Knowledge: Clotting and its disorders.........

Fibrin (also called Factor Ia) is a fibrous, non-globular protein, synthesized by the liver involved in the clotting of blood. It is formed by the action of the protease thrombin on fibrinogen.

(http://mymedicalstudentnotes.com/coagulation/)

The formation of fibrin clots that are relatively resistant to lysis represents the final step in blood coagulation. Factor XIII or fibrin stabilizing factor is an enzyme (EC 2.3.2.13) of the blood coagulation system that crosslinks  fibrin.

(http://www.thrombocyte.com/what-is-fibrin/)

Factor XIII deficiency is a rare clotting protein deficiency which can cause intracranial hemorrhage,  mucosal bleeding, miscarriage, and poor wound healing. Acquired factor XIII deficiency can occur by abnormal activation of the immune system, which produces autoantibodies.

The majority of the deficient patients lack the A subunits which carry the catalytic site.  Patients with B subunit deficiency have a less severe bleeding diathesis, but their FXIII survival is very short,

While traditional Sanger sequencing of exons is currently the standard of care, it has limitations

Sequencing libraries are prepared using a bait capture approach, and sequencing is conducted on a MiSeq platform by Illumina

The library panel was designed  to cover all exons and over 500 nucleotides adjacent to each exon, thus including splice sites, and regulatory regions.  Additionally, a total of 12 introns are completely covered by the sequencing design, as well as 1500 nucleotides both upstream and downstream from the A and B subunit genes

the NGS assay was able to identify a series of mutations that were not picked up using Sanger technology, including mutations in intronic regions, and large entire exonic deletions which were previously suspected

Among the 45 patients, 21 had a homozygous mutation pattern, 19 were compound heterozygotes and there was no new exon coding mutations found in 5 patients

Of the 40 mutations identified, 21 were missense, 5 resulted in stop codon, 3 resulted in frameshift, 1 was a splice site mutation, and 4 had large deletions covering many exons.  Of these mutations, only 12 had been previously reported in the literature.

multiple new variants in the intron and flanking sequences, including insertions and deletions in all patients including those without a mutation detected on Sanger sequencing were found