Abstract 16134: Dysregulated Splicing of DICER1 mRNA in Myocardium From Infants With Tetralogy of Fallot Results in Altered Protein Isoforms

Introduction: Congenital heart defects (CHDs) are the most common birth defect and represent a substantial health care burden. Research in the last 10 years has led to improved understanding of the genetic regulation of vertebrate heart development, but despite this effort, approximately 70% of all CHD cases still have an unknown etiology. We recently showed that splicing is dysregulated in pathways that regulate heart development in cardiac tissue from infants with tetralogy of Fallot (TOF). Hypothesis: Alternative splicing during cardiogenesis results in alternative protein isoforms. Methods: Right ventricular (RV) tissue was obtained at the time of surgical repair of TOF and control RV tissue was obtained from donor pulmonary valve samples as we have previously described. In addition, 5 controls were obtained from the Brain and Tissue Bank. Protein was extracted using T-PER Tissue Extraction Reagent from Thermo-Fisher Scientific according to manufacturer’s specifications. The protein concentration of the lysed tissue was quantified using the Pierce BCA Protein Assay Kit from Thermo-Fisher Scientific according to company specifications. Western Blots were performed using the Wes 66-440kDa Master Kit with Split Running Buffer. The primary antibody was specific for DICER1 (Abiocode, cat # R2388-2), and we used an anti-Rabbit secondary antibody following WES system (Protein Simple, Inc.) standard protocols. Results: We focused our preliminary analysis on DICER1 as it has been shown to be an integral regulator of splicing in vertebrate heart development and our previous data showed that it was alternatively spliced in myocardium from infants with TOF. Control samples had two isoforms of DICER1, a very prominent lower band (~190 kDa) and faint upper band (~220 kDa). The protein from the TOF RV samples had no visible lower band but their upper band was approximately 7 fold more intense than the corresponding upper band in the control lanes. Conclusions: Thus mRNA splicing variants in DICER1 in TOF myocardium resulted in corresponding protein isoform synthesis and resulted in changed protein variant concentrations. Therefore, failure of precise splicing that leads to changes in protein isoforms may be a contributing factor to TOF.