Insights into the molecular mechanism of pulmonary vein stenosis in pediatric patients with congenital heart disease

Pulmonary vein specimens were collected for PVS patient and bulk-RNA sequencing conducted.

Figure 1. A, Study design: experimental Subgroups: We included primary and postrepair PVS and for both groups had samples for both aggressive and nonaggressive phenotype. B, Experimental design: pulmonary vein stenosis (PVS) samples were collected from the biobank and RNA isolation conducted and checked for quality control. Bulk RNA sequencing was performed, and the analysis consisted of principal component analysis (PCA), differential gene expression, Gene Ontology analysis, and Kyoto Encyclopedia of Genes and Genomes and Reactome analysis. C, Sample identification: 6 samples were used for this study and labeled as A-F and categorized as primary PVS, postrepair PVS, and further categorized as aggressive or nonaggressive. D, Group identification: 6 groups were identified, as listed in Table 1, and the respective samples for each group also are identified in Table 1. E, Study comparisons: 3 comparisons were studies and identified in Table 1 and the specific comparison groups are also identified in Table 1.

Figure 2. Principal component analysis (PCA) plots are shown for the following comparisons: (A) AggPPVS versus NonAggPPVS; (B) AllAgg versus AllNonAgg; and (C) AllPPVS versus AllPRPVS. Agg, Aggressive; NonAgg, nonaggressive; PPVS, primary pulmonary vein stenosis; PRPVS, post-repair pulmonary vein stenosis.

Figure 3. Differentially expressed genes. Volcano plots are shown for the following comparisons: (A) AggPPVS versus NonAggPPVS; (B) AllAgg versus AllNonAgg; and (C) AllPPVS versus AllPRPVS. Red dots represent genes fold change (FC) greater than 1.5 whereas the black dots are genes with fold change less than 1.5. Agg, Aggressive; NonAgg, nonaggressive; PPVS, primary pulmonary vein stenosis; PRPVS, post-repair pulmonary vein stenosis.

Figure 4. Heatmaps showing top 50 genes. The top 50 upregulated, downregulated and most significant genes are presented for the 4 groups: (A) AggPPVS versus NonAggPPVS; (B) AllAgg versus AllNonAgg; (C) AggPPVS versus AllPRPVS. Agg, Aggressive; NonAgg, nonaggressive; PPVS, primary pulmonary vein stenosis; PRPVS, post-repair pulmonary vein stenosis.

Figure 5. Gene Ontology analysis for AggPPVS versus NonAggPPVS. A, Biological processes. B, Cellular components. C, Molecular functions. Left panel shows the top 50 most significant pathways. Right panel shows the number of differentially expressed genes for each pathway. Agg, Aggressive; NonAgg, nonaggressive; PPVS, primary pulmonary vein stenosis; PRPVS, post-repair pulmonary vein stenosis.

Figure 6. A, Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis for AggPPVS versus NonAggPPVS. B, Reactome analysis for AggPPVS versus NonAggPPVS. For both (A) and (B) the top 50 most significant pathways are shown. Agg, Aggressive; NonAgg, nonaggressive; PPVS, primary pulmonary vein stenosis; PRPVS, post-repair pulmonary vein stenosis.

Figure 7. Selected Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway for AggPPVS versus NonAggPPVS. A, Cardiac muscle contraction. B, Calcium signaling pathway. Agg, Aggressive; NonAgg, nonaggressive; PPVS, primary pulmonary vein stenosis.