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.

Hemophilia A: different phenotypes may be explained by multiple and variable effects of the causative mutation in the F8 gene

“…This is probably a general feature of several other hematologic disorders frequently associated with mutations that are defined simply as "missense" without the interplay among several molecular mechanisms producing the disease being known. However, the results obtained by Donadon et al 1 suggest slightly milder hemophilia phenotypes than those observed in patients by clotting assays (see Table 1 in Donadon et al 1 ). It is tempting to speculate that other components could have a role in influencing the phenotype in addition to the mechanisms highlighted by Donadon et al 1 For example, studies evaluating the half life of different missense mutations are very rare and yet these are needed in order to assess the impact of clearance mechanisms on these mutant proteins.…”

“…The mutated c.6046C>T associated with the mutation was found to alter splicing and to decrease the proportion of correct transcript to approximately 75% of wild type by inducing a variable degree of skipping of exon 19. Surprisingly, they found that several other missense changes in the same exon 19 (p.G2013R, p.E2018G, and p.N2038S) are responsible for variable splicing alterations, and that only the combination of altered RNA processing and abnormal protein biology produced a clinically relevant defect (see Figure 4 in Donadon et al 1 ). This is probably a general feature of several other hematologic disorders frequently associated with mutations that are defined simply as "missense" without the interplay among several molecular mechanisms producing the disease being known.…”

“…I n this issue of Haematologica, Donadon et al 1 investigated at molecular level the effect of a mutation in the frequent F8 gene (p.R2016W) in determining the circulating Factor VIII (FVIII) level in patients with hemophilia A carrying this missense mutation. The p.R2016W change was expressed and found to impair both FVIII secretion and activity.…”

“…2,3 Donadon et al 1 were able to quantitatively evaluate the pleiotropic effects of a nucleotide change at the RNA and protein (codon) levels. To do so, they chose the p.R2016W F8 mutation which is very frequent in Italy 4 and has been putatively suggested to induce also mRNA splicing impairment.…”

“…5 Very few hemophilia A missense mutations have so far been characterized because of the very low secretion efficiency of recombinant full-length FVIII. Donadon et al 1 used a lentiviral-mediated delivery of expression cassette consisting of the codon-optimized FVIII cDNA lacking the B domain. By this means, they showed that the p.R2016W mutation impairs both FVIII secretion and function.…”