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Background Large-scale “omics” datasets have not been leveraged and integrated with functional analyses to discover potential drivers of cardiomyopathy. This study addresses the knowledge gap. Methods We coupled RNA sequence (RNA-Seq) variant detection and transcriptome profiling with pathway analysis to model drug refractory dilated cardiomyopathy (drDCM) using the BaseSpace sequencing hub and Ingenuity Pathway Analysis. We used RNA-Seq case-control datasets (n = 6 cases, n = 4 controls), exome sequence familial DCM datasets (n = 3 Italians, n = 5 Italians, n = 5 Chinese), and controls from the HapMap project (n = 5 Caucasians, and n = 5 Asians) for disease modeling and putative mutation discovery. Variant replication datasets: n = 128 cases and n = 15 controls. Source of datasets: NCBI Sequence Read Archive. Statistics: Pairwise differential expression analyses to determine differentially expressed genes and t-tests to calculate p-values. We adjusted for false discovery rates and reported q-values. We used chi-square tests to assess independence among variables, the Fisher’s Exact Tests and overlap p-values for the pathways and p-scores to rank network. Results Data revealed that ECHS1(enoyl-CoA hydratase, short chain 1(log2(foldchange) = 1.63329) hosts a mirtron, MIR3944 expressed in drDCM (FPKM = 5.2857) and not in controls (FPKM = 0). Has-miR3944-3p is a putative target of BAG1 (BCL2 associated athanogene 1(log2(foldchange) = 1.31978) and has-miR3944-5p of ITGAV (integrin subunit alpha V(log2(foldchange) = 1.46107) and RHOD (ras homolog family member D(log2(foldchange) = 1.28851). There is an association between ECHS1:11 V/A(rs10466126) and drDCM (p = 0.02496). The interaction (p = 2.82E-07) between ECHS1:75 T/I(rs1049951) and ECHS1:rs10466126 is associated with drDCM (p

Figure S2. Expression profiles for ECHS1 and has-mir-3944 in normal heart. Legend: Panel 1: ECHS1 expression profiles in 6 different tissues. Panel 2: has-mir-3944 expression profiles in 6 different tissues. Panel 3: Expression correlations between has-mir-3944-5p and the ECHS1 in the 6 different tissues. (PPTX 248 kb)

Figure S3. Predicted conservation: Putative target region and has-mir-3944. Legend: A Conservation for RHOD. B Conservation for ITGAV. C Conservation for BAG1. (PPTX 545 kb)

Lung side population (SP) cells are resident lung precursor cells with both epithelial and mesenchymal potential that are believed to play a role in normal lung development and repair. Neonatal hyperoxic exposure impairs lung development leading to a long-term decrease in gas exchange surfaces. The hypothesis that lung SP cells are altered during impaired lung development has not been studied. To address this issue, we characterized the endothelial potential of neonatal lung SP and subsets of lung SP from neonatal mice following hyperoxic exposure during room air recovery. Lung SP cells were isolated and sorted on the basis of their capacity to efflux Hoechst 33342. The lung SP was further sorted based on expression of Flk-1 and CD45. In vitro, both CD45pos/Flk-1pos and CD45neg/Flk-1pos bind isolectin B4 and incorporate LDL and form networks in matrigel, indicating that these populations have endothelial cell characteristics. Hyperoxic exposure of neonatal mice resulted in subtle changes in vascular and alveolar density on P13, which persisted with room air recovery to P41. During room air recovery, a decrease in lung SP cells was detected in the hyperoxic-exposed group on postnatal day 13 followed by an increase on day 41. Within this group, the lung SP subpopulation of cells expressing CD45 increased on day 21, 41, and 55. Here, we show that lung SP cells demonstrate endothelial potential and that the population distribution changes in number as well as composition following hyperoxic exposure. The hyperoxia-induced changes in lung SP cells may limit their ability to effectively contribute to tissue morphogenesis during room air recovery.

Background & Aims Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in the United States. The etiology is believed to be multifactorial with a substantial genetic component; however, the heritability of NAFLD is undetermined. Therefore, a familial aggregation study was performed to test the hypothesis that NAFLD is highly heritable. Methods Overweight children with biopsy-proven NAFLD and overweight children without NAFLD served as probands. Family members were studied, including the use of magnetic resonance imaging to quantify liver fat fraction. Fatty liver was defined as a liver fat fraction of 5% or higher. Etiologies for fatty liver other than NAFLD were excluded. Narrow-sense heritability estimates for fatty liver (dichotomous) and fat fraction (continuous) were calculated using variance components analysis adjusted for covariate effects. Results Fatty liver was present in 17% of siblings and 37% of parents of overweight children without NAFLD. Fatty liver was significantly more common in siblings (59%) and parents (78%) of children with NAFLD. Liver fat fraction was correlated with body mass index, although the correlation was significantly stronger for families of children with NAFLD than those without NAFLD. Adjusted for age, sex, race, and body mass index, the heritability of fatty liver was 1.000 and of liver fat fraction was 0.386. Conclusions Family members of children with NAFLD should be considered at high risk for NAFLD. These data suggest that familial factors are a major determinant of whether an individual has NAFLD. Studies examining the complex relations between genes and environment in the development and progression of NAFLD are warranted.

Figure S5. Proposed schematic: Dysfunction in the mitochondria. Legend: Arrows (red) pointing up represent up-regulated genes and arrows (green) pointing down, down-regulated genes. Printed in blue indicates putative modifiers and in red mutations. In bold print indicates genes carrying a variant. Orange arrows indicate the flow of catalysis in the fatty acid beta-oxidation pathway. Green arrows show the flow of catalysis in the Leucine degradation 1 pathway. Purple arrows show the flow of catalysis in the Valine degradation 1 pathway. Blue arrows indicate the flow of catalysis in the Isoleucine degradation 1 pathway. In boxes at the end of each pathway are proposed products of each pathway. (PPTX 583 kb)

Figure S5. Proposed schematic: Dysfunction in the mitochondria. Legend: Arrows (red) pointing up represent up-regulated genes and arrows (green) pointing down, down-regulated genes. Printed in blue indicates putative modifiers and in red mutations. In bold print indicates genes carrying a variant. Orange arrows indicate the flow of catalysis in the fatty acid beta-oxidation pathway. Green arrows show the flow of catalysis in the Leucine degradation 1 pathway. Purple arrows show the flow of catalysis in the Valine degradation 1 pathway. Blue arrows indicate the flow of catalysis in the Isoleucine degradation 1 pathway. In boxes at the end of each pathway are proposed products of each pathway. (PPTX 583 kb)

Figure S4. Pathways associated with the ECHS1 gene. (PPTX 849 kb)

Figure S1. Transcription factors that bind to the ECHS1 gene. (PPTX 202 kb)

Figure S1. Transcription factors that bind to the ECHS1 gene. (PPTX 202 kb)

Figure S4. Pathways associated with the ECHS1 gene. (PPTX 849 kb)