Abstract 12944: Functional Assessment of Potential Splice Site Variants in Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy
Introduction: Interpretation of genetic screening results in Arrhythmogenic Right Ventricular Dysplasia/Cardiomyopathy (ARVD/C) is often difficult. Pathogenicity of variants with uncertain significance may be predicted by software algorithms. However, functional assessment can unambiguously demonstrate the effect of such variants.
Aim: Functional analysis of putative splice site variants identified in ARVD/C patients.
Methods: Eleven variants in desmosomal (PKP2, JUP, DSG2, DSC2) and non-desmosomal (TMEM43) genes with potential RNA splicing effect (predicted by SpliceSiteFinder-like, MaxEntScan, NNSPLICE, GeneSplicer, and Human Splicing Finder) were analyzed. The variants were found in patients with ARVD/C (fulfilment of 2010 Task Force Criteria) or suspected ARVD/C (3 patients: DSC2 c.1350A>G, JUP, TMEM43 variant). Total RNA was isolated from fresh blood samples and subjected to rtPCR. Obtained cDNA products were amplified by PCR with exonic primers specific for fragments of interest. All cDNA fragments were separated according to size using gel electrophoresis. PCR fragments were subjected to direct sequence analysis.
Results: Of the 11 variants, 6 were intronic and 5 exonic. Eight variants, including 2 missense variants, had a functionally deleterious effect on mRNA splicing by causing exon skipping, generating new splice sites, or activating cryptic sites (Table 1). All 6 intronic variants affected mRNA splicing. Two of 5 exonic variants severely impaired pre-mRNA processing. The remaining 3 exonic variants had no detectable effect on splicing, heterozygous presence in mRNA confirmed biallelic expression in these cases.
Conclusion: Eight variants of uncertain significance had a functional effect on mRNA splicing, indicative of being ARVD/C related pathogenic splice site mutations. This highlights the importance of functional assessment of potential splice site variants to enhance patient care and facilitate cascade screening.
- © 2013 by American Heart Association, Inc.