Functional Studies on Novel Arrhythmogenic Right Ventricular Dysplasia-related DSG2 Mutations

背景： 促心律不整右心室發育不全（ARVD）是一種遺傳性心肌病變，其特徵為心肌細胞會被纖維脂肪組織所取代。目前已知plakoglobin(JUP)、desmoplakin (DSP)、 plakophilin-2 (PKP2)、desmoglein-2 (DSG2)以及desmocollin-2(DSC2)的突變與ARVD有關，因此認為ARVD的形成與胞橋小體的缺陷有關。我們在台灣地區ARVD病人中發現三個相關的DSG2錯意突變。為了了解DSG2錯意突變與ARVD的關係，我們對突變的DSG2進行功能性研究。 方法與結果： 我們針對從台灣地區ARVD病人新發現的DSG2突變：Asn330Asp, Phe531Cys 與 Glu713Lys，研究其在細胞的分佈及與其他蛋白的組合特性是否會有所變化。DSG2基因建構於會表現綠螢光蛋白（GFP）的pEGFP-N2 vector，再送入會表現胞橋小體的HEK293T 或PC-3細胞。共軛焦顯微鏡觀察的結果顯示這三個DSG2突變型的分佈位置與野生型相比均無明顯差異。我們再利用免疫沈澱的方式了解DSG2與其他胞橋小體蛋白的連結情形。結果發現突變型DSG2與DSC2/3以及細胞固有的DSG2之連結情形與野生型相比沒有差異，均無法被免疫沈澱下；PKP2可被野生型或三種突變型的DSG2免疫沈澱下，突變型DSG2與PKP2的連結與野生型相比雖有變化，但無顯著差異；JUP亦可被野生型或三種突變型的DSG2免疫沈澱下，其中Asn330Asp和Phe531Cys突變型和JUP的連結與野生型相比無明顯變化，但Glu713Lys突變型與JUP的連結和野生型相比有明顯變弱的現象。 結論： 在台灣地區ARVD病患中所找出的Asn330Asp, Phe531Cys 與 Glu713Lys突變並不影響DSG2的分佈，但Glu713Lys突變型與JUP的連結會明顯變弱。這可能會進而影響胞橋小體的黏附作用而導致ARVD的形成。Background: Arrhythmogenic right ventricular dysplasia（ARVD）is an inherited cardiomyopathy characterized by progressive myocardial loss with fibrofatty replacement. Identification of causative mutations in plakoglobin(JUP), desmoplakin (DSP), plakophilin-2 (PKP2) , desmoglein-2 (DSG2)and desmocollin-2(DSC2) genes laeds to the hypothesis that ARVC is due to desmosomal defects. Three novel ARVD related DSG2 missense mutations were found in Taiwanese patients. Functional studies were performed to establish a causal relationship between DSG2 missense mutations and ARVD. Methods and Results: We studied the trafficking and assembly properties of three novel DSG2 mutations identified in patients with ARVD in Taiwan: Asn330Asp, Phe531Cys and Glu713Lys. DSG2 gene was cloned into pEGFP-N2 vector, which expressed a green fluorescent protein (GFP) fusion protein. The vector was transfected to desmosome-forming cell line: HEK293T or PC-3 cells. Confocal microscopic studies showed that all these mutants are trafficking competent. In order to investigate the effects of these mutations on the interactions between DSG2 and other desmosomal proteins, co-immunoprecipitation assay was performed. No changes were observed in the immunoprecipitation of DSC2/3 and DSG2; PKP2 was found to be immunoprecipitated by wild type and three mutant DSG2, the interactions between mutant DSGs and PKP2 are not different from that between wild type DSGs and PKP2; JUP was also found to be immunoprecipitated by wild type and three mutant DSG2, while the interactions between JUP and Glu713Lys mutant form decreased significantly. Conclusions: Three DSG2 mutations identified in patients with ARVD in Taiwan: Asn330Asp, Phe531Cys and Glu713Lys are all trafficking competent, while the interactions between JUP and Glu713Lys mutant form are much weaker. This may affect the adhesive capacity of desmosomes and finally results in ARVD