Genome-wide association identifies a deletion in the 3' untranslated region of striatin in a canine model of arrhythmogenic right ventricular cardiomyopathy

Abstract

Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a familial cardiac disease characterized by ventricular arrhythmias and sudden cardiac death. It is most frequently inherited as an autosomal dominant trait with incomplete and age-related penetrance and variable clinical expression. The human disease is most commonly associated with a causative mutation in one of several genes encoding desmosomal proteins. We have previously described a spontaneous canine model of ARVC in the boxer dog. We phenotyped adult boxer dogs for ARVC by performing physical examination, echocardiogram and ambulatory electrocardiogram. Genome-wide association using the canine 50k SNP array identified several regions of association, of which the strongest resided on chromosome 17. Fine mapping and direct DNA sequencing identified an 8-bp deletion in the 3' untranslated region (UTR) of the Striatin gene on chromosome 17 in association with ARVC in the boxer dog. Evaluation of the secondary structure of the 3' UTR demonstrated that the deletion affects a stem loop structure of the mRNA and expression analysis identified a reduction in Striatin mRNA. Dogs that were homozygous for the deletion had a more severe form of disease based on a significantly higher number of ventricular premature complexes. Immunofluorescence studies localized Striatin to the intercalated disc region of the cardiac myocyte and co-localized it to three desmosomal proteins, Plakophilin-2, Plakoglobin and Desmoplakin, all involved in the pathogenesis of ARVC in human beings. We suggest that Striatin may serve as a novel candidate gene for human ARVC.

Figure 1

Genome-wide association mapping of canine ARVC. A 10 Mb region on canine chromosome 17 exhibited genome-wide significant association (p genome< 0.04) (A). Close up of the chromosome 17 peaks (B).

Figure 1

Genome-wide association mapping of canine ARVC. A 10 Mb region on canine chromosome 17 exhibited genome-wide significant association (p genome< 0.04) (A). Close up of the chromosome 17 peaks (B).

Figure 2

An 8 base pair deletion (control (A), homozygous ARVC (B)) in the 3’ untranslated region of the canine Striatin gene was associated with the development of canine ARVC (p=0.005). The black bar above the control sequence (A) indicates the sequence deleted in affected dogs.

Figure 3

Electrocardiogram from a boxer dog with canine ARVC (A). The black bar indicates a run of ventricular tachycardia that occurred after several normal sinus complexes. Dogs that were homozygous for the mutation had more severe disease based on ventricular premature complex number in comparison to dogs that were heterozygous for the mutation (p=0.001) (B).

Figure 4

Secondary structure prediction of the 3’ untranslated region determined an alteration in both stem and loop formation in the ARVC dog in comparison to the control. (A, control dog; B, ARVC dog; arrow indicates region of deletion)

Figure 5

Expression analysis of mRNA levels in cardiac tissue identified a significant reduction (p=0.04) in Striatin between 2 non-Boxer controls and 4 ARVC dogs. The length of each box indicates the range of values and the horizontal line indicates mean for the population.

Figure 6

Representative image of protein analysis of Striatin in right ventricular myocardial sections. Lane 1 contains a non-boxer control; lane 2 contains a heterozygous ARVC dog; lane 3 contains a homozygous ARVC dog. Striatin protein as measured by corrected Optical Density was significantly reduced between ARVC cases (n=4) and controls (n=2) (p=0.03). Quantification of protein levels for 2 non-boxer controls and 2 heterozygous and 2 homozygous ARVC boxers is expressed at the bottom as a percentage of control.

Figure 7

Striatin colocalizes with three desmosomal proteins. Confocal microscopy of normal beagle myocardium. Immunolabeling for striatin polyclonal antibody is green, red for desmosomal proteins (plakophilin 2, plakoglobin and desmoplakin) and the nuclei in the merged image are stained blue with DAPI. A no primary negative control is included for non-specific labeling. Bar=5µm.

Figure 8

Representative images of immunofluorescence studies demonstrating Striatin (green) and Plakophilin-2 (red) colocalizing in the desmosomal region of the cardiac myocyte in a control non-boxer dog (A), heterozygous ARVC dog (B) and homozygous ARVC dog (C).