Screening Of Desmosome Genes In An Extendted Chinese Family And The Mechanism Of DSG2F531C Mutation In Arrhythmogenic Right Ventricular Cardiomyopathy

Part I:Screening of desmosome genes in a Chinese family with Arrhythmogenic right ventricular cardiomyopathyBackground:Arrhythmogenic right ventricular cardiomyopathy (ARVC), characterized by right ventricular fibrofatty replacement and malignant ventricular arrhythmias, is a common reason to premature death. Mutations in the desmosome-encoding genes have been reported in patients with ARVC. However, there are hardly any genetic studies in Asians. In this study, we aimed to investigate the clinical characteristics, cardiac manifestations and desmosome gene mutations in one extended Chinese ARVC family.Objectives:The aim of this study was to investigate the phenotype of Chinese ARVC family and to screen the mutations in desmosome-encoding genes in this family.Methods:The family members were evaluated by12-lead ECG, two-dimensional echocardiography (2-DE) and24-hour Holter. Genomic DNA of subjects in this study was extracted from peripheral whole blood and the mutational analysis of desmosmes was performed using polymerase chain reaction (PCR) and direct sequencing. The DNA sequence variants in this study were searched in the Single-nucleotide Polymorphism (SNP) Database and the Inherited Arrhythmias Database to detect whether the variants had been reported previously. New nonsynonymous sequence variant would be assessed in a population of200ethnically matched normal controls (400allels) to exclude the possibility of SNP.Results:Thirteen out of32members were recruited for this study, seven of which were diagnosed ARVC. All patients presented with palpitations and chest dyspnea, and the eldest sister of the index case died suddenly at the age of50. The age of onset of symptomatic ARVC was50.5±6.38years. Regional wall motion abnormalities were identified by2-DE in right ventricular free wall in all patients. During follow-up,2-DE revealed biventricular dilation with RV aneurysm in the proband’s elder sister. Despite the systolic impairment in proband’s younger brother, the NYHA functional class remained II. Twelve-lead ECG demonstrated extended T wave inversion. Except for the youngest sister, all patients presented with Epsilon wave after the QRS complex and the terminal activation delay was90.5±18.8(65-120) ms. The eldest brother had no epsilon waves upon S-ECG, when initially diagnosed ARVC. Four years later, QRS complex was widen and Epsilon waves were detected in precordial leads. In this family, all were homoozygous for the F531C mutation in DSG2, and this mutation was highly conservative in different species.Conclusion: Mutations in DSG2display a high degree of penetrance. Dynamic changes of ECG and2-DE suggest ARVC is a progressive disease with LV involvement as a prominent feature in this family. Part Ⅱ:Transcriptome profiling and molecular biological study reveals a major role for DSG2in the pathogenesis of ARVCBackground:Desmoglein-2(DSG2) is transmembrane cell adhesion protein of desmosomes. Mutations of DSG2have been reported in arrhythmogenic right ventricular cardiomyopathy (ARVC), suggesting that DSG2may play a role in the pathogenesis of ARVC. In our previous study, we found the loss of DSG2recapitulate the molecular changes of ARVC. However, no study has examined the mechanistic contribution of DSG2deficiency to ARVC.Objectives:The aim of our study is to investigate the loss of DSG2on the apoptosis of HL-1cell and changes of transcriptome profiling.Methods:A set of DSG2-specific siRNAs were synthesized and cloned into the vectors pGPU6/GFP/Neo. The recombinant plasmids pGPU6/GFP/Neo-ShDSG2(ShDSG2) were transfected into HL-1cells, and the positive cells clones were screened with G418. The suppression effect of DSG2mRNA and protein was measured by RT-PCR and Western blot to select the optimal cell line. NimbleGen mRNA microarray were used to examine differentially expressed genes between normal HL-1cells and DSG2silenced HL-1cells.Results:Four recombinant ShDSG2plasmids were constructed successfully and transfected into HL-1cells to induce RNAi, and stable cell lines were enstablished. ShDSG2-273group could effectively knockdown the DSG2gene expression in mRNA level (71.67%, P=0.004) and in protein level (51.72%, P<0.001), as compared with the normal HL-1cell line. We found2282differential expressed gene (up-regulated820genes, down-regulated1404genes). Meanwhile, Gene Ontology analysis showed significant alteration of gene functions such as cardiac muscle fiber development, actomyosin structure organization and regulation of heart contraction. Cardiac ion channels composed of potassium and calcium were significantly decreased after DSG downregulation. In addition, KEGG Pathway analysis demonstrated deregulation of many important signaling pathways including Tyrosine metabolism, MAPK signaling pathway and Cell Communication. Moreover, we found that loss of DSG2activated AKT/beta-catenin. Conclusion:Loss of DSG2in HL-1cell produced broad transcription changes that were similar to human ARVC. The occurrence of apoptosis, remodeling of Cx43and deregulation of ion channels may contribute to the pathogenesis of ARVC. Part Ⅲ:The estabilishement and analysis the phenotype of cardiac-specific DSG2F536c transgenic miceBackground:In our previous study, we found a highly conservative mutation F531C in DSG2gene associated arrhythmogenic right ventricular cardiomyopathy (ARVC). However, the relationship between this mutation and ARVC is unclear.Objective:To generate the cardiac-specific DSG2F536C expression transgenic mice and to determine the cardiac functional and morphological changes of the mice.Methods:The transgenic vector was constructed by inserting the mouse DSG2F536C gene into the downstream of α-MHC promoter. The transgenic mice were created by the method of micro injection. The genotype of the transgenic line was identified by PCR and the expression level of the gene was determined by Western blot. The pathologic changes were analyzed with echocardiography and Masson staining.Results:Three lines of B6CBF1DSG2F536C and two lines of DSG2WT transgenic mice were established. The high expressed line was smaller than the littermates after birth. The histological examination of Tg-H transgenic mice showed increased size of cardiomyocytes and decreased percent fractional shortening (FS%) compared with the DSG2WTtransgenic mice by echocardiography analysis. Myocardial hypertrophy and fibrosis were observed by histological analysis.Conclusions The DSG2F536C transgenic mice produce a similar pathological phenotype with the human arrhythmogenic right ventricular cardiomyopathy (ARVC). The transgenic mice could be a useful animal model to explore the mechanisms of mutation in DSG2causing ARVC.