Identification and Characterization of L1-Mediated Large Tandem Duplication, Spanning Exon 4 to 10 of the F13A1, In a Patient with Congenital Factor XIII Deficiency.

Abstract 3659

Blood coagulation factor XIII (FXIII) is a plasma glycoprotein that plays an important role in the stabilization of fibrin clot in the final stage of blood coagulation. The FXIII circulates as a heterotetramer composed of two A and two B subunits in plasma. The A subunit (FXIIIA) possesses catalytic activity and this catalytic subunit is carried and protected by the B subunit (FXIIIB). Inherited deficiency of FXIII is a rare autosomal recessive bleeding disorder. Based on the genotype, it is classified into two types: FXIIIA deficiency (>95% of all cases), characterized by mutations in the F13A1 gene, and FXIIIB deficiency, characterized by mutations in the F13B gene. At the time of writing, at least 86 different mutations, most of which are point mutations, have been identified and registered in the F13-database. Here we show a novel, large tandem duplication in the F13A1 gene of a patient with congenital factor XIII deficiency. A female patient, born to consanguineous parents, suffered from severe bleeding diathesis, including menorrhagia, intracranial hemorrhage and ovarian hemorrhage, from childhood. Bleeding manifestations had been successfully controlled by monthly prophylactic replacement therapy using factor XIII concentrate (Fibrogammin). Trough levels of both factor XIII activity and antigen were 19% (Berichrom FXIII) and <10% (ELISA method), respectively. No pathogenic mutations associated with FXIII deficiency were detected from nucleotide sequencing of the coding region, 5′-UTR and 3′-UTR of both F13A1 and F13B. However, from an observation of the RT-PCR amplification state, the F13A1 mRNA level of the patient was apparently lower than that of healthy individuals. This result suggested the existence of abnormalities in the patient's F13A1. Relative exon copy number analysis using real-time PCR revealed two times as many of the continuing 7 exons (exon 4–10) as in the remaining region of the F13A1. This is likely due to some genomic rearrangement, probably homologous recombination in the F13A1 gene. IVS-3 and IVS-10 of F13A1 were very large and contained many repetitive sequences. The existence of an almost full-length (≂f6kb) L1 element, a well known long interspersed repetitive element (LINE) in humans, in both introns suggests that the recombination might arise from the L1 element. The provision of the PCR product (amplified by an IVS-10 specific forward primer and an IVS-3 specific reverse primer) confirmed that IVS-10 connected to IVS-3 with L1 as the boundary. Furthermore, sequencing this PCR product identified the 15bp sequence in the L1 element as an actual breakpoint. Taken together with the results of the analysis of the genomic DNA, this confirmed that the L1-mediated large (≂f109kb) tandem duplication was located in the patient's F13A1. In order to quantify the F13A1 mRNA, a relative real-time PCR quantification was performed using TaqMan Gene Expression Assays. Three different positions: one upstream of duplication (Exon2-3), one at duplication (Exon5-6) and one downstream of the duplication (Exon13-14) were used. The mRNA level of the patient was a markedly low compared to the normal control and the duplicated region (28% of normal) was clearly higher than both the upstream and downstream positions (9% of normal for each). This result reflected that the mRNA was probably maintaining the duplication. In order to analyze the mRNA splicing of the joint between the two duplicated regions, RT-PCR using a forward primer in exon 9 and a reverse primer in exon 4 of F13A1 was performed. Two major transcripts were amplified. The larger transcript was the product that keeps the genomic exon order and connects exon 10 to 4. However, the transcript is thought to lead to frameshift and to generate premature termination codon in exon 4. The other was the product that was skipping exon 10 and connected exon 9 to 4. This transcript is thought to escape frameshift and may translate to the unusual extra large FXIIIA. However, it is unlikely that the protein translated from the extra large mRNA circulates in blood. In conclusion, we identified an L1-mediated large tandem duplication, spanning exon 4 to 10 of the F13A1 gene, as an etiology of the congenital FXIII deficiency.