scholarly journals Case Report: BMPR2-Targeted MinION Sequencing as a Tool for Genetic Analysis in Patients With Pulmonary Arterial Hypertension

2021 ◽  
Vol 8 ◽  
Author(s):  
Tomoya Takashima ◽  
Sophie Brisset ◽  
Asuka Furukawa ◽  
Hirohisa Taniguchi ◽  
Rika Takeyasu ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Genetic Analysis ◽  
Arterial Hypertension ◽  
Genetic Diagnosis ◽  
Variant Calling ◽  
Read Length ◽  
Sequencing Analysis ◽  
Bioinformatics Pipeline ◽  
Pulmonary Arterial ◽  
Nanopore Sequencer

Background: Mutations in the bone morphogenetic protein receptor type 2 gene (BMPR2) represent a major genetic cause of pulmonary arterial hypertension (PAH). Identification of BMPR2 mutations is crucial for the genetic diagnosis of PAH. MinION nanopore sequencer is a portable third-generation technology that enables long-read sequencing at a low-cost. This nanopore technology-based device has not been used previously for PAH diagnosis. This study aimed to determine the feasibility of using MinION nanopore sequencing for the genetic analysis of PAH patients, focused on BMPR2.Methods: We developed a protocol for the custom bioinformatics pipeline analysis of long reads generated by long-PCR. To evaluate the potential of using MinION sequencing in PAH, we analyzed five samples, including those of two idiopathic PAH patients and a family of three members with one affected patient. Sanger sequencing analysis was performed to validate the variants.Results: The median read length was around 3.4 kb and a good mean quality score of approximately 19 was obtained. The total number of reads generated was uniform among the cases and ranged from 2,268,263 to 3,126,719. The coverage was consistent across flow cells in which the average number of reads per base ranged from 80,375 to 135,603. We identified two polymorphic variants and three mutations in four out of five patients. Certain indel variant calling-related errors were observed, mostly outside coding sequences.Conclusion: We have shown the ability of this portable nanopore sequencer to detect BMPR2 mutations in patients with PAH. The MinION nanopore sequencer is a promising tool for screening BMPR2 mutations, especially in small laboratories and research groups.

scholarly journals EXPRESS: Genetic basis of pulmonary arterial hypertension: A prospective study from a highly inbred population

2021 ◽  
pp. 204589402110320
Author(s):  
Abdullah Aldalaan ◽  
Khushnooda Ramzan ◽  
Sarfraz Saleemi ◽  
Ihab Weheba ◽  
Laila Alquait ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Genetic Basis ◽  
Genetic Diagnosis ◽  
Saudi Arabian ◽  
High Rate ◽  
Risk Genes ◽  
Entire Cohort ◽  
Pulmonary Arterial ◽  
A Prospective Study

Pulmonary arterial hypertension (PAH), whether idiopathic PAH (IPAH), heritable PAH, or associated with other conditions, is a rare and potentially lethal disease characterized by progressive vascular changes. To date, there is limited data on the genetic basis of PAH in the Arab region, and none from Saudi Arabian patients. This study aims to identify genetic variations and to evaluate the frequency of risk genes associated to PAH, in Saudi Arabian patients. Adult PAH patients, diagnosed with IPAH and pulmonary veno-occlusive disease (PVOD), of Saudi Arabian origin, were enrolled in this study. Forty-eight patients were subjected to whole exome sequencing (WES), with screening of 26 genes suggested to be associated with the disease. The median age at diagnosis was 29.5 years of age, with females accounting for 89.5% of our cohort population. Overall, we identified variations in 9 genes previously associated with PAH, in 16 patients. Fourteen of these variants have not been described before. Plausible deleterious variants in risk genes were identified in 33.3% (n=16/48) of our entire cohort and 25% of these cases carried variants in BMPR2 (n=4/16). Our results highlight the genetic etiology of PAH in Saudi Arabia patients and provides new insights for the genetic diagnosis of familial and IPAH, as well as for the identification of the biological pathways of the disease. This will enable the development of new target therapeutic strategies, for a disease with a high rate of morbidity and mortality.

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

2015 ◽  
Vol 3 (4) ◽  
pp. 354-362 ◽  
Author(s):  
Sanna Vattulainen ◽  
Joonas Aho ◽  
Pertteli Salmenperä ◽  
Siina Bruce ◽  
Jonna Tallila ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Genetic Diagnosis ◽  
Pulmonary Arterial ◽  
Selective Sequencing

Abstract 14257: Endothelial SOX17 Deficiency Induces Pulmonary Arterial Hypertension

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Zhiyu Dai ◽  
Dan Yi ◽  
BIN LIU ◽  
Shuai Li
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Vascular Remodeling ◽  
Molecular Mechanisms ◽  
Association Studies ◽  
Genome Wide Association Studies ◽  
Sequencing Analysis ◽  
Pulmonary Arterial

Introduction: Pulmonary arterial hypertension (PAH) is a disaster disease characterized by obliterative vascular remodeling and persistent increase of vascular resistance, leading to right heart failure and premature death. Understanding the cellular and molecular mechanisms will help develop novel therapeutic approaches for PAH patients. Hypothesis: Human genome-wide association studies identified that SOX17 locus variants are associated with PAH. SOX17 mutation is also found in patients with PAH. We hypothesis that endothelial SOX17 deficiency contributes to the pathogenesis of PAH. Methods: Mice with EndoSCL-CreERT mediated deletion of Sox17 ( Sox17 iCKO ) were generated. Sox17 iCKO and Sox17 f/f mice after tamoxifen injection were incubated with hypoxia (10% O 2 ) for 3 weeks to induced PAH. Hemodynamics and histological examination were measured to determine the PAH phenotypes and vascular remodeling. EC proliferation and apoptosis were assessed in SiRNA-mediated SOX17 knockdown in human lung microvascular endothelial cells (hLMVECs). The RNA-sequencing analysis was performed to understand the molecular mechanisms of SOX17 deficiency in ECs. Results: Sox17 iCKO mice exhibited exaggerative PAH evident by the increase of RVSP and RV hypertrophy after hypoxia treatment compared to Sox17 f/f WT mice. SOX17 knockdown in hLMVECs induced cell proliferation and reduced starvation-induced apoptosis. RNA-seq analysis and DAVID pathway analysis demonstrated that there was dysregulation of cell proliferation-related genes, which are enriched in the pathways related to cell cycle, cell division, and mitotic cell cycle. Transcriptional factor, target, and motif discovery analysis of the dysregulated gene set revealed the involvement of transcriptional factors FOXM1 and E2F1. siRNA mediated knockdown of E2F1 but not FOXM1 normalized SOX17 deficiency-induced hLMVECs proliferation and anti-apoptosis. Conclusions: Our study demonstrated that endothelial SOX17 deficiency exaggerates hypoxia-induced PAH. Loss of SOX17 promotes EC proliferation and anti-apoptosis via the upregulation of transcription factor E2F1.

Abstract 15544: Implication of the Histone Methyltransferase “G9a” in Pulmonary Arterial Hypertension

Circulation ◽  
2020 ◽  
Vol 142 (Suppl_3) ◽  
Author(s):  
Charifa awada ◽  
Karima Habbout ◽  
Valerie Nadeau ◽  
Sandra Breuils-Bonnet ◽  
Roxane Paulin ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Cholesterol Metabolism ◽  
Cholesterol Biosynthesis ◽  
Histone Methyltransferase ◽  
Sequencing Analysis ◽  
Apoptosis Resistance ◽  
Cancer Pathogenesis ◽  
Therapeutic Benefits ◽  
Pulmonary Arterial

Rationale: Pulmonary arterial hypertension (PAH) is a cardiopulmonary disorder characterized by elevation of pulmonary arterial (PA) pressure and premature death. PA smooth muscle cells (PASMCs) from PAH patients present a cancer-like hyperproliferative and apoptosis-resistant phenotype contributing to remodeling of distal PAs. Although epigenetic alterations contribute to PAH development, one important challenge is defining which genes are the drivers . A growing body of literature points to the role of an epigenetic factor called G9a in cancer pathogenesis. Indeed, G9a is a histone methyltransferase overexpressed in many cancers promoting cell proliferation and survival. Given the similarities between PAH and cancer, it is of interest to determine whether G9a is implicated in PAH. We thus hypothesized that G9a inhibition reduces the pro-proliferative and apoptosis resistance phenotype of PAH-PASMCs. Methods and Results: Using Western blot (WB) and immunofluorescence (IF), we showed that G9a is overexpressed in distal PAs and isolated PASMCs from PAH patients (n= 6-14, p<0.01). Similarly, G9a was increased (WB and IF, p<0.05) in two models mimicking the disease; namely the monocrotaline rat and mice exposed to chronic hypoxia. In vitro, we found that pharmacological inhibition of G9a using BIX01294 and UNC0642 reduces PAH-PASMC proliferation (Ki67 and EdU assays, p<0.001) and survival (Annexin V assay p<0.001). Through RNA sequencing analysis conducted in PAH-PASMCs treated or not with BIX01294, we found that upregulated differentially expressed genes (DEGs) were enriched in cholesterol biosynthesis, autophagy-lysosome and ER stress-induced apoptotic pathways. However, downregulated DEGs were involved in cell cycle and fibrosis-related processes. Consistently, inhibition of G9a generates numerous cytoplasmic vacuoles positive for LC3-II and p62 (WB, IF), thus suggesting that the inhibition of G9a induces cell death by altering cholesterol metabolism-dependent autophagy. Conclusion: We showed for the first time that G9a is overexpressed in PAH contributing to the pro-proliferative and anti-apoptotic phenotype of PAH-PASMCs. Current experiments aim to determine whether G9a inhibition provides therapeutic benefits in PAH.

scholarly journals Pre-implantation genetic diagnosis in pulmonary arterial hypertension due toBMPR2mutation: Figure 1–

2012 ◽  
Vol 39 (6) ◽  
pp. 1534-1535 ◽  
Author(s):  
Nelly Frydman ◽  
Julie Steffann ◽  
Barbara Girerd ◽  
René Frydman ◽  
Arnold Munnich ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Genetic Diagnosis ◽  
Pulmonary Arterial

scholarly journals Genetics and genomics of pulmonary arterial hypertension

2019 ◽  
Vol 53 (1) ◽  
pp. 1801899 ◽  
Author(s):  
Nicholas W. Morrell ◽  
Micheala A. Aldred ◽  
Wendy K. Chung ◽  
C. Gregory Elliott ◽  
William C. Nichols ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Genetic Diagnosis ◽  
Molecular Classification ◽  
Incomplete Penetrance ◽  
Therapeutic Approaches ◽  
Genomic Studies ◽  
Pulmonary Arterial ◽  
The Impact

Since 2000 there have been major advances in our understanding of the genetic and genomics of pulmonary arterial hypertension (PAH), although there remains much to discover. Based on existing knowledge, around 25–30% of patients diagnosed with idiopathic PAH have an underlying Mendelian genetic cause for their condition and should be classified as heritable PAH (HPAH). Here, we summarise the known genetic and genomic drivers of PAH, the insights these provide into pathobiology, and the opportunities afforded for development of novel therapeutic approaches. In addition, factors determining the incomplete penetrance observed in HPAH are discussed. The currently available approaches to genetic testing and counselling, and the impact of a genetic diagnosis on clinical management of the patient with PAH, are presented. Advances in DNA sequencing technology are rapidly expanding our ability to undertake genomic studies at scale in large cohorts. In the future, such studies will provide a more complete picture of the genetic contribution to PAH and, potentially, a molecular classification of this disease.

scholarly journals Regulation of The Methylation and Expression Levels of the BMPR2 Gene by SIN3a As A Novel Therapeutic Mechanism in Pulmonary Arterial Hypertension

Circulation ◽  
2021 ◽  
Author(s):  
Malik Bisserier ◽  
Prabhu Mathiyalagan ◽  
Shihong Zhang ◽  
Firas Elmastour ◽  
Peter Dorfmüller ◽  
...  
Keyword(s):  
Pulmonary Arterial Hypertension ◽  
Arterial Hypertension ◽  
Transcriptional Regulator ◽  
Ctcf Binding ◽  
Sequencing Analysis ◽  
Epigenetic Mechanisms ◽  
Rodent Models ◽  
Virus Serotype ◽  
Pulmonary Arterial

Background: Epigenetic mechanisms are critical in the pathogenesis of pulmonary arterial hypertension (PAH). Previous studies have suggested that hypermethylation of the Bone Morphogenetic Protein Receptor Type 2 (BMPR2) promoter is associated with BMPR2 downregulation and progression of PAH. Here, we investigated for the first time the role of Switch-Independent 3a (SIN3a), a transcriptional regulator, in the epigenetic mechanisms underlying hypermethylation of BMPR2 in the pathogenesis of PAH. Methods: We used lung samples from PAH patients and non-PAH controls, preclinical mouse and rat PAH models, and human pulmonary arterial smooth muscle cells (hPASMC). Expression of SIN3a was modulated using a lentiviral vector or a siRNA in vitro and a specific Adeno-Associated Virus serotype 1 (AAV1) or a lentivirus encoding for human SIN3a in vivo . Results: SIN3a is a known transcriptional regulator; however, its role in cardiovascular diseases, especially PAH, is unknown. Interestingly, we detected a dysregulation of SIN3 expression in patients and in rodent models, which is strongly associated with decreased BMPR2 expression. SIN3a is known to regulate epigenetic changes. Therefore, we tested its role in the regulation of BMPR2 and found that BMPR2 is regulated by SIN3a. Interestingly, SIN3a overexpression inhibited hPASMC proliferation and upregulated BMPR2 expression by preventing the methylation of the BMPR2 promoter region. RNA sequencing analysis suggested that SIN3a downregulated the expression of DNA and histone methyltransferases such as DNMT1 and EZH2 while promoting the expression of the DNA demethylase TET1. Mechanistically, SIN3a promoted BMPR2 expression by decreasing CTCF binding to the BMPR2 promoter. Finally, we identified intratracheal delivery of AAV1.hSIN3a to be a beneficial therapeutic approach in PAH- by attenuating pulmonary vascular and RV remodeling, decreasing RVSP and mPAP pressure, and restoring BMPR2 expression in rodent models of PAH. Conclusions: Altogether, our study unveiled the protective/beneficial role of SIN3a in pulmonary hypertension. We also identified a novel and distinct molecular mechanism by which SIN3a regulates BMPR2 in hPASMC. Our study also identified lung-targeted SIN3a gene therapy using AAV1 as a new promising therapeutic strategy for treating patients with PAH.

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