Molecular Genetics of Hereditary Prothrombin Deficiency in Indian Patients: Identification of a Novel Ala362→Thr (Prothrombin Vellore 1) Mutation by Conformation Sensitive Gel Electrophoresis.

Abstract Prothrombin deficiency is a rare (1:200000) autosomal recessive disorder caused by diverse mutations in prothrombin gene. We have studied the molecular basis of this disorder in 4 unrelated Indian patients. The clinical features of these patients included easy bruisability, post-traumatic bleeds, hemarthroses (50% each) and epistaxis, gum bleedings, menorrhagia, hematemesis and post-surgical bleeding (25% each). The diagnosis was based on prolonged prothrombin and activated partial thromboplastin times and low factor II coagulant activity (FII: C) measured using a prothrombin time based assay. FII: C levels ranged between 4.7–17.5%. Genomic DNA was screened for prothrombin gene mutations by a novel PCR and conformation sensitive gel electrophoresis (CSGE) strategy. Fourteen exonic and their flanking intronic regions and 3′ untranslated region of prothrombin gene were amplified by 12 pairs of primers designed by Primer3 software. CSGE was performed in a mildly denaturing gel containing 10% acrylamide. Samples displaying abnormal CSGE profiles were sequenced by the Big Dye Terminator cycle sequencing kit to confirm the nature of nucleotide change. A novel missense mutation was studied based on the coordinates (identifier 1ppb) for the human ∞-thrombin (1.92Å resolution) three-dimensional structure. The evolutionary conservation of this aminoacid, mutated by missense change, was studied in the prothrombin sequence in 17 different species and 7 related proteases obtained from SwissProt and Trembl databases using PSI-BLAST. Mutations were identified in all the four patients. Five different causative mutations including 4 (80%) missense and an in-frame deletion (20%) were identified. One of them was a novel, Ala362→Thr aminoacid change affecting ‘B’ chain of ∞-thrombin. This mutation was identified in a compound heterozygous state with a previously reported Arg-1→Gln missense change affecting pro-peptide cleavage site. Ala362→Thr occurred at a codon, evolutionarily conserved in all the 24 different prothrombins or its related serine proteases studied. This indicates its importance to the structure of α-thrombin. Molecular modeling of this Ala362→Thr mutation was found to cause a conformational change around the region involving a catalytic triad residue His363 and a cysteine residue at codon 364 due to the accommodation of a larger and polar side chain of threonine. The FII: C level in this patient was 17.5%. Three other previously reported mutations were also detected in the homozygous state: Arg271→Cys missense mutation in Kringle-2 region, a Glu309→Lys missense mutation in ‘A’ chain of ∞-thrombin and an in-frame deletion of 3bp (AAG) leading to Del Lys301/302 in ‘A’ chain of ∞-thrombin. This is the first report of the molecular basis of prothrombin deficiency in Indian patients and we suggest the eponym ‘Prothrombin Vellore 1′ for Ala362→Thr mutation.