Structural and functional compatibility of a novel mutation Asp413Asn and the Gly420Arg mutation in the factor X gene. رسالة ماجستير

Abstract

Factor X is a vitamin k dependent serine protease; produced in the Liver and has a pivotal role in the coagulation cascade. It is activated by either extrinsic or the intrinsic pathways. It constitutes a major component of the prothrombinase complex, which converts prothrombin into thrombin. Factor X is encoded by a gene on chromosome 13 consisting of 8 exons encoding a 488 amino acids protein. More than 130 mutations have been reported in F10 gene, represented mostly by missense mutations. FX deficiency is classified as decrease in level and function (type1) or only dysfunction (type2) of the protein. Recently, two novel mutations were identified in the FX gene in our group, one a substitution (c.1237 G>A) leading to missense p.Asp413Asn mutation. Another missense mutation (p.Gly420Arg) in the F10 gene (c.1258 G>A) was reported in a previous study. Our study aimed to understand the functional and structural implications of both missense mutations on FX protein. The full-length wild type F10 cDNA was obtained commercially and used to generate both indicated missense mutations using site directed mutagenesis. The wild type and mutant constructs were transfected in HEK293t cells. Western blot analysis of the lysates from transfected cells showed no significant reduction in the FX protein level in both mutants compared to the wild type. FX activity (prothrombin time) was measured in the media of transfected cells and showed significant lower activity of both mutants compared to the wild type. Wild type and mutants F10 mRNA levels were measured in transfected cells and showed no significant difference. Bioinformatic analysis showed the both Asp413 and Gly420 are highly conserved in the catalytic domain of the protein among different species which indicates that VI both amino acids are crucial for FX protein activity. Molecular modeling analysis showed noticeable structural change in the protein. This change was more apparent in the Gly420Arg mutant than Asp413Asn mutant, which is predicted from the nature of amino acid change; however, both mutants were predicted to be damaging in the protein structure. This is consistent with the FX protein and mRNA levels and loss of FX activity described above