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Clustered F8 missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis and activity.

ABSTRACT: Dissection of pleiotropic effects of missense mutations, rarely investigated in inherited diseases, is fundamental to understanding genotype-phenotype relationships. Missense mutations might impair mRNA processing in addition to protein properties. As a model for hemophilia A, we investigated the highly prevalent F8 c.6046c>t/p.R2016W (exon 19) mutation. In expression studies exploiting lentiviral vectors, we demonstrated that the amino acid change impairs both Factor VIII (FVIII) secretion (antigen 11.0±0.4% of wild-type) and activity (6.0±2.9%). Investigations in patients' ectopic F8 mRNA and with minigenes showed that the corresponding nucleotide change also decreases correct splicing to 70±5%, which is predicted to lower further FVIII activity (4.2±2%), consistently with patients' levels (<1-5%). Masking the mutated exon 19 region by antisense U7snRNA supported the presence of a splicing regulatory element, potentially affected by several missense mutations causing hemophilia A. Among these, the c.6037g>a (p.G2013R) reduced exon inclusion to 41±3% and the c.6053a>g (p.E2018G) to 28±2%, similarly to a variant affecting the 5' splice site (c.6113a>g, p.N2038S, 26±2%), which displayed normal protein features upon recombinant expression. The p.G2013R reduced both antigen (7.0±0.9%) and activity (8.4±0.8%), while the p.E2018G produced a dysfunctional molecule (antigen: 69.0±18.1%; activity: 19.4±2.3%). In conclusion, differentially altered mRNA and protein patterns produce a gradient of residual activity, and clarify genotype-phenotype relationships. Data detail pathogenic mechanisms that, only in combination, account for moderate/severe disease forms, which in turn determine the mutation profile. Taken together we provide a clear example of interplay between mRNA and protein mechanisms of disease that operate in shaping many other inherited disorders.

SUBMITTER: Donadon I

PROVIDER: S-EPMC5792279 | biostudies-literature | 2018 Feb

REPOSITORIES: biostudies-literature

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Clustered <i>F8</i> missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis and activity.

Donadon Irving I McVey John H JH Garagiola Isabella I Branchini Alessio A Mortarino Mimosa M Peyvandi Flora F Bernardi Francesco F Pinotti Mirko M

Haematologica 20171123 2

Dissection of pleiotropic effects of missense mutations, rarely investigated in inherited diseases, is fundamental to understanding genotype-phenotype relationships. Missense mutations might impair mRNA processing in addition to protein properties. As a model for hemophilia A, we investigated the highly prevalent <i>F8</i> c.6046c>t/p.R2016W (exon 19) mutation. In expression studies exploiting lentiviral vectors, we demonstrated that the amino acid change impairs both Factor VIII (FVIII) secreti ...[more]

PMID: 29170251

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Project description:BackgroundMissense 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.ResultsSIFT, 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.ConclusionsA 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.

Dataset Information

Clustered F8 missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis and activity.

Publications

Clustered <i>F8</i> missense mutations cause hemophilia A by combined alteration of splicing and protein biosynthesis and activity.

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