Factor XI (FXI) is the zymogen of a trypsin-like serine protease that catalyzes the activation of FIX in the consolidation phase of blood coagulation through a thrombin-generated feedback loop. The human FXI gene is located on the long arm of chromosome 4 (4q35) . It is composed of 15 exons and spread over approximately 23 kilobases . Mutations in this gene result in a relatively mild and variable bleeding diathesis . Although FXI deficiency is common in well-defined populations, including Ashkenazi Jews and French Basques [4,5], only 50 different mutations in this gene have been reported world-wide . An increased frequency of such disorders has been expected in Middle Eastern countries and Southern India where consanguineous marriages are relatively common . We report here for the first time, the molecular data in three South Indian patients with FXI deficiency.
Patients with clinical bleeding or a prolonged activated partial thromboplastin time (APTT) on preoperative screening were further evaluated. A diagnosis of FXI or FIX deficiency was based on a prolonged APTT and a low FIX or FXI coagulant activity (FIX/FXI:C). Genomic DNA was obtained from the peripheral blood of these patients. FXI gene was amplified by 14 pairs of primers (Primer3, http://www.broad.mit.edu/cgi-bin/primer/primer3_http://www.cgi) designed to cover all 15 exons and intron–exon boundaries (Table 1). Mutations were screened by conformation-sensitive gel electrophoresis (CSGE) as previously described . In the patient with a double coagulation defect, FIX gene defects were identified by a multiplex polymerase chain reaction (PCR) and CSGE strategy as reported earlier . Samples displaying abnormal CSGE patterns were sequenced using the Big Dye Terminator cycle sequencing kit (Applied Biosystems, Warrington, UK) on an ABI 310 genetic analyzer (PE Applied Biosystems, Foster City, CA, USA). PCR–restriction enzyme analysis (PCR-REA) was carried out with BsaAI (New England Biolabs, Hitchin, UK) and BamHI (MBI Fermentas, St Leon Rot, Germany) to confirm the novel mutations identified in exons 8 and 9 of the FXI gene and to screen for their presence in 50 normal chromosomes of south Indian descent. Mutations at or near the splice junction consensus sequences were analyzed using the ‘Splice Site Prediction program’ (http://www.fruitfly.org/seq_tools/splice.html) to predict changes in RNA splicing. FXI amino acid sequences from 10 different species/related serine proteases were obtained from the SwissProt and Trembl databases (http://us.expasy.org/sprot/) by psi-blast to study the conservation of novel missense substitutions identified.
|Exon||Forward primer (5′−3′)||Reverse primer (5′−3′)||Amplicon |
The clinical and hematological data for these patients (Table 2), show that those patients with severe FXI deficiency alone, had no spontaneous bleeding. The measurement of plasma levels of factor activity usually helps to predict the severity and frequency of clinical manifestations, but for FXI deficiency this relationship is poor as is evident from this study and as reported previously . However, the patient with combined deficiency (FIX and FXI) had a clinical phenotype consistent with severe hemophilia. It is of interest to note that replacement of FIX concentrates alone relieved the bleeding symptoms of hemarthroses in this patient.
Mutations in FXI or FIX genes were identified in all three subjects by PCR and CSGE. In all, four different causative missense mutations (FXI, n = 3; FIX, n = 1) were detected of which three were novel (Table 2). Two previously reported  polymorphisms in FXI gene, −138A→C in intron A and Gly379Gly (GGT→GGC) were also identified in a heterozygous (BL-86) or a homozygous state (BL-154) in two patients. In the patient (BL-154) with a double coagulation defect, a novel T→G transversion at nucleotide 31166 of FIX gene (http://www.kcl.ac.uk/ip/petergreen/haemBdatabase.html//) resulted in a phenylalanine to valine missense substitution at codon 349 affecting the FIX catalytic domain. A Phe394Ile missense change has been shown  to be causative of hemophilia B. Both these amino acids differ in size and shape and Phe349 is present in a disulfide loop formed by Cys336 and Cys350. The Phe349Val missense change identified in this study may cause hemophilia B by a similar mechanism, as both isoleucine and valine mutants are structurally similar aliphatic amino acids. This mutation was coinherited with a previously reported, homozygous, Gly460Arg missense change in the catalytic domain of FXI and has been shown to be causative of FXI deficiency .
In another patient (BL-86), the Gly460Arg mutation was detected in a compound heterozygosity with a novel, Val271Leu (GTG→CTG) amino acid change affecting the apple-3 domain of FXI. This substitution is also likely to be disease causative. As Val271 is the last codon of exon 8, located at its 3′ splice donor junction, a mutation at this consensus splice sequence (CA G-GTA→CA C-GTA) is predicted to abolish (calculated splice site consensus value 0) the physiological donor splice site (normal splice consensus value 0.91) for exon 8 and probably results in an abnormal FXI transcript. Evaluation of 50 normal chromosomes from the same geographic region did not reveal this sequence change (data not shown).
A novel A→C transversion in exon 10 causing a Tyr351Ser (TAC→TCC) missense change was found in patient BL-166. This sequence change was absent in the 50 normal chromosomes screened. Tyr351 is highly conserved in the FXI amino acid sequences or related proteases in the 10 different species studied (Table 2), indicating the importance of this residue to the structure of apple-4 domain in FXIa. Apple-4 domain is important for the dimerization of FXIa . A missense substitution at the adjoining residue Gly350Glu (factor XI Nagoya II) has been demonstrated  by expression studies to have a profound effect on FXIa dimerization. It is possible that Tyr351Ser could have a similar effect on FXIa dimerization but further studies are required to demonstrate the functional effect of this mutation.
In summary, we report for the first time the molecular basis of severe FXI deficiency in three unrelated Indian patients. Combined deficiency of coagulation factors is rare. Many of them represent a chance inheritance of disease genes , with implications for genotype–phenotype correlations . Only two patients [15,16] with a combined deficiency of factors IX and XI deficiency have been described but without data of genetic evaluation. The molecular data provided in this report on combined FIX and FXI deficiency therefore acquires significance.