Combined homology modelling and evolutionary significance evaluation of missense mutations in blood clotting factor VIII to highlight aspects of structure and function

Abstract

Most small lesions in the factor VIII (FVIII) gene that cause haemophilia A (HA) are single nucleotide substitutions resulting in amino acid replacing (missense) mutations and leading to various phenotypes, ranging from mild to severe. We took a combined approach of homology modelling and quantitative evaluation of evolutionary significance of amino acid replacing alterations using the Grantham Matrix Score (GMS) to assess their structural effects and significance of pathological expression. Comparative homology models of all amino acid substitutions summarized in the FVIII mutations database plus these identified and reported lately by us or by our collaborators were evaluated. Altogether 640 amino acid replacing mutations were scored for potential distant or local conformation changes, influence on the molecular stability and predicted contact residues, using available FVIII domain models. The average propensity to substitute amino acid residues by mutation was found comparable to the overall probability of de novo mutations. Missense changes reported with various HA phenotypes were all confirmed significant using GMS. The fraction of these, comprising residues apparently involved in intermolecular interactions, exceeds the average proportion of such residues for FVIII. Predicted contact residues changed through mutation were visualized on the surface of FVIII domains and their possible functional implications were verified from the literature and are discussed considering available structural information. Our predictive modelling adds on the current view of domain interface molecular contacts. This structural insight could aid in part to the design of engineered FVIII constructs for therapy, to possibly enhance their stability and prolong circulating lifetime.

Factor VIII mutation and desmopressin‐responsiveness in 62 patients with mild haemophilia A

“…The vast majority of responsible mutations are point mutations that result in an amino acid substitution and cause subtle changes in the local environment, minor conformational changes in position of the protein backbone, or direct gain or loss of ionic or hydrogen bonding interactions reducing protein stability or disrupting cofactor activity. [10][11][12][13] Mutations that affect the stability of the protein, the thrombin activation cleavage site and/or interfere with secretion of the protein appear to be less likely to respond to DDAVP administration. [14,15] (see Table 4) Presumably, these patients may be unable to manufacture adequate stores of the protein or the functional aspect of the protein is impaired in such a way that increasing the amount of protein does little to enhance its function.…”

Computational prediction of phenotype in haemophilia A

“…Several previous studies have used multiple sequence alignment and homology modelling to assess structural and functional impact of F8 SNPs, attempting to correlate phenotype and genotype. A large study of 640 point mutations, including mutations associated with variable phenotype, identified structural alterations that generally correlated with a severe or non-severe phenotype using homology modelling and evaluation of evolutionary significance [3]. A large study of 640 point mutations, including mutations associated with variable phenotype, identified structural alterations that generally correlated with a severe or non-severe phenotype using homology modelling and evaluation of evolutionary significance [3].…”

“…When occurring within a given coding region, a SNP can result in either change (non-synonymous) or no-change (synonymous) of protein primary structure [1]. Variation in phenotype is also seen at the same amino acid position, dependent on the substituted residue (homonymous replacement) [3]. Interestingly, these mutations are associated with substantial variation in their effect on FVIII function and resultant bleeding phenotype.…”

“…Much of the early understanding of the functional regions of the FVIII protein developed through the in vitro introduction of point mutations via site directed mutagenesis [4]. A large study of 640 point mutations, including mutations associated with variable phenotype, identified structural alterations that generally correlated with a severe or non-severe phenotype using homology modelling and evaluation of evolutionary significance [3]. A recent survey of 13 international treatment centres described genotype testing in >75% HA patients in 62% (8/13) of surveyed centres [5].…”

Ten novel factor VIII (F8C) mutations in eighteen haemophilia A families detected in Singapore

“…To assess potential structural effects on identified missense mutations, molecular modelling in silico was performed essentially as described in Ref. [5].…”