A substitution mutation in cardiac ubiquitin ligase, FBXO32, is associated with an autosomal recessive form of dilated cardiomyopathy

Abstract

Background: Familial dilated cardiomyopathy (DCM) is genetically heterogeneous. Mutations in more than 40 genes have been identified in familial cases, mostly inherited in an autosomal dominant pattern. DCM due to recessive mutations is rarely observed. In consanguineous families, homozygosity mapping and whole exome sequencing (WES) can be utilized to identify the genetic defects in recessively inherited DCM.

Methods: In a consanguineous family with four affected siblings with severe DCM, we combined homozygosity mapping, linkage analysis and WES, to uncover the genetic defect.

Results: A region of homozygosity (ROH) on chromosome 8q24.13-24.23 was found to be shared by all of the four affected siblings. WES detected ~47,000 variants that were filtered to a homozygous mutation (p.Gly243Arg) in the FBXO32 gene, located within the identified ROH. The mutation segregated with the phenotype, replaced a highly-conserved amino acid, and was not detected in 1986 ethnically-matched chromosomes. FBXO32, which encodes a muscle-specific ubiquitin ligase, has been implicated in the pathogenesis of cardiomyopathy through the ubiquitin proteasome system (UPS). In addition, FBXO32-knockout mice manifest with cardiomyopathy. Screening the index patient for all of the WES variants in 48 genes known to be implicated in hypertrophic and dilated cardiomyopathy was negative.

Conclusions: Our data suggest that FBXO32 is a candidate gene for recessive DCM. Acting as a cardiac ubiquitin ligase, mutated FBXO32 could perturb the degradation of target proteins in the UPS, the impairment of which has been observed in cardiomyopathy. Our work proposes that genes encoding other ubiquitin ligases could also be implicated in familial cardiomyopathy.

Fig. 1

M-mode echocardiographic recordings from the index patient showing severe dilatation of the left ventricle (LVIDs: Left ventricle internal diameter during systole; LVIDd: Left ventricle internal diameter in diastole; both are markedly increased) and poor contractility with an ejection fraction (EF) of 12.5 %

Fig. 2

Family pedigree showing the four affected siblings (filled symbols). Haplotypes spanning 8q24.12–q24.21 are shown under each symbol with two black arrows indicating the shared homozygous region between the affected individuals. The presence of the mutant allele (c.727G > C) is indicated by (+). The index case is indicated by the red arrow. The number below the symbol represents the age (in years). Echocardiogram was performed on the parents of the index case and all of their offspring

Fig. 3

Flowchart showing the several steps of filtering the variants identified by whole exome sequencing. ROH: region of homozygosity

Fig. 4

a Analysis of the SNP data using AutoSNPa software identified a block of homozygosity (arrow) of about 17 Mb on chromosome 8q24.13–q24.23 (8:122,185,539-139,111,674) that was shared by all of the four affected siblings (left) and not found in their parents and unaffected siblings (right). The horizontal axis indicates the chromosome numbers. The vertical axis indicates the LOD score. b GeneHunter Easy Linkage analysis showing the identified locus on 8q24 with a maximum LOD score of 3.37. Homozygous variants are displayed in black. Heterozygous variants are displayed in yellow

Fig. 5

a The homozygous mutation (c.727G > C, p.G243R) is indicated by the arrow in the upper panel. The normal allele is shown in the lower panel. b Protein sequence alignment of FBXO32 orthologs demonstrating that the glycine residue is a conserved amino acid down to zebrafish (D. rerio). c In Silico Protein model generated using Expasy-based modeling tools. On the left, the color of the model is based on the size properties of the amino acids. The color-coded polarity and hydrophobicity for the variant is given below the figure. The variant is predicted to destabilize the structure

Fig. 6

a Hematoxylin and eosin (H&E) and (b) trichrome staining of a left ventricular myocardial biopsy from the explanted heart of the proband showing hypertrophied myocytes, often with bizarrely shaped hyperchromatic nuclei and waving of the myocytes. c H&E and (d) trichrome staining from the control myocardium showing no cardiomyopathic changes, disarray, or fibrosis. e and (f) Immunohistochemical analysis of FBXO32 showing cytoplasmic staining with reduced protein expression in a section from the left ventricle of the proband (e) in comparison to a control myocardial specimen (f)