Genetic Background in Patients with Severe Factor XIII A-Subunit Deficiency Treated with Recombinant FXIII

Congenital Factor XIII (FXIII) deficiency is a rare, autosomal recessive bleeding disorder, with a significant majority of patients showing defects in the FXIII-A subunit. The disease is caused by a variety of F13A1 gene mutations resulting in a severe quantitative FXIII-A type I deficiency. Here, we report a wide spectrum of mutations identified in 41 severe Factor XIII-A deficiency patients (≥6 years of age, mean, 26.4; range, 7–60).

A total of 41 patients were recruited in a multinational (23 centers, 11 countries), open-label, single-arm, phase 3, prophylaxis trial for the evaluation of the efficacy and safety of a novel recombinant FXIII (rFXIII). Eleven of the 41 patients (Israel [n=8], Switzerland [n=2] and Italy [n=1]) already had established genetic backgrounds and carried previously reported F13A1 mutations. Mutational screening in the remaining 30 patients who had unknown genetic status was done using direct sequencing on an ABI Prism 3130TL (Applied Biosystems, Weiterstadt, Germany). For two patients with splice-site mutations, cDNA analysis was done with RT-PCR (Quiagen One-step RT-PCR kit). The crystallographic model of the recombinant human cellular coagulation FXIIIA zymogen (EC: 2.3.2.13, resolution solved to 2.1Å) was downloaded from the Protein Data Bank (data file 1F13) for viewing, analysis and graphical rendering using YASARAver11.11.2. Classic molecular dynamic simulation approaches were used on the FXIII-A crystal structure to evaluate the effect of the novel missense mutations on protein structure.

In total, 31 distinct mutations in 41 patients have been identified revealing 13 missense mutations, seven small deletions, six splice-site mutations, three nonsense mutations, one large deletion and one small insertion. Amongst this cohort of mutations, 16 mutations were novel. In one patient, a heterozygous missense mutation was detected in spite of severe deficiency symptoms shown by the patient. We assume that the other mutation could not be detected within the scope of our screening set up. The IVS5–1G>A (c.691–1G>A) splice-site mutation was the most commonly occurring (n=9 [21.9%]) mutation in this cohort. Two siblings carried a missense mutation in F13A1 gene (Ser295Arg) and in combination with a novel variant in F13B gene (Ser634Phe). This variant does not seem to significantly affect the B-subunit stability, since it is located in the terminal part of the molecule. Missense mutations are of special interest since they help to better understand the structure and function of FXIII A-subunit. The MD simulation of four novel missense mutations predicted a damaging effect on the protein structure for three of the missense mutations (Glu229Arg, Leu275Phe, and Arg703Trp) based on changes in bonding patterns, free energy calculations, change in local secondary structure etc. The Leu588Gln located on the surface of barrel 1 domain was not observed to cause a significant change in local structure, nevertheless, owing to its surface presentation it might influence the interaction with FXIII B-subunit. The analysis for the two novel splice-site mutations (IVS7+1G>A, IVS12+1G>A) did not show any aberrant mRNA product. Inhibitor development is the most undesirable side-effect of treatment with plasma-derived or rFXIII products. Overall, the incidence of inhibitors in patients with congenital FXIII deficiency is unknown, but is expected to be much lower compared with other coagulopathies, e.g. hemophilia A. In the present study, none of the patients treated with rFXIII (a mean treatment period of 322 days) developed FXIII neutralizing antibodies. Four patients developed transient low-titer anti-rFXIII antibodies that had no neutralizing activity. Interestingly, two patients (male and female) were siblings carrying the same common splice-site mutation in intron 5 (IVS5-1 G>A). The third patient was compound heterozygous for two missense mutations (Gln229Arg; Ser413Leu). The fourth patient was also found to be compound heterozygous for two missense mutations (Arg77His; Leu275Phe).

To conclude, the identified mutations, including 16 novel mutations, are implicated in the causality of severe FXIII deficiency. However, none of these mutations were associated with the development of inhibitory antibodies in the context of treatment with rFXIII.

Tehranchi:Novo Nordisk: Employment, Equity Ownership. Oldenburg:Biogen Idec, Swedish Orphan Biovitrum: Honoraria; Baxter, Bayer, Biotest, CSL Behring, Grifols, Inspiration, Novo Nordisk, Octapharma, Pfizer: Honoraria, Research Funding.