Six Novel Mutations Including Triple Heterozygosity for Phe31Ser, 514delT and 516T→G Mutations in Factor X Gene Is Responsible for Congenital Factor X Deficiency in Patients of Indian and Nepali Origin.

Factor X (FX) deficiency is a rare (1:100000) autosomal recessive disorder caused by heterogeneous mutations in FX gene. We have studied the molecular basis of this disease in 6 Indian and 1 Nepali patients. The clinical features included easy bruisability (71%), epistaxis, hemarthroses, gum bleeding, gastro-intestinal and central nervous system bleeding (28% each), echymoses, haematuria and umbilical cord bleeding (14% each). Diagnosis was confirmed by measuring the FX coagulant activity (FX: C) using a PT based assay. Six of them had a FX: C of <1% and one patient had 24% activity. FX gene mutations were screened by a PCR and conformation sensitive gel electrophoresis strategy using genomic DNA. FX aminoacid sequences from 12 different species and 5 related serine proteases were obtained from SwissProt and Trembl databases to study the conservation of an aminoacid mutated by missense change. Novel missense mutations were studied based on the normal type FXa three-dimensional structure (PDB: 1hcg). Mutations were identified in all the 7 patients. These included 8 (88.8%) missense and 1-frame shift (11.2%) mutations of which six were novel. Three of the novel mutations, Phe31Ser affecting ‘Gla’ domain, 514delT and 516T→G mutations affecting Cys132 in ‘connecting region’ were identified in a triple compound heterozygous state in a Nepali patient presenting with a severe phenotype. PCR-restriction enzyme analysis with DdeI and SmaI, confirmed the paternal origin of Phe31Ser mutation, while 514delT and 516T→G mutations were derived from a single allele from the mother. 514delT and 516T→G double mutation predicts a frame shift from Cys132 to cause a premature termination codon ‘TGA’ at residue 226 in the catalytic region. It also abolishes a disulphide bond between Cys132-Cys302 thereby destabilizing the link between Ile125-Arg139 of light chain of both FX and FXa to their heavy chain. The substitution of Phe31 an aromatic hydrophobic residue conserved in 16 out of 17 FX protein species examined, by the hydroxylic group of serine may open a cavity inside the hydrophobic core to probably change the secondary structure or result in partial misfolding of ‘Gla’ domain. Two other novel mutations, Gly133Arg, may affect the disulphide bridge between Cys132-Cys302 in the connecting region while Gly223Arg may perturb the catalytic triad (His236, Asp282 and Ser379). The other novel mutation, Ser354Arg, involves the replacement of a small buried residue by a large basic aminoacid and is likely to have steric or electrostatic effects in the pocket involving Lys351-Arg347-Lys414 that contributes to the core epitope of FXa for binding to FVa. Three previously reported mutations, Thr318Met; Gly323Ser; Gly366Ser were also identified. This is the first report of the molecular basis of FX deficiency in patients from the Indian subcontinent.