FBXO32, encoding a member of the SCF complex, is mutated in dilated cardiomyopathy

Abstract

Background: Dilated cardiomyopathy (DCM) is a common form of cardiomyopathy causing systolic dysfunction and heart failure. Rare variants in more than 30 genes, mostly encoding sarcomeric proteins and proteins of the cytoskeleton, have been implicated in familial DCM to date. Yet, the majority of variants causing DCM remain to be identified. The goal of the study is to identify novel mutations causing familial dilated cardiomyopathy.

Results: We identify FBXO32 (ATROGIN 1), a member of the F-Box protein family, as a novel DCM-causing locus. The missense mutation affects a highly conserved amino acid and is predicted to severely impair binding to SCF proteins. This is validated by co-immunoprecipitation experiments from cells expressing the mutant protein and from human heart tissue from two of the affected patients. We also demonstrate that the hearts of the patients with the FBXO32 mutation show accumulation of selected proteins regulating autophagy.

Conclusion: Our results indicate that abnormal SCF activity with subsequent impairment of the autophagic flux due to a novel FBXO32 mutation is implicated in the pathogenesis of DCM.

Fig. 1

Identification of a novel genetic mutation in dilated cardiomyopathy. a Linkage analysis reveals a single peak indicated by the red arrow. b Stacked Venn diagram showing the filtering strategy to narrow down the novel locus

Fig. 2

Location and conservation of the mutated amino acid. a DNA sequencing chromatograms showing the novel missense mutation in all affected members with the site of mutation marked by an inverted red triangle. b Top: Schematic representation of FBXO32 with the different domains of the protein and the G243R mutation in the F-Box domain. Bottom: multisequence alignment orthologs showing conservation of the mutation (p. G243) across species. c Conservation across the sub-family of human F-Box proteins. The affected amino acid residue is boxed in red. NLS = nuclear localization signals. PD = PDZ domain. LZ = leucine-zipper domain. LCD = leucine-charged residue-rich domain. F-Box = F-Box domain

Fig. 3

FBXO32 mutation affects the molecular interaction with components of the SCF complex. a Homology model of FBXO32-SKP1 interaction (right panel) based on the FBW7-SKP1 structure (left panel, PDB ID: 2OVR). FBXO32 is in ribbon representation (cyan) and SKP1 is in surface representation (green). The Gly to Arg mutation, shown as white sticks leads to clashes (marked by red crosses) with neighboring helices in the F-Box domain. b HEK293 cells co-transfected with the indicated plasmids. Equal amounts of protein lysates were co-immunoprecipitated with Flag resines and analyzed by immunoblotting with the indicated antibodies. Blots are representative of three independent experiments. c Immunoblot analysis after IP in patients IV.5 and IV.7 with FDC compared to Ctr (control) and IDC. IP was performed with control IgG antibody used a negative control

Fig. 4

The FBXO32 mutation impairs the expression of selected proteins of the autophagy/lysosomal system. Immunoblot blot analysis of the indicated autophagy and lysosomal marker in FDC patients IV.5 and IV.7 carrying the FBXO32 variant, in a cardiomyopathic heart from another family (FDC2), in idiopathic dilated hearts (IDC) and in control hearts (Ctr)

Fig. 5

Hypothetical model of impaired autophagy flux mediated by the mutated FBXO32. a SCF complex containing FBXO32 induces the ubiquitination and subsequent degradation of target proteins. b Mutation in FBXO32 results in an inactive SCF complex, which stabilizes selective proteins regulating autophagy resulting in heart failure