Novel Missense Mutations Associated with FXIII Deficiency and Bleeding.

Abstract 4201 Introduction Factor XIII deficient patients may present at any age, with a variety of bleeding symptoms, poor wound healing, and in females, frequent miscarriages. Factor XIII (FXIII) is a transglutaminase enzyme that was first discovered as a clotting protein in the coagulation cascade, but it is now understood that it cross-links proteins in the plasma, vascular matrix, endothelial cells, platelets and monocytes. In addition to maintaining normal hemostasis, FXIII plays a role in atherosclerosis, wound healing, inflammation, and pregnancy. FXIII circulates in plasma as a tetramer protein (FXIII-A 2 B 2 ) held together by non-covalent bonds. FXIII has two catalytic A subunits (FXIII-A 2 ) of 83kd and two non-catalytic B subunits or carrier subunits (FXIII-B 2 ) of 79kd. Mutations have been identified in almost every exon of the FXIIIA subunit and often are unique to a particular cohort or family with Factor XIII deficiency. Our center has been characterizing patients with FXIII deficiency and has an IRB approved study to characterize bleeding phenotype in relation to genotype or mutational analysis. As part of this effort, we have identified 3 novel missense mutations, which we have not found in the FXIII database ( ) or in previous publications. Methods After obtaining informed consent, venous blood was collected in EDTA tubes for DNA isolation, PCR and ultimately DNA sequencing. DNA was isolated using QIAamp DNA Blood Midi Kit (Qiagen, Germantown, MD). Customary PCR was used to amplify the 15 exons for subunit A and the 12 exons for subunit B, using sequence specific primers based on previous publication and created to initiate outside of the encoding sequence. The nucleotide sequencing of amplified products was obtained via ABI 3730 DNA Analyzer (UCLA Sequencing and Genotyping Core). Results All three novel mutations were found in three, separate, unrelated individuals, with FXIII deficiency diagnosed early in life with a moderate to severe bleeding. Using the methods described above, the DNA sequencing and analysis for all exons for both the A and B subunits revealed three novel mutations, two on exon 12 subunit A and one on exon 10 subunit A. Patient 1 has a novel missense mutation in exon 10 at the 427 amino acid position, changing the aspartic acid into an asparagine (Asp427Asn) in the catalytic core. Patient 2 and 3 each had a unique mutation in exon 12. Exon 12 covers the transition from the catalytic core region and the Barrel 1 region of the FXIIIA molecule thus including portions of each functional region. Patient 2: An exon 12 missense mutation in aa 501 resulting in a change from glycine to an arginine (Gly501Arg) still in the catalytic core region. Patient 3 also had a mutation in exon 12 but in position 576 resulting in an amino acid change from threonine to methionine (Thr576Met) now in the Barrel 1 region. Patient 2 also had a missense mutation that has been previously reported in exon 3 (Arg77His) in the β-sandwich region. Three new missense mutations have been identified in patients with severe Factor XIII deficiency and a bleeding disorder. Previous reports of other point mutations in the FXIIIA catalytic core and barrel 1 regions have also been described in association with a hemorrhagic state in deficient patients. Ongoing protein expression studies will aid in our understanding of how these single amino acid substitutions result in such a serious bleeding diathesis. Disclosures: No relevant conflicts of interest to declare.