In silico analysis of missense mutations in exons 1-5 of the F9 gene that cause hemophilia B

Abstract

Background: Missense mutations in the first five exons of F9, which encodes factor FIX, represent 40% of all mutations that cause hemophilia B. To address the ongoing debate regarding in silico identification of disease-causing mutations at these exons, we analyzed 215 missense mutations from www.factorix.org using six in silico prediction tools, which are the most common used programs for analysis prediction of impact of mutations on the protein structure and function, with further advantage of using similar approaches. We developed different algorithms to integrate multiple predictions from such tools. In order to approach a structural analysis on FIX we performed a modeling of five selected pathogenic mutations.

Results: SIFT, PolyPhen-2 HumDiv, SNAP2, and MutationAssessor were the most successful in identifying true non-causative and causative mutations. A proposed function integrating these algorithms (wgP4) was the most sensitive (90.1%), specific (22.6%), and accurate (87%) than similar functions, and identified 187 variants as deleterious. Clinical phenotype was significantly associated with predicted causative mutations at all five exons. However, PolyPhen-2 HumDiv was more successful in linking clinical severity to specific exons, while functions that integrate 4-6 predictions were more successful in linking phenotype to genotypes at the light chain (exons 3-5). The most important value of integrating multiple predictions is the inclusion of scores derived from different approaches. Modeling of protein structure showed the effects of pathogenic nsSNPs on structure and function of FIX.

Conclusions: A simple function that integrates information from different in silico programs yields the best prediction of mutated phenotypes. However, the specificity, sensitivity, and accuracy of genotype-phenotype predictions depend on specific characteristics of the protein domain and the disease of interest as we validated by the structural analysis of selected pathogenic F9 mutations. The proposed function integrating algorithm (wgP4) might be useful for the analysis of nsSNPs impact on other genes.

Keywords: F9 exons 1–5; Genotype-phenotype correlation; Hemophilia B; In silico analysis.

Fig. 1

Formulas for combined predictions. (1 – SIFT), as SIFT scores are inverse to PolyPhen-2 scores, they were scaled by subtracting from 1. PolyPhen, score obtained from PolyPhen-2 HumDiv. (SNAP2/100)2, SNAP2 scores may be positive and negative percentages, they were scaled to PolyPhen-2 scores by dividing by 100 and squaring. MutationAssessor, scores range from 4 to − 2. Mutations scoring below 1.9 are considered benign, and so are coded as 1. Predicted values were log-transformed at base 5 to obtain values between 0 and 1. PANTHER and PROVEAN, predictions are categorized as deleterious or benign, and are coded 1 and 0 respectively. n, number of programs used in combined analysis. In the functions wgP6 and wgP4, n is substituted by the weight for each program. In B and C, predicted values in the numerator are multiplied by the weight

Fig. 2

Sensitivity, specificity, and accuracy for five factor IX domains. The first five domains encoded by exons 1–5 were analyzed as one unit using individual tools. See text for more details

Fig. 3

Sensitivity, specificity, and accuracy for each factor IX domain. The (a) signal peptide at exon 1, (b) propeptide at exon 2, (c) Gla domain at exon 3, (d) EGF-1 domain at exon 4, and (e) EGF-2 domain at exon 5 were analyzed by individual tools. See text for more details

Fig. 4

Analysis of factor IX secondary structure by the FFPRED tool in PSIPRED. Analysis of predicted changes in (a) percentage alpha helix, extended strand, and random coil, as well as in (b) aliphatic index, hydrophobicity, surface area, and addition or deletion of phosphorylation sites. Domains are depicted in different shades of gray

Fig. 5

Comparison of the local environment of severe and mild mutations in the EGF domains of FIX. The protein backbone is shown in silver ribbons, interacting amino acids as a black licorice and the calcium ion as a white sphere. a, c, e, g, i correspond to wild type FIX. b, d, f, h, j correspond to mutant FIX. a Location of Gly105 in EGF-1 (from PDB ID 1PFX); the N-terminus of the domain is labeled. b Location of Asp105 in EGF-1; the N-terminus of the domain is labeled c Neighboring residues for Gln143 (from PDB ID 6MV4), labeled. d Neighboring residues for Arg143, clashing with the disulfide bond between Cys157 and Cys170, Tyr161 and Phe423. e Salt bridge between Glu124 in EGF-1 and Arg140 in EGF-2; neighboring positive residue also labeled (from PDB ID 1PFX). f Group of nearby positive charges in the Glu124Lys mutant. g Selected residues close to Val153 (from PDB ID 1PFX). h Residues that clash with Met153. i Residues coordinating the calcium ion in EGF-1; the residues that contribute their side chains are labeled (from PDB ID 1EDM). j Location of Pro96 as a first coordination shell residue for calcium