In this work, we combined homozygosity mapping, linkage analysis, and WES, to identify the genetic alteration in a consanguineous family with a recessive form of DCM. The identified homozygous mutation in the FBXO32 gene was uncovered after homozygosity mapping, an approach that we adopted in this case in view of the high probability of identifying a homozygous mutation in a consanguineous family. The parents were asymptomatic with normal echocardiograms, and the disease affected both sexes.
The phenotype in the proband was remarkable, with severe CMP progressing to heart failure that necessitated heart transplantation. Screening family members by echocardiogram revealed variable presentation: two siblings were asymptomatic while the youngest affected child had suggestive cardiac symptoms. This observation underscores the importance of screening first-degree relatives in cases of DCM.
The FXO32 gene is one of more than 38 members of the FBXO (F-Box Only) family of proteins that have an F-box domain characterized by approximately 50 amino acids, which functions as a site for protein-protein interaction . FBXO32 is one of the components of the E3 ubiquitin ligase SCF (Skp1, Cullin-1, F-box), which binds target proteins for degradation by the UPS. The F-box domain links the F-box protein to the other SCF components by binding Skp1, leading to the destruction of the target proteins. In animals, through enhancing protein degradation, Fbxo32 was initially discovered to play a critical role in inducing muscle atrophy [8, 9].
Serving as a cardiac ubiquitin ligase, FBXO32 has been implicated in the pathogenesis of CMP.6,7 FBXO32 has been shown to repress calcineurin through assembling with the UPS complex, which promotes cardiac hypertrophy in response to pathologic stimuli . Li et al. have also found that over-expressed Fbxo32 in neonatal rat cardiomyocytes disrupts the Akt-dependent pathway responsible for physiological cardiac hypertrophy.11 Interestingly, down-regulation of Fbxo32 in knockout mice produces the opposite effect, with the inhibition of cardiac hypertrophy in response to pressure overload , suggesting that there are different mechanisms by which FBXO32 induces atrophy and hypertrophy in cardiomyocytes . In a recent study on atrogin-1 knockout mice, charged multivesicular body protein 2B (CHMP2B), which is part of an endosomal sorting complex required for autophagy, has been identified as a target of atrogin-1-mediated degradation.12 Mice lacking atrogin-1 fail to degrade CHMP2B, resulting in autophagy impairment, intracellular protein aggregate accumulation, unfolded protein response activation and subsequent cardiomyocyte apoptosis, leading, ultimately, to CMP and premature death.
Furthermore, in support of a potential link between FBXO32 and DCM in this family, other cardiac ubiquitin ligases (MuRF1, MuRF2, MuRF3, CHIP, MDM2) have also been implicated in the pathogenesis of cardiac hypertrophy, atrophy, and ischemia reperfusion injury.5 Recently, mutations in the cardiac ubiquitin ligase TRIM63, which encodes MuRF1, have been identified in patients with familial hypertrophic CMP . The expression of mutant TRIM63 was associated with impaired UPS-mediated protein degradation in cardiomyocytes. This finding, together with our work, suggests that the dysfunction of other proteins in the UPS complexes may also be implicated in the pathogenesis of Mendelian forms of CMP.
Although causality is yet to be established, several factors pertinent to the identified mutation (p.Gly243Arg) support the suggestion that FBXO32 is a likely candidate gene for DCM. There was a clear co-segregation of the mutation with the clinical phenotype. All of the affected patients, but none of their parents and unaffected siblings, were homozygous for the mutant allele. In addition, the mutation, which replaces a highly-conserved aliphatic non-polar amino acid with a polar positively charged one, was not present in 1986 chromosomes, and was predicted to destabilize the protein.
The identified homoallelic variant in the F-box domain is likely to be a loss-of-function mutation. Being within the F-box domain, this mutation may cause loss of substrate specificity, leading to the premature degradation of functional proteins. It may also cause loss of efficient recruitment of the components of the UPS. However, a gain-of-function mechanism is still a possibility, where the mutant allele could lead to the accumulation of proteins that are damaging. Both mechanisms would theoretically perturb the well-controlled system of protein homeostasis within cardiomyocytes, and lead to CMP.
It is noteworthy that none of the affected patients had skeletal muscle weakness. The mutation identified in this family may have damaged a cardiac-specific protein, and hence the skeletal muscles are spared. However, further studies in cell or animal models harboring this particular mutation may provide a plausible explanation of the mechanism of cardiac disease and of the apparent lack of skeletal involvement.
The methodology that we have adopted in this study has recognizable limitations. The family pedigree was highly suggestive of a recessive pattern of inheritance, and therefore we implemented the approach of homozygosity mapping and WES, followed by excluding non-homozygous variants. Although the recessive inheritance conforms to the segregation of the disease in this family, a dominant pattern with reduced penetrance remains a possibility. In addition, the unaffected individuals may still be in a presymptomatic phase of a late-onset CMP. A long-term follow-up with periodic evaluation may clarify if the identified variant is not fully penetrant. Last, as our study was conducted in a single family, screening FBXO32 in other families with CMP of unknown etiology may provide more insight and establish the causal relationship between FBXO32 and CMP.