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.

Figure 1.

The splicing-defective F8 missense mutations differentially impair Factor VIII (FVIII) protein secretion and function. (A) Schematic representation (upper part) of the lentiviral vector backbone harboring the codon-optimized cDNA of human FVIII lacking the B-domain (coFVIII) and sequence of the affected region and of the investigated mutations (middle part). The alignment of FVIII sequence across species is reported (lower part) together with affected residues (red). (B) Secreted FVIII antigen (upper) and co-factor activity (lower) levels of rFVIII variants expressed as % of rFVIIIwt. Secreted protein levels were normalized on virus copy number per cell determined by qPCR. Results are reported as mean±Standard Deviation from three independent experiments. (C) Structure of the human FVIII (PDB: 2R7E). Domain overview (A1-A2-A3-C1-C2 domains, inset) and interface between the A1 (blue) and A3 (white) domains (ribbon). The clustered residues under investigation (HGVS numbering) are represented by ball and stick. The R2016 residue is shown in space-filling.

Figure 2.

Features of the F8 minigene and of the sequences under investigation. Schematic representation (bottom) of the F8 exon 19 region cloned into the pTB vector through the NdeI restriction sites (indicated). The sequences of the exon-intron boundary and of the engineered U1snRNA (U1 F8ex19) are reported together with 3′ss and 5′ss scores (numbers) (splice site prediction by neural network; www.fruitfly.org). (Top) The region masked by the engineered U7 F8exon19 is magnified. The investigated mutations are indicated as nucleotide and amino acid changes.

Figure 3.

The c.6046c>t mutation as well as other adjacent missense changes promote exon 19 skipping differentially. (A) (Top) F8 splicing patterns in white blood cells from a normal control (N) and from 6 representative hemophilia A (HA) patients (P1-6) affected by the c.6046c>t mutation. The RT-PCR was conducted with primers in exons 17 and 22, and amplified fragments were separated on 2% agarose gel. (Bottom) Splicing assays with the wild-type (wt) and the mutated (c.6046c>t) minigenes. Effects of the antisense U7 F8ex19 (+) or the compensatory U1 F8ex19 (+) and of the strengthened 5′ss (5′ssHC) are shown. The RT-PCR was conducted with primers alpha 2,3 and Bra2 in the pTB construct (see arrows in Figure 2). Amplified fragments were separated on 2% agarose gel. Numbers report the percentage of exon 19 inclusion measured in at least three independent experiments; expressed as mean ± Standard Deviation. The percentage of exon 19 inclusion was estimated by densitometric analysis of bands. n: normal; s: exon 19 skipped. (B) (Top) Comparison of the exon 19 inclusion rates measured in 20 HA patients affected by the c.6046c>t mutation (light gray column) and in HepG2 cells expressing F8 minigene variants (dark gray columns). (Bottom) Representative example of splicing patterns of the selected missense variants. RT-PCR and analysis was conducted as in (A).

Figure 4.

Pleiotropic effects of F8 variants. Detrimental effects of variants on F8 splicing and on Factor VIII (FVIII) protein expressed as % reduction extent of wild-type (wt) of correct transcripts (top: splicing impairment) or of co-factor activity (bottom: protein impairment).