A circular RNA based biomarker for transposition of the great arteries(TGA) hsa-PHACTR4_0009 upregulates cell migration during TGA pathogenesis
N Rawal, A Gurgela, S Kumari, M K Rohit, A Bahl, A MittalAbstract
Background
Dextro-transposition of the great arteries (d-TGA) is a serious cyanotic congenital heart defect caused by anatomical reversal of the aorta and pulmonary artery, resulting in parallel circulation. Despite advances in surgery, neonatal morbidity and mortality remain high. Prenatal diagnosis by foetal echocardiography has variable sensitivity, and the molecular mechanisms underlying d-TGA are poorly understood. Given its complex gene–environment and polygenic etiology, epigenetic regulators such as circular RNAs (circRNAs) may contribute to disease pathogenesis. Besides important regulatory functions, circRNAs also act as potentially better candidates as biomarkers due to their higher stability in plasma than any linear non-coding RNA.
Objectives
This study aimed to identify plasma-derived circRNAs dysregulated in neonates with d-TGA, evaluate their diagnostic potential, and explore their functional and molecular roles in disease development.
Methods
We performed whole transcriptomics with circRNA enrichment on plasma samples from TGA neonates and age-matched controls. Through bioinformatic analysis, we identified differentially expressed circRNAs and later validated their expression in a larger cohort through qRT-PCR. Diagnostic performance of the candidate circRNA was evaluated using ROC curve analysis, with foetal echocardiography as the reference standard. Associations between circRNA expression and disease severity were analyzed based on cardiac anatomical features. To elucidate circRNA-associated gene networks, we conducted transcriptomic pathway analysis, and functional effects were examined through circRNA overexpression and migration assays in H9C2 cardiomyocytes.
Results
Several circRNAs were significantly dysregulated in d-TGA plasma samples. Among them, hsa-PHACTR4_0009 was markedly upregulated and consistently validated in a larger cohort. ROC curve analysis revealed 85% sensitivity, 80% specificity, and an AUC of 0.900, indicating good diagnostic performance of this circular RNA as a non-invasive biomarker. hsa-PHACTR4_0009 levels positively correlated with disease severity and were higher in neonates with intact interventricular and interatrial septa. Analysis of RNA-Seq data showed that the DEGs are linked to focal adhesion and cytoskeletal organization, correlating with elevated hsa-PHACTR4_0009 expression. Functional assays showed that hsa-PHACTR4_0009 overexpression significantly enhanced cardiomyocyte migration.
Conclusion
circRNA hsa-PHACTR4_0009 serves as a plasma biomarker with high diagnostic accuracy for d-TGA and a strong association with disease severity. Functional and transcriptomic findings suggest its involvement in d-TGA pathogenesis by regulating cell migration, a known mechanism that leads to faulty outflow tract development during cardiogenesis. Therefore, this study also offers new mechanistic insights to improve TGA prognosis.Graphical abstract