromboplastin time.

A linkage analysis pointed toward a locus near the antithrombin gene that differed between those with and without the disorder, though no variant was identified at first. Researchers subsequently narrowed in on a region of the F5 gene and then further to a variant in exon 13. Coagulation factor V, or FV, which is encoded by the F5 gene, can both promote and inhibit coagulation by interacting with thrombin and active factor X.

But that finding was presumed to not be disease-causing as the domain where the mutation was located is not needed for FV activity and because affected family members had normal FV clotting findings. It was, researchers surmised then, a private, familial variant.

However, in this new study, UTHSC's Lisa Vincent and her colleagues noticed that affected family members with the F5 variant had shorter FV proteins as well as shorter FV transcripts, suggesting that the variant might not be so benign after all.

"We have identified what we believe is the first autosomal dominant bleeding disorder due to a gain-of-function mutation in the gene encoding coagulation FV," Vincent and her colleagues wrote.

In addition, they noted that since the shortened FV isoform binds to and keeps TFPIα — a regulator of coagulation — in circulation, current work on TFPI inhibitors could offer a possible therapy for the affected family members.

Through an immunoblotting assay, Vincent and her colleagues determined that affected family members had shorter-than-usual factor V proteins while unaffected family members had usual-length proteins, a finding they confirmed through a mass spec analysis.

Only family members with the A2440G variant in the F5 gene had that short version of factor V. Further, RT-PCR analysis indicated that unaffected family members had the expected 2,946-basepair transcript while affected family members had a smaller, 840-basepair transcript. Some unaffected family members did have low levels of the smaller transcript, the researchers noted.

By sequencing the smaller transcript, the researchers uncovered an in-frame deletion of some 2,100 basepairs, indicating that FV-short is due to a splice variant.

The A2440G variant appeared to lead to the upregulation of that alternatively spliced transcript, the investigators added. As compared to controls, individuals with the A2440G variant had an average 22-fold increase of FV-short transcript expression levels, as determined by RT-qPCR, the researchers noted.

"[O]ur results support the conclusion that a splicing event in exon 13 occurs normally to a low degree in controls and the efficiency of this splicing event is increased by the presence of the A2440G mutation," Vincent and her colleagues noted.

On the protein side, the splicing variant and truncated transcripts lead to the deletion of 702 amino acids from the factor V protein. The protein, though, the researchers pointed out, can still be activated by thrombin.

Instead, FV-short works indirectly to inhibit coagulation, and it associates with TFPIα in affected family members, the researchers said. Indeed, TFPIα appeared to bind preferentially to FV-short rather than FV-long. This complex then lingers in the bloodstream.

"Presumably the binding of TFPIα to FV-short in affected family members’ plasma results in its retention in circulation, thus causing the higher TFPIα plasma concentrations," the researchers added.

Higher TFPIα levels in the plasma — which the investigators reported could be up to 10 times higher in cases than in controls — likely leads to the inhibition of coagulation.

The identification of TFPIα as part of the chain of events leading to the bleeding disorder could offer some hope for the east Texas family. "[T]he elucidation of the underlying pathophysiology suggests that TFPI inhibitors currently in development may provide a means of treatment," added George Broze and Thomas Girard from the Washington University School of Medicine in St. Louis in an accompanying commentary in JCI.