A case of Fibrodysplasia Ossificans Progressiva associated with a novel variant of the ACVR1 gene

Abstract

Background: Fibrodysplasia Ossificans Progressiva (FOP) is a rare autosomal dominant disease characterized by congenital malformation of the great toes and progressive heterotopic ossification of soft tissues leading to cumulative disability. The genetic cause of FOP are mutations in the ACVR1 gene that encodes a type I receptor of Bone Morphogenetic Proteins. The most recurrent mutation in FOP patients is R206H affecting the Glycine-Serine rich domain and causing the hyper-activation of the receptor and the responsivity to the non-canonical ligand, Activin A. In the present study, we described a 3-years old child with early and highly suggestive clinical features of FOP who was found negative for the recurrent p.R206H substitution.

Methods: Molecular screening of the whole ACVR1 coding sequence and functional characterization in transfection-based assays.

Results and conclusions: We identified a novel, de novo variant in the fifth ACVR1 coding exon (NM_001111067.4:c.772A>T; NP_001104537.1:p.(R258W)). This substitution, never reported in association with FOP, affects a conserved arginine residue in the kinase domain of the protein. In silico analysis predicted the pathogenicity of this substitution, demonstrated by in vitro assays showing that the p.R258W ACVR1 mutated receptor acquires the ability to transduce the aberrant Activin A-mediated signaling, as observed for the gene variants associated with FOP.

Keywords: ACVR1; Activin A; BMP signaling; Fibrodysplasia Ossificans Progressiva; p.R258W.

FIGURE 1

Clinical features of the proband. (a,b) Bilateral malformation of the big toes. (c) MRI of the head region showing infiltration of fascia in the site of the lumps (fasciitis). (d) Hard lesions indicating the presence of para‐vertebral heterotopic ossifications of the back

FIGURE 2

Novel variant of the ACVR1 gene associated with Fibrodysplasia Ossificans Progressiva. (a) Schematic representation of the ACVR1 gene and protein. ACVR1 gene consists of two untranslated (UTR) and nine coding exons. Mutations of the gene associated with FOP are indicated. EC, extra‐cellular domain; TM, trans‐membrane domain; GS, Glycine‐Serine rich domain; KD, kinase domain. (b) Electropherograms showing the region of the fifth ACVR1 coding exon containing the heterozygous c.772A>T (RefSeq NM_001111067.4) in the proband (Fop85.1) and absent in his parents (Fop85.2 and Fop85.3)

FIGURE 3

The novel p.R258W variant perceives Activin A as an agonist. U2OS cells were transiently transfected with the empty pCMV expression vector (EV) or carrying the wild‐type (WT), R206H, R258S, and R258W ACVR1 cDNAs, then evaluated in basal condition or upon Activin A or BMP2 treatment. A and B, ActA increased the BRE‐Luc activity (a) and the expression of the ID1 target gene (b) in cells expressing mutated cDNA but not in cells transfected with the WT nor with the EV. Histograms represent the fold activation compared to the activity measured in cells transfected with wild‐type ACVR1 construct in basal condition set as 1. (c) ActA induced phosphorylation of SMAD1/5/9 (p‐SMAD) in U2OS cells transiently expressing mutant R206H, R258S and the R258W ACVR1 cDNAs but not in cells transfected with the WT form, or the EV. A representative image of the Western blot (WB) analysis is shown. (d) Densitometric analysis of relative p‐SMAD1/5/9 phosphorylation levels corrected over total SMAD1. The histograms represent the densitometric mean values (±SD) obtained in three different WB experiments. As expected, in all the presented experiments, cells expressing both the WT and mutated ACVR1 cDNAs can transduce signaling upon BMP2 treatment. UN, untreated cells (white bars); ActA, cells treated with Activin A (grey bars); BMP2, cells treated with BMP2 (black bars). Results are the mean ± SD. n = 3–4 (A and B), n = 3 (D) *p < .05; **p < .01; ***p < .001 by Unpair t test compared with untreated cells transfected with WT ACVR1 cDNA