The Research Of The Molecular Disease Mechanism Of Arrhythmogenic Right Ventricular Cardiomyopathy

BackgroundArrhythmogenic Right Ventricular Cardiomyopathy (ARVC) is a inherited heart-muscle disease characterized by progressive loss of myocytes and fibrofatty tissue replacement, resulting in electrical instability of heart. The most important symptoms are ventricular arrhythmia and sudden cardiac death. Approximately 50% of the patients with ARVC have one or more mutations in their desmosomal genes; hence, ARVC is referred to as’a disease of he desmosome’. In addition to desmosomal genes, mutations in non-desmosomal genes such as DES、RYR2、TMEM43、TGFB3 are considered to be pathogenic. Since the genetic heterogeneity and phenotypic diversity of AC, modifier genes and environmental factors have an important role in disease expression. Parts of ARVC patients do not carry identifiable pathogenic mutations, so we speculate that there would be other pathogenic genes to be found. We plan to search for new pathogenic genes and study molecular disease mechanism of ARVC making use of new methods, and provide theoretical basis for the diagnosis and therapy of ARVC.Objective1) Searching for new pathogenic mutations for ARVC by high-throughout sequencing.2) Study the function of pathogenic mutation and pathogenic mechanism of ARVC.Methods and results1) The clinical characteristics and pathologic features of ARVC patientsWe corrected 25 ARVC patients who underwent heart transplantation in our hospital from 2006-2013, among them, there are 18 males and 7 females, the mean age is 41.4±13.8. Besides, there are 2 patients have family history, and 23 patients are sporadic cases. The clinical symptoms are palpitation, chest distress, and dyspnea. The results of pathology showed that the myocardium was replaced by fibrofatty tissue, but there were no apoposis in myocardium. These pathological changes occurred primarily in right ventricle, and 3 cases were left ventricular involvement.2) Searching for new pathogenic genes by high-throughout sequencingFirstly, we collected six typical ARVC patients for exome sequencing, and then, did target area capture sequencing in 25 ARVC patients. The sequencing results were verified by Sanger sequencing. Our result showed that in 25 patients,5 patients carried pathogenic gene mutation (DSG2, PKP2, DSP),1 patient carried cytoskeletal gene mutation (DES). In addition,10 patients carried FAT4 mutation which correlated with Hippo pathway; 4 patients carried TBX gene mutation; and 4 carried SYNE1 mutation which involved in the function of nucleoprotein. According to the function prediction, these mutations were considered to be pathogenic.3) Study of the function of pathogenic mutation and new potential disease mechanism of ARVCWe detected the location and expression of related protein by immunohistochemistry and western blot, the results showed that the location and expression of DSG2 are abnormal in patients with DSG2 p.F531C, in one of the twp patients, the location and the expression of Cx43 and JUP are also abnormal. These results are consistent in cell experiment. DSG2 p.L797Q and DSP p.K1937M do not affect the location and expression of DSG2 and DSP, the expression of PKP2 is decreased significantly in myocardium of patients with PKP2 p.S249T �'� E808fsX30. Moreover, the location of DES, JUP and Cx43 are abnormal in myocardium of patients with DES p.S12F.We detected the activity and expression of RhoA in myocardium of ARVC by western blot, and the results showed that the activity and expression of RhoA increased in all ARVC patients except one who carried PKP2 mutation, in addition, the expression of Cx43 was also increased. The results in vitro showed that the increase of activity of RhoA lead to the increase of expression of Cx43, however, it did not influence cytoskeleton and cell-cell adhesion in myocardiocytes.ConclusionARVC is a primarily inherited cardiomyopathy and related with genetic background. The pathological features of the patients in our study are consistant with previous reports. And desmosomal genes were the main pathogenic gene. In addition, the active RhoA may involve in the process of ARVC.Though desmosomal genes are the most common disease-causing genes, the analysis results of high-throughout sequencing in our study suggested that the mutation of other genes, FAT4, TBX2, TBX20, and SYNE1 are possibly related with the disease mechanism of ARVC.