Functional assessment of potential splice site variants in arrhythmogenic right ventricular dysplasia/cardiomyopathy

• Published in: Heart rhythm Vol. 11; no. 11; pp. 2010 - 2017
• Main Authors: Groeneweg, Judith A., MD, Ummels, Amber, Mulder, Marcel, Bikker, Hennie, PhD, van der Smagt, Jasper J., MD, van Mil, Anneke M, Homfray, Tessa, MD, Post, Jan G., MD, Elvan, Arif, MD, PhD, van der Heijden, Jeroen F., MD, PhD, Houweling, Arjan C., MD, PhD, Jongbloed, Jan D.H., PhD, Wilde, Arthur A.M., MD, PhD, van Tintelen, J. Peter, MD, PhD, Hauer, Richard N., MD, PhD, Dooijes, Dennis, PhD
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 01-11-2014
• Subjects: Adolescent; Adult; Aged; Algorithms; Alleles; Arrhythmogenic Right Ventricular Dysplasia - genetics; Arrhythmogenic Right Ventricular Dysplasia - physiopathology; Arrhythmogenic right ventricular dysplasia/cardiomyopathy; Calcium-Binding Proteins - genetics; Cardiomyopathies - genetics; Cardiomyopathies - physiopathology; Cardiovascular; Desmocollins - genetics; Desmoglein 2 - genetics; Desmoplakins - genetics; Desmosomes; Electrocardiography; Exons; Female; gamma Catenin; Genetic Variation; Genetics; Humans; Introns; Male; Membrane Proteins - genetics; Middle Aged; Mutation; Pedigree; Plakophilins - genetics; Reverse Transcriptase Polymerase Chain Reaction; RNA analysis; RNA Splice Sites; RNA, Messenger - blood; Software; Splice site mutations; Ventricular arrhythmias; desmoplakin; Ventricular arrhythmias; RV; ARVD/C; left ventricle; plakophilin-2; Desmosomes; LV; transmembrane protein-43; TFC; Splice site mutations; PLN; TMEM43; LBBB; RT-PCR; reverse transcriptase polymerase chain reaction; Genetics; Task Force Criteria for ARVD/C diagnosis; DSP; variants of uncertain clinical significance; desmocollin-2; JUP; arrhythmogenic right ventricular dysplasia/cardiomyopathy; desmoglein-2; left bundle branch block; phospholamban; RNA analysis; MAF; plakoglobin; minor allele frequency; right ventricle; ventricular tachycardia; VUS; PKP2; DSG2; DSC2; VT; Arrhythmogenic right ventricular dysplasia/cardiomyopathy
• ISSN: 1547-5271; 1556-3871
• DOI: 10.1016/j.hrthm.2014.07.041

Background Interpretation of genetic screening results in arrhythmogenic right ventricular dysplasia/cardiomyopathy (ARVD/C) often is difficult. Pathogenicity of variants with uncertain clinical significance may be predicted by software algorithms. However, functional assessment can unambiguously demonstrate the effect of such variants. Objective The purpose of this study was to perform functional analysis of potential splice site variants in ARVD/C patients. Methods Nine variants in desmosomal ( PKP2 , JUP , DSG2 , DSC2 ) genes with potential RNA splicing effect were analyzed. The variants were found in patients who fulfilled 2010 ARVD/C Task Force Criteria (n = 7) or had suspected ARVD/C (n = 2). Total RNA was isolated from fresh blood samples and subjected to reverse transcriptase polymerase chain reaction. Results An effect on splicing was predicted by software algorithms for all variants. Of the 9 variants, 5 were intronic and 4 exonic. RNA analysis showed a functional effect on mRNA splicing by exon skipping, generation of new splice sites, or activation of cryptic sites in 6 variants. All 5 intronic variants tested severely impaired splicing. Only 1 of 4 exonic potential splice site variants was shown to have a deleterious effect on splicing. The remaining 3 exonic variants had no detectable effect on splicing, and heterozygous presence in mRNA confirmed biallelic expression. Conclusion Six variants of uncertain clinical significance in the PKP2 , JUP , and DSG2 genes showed a deleterious effect on mRNA splicing, indicating these are ARVD/C–related pathogenic splice site mutations. These results highlight the importance of functional assessment of potential splice site variants to improve patient care and facilitate cascade screening.