Next-generation sequencing identified novel Desmoplakin frame-shift variant in patients with Arrhythmogenic cardiomyopathy

Abstract

Background: Arrhythmogenic cardiomyopathy (AC) is one of the leading causes for sudden cardiac death (SCD). Recent studies have identified mutations in cardiac desmosomes as key players in the pathogenesis of AC. However, the specific etiology in individual families remains largely unknown.

Methods: A 4-generation family presenting with syncope, lethal ventricular arrhythmia and SCD was recruited. Targeted next generation sequencing (NGS) was performed and validated by Sanger sequencing. Plasmids containing the mutation and wild type (WT) were constructed. Real-time PCR, western-blot and immunofluorescence were performed to detect the functional change due to the mutation.

Results: The proband, a 56-year-old female, presented with recurrent palpitations and syncope. An ICD was implanted due to her family history of SCD/ aborted SCD. NGS revealed a novel heterozygous frame-shift variant (c.832delG) in Desmoplakin (DSP) among 5 family members. The variant led to frame-shift and premature termination, producing a truncated protein. Cardiac magnetic resonance (CMR) of the family members carrying the same variant shown myocardium thinning and fatty infiltration in the right ventricular, positive bi-ventricular late gadolinium enhancement and severe RV dysfunction, fulfilling the diagnostic criteria of AC. HEK293T cells transfected with mutant plasmids expressed truncated DSP mRNA and protein, upregulation of nuclear junction plakoglobin (JUP) and downregulation of β-catenin, when compared with WT.

Conclusion: We infer that the novel c.832delG variant in DSP was associated with AC in this family, likely through Wnt/β-catenin signaling pathway.

Keywords: Arrhythmogenic cardiomyopathy; Desmoplakin; Genetic variant; Next generation sequencing.

Fig. 1

Genetic analysis and in silico prediction. a A heterozygous frame-shift variant DSP c.832delG was identified through targeted next generation sequencing; b Pedigree and genotype. Family members in the red frame were genotyped. Arrow indicates the proband; squares indicate male family members; circles indicate female members; black filled indicate family members diagnosed with AC or experienced sudden cardiac death; diagonal lines indicate deceased family member; c Schematic diagram of the location of DSP p.A278Pfs*39

Fig. 2

Representative cardiac magnetic resonance images. Myocardium thinning and fatty infiltration (arrow) in the right ventricular and positive bi-ventricular late gadolinium enhancement were detected in III5 and IV3. Myocardium thinning and fatty infiltration (arrow) were detected in the right apical region in III-3. LGE, late gadolinium enhancement

Fig. 3

DSP mRNA expression, total DSP and JUP protein expression. HEK293T cells were transfected with either blank, wild type or mutant plasmids. Blank plasmids without DSP gene served as control group. a-b qPCR analysis for DSP mRNA levels in the N-terminal and C-terminal of the c.832delG mutation site. There were no significant differences between mutant and wild-type in mRNA levels in the N-terminal side of DSP mutation, whereas, mRNA level in C-terminal side of DSP mutation was only elevated in cells transfected with wild type plasmid transfection; c-dGFP antibody was used to exam the length of protein expressed in whole cell lysates. Mutant DSP protein was much shorter than wild type, suggesting truncation effect of the mutation. JUP expression was significantly increased in the mutant group. GAPDH served as an internal control. DSP, Desmoplakin; JUP, Junction plakoglobin; WT, wild type

Fig. 4

JUP and β-catenin expression in cytoplasm and nuclear, separately. HEK293T cells were transfected with either wild type or mutant plasmids. a-b JUP was significantly upregulated and a-c β-catenin was downregulated in cells transfected with mutant type in the nuclear, rather than cytoplasm, when compared with wild type DSP. GAPDH served as an internal control in the cytoplasm and Lamin B served as an internal control in the nuclear. Blank plasmids carrying no DSP gene served as control group. DSP, Desmoplakin; JUP, Junction plakoglobin; WT, wild type

Fig. 5

Immunofluorescent staining examined JUP expression levels with either wild type or mutant DSP. HEK293T cells were transfected with either wild type or mutant plasmids. Blue indicate nuclear (DAPI) and red indicate JUP. a Representative images of the immunofluorescent staining of transfected HEK293T cells; b Confocal microscopic detection of the colocalization of JUP with DAPI; c Colocalization analysis of JUP and DAPI (n = 10). DSP, Desmoplakin; JUP, Junction plakoglobin; WT, wild type