Gene Analysis of 45 FXIII Deficient Patients in the US: An in Depth Whole Gene NGS Sequencing Analysis of FXIII a and B Subunit Mutations

Factor XIII deficiency is a rare clotting protein deficiency characterized by a very high risk of spontaneous intracranial hemorrhage (ICH), mucosal bleeding, miscarriage, and poor wound healing. The majority of FXIII 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, reduced to 6-8 hours as compared to a normal FXIII half-life of 6-10 days.

The striking finding for FXIII deficiency is that nearly every patient appears to have unique combinations of mutations resulting in a wide spectrum of bleeding severity, even within the same family. This is particularly true in the US where about a third of the families demonstrate a compound heterozygous pattern and only 50% are homozygous for the same mutation, usually associated with a history of consanguinity or geographic isolation. Sequencing of FXIII A and B subunit mutations is used clinically to initiate appropriate treatment and as a research tool to further our understanding of genotype-phenotype associations. While traditional Sanger sequencing of exons is currently the standard of care, this approach is time consuming, expensive, and 15-20% of FXIII deficient patients do not show any mutations in the exon coding regions. Therefore, we evaluated a whole gene sequencing approach to improve accuracy and turn-around time to diagnosis.

Methods: Generation of Next Generation Sequencing panel for FXIII A and B subunits. Sequencing libraries were prepared using a bait capture approach, and sequencing was conducted on a MiSeq platform by Illumina using standard methodologies. 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.

Results: We compared the results of 45 FXIII deficient patients previously sequenced in our laboratory using a Sanger approach to those results obtained using the NGS assay. The NGS assay confirmed all exonic mutations previously detected using Sanger sequencing. Additionally, 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, but could not be confirmed using Sanger sequencing alone. 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.

Conclusion: We report the results of the largest group of USA FXIII deficient patients to be sequenced thus far, and compare the genotypes obtained from both a Sanger Sequencing and NGS approach. Using NGS whole gene sequencing , we found 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. These are currently being analyzed as possible causative mutations in the 5 patients without exonic mutations and as potential modifying variants in patients with heterozygous mutations. NGS whole gene sequencing will give us more information than currently available on FXIII genome sequence and provide a rapid, cost effective assay to analyze future patients.

Funded in part through an Investigator Initiated Award to Dr Diane Nugent from Novo Nordisk