Genetics and genotype-phenotype correlations in Finnish patients with dilated cardiomyopathy.

AIMS: Despite our increased understanding of the genetic basis of dilated cardiomyopathy (DCM), the clinical utility and yield of clinically meaningful findings of comprehensive next-generation sequencing (NGS)-based genetic diagnostics in DCM has been poorly described. We utilized a high-quality oligonucleotide-selective sequencing (OS-Seq)-based targeted sequencing panel to investigate the genetic landscape of DCM in Finnish population and to evaluate the utility of OS-Seq technology as a novel comprehensive diagnostic tool. METHODS AND RESULTS: Using OS-Seq, we targeted and sequenced the coding regions and splice junctions of 101 genes associated with cardiomyopathies in 145 unrelated Finnish patients with DCM. We developed effective bioinformatic variant filtering strategy and implemented strict variant classification scheme to reveal diagnostic yield and genotype-phenotype correlations. Implemented OS-Seq technology provided high coverage of the target region (median coverage 410× and 99.42% of the nucleotides were sequenced at least 15× read depth). Diagnostic yield was 35.2% (familial 47.6% and sporadic 25.6%, P = 0.004) when both pathogenic and likely pathogenic variants are considered as disease causing. Of these, 20 (53%) were titin (TTN) truncations (non-sense and frameshift) affecting all TTN transcripts. TTN truncations accounted for 20.6% and 14.6% of the familial and sporadic DCM cases, respectively. CONCLUSION: Panel-based, high-quality NGS enables high diagnostic yield especially in the familial form of DCM, and bioinformatic variant filtering is a reliable step in the process of interpretation of genomic data in a clinical setting.

Suppression of endothelial CD39/ENTPD1 is associated with pulmonary vascular remodeling in pulmonary arterial hypertension.

Endothelial cell (EC) dysfunction plays a role in the pathobiology of occlusive vasculopathy in pulmonary arterial hypertension (PAH). Purinergic signaling pathways, which consist of extracellular nucleotide and nucleoside-mediated cell signaling through specific receptors, are known to be important regulators of vascular tone and remodeling. Therefore, we hypothesized that abnormalities in the vascular purinergic microenvironment are associated with PAH. Enzymatic clearance is crucial to terminate unnecessary cell activation; one of the most abundantly expressed enzymes on the EC surface is E-NTPDase1/CD39, which hydrolyzes ATP and ADP to AMP. we used histological samples from patients and healthy donors, radioisotope-labeled substrates to measure ectoenzyme activity, and a variety of in vitro approaches to study the role of CD39 in PAH. Immunohistochemistry on human idiopathic PAH (IPAH) patients' lungs demonstrated that CD39 was significantly downregulated in the endothelium of diseased small arteries. Similarly, CD39 expression and activity were decreased in cultured pulmonary ECs from IPAH patients. Suppression of CD39 in vitro resulted in EC phenotypic switch that gave rise to apoptosis-resistant pulmonary arterial endothelial cells and promoted a microenvironment that induced vascular smooth muscle cell migration. we also identified that the ATP receptor P2Y11 is essential for ATP-mediated EC survival. Furthermore, we report that apelin, a known regulator of pulmonary vascular homeostasis, can potentiate the activity of CD39 both in vitro and in vivo. we conclude that sustained attenuation of CD39 activity through ATP accumulation is tightly linked to vascular dysfunction and remodeling in PAH and could represent a novel target for therapy.

Loss of bone morphogenetic protein receptor 2 is associated with abnormal DNA repair in pulmonary arterial hypertension.

Occlusive vasculopathy with intimal hyperplasia and plexogenic arteriopathy are severe histopathological changes characteristic of pulmonary arterial hypertension (PAH). Although a phenotypic switch in pulmonary endothelial cells (ECs) has been suggested to play a critical role in the formation of occlusive lesions, the pathobiology of this process is poorly understood. The goal of this study was to identify novel molecular mechanisms associated with EC dysfunction and PAH-associated bone morphogenetic protein receptor 2 (BMPR2) deficiency during PAH pathogenesis. A bioinfomatics approach, patient samples, and in vitro experiments were used. By combining a metaanalysis of human idiopathic PAH (iPAH)-associated gene-expression microarrays and a unique gene expression-profiling technique in rat endothelium, our bioinformatics approach revealed a PAH-associated dysregulation of genes involving chromatin organization, DNA metabolism, and repair. Our hypothesis that altered DNA repair and loss of genomic stability play a role in PAH was supported by in vitro assays where pulmonary ECs from patients with iPAH and BMPR2-deficient ECs were highly susceptible to DNA damage. Furthermore, we showed that BMPR2 expression is tightly linked to DNA damage control because excessive DNA damage leads to rapid down-regulation of BMPR2 expression. Moreover, we identified breast cancer 1 (BRCA1) as a novel target for BMPR2 signaling and a novel modulator of pulmonary EC homeostasis. We show here that BMPR2 signaling plays a critical role in the regulation of genomic integrity in pulmonary ECs via genes such as BRCA1. We propose that iPAH-associated EC dysfunction and genomic instability are mediated through BMPR2 deficiency-associated loss of DNA damage control.

Accurate genetic diagnosis of Finnish pulmonary arterial hypertension patients using oligonucleotide-selective sequencing.

The genetic basis of pulmonary arterial hypertension (PAH) among Finnish PAH patients is poorly understood. We adopted a novel-targeted next-generation sequencing (NGS) approach called Oligonucleotide-Selective Sequencing (OS-Seq) and developed a custom data analysis and interpretation pipeline to identify pathogenic base substitutions, insertions, and deletions in seven genes associated with PAH (BMPR2, BMPR1B, ACVRL1, ENG, SMAD9, CAV1, and KCNK3) from Finnish PAH patients. This study represents the first clinical study with OS-Seq technology on patients suffering from a rare genetic disorder. We analyzed DNA samples from 21 Finnish PAH patients, whose BMPR2 and ACVRL1 mutation status had been previously studied using Sanger sequencing. Our sequencing panel covered 100% of the targeted base pairs with >15× sequencing depth. Pathogenic base substitutions were identified in the BMPR2 gene in 29% of the Finnish PAH cases. Two of the pathogenic variant-positive patients had been previously tested negative using Sanger sequencing. No clinically significant variants were identified in the six other PAH genes. Our study validates the use of targeted OS-Seq for genetic diagnostics of PAH and revealed pathogenic variants that had been previously missed using Sanger sequencing.

FOXL2, GATA4, and SMAD3 co-operatively modulate gene expression, cell viability and apoptosis in ovarian granulosa cell tumor cells.

Aberrant ovarian granulosa cell proliferation and apoptosis may lead to granulosa cell tumors (GCT), the pathogenesis of which involves transcription factors GATA4, FOXL2, and SMAD3. FOXL2 gene harbors a point mutation (C134W) in a vast majority of GCTs. GATA4 is abundantly expressed in GCTs and its expression correlates with poor prognosis. The TGF-ß mediator SMAD3 promotes GCT cell survival through NF-κB activation, and interacts with FOXL2. Here, we find that the expression patterns of these factors overlap in the normal human ovary and 90 GCTs, and positively correlate with each other and with their mutual target gene CCND2, which is a key factor for granulosa cell proliferation. We have explored the molecular interactions of FOXL2, GATA4, and SMAD3 and their roles in the regulation of CCND2 using co-immunoprecipitation, promoter transactivation, and cell viability assays in human GCT cells. We found that not only SMAD3, but also GATA4 physically interact with both wild type and C134W-mutated FOXL2. GATA4 and SMAD3 synergistically induce a 8-fold increase in CCND2 promoter transactivation, which is 50% reduced by both FOXL2 types. We confirmed that wild type FOXL2 significantly decreases cell viability. Interestingly, GATA4 and SMAD3 caused a marked reduction of GCT cell apoptosis induced by wild type FOXL2. Thus, the effects of GATA4 and SMAD3 on both cell viability and apoptosis are distinct from those of wild type FOXL2; a perturbation of this balance due to the oncogenic FOXL2 mutation is likely to contribute to GCT pathogenesis.