Severe factor XI deficiency caused by compound heterozygosity


Hereditary factor XI (FXI) deficiency is an autosomal bleeding disorder, which is highly prevalent among Ashkenazi Jews (Asakai et al, 1991). Over 96% of FXI deficiency is accounted for by two of the four common mutations found in Ashkenazi Jews, namely type II (a nonsense mutation in exon 5) and type III (a missense mutation in exon 9). However, in non-Ashkenazi Jews, 30 different causative mutations have been identified with no single mutation predominating (Krawczak & Cooper, 1997; These mutations occur throughout the FXI gene, interfering in FXI protein expression (Pugh et al, 1995).

We report a new type of severe FXI deficiency in two related patients of North European Caucasian extraction. These two patients had no active bleeding problems following laporoscopic sterilization. Preoperative laboratory tests revealed a prolonged activated partial thromboplastin time. Further investigation revealed a severe FXI deficiency with FXI coagulant activity (FXI:C) of 4–4·5 U/dl. DNA sequence analysis of the FXI gene (Alhaq et al, 1999) revealed a novel missense mutation in exon 8 (AAA → ATA, FXI-Lys252Ile) resulting in the substitution of isoleucine for lysine at position 252 in the third apple domain. This mutation was found in compound heterozygosity with a nonsense mutation at codon 128 in exon 5 (TGC → TGA, FXI-Cys128Stop).

The nonsense mutation FXI-Cys128Stop results in the production of a truncated protein, which partially accounted for the FXI deficiency (Imanaka et al, 1995). To investigate the contribution of the missense mutation FXI-Lys252Ile to the FXI deficiency, in vitro expression experiments were performed. Human FXI cDNA in pZEM229FXI vector (gift from Dr Dominic Chang, University of Washington) was subcloned into the EcoRI site of the plasmid pZeoSV (Invitrogen, Paisley, UK). Mutant FXI-Lys252Ile cDNA was generated using a Quick change site-directed mutagensis kit (Stratagene, Amsterdam, the Netherlands) according to the manufacturer's instructions. The integrity of mutant cDNA was verified by sequence analysis. Expression plasmids were transfected into Baby hamster kidney cells 570 (BHK cells) using the calcium phosphate precipitation method as previously described (Dai et al, 2003). Stably transfected cells were selected and grown in culture medium containing Zeocin (250 μg/ml) for 2 weeks. The cultured medium and cell lysates were collected from the stably transfected cells (1 × 106) following incubation with 5 ml of OPTI-MEM (Invitrogen) for 24 h. FXI antigen levels in the cultured medium and cell lysates was determined by an enzyme-linked immunosorbent assay (Alhaq et al, 1999). The expression of mutant FXI-Lys252Ile in stably transfected cells showed a 73% reduction as compared with normal FXI in the cultured medium (Table I). However, the expression of mutant FXI in cell lysates was similar to normal FXI.

Table I.  Factor XI (FXI) produced by Baby hamster kidney cells transfected with normal and mutant cDNAs.
Recombinant cDNANo. of transfectantsFXI secreted in medium (ng/ml)Intracellular FXI (ng/ml)
  1. Expression of FXI by stably transfected cells in the cultured medium and cell lysates was determined using ELISA. The results are expressed as mean ± SD of three transfectants.

Wild type353·6 ± 9·921·6 ± 3·7
Lys252Ile318·3 ± 7·322·5 ± 2·8

The nonsense mutation FXI-Cys128Stop is thought to cause FXI deficiency by mRNA instability and/or incorrect folding of the truncated protein (Imanaka et al, 1995). The missense mutation FXI-Lys252Ile results in an amino acid substitution in the third apple domain. The expression studies indicated that the mutant protein was synthesized in transfected BHK cells, whereas the secretion of the mutant protein was reduced. The substitution of isoleucine (a neutrally charged hydrophobic amino acid) for lysine (a positively charged hydrophilic amino acid) alters charge and polarity within the region of the third apple domain. This may cause unstable conformation of FXI molecule, interfering with secretion of FXI.