Characterization of the genetic basis of FXI deficiency in two Turkish patients

Factor XI (FXI) is a haemostatic plasma glycoprotein circulating as a zymogen of the serine protease FXIa in non-covalent interaction with cofactor high molecular weight kininogen. FXIa is required for the activation of factor IX (FIX) to maintain thrombin production during the coagulation process. FXI circulates in plasma as a disulphide-bound homodimer (160 kDa); each monomer is composed of four apple domains (A1–A4) and a C-terminal catalytic domain (SP). The FXI gene (F11) maps on chromosome 4 (4q35.2), and is composed of 15 exons, spanning a genomic region of about 23 kb. Congenital FXI deficiency (MIM *264900) is an autosomal disorder characterized by bleeding, mainly after trauma or surgery. While rare in most populations (1:1 000 000), the disease is frequent among Ashkenazi Jews. Patients typically present with decreased FXI functional activity associated with normal PT and platelet count [1]. So far, more than 180 mutations have been described in F11 (see http://www.isth.org/Publications/RegistriesDatabases/MutationsRareBleedingDisorders/FactorXI.aspx, http://www.wienkav.at/kav/kar/texte_anzeigen.asp?ID=7137 and http://www.factorxi.org); however, there are only nine registered FXI-deficient patients in Turkey and no attempt has been carried out to reveal the genetic basis of FXI deficiency in the Turkish population. In this study, we report the molecular analysis of FXI deficiency in two unrelated Turkish patients. A comprehensive mutational screening of F11 led to the identification of the previously reported Trp501X (or p.Trp519X, according to the HGVS nomenclature) [2] and Thr33Pro (or p.Thr51Pro) [3] mutations as well as that of the novel Gly217Ser (or p.Gly235Ser) substitution (Table 1). Moreover, the pathogenic role of the identified missense mutations was analysed by using expression experiments in eukaryotic cells. The Trp501X mutation was previously described in an asymptomatic Japanese patient with severe FXI deficiency [2]. Although the predicted protein truncation would probably impair secretion, and the truncated protein will also lose its catalytic properties, the well-documented sensitivity of F11 mRNAs bearing premature termination codons to the nonsense-mediated mRNA decay pathway [1] suggests that Trp501X F11 transcripts are likely to be degraded after the first round of translation. The patient bearing the Trp501X mutation had prolonged APTT (42 s) and FXI coagulant activity (FXI:C 22%) and antigen level (FXI:Ag 26%) lower than the expected 50% reduction for a single null allele. In fact, mutational screening revealed a second missense variation (Gly217Ser) on the other chromosome (Table 1). The Gly217 mutation lies in a protein region involved in FIX and platelet binding, and is highly conserved among mammals; however, the presence of a serine at the corresponding position in bovine and canine FXI might suggest that the Gly217Ser substitution could be tolerated. Analysis of the effect of this amino acid variation on FXI biosynthesis and secretion by expression studies in COS-1 cells revealed that the mutation causes a slight but significant reduction in FXI secretion (1, 2a) without affecting the functional properties of the molecule (Fig. 2b). In fact, the Gly217Ser FXI levels in conditioned media, measured using an ELISA, were reduced to 80% of the wild type (homozygous condition); co-expression of Gly217Ser and wild-type FXI resulted in a FXI antigen level not significantly different from that of the wild-type one (Fig. 2a). Metabolic labelling of wild-type, Thr33Pro and Gly217Ser recombinant FXIs in transiently transfected COS-1 cells. Cells were labelled with [35S]-cysteine and methionine mixture 24 h after transfection. Radiolabelled FXIs were immunoprecipitated from cell lysates and media using goat anti-human FXI. Immunoprecipitated FXIs were analysed on 8% reducing (lower panel) and 6% non-reducing (upper panel) SDS–PAGE and visualized by scanning phosphor image screens. The pcDNA3.1 empty vector was used as negative control (mock). +: presence of goat anti-human FXI antibody, −: absence of goat anti-human FXI antibody. Transient expression of wild-type and mutant FXI proteins. pCDNA3.1/FXI, pCDNA3.1/T33P, pCDNA3.1/G217S or equimolar amounts of wild-type and each of the mutant plasmids (heterozygous condition) were transiently transfected in COS-1 cells. (Left panel) Antigen levels of recombinant FXI were measured in conditioned media using ELISA [5]. Bars represent relative concentrations of protein in media compared with the mean antigen level measured in the wild type, set as 100%. Results are given as mean ± SEM. (Right panel) The specific activities of recombinant proteins were determined by calculating the ratio between FXI activity (measured using a one-stage method based on a modified partial thromboplastin time) and FXI antigen levels [5], both assayed in about 15x concentrated media. Bars represent mean ± SEM of three independent experiments, each performed in duplicate. The mean value of wild-type FXI was set as 100%. The results were analysed using unpaired t-test (***P < 0.001); nd, not determined. The Thr33Pro mutation was detected in a symptomatic heterozygous patient who had decreased FXI:C (42%) and FXI:Ag (46%) levels (Table 1). This substitution is a non-conservative change from a medium-sized hydrophilic neutral amino acid to a large hydrophobic residue. Metabolic labelling experiments demonstrated that the mutation does not affect the biosynthesis but rather impairs secretion (Fig. 1). As co-expression of wild-type and Thr33Pro FXIs led to a quite more marked reduction in protein secretion than the expected 50% (Fig. 2a), the Thr33Pro mutation might also exert a dominant negative effect. The existence of a dominant negative effect on FXI secretion, mediated by the retention in the endoplasmic reticulum of heterodimeric FXI molecules, is further confirmed by the observation that the FXI specific activities measured in media of cells co-expressing the wild-type and the mutant protein and in media of cells expressing only the wild-type FXI were comparable (Fig. 2b). This is an indirect evidence that there is little or no secretion of heterodimeric molecules. Thr33 is involved in high molecular weight kininogen binding and lies in a structurally conserved region in mammalian FXI sequences. The adjacent Cys32 residue is involved in the Cys32–Cys38 bond, which forms the small loop in the A1 domain. A similar disulphide bond arrangement is present in the other three A domains. No mutation has been reported in the corresponding Thr residues in the A2 and A3 domains of FXI, whereas in the A4 domain, Thr at the 33rd position (Thr304) was found mutated to Ile in patients with quantitative FXI deficiency. This substitution was demonstrated to cause reduced secretion and intracellular retention of the mutant protein, probably by interfering with the Cys303–Cys309 disulphide bond [4]. The observed lack of secretion competence of Thr33Pro FXI is likely to result from a perturbation of the A1 domain folding caused by the disruption of the alpha helix structure and of the Cys32–Cys38 bridge. In conclusion, we have identified the genetic basis of FXI deficiency in two Turkish patients and demonstrated the pathogenic mechanism underlying the Thr33Pro and Gly217Ser mutations. In vitro characterization of the Thr33Pro mutation adds to the small number of missense mutations for which a clear dominant negative effect was experimentally verified. SD is a recipient of a Bayer Hemophilia Early Career Investigator Award 2006. This work was also supported by Boğaziçi University Research Foundation Project no 08HB105 and by MIUR (Ministero dell’Istruzione, dell’Università e della Ricerca): FIRST 2006 (Fondo per gli Investimenti Ricerca Scientifica e Tecnologica). The authors state that they have no interests that might be perceived as causing a conflict or bias.