Genetic analysis in a large cohort of unrelated consecutive patients with Arrhythmogenic Right Ventricular Cardiomyopathy/Dysplasia

Introduction - Arrhythmogenic right ventricular cardiomyopathy/dysplasia (ARVC/D) is an inherited heart muscle disorder that primarily affects the right ventricular myocardium early in the course of disease with later-onset left ventricular involvement. Clinically, it is characterized by ventricular arrhythmias of right ventricular origin, as noted by ventricular tachycardia with a left bundle branch block morphology, commonly associated with syncope or sudden cardiac death in particular in teenagers and in young adults. To date, mutations in 7 genes, including 5 encoding desmosomal proteins, Junctional plakoglobin (JUP), Desmoplakin (DSP), Plakophilin-2 (PKP2), Desmoglein-2 (DSG2) and Desmocollin-2 (DSC2), have been identified in ARVC/D patients. The study of genetically engineered mice models of ARVC/D, generated through transgenesis and gene targeting, recapitulates the pathogenic characteristics of the disease. Methods - The study involved a cohort of 110 unrelated consecutive index cases and their available family members. Clinical diagnosis of ARVC/D was based on major and minor criteria established by an international Task Force. Mutation screening in four desmosomal protein genes (PKP2, DSP, DSG2 and DSC2) was performed by denaturing high-performance liquid chromatography (DHPLC) and direct sequencing in ARVC/D index cases. Desmin (DES) and plakophilin-4 (PKP4) candidate genes were screened in 80 ARVC/D index cases, by DHPLC and direct sequencing as well. In order to generate a knock-in mouse carrying a targeted mutation in DSG2, the mouse dsg2 gene was isolated from the l FIX II 129/SVJ library. A 7041bp genomic fragment was subcloned in the targeting vector, and three nucleotide mutations (G105R, N271S, and K299E) were introduced in mouse dsg2 exons 4, 7, and 8 by site-directed mutagenesis. Neomycin resistance cassette (Neo) and Thymidine kinase cassette (TK) were cloned in the targeting vector, thus allowing positive-negative selection of the recombination events. Results - Analysis of coding sequences of PKP2, DSP, DSG2 and DSC2 genes was performed on genomic DNA of 110 ARVC/D index cases. One PKP2 mutation was detected in 16 probands (14.6%), one DSP mutation in 11 (10.0%), one DSG2 mutation in 8 (7.3%), and one DSC2 mutation in 3 subjects (2.7%). Compound or double heterozygosity was identified in 14 probands (12.7%). Available family members of 19 index cases were screened for the detected mutations and clinical investigation showed that clinical expression of ARVC/D mutations is heterogeneous even among relatives, ranging from a complete lack of symptoms and/or clinical manifestations to severe disease phenotype. According with the hypothesis that ARVC/D is due to desmosomal defects, DES and PKP4 genes were screened in 80 ARVC/D probands. Two variations (K241E and c.736-11A>G) were detected in DES gene in two subjects. Three PKP4 variations (c.245+101A>G, A479A, and P797P) were detected in three subjects. None of the nucleotide changes was found in 300 control subjects from the same population. To establish a cause and effect relationship between DSG2 mutations and ARVC/D, a knock-in mouse model will be generated. A dsg2 genomic fragment was cloned in the targeting vector that will be used; by site-directed mutagenesis three dsg2 pathogenic mutations (G105R, N271S, and K299E) were introduced; also Neo and TK cassettes were cloned. After linearization, the vector will be transfected into the murine embryonic stem cells. Discussion - ARVC/D is a recognized cause of sudden cardiac death, which may be prevented by timely detection and intervention. Since mutations causing ARVC/D have been identified so far in genes encoding desmosomal proteins, this cardiomyopathy might be considered as "a disease of the desmosome". Mutation screening of the four desmosomal genes PKP2, DSP, DSG2 and DSC2 in 110 ARVC/D unrelated individuals allowed successful genotyping of 52 (47.3%). Emerging data suggest that an important minority of ARVC/D patients are compound heterozygous or double heterozygous (12.7%). Clinical comparison of patients carrying single and multiple mutations showed no significant differences in terms of electrocardiographic and structural abnormalities, major events and disease expression. The only significant difference was that patients carrying DSG2 mutations were found older at diagnosis and at the time of major arrhythmic symptoms than DSP and PKP2 carriers. On the ground of these data, it is impossible to clinically differentiate different forms of ARVC/D due to mutations in different genes. Since no causing mutations have been identified in more than 50% of patients, additional components of the desmosome-intermediate filament complex and associated proteins were considered the primary candidates disease-genes. The coding regions of DES and PKP4 genes were screened in 80 ARVC/D index cases. Most of the detected nucleotide changes were intronic and synonymous variations that do not change the sequence of the gene product, but might affect splicing (by activating a cryptic splice site). On the basis of present data, it is not possible to exclude the involvement of these genes in the pathogenesis of ARVC/D; thus, mutation screening in ARVC/D genes on DNA of probands should be planned only on the basis of relative prevalence of mutations in different genes. The identification of the primary genetic causes of ARVC/D has opened the possibility to generate animal models where the events underlying the pathophysiology of this disease can be studied in detail. Gene transfer technology allows the creation of specific mutant genotypes in animals thereby increasing their chance of resembling human diseases at the genetic and phenotypic levels. The generated targeting vectors will be transfected into the murine embryonic stem cells to create a DSG2 knock-in mouse model. In perspective, such models should prove useful for investigating cellular mechanisms involved in the molecular pathogenesis of ARVC/D and for assessing the effects of selected pharmacological treatments.