ABSTRACT
Pulmonary arterial hypertension (PAH) is a progressive disease in which pulmonary arterial (PA) endothelial cell (EC) dysfunction is associated with unrepaired DNA damage. BMPR2 is the most common genetic cause of PAH. We report that human PAEC with reduced BMPR2 have persistent DNA damage in room air after hypoxia (reoxygenation), as do mice with EC-specific deletion of Bmpr2 (EC-Bmpr2-/-) and persistent pulmonary hypertension. Similar findings are observed in PAEC with loss of the DNA damage sensor ATM, and in mice with Atm deleted in EC (EC-Atm-/-). Gene expression analysis of EC-Atm-/- and EC-Bmpr2-/- lung EC reveals reduced Foxf1, a transcription factor with selectivity for lung EC. Reducing FOXF1 in control PAEC induces DNA damage and impaired angiogenesis whereas transfection of FOXF1 in PAH PAEC repairs DNA damage and restores angiogenesis. Lung EC targeted delivery of Foxf1 to reoxygenated EC-Bmpr2-/- mice repairs DNA damage, induces angiogenesis and reverses pulmonary hypertension.
Subject(s)
Hypertension, Pulmonary , Pulmonary Arterial Hypertension , Mice , Humans , Animals , Pulmonary Arterial Hypertension/genetics , Hypertension, Pulmonary/genetics , Hypertension, Pulmonary/metabolism , Familial Primary Pulmonary Hypertension/metabolism , Pulmonary Artery/metabolism , DNA Damage , Bone Morphogenetic Protein Receptors, Type II/genetics , Forkhead Transcription Factors/genetics , Forkhead Transcription Factors/metabolismABSTRACT
Reduced expression and/or activity of Kv1.5 channels (encoded by KCNA5) is a common hallmark in human or experimental pulmonary arterial hypertension (PAH). Likewise, genetic variants in KCNA5 have been found in patients with PAH, but their functional consequences and potential impact on the disease are largely unknown. Herein, this study aimed to characterize the functional consequences of seven KCNA5 variants found in a cohort of patients with PAH. Potassium currents were recorded by patch-clamp technique in HEK293 cells transfected with wild-type or mutant Kv1.5 cDNA. Flow cytometry, Western blot, and confocal microscopy techniques were used for measuring protein expression and cell apoptosis in HEK293 and human pulmonary artery smooth muscle cells. KCNA5 variants (namely, Arg184Pro and Gly384Arg) found in patients with PAH resulted in a clear loss of potassium channel function as assessed by electrophysiological and molecular modeling analyses. The Arg184Pro variant also resulted in a pronounced reduction of Kv1.5 expression. Transfection with Arg184Pro or Gly384Arg variants decreased apoptosis of human pulmonary artery smooth muscle cells compared with the wild-type cells, demonstrating that KCNA5 dysfunction in both variants affects cell viability. Thus, in addition to affecting channel activity, both variants were associated with impaired apoptosis, a crucial process linked to the disease. The estimated prevalence of dysfunctional KCNA5 variants in the PAH population analyzed was around 1%. The data indicate that some KCNA5 variants found in patients with PAH have critical consequences for channel function, supporting the idea that KCNA5 pathogenic variants may be a causative or contributing factor for PAH.
Subject(s)
Hypertension, Pulmonary , Pulmonary Arterial Hypertension , Humans , Pulmonary Arterial Hypertension/metabolism , HEK293 Cells , Hypertension, Pulmonary/metabolism , Kv1.5 Potassium Channel/genetics , Kv1.5 Potassium Channel/metabolism , Familial Primary Pulmonary Hypertension/metabolism , Pulmonary Artery/pathologyABSTRACT
Rationale: Despite the increased recognition of TBX4 (T-BOX transcription factor 4)-associated pulmonary arterial hypertension (PAH), genotype-phenotype associations are lacking and may provide important insights. Objectives: To compile and functionally characterize all TBX4 variants reported to date and undertake a comprehensive genotype-phenotype analysis. Methods: We assembled a multicenter cohort of 137 patients harboring monoallelic TBX4 variants and assessed the pathogenicity of missense variation (n = 42) using a novel luciferase reporter assay containing T-BOX binding motifs. We sought genotype-phenotype correlations and undertook a comparative analysis with patients with PAH with BMPR2 (Bone Morphogenetic Protein Receptor type 2) causal variants (n = 162) or no identified variants in PAH-associated genes (n = 741) genotyped via the National Institute for Health Research BioResource-Rare Diseases. Measurements and Main Results: Functional assessment of TBX4 missense variants led to the novel finding of gain-of-function effects associated with older age at diagnosis of lung disease compared with loss-of-function effects (P = 0.038). Variants located in the T-BOX and nuclear localization domains were associated with earlier presentation (P = 0.005) and increased incidence of interstitial lung disease (P = 0.003). Event-free survival (death or transplantation) was shorter in the T-BOX group (P = 0.022), although age had a significant effect in the hazard model (P = 0.0461). Carriers of TBX4 variants were diagnosed at a younger age (P < 0.001) and had worse baseline lung function (FEV1, FVC) (P = 0.009) than the BMPR2 and no identified causal variant groups. Conclusions: We demonstrated that TBX4 syndrome is not strictly the result of haploinsufficiency but can also be caused by gain of function. The pleiotropic effects of TBX4 in lung disease may be in part explained by the differential effect of pathogenic mutations located in critical protein domains.
Subject(s)
Gain of Function Mutation , Lung Diseases , Humans , T-Box Domain Proteins/genetics , Bone Morphogenetic Protein Receptors, Type II/genetics , Phenotype , Lung Diseases/genetics , Mutation/genetics , GenotypeABSTRACT
Pulmonary Arterial Hypertension (PAH) is a rare disease caused by the obliteration of the pulmonary arterioles, increasing pulmonary vascular resistance and eventually causing right heart failure. Endothelin-1 (EDN1) is a vasoconstrictor peptide whose levels are indicators of disease progression and its pathway is one of the most common targeted by current treatments. We sequenced the EDN1 untranslated regions of a small subset of patients with PAH, predicted the effect in silico, and used a luciferase assay with the different genotypes to analyze its influence on gene expression. Finally, we used siRNAs against the major transcription factors (TFs) predicted for these regions [peroxisome proliferator-activated receptor γ (PPARγ), Krüppel-Like Factor 4 (KLF4), and vitamin D receptor (VDR)] to assess EDN1 expression in cell culture and validate the binding sites. First, we detected a single nucleotide polymorphism (SNP) in the 5' untranslated region (UTR; rs397751713) and another in the 3'regulatory region (rs2859338) that altered luciferase activity in vitro depending on their genotype. We determined in silico that KLF4/PPARγ could bind to the rs397751713 and VDR to rs2859338. By using siRNAs and luciferase assays, we determined that PPARγ binds differentially to rs397751713. PPARγ and VDR Knock-Down (KD) increased the EDN1 mRNA levels and EDN1 production in porcine aortic endothelial cells (PAECs), while PPARγ and KLF4 KD increased the EDN1 production in HeLa. In conclusion, common variants in EDN1 regulatory regions could alter EDN1 levels. We were able to validate that PPARγ binds in rs397751713 and is a key regulator of EDN1. In addition, KLF4 and VDR regulate EDN1 production in a cell-dependent manner, but VDR does not bind directly to the regions we studied.
ABSTRACT
Pulmonary Arterial Hypertension (PAH) is a severe complication of Connective Tissue Disease (CTD), with remarkable morbidity and mortality. However, the molecular and genetic basis of CTD-PAH remains incompletely understood. This study aimed to screen for genetic defects in a cohort of patients with CTD-PAH, using a PAH-specific panel of 35 genes. During recruitment, 79 patients were studied, including 59 Systemic Sclerosis patients (SSc) and 69 females. Disease-associated variants were observed in nine patients: 4 pathogenic/likely pathogenic variants in 4 different genes (TBX4, ABCC8, KCNA5 and GDF2/BMP9) and 5 Variants of Unknown Significance (VUS) in 4 genes (ABCC8, NOTCH3, TOPBP1 and CTCFL). One patient with mixed CTD had a frameshift pathogenic variant in TBX4. Two patients with SSc-PAH carried variants in ABCC8. A patient diagnosed with Systemic Lupus Erythematous (SLE) presented a pathogenic nonsense variant in GDF2/BMP9. Another patient with SSc-PAH presented a pathogenic variant in KCNA5. Four patients with SSc-PAH carried a VUS in NOTCH1, CTCFL, CTCFL and TOPBP1, respectively. These findings suggest that genetic factors may contribute to Pulmonary Vascular Disease (PVD) in CTD patients.
Subject(s)
Lupus Erythematosus, Systemic , Mutation , Pulmonary Arterial Hypertension , Scleroderma, Systemic , Female , Humans , Lupus Erythematosus, Systemic/complications , Lupus Erythematosus, Systemic/genetics , Male , Middle Aged , Pulmonary Arterial Hypertension/etiology , Pulmonary Arterial Hypertension/genetics , Scleroderma, Systemic/complications , Scleroderma, Systemic/geneticsABSTRACT
In this study, we aimed to evaluate the role of ALMS1 in the morphology of primary cilia and regulation of cellular signaling using a knockdown model of the hTERT-RPE1 cell line. ALMS1 depletion resulted in the formation of longer cilia, which often displayed altered morphology as evidenced by extensive twisting and bending of the axoneme. Transforming growth factor beta/bone morphogenetic protein (TGF-ß/BMP) signaling, which is regulated by primary cilia, was similarly affected by ALMS1 depletion as judged by reduced levels of TGFß-1-mediated activation of SMAD2/3. These results provide novel information on the role of ALMS1 in the function of primary cilia and processing of cellular signaling, which when aberrantly regulated may underlie Alström syndrome.
ABSTRACT
Pulmonary arterial hypertension is a very infrequent disease, with a variable etiology and clinical expressivity, making sometimes the clinical diagnosis a challenge. Current classification based on clinical features does not reflect the underlying molecular profiling of these groups. The advance in massive parallel sequencing in PAH has allowed for the describing of several new causative and susceptibility genes related to PAH, improving overall patient diagnosis. In order to address the molecular diagnosis of patients with PAH we designed, validated, and routinely applied a custom panel including 21 genes. Three hundred patients from the National Spanish PAH Registry (REHAP) were included in the analysis. A custom script was developed to annotate and filter the variants. Variant classification was performed according to the ACMG guidelines. Pathogenic and likely pathogenic variants have been found in 15% of the patients with 12% of variants of unknown significance (VUS). We have found variants in patients with connective tissue disease (CTD) and congenital heart disease (CHD). In addition, in a small proportion of patients (1.75%), we observed a possible digenic mode of inheritance. These results stand out the importance of the genetic testing of patients with associated forms of PAH (i.e., CHD and CTD) additionally to the classical IPAH and HPAH forms. Molecular confirmation of the clinical presumptive diagnosis is required in cases with a high clinical overlapping to carry out proper management and follow up of the individuals with the disease.
Subject(s)
Familial Primary Pulmonary Hypertension/diagnosis , Familial Primary Pulmonary Hypertension/genetics , High-Throughput Nucleotide Sequencing , Cohort Studies , Connective Tissue Diseases/genetics , Female , Genetic Association Studies , Genetic Predisposition to Disease , Genetic Testing , Heart Defects, Congenital/genetics , Humans , Inheritance Patterns , Male , Mutation , Pedigree , Pulmonary Veno-Occlusive Disease/geneticsABSTRACT
Pulmonary Arterial Hypertension (PAH) is a rare and fatal disease where knowledge about its genetic basis continues to increase. In this study, we used targeted panel sequencing in a cohort of 624 adult and pediatric patients from the Spanish PAH registry. We identified 11 rare variants in the ATP-binding Cassette subfamily C member 8 (ABCC8) gene, most of them with splicing alteration predictions. One patient also carried another variant in SMAD1 gene (c.27delinsGTAAAG). We performed an ABCC8 in vitro biochemical analyses using hybrid minigenes to confirm the correct mRNA processing of 3 missense variants (c.211C > T p.His71Tyr, c.298G > A p.Glu100Lys and c.1429G > A p.Val477Met) and the skipping of exon 27 in the novel splicing variant c.3394G > A. Finally, we used structural protein information to further assess the pathogenicity of the variants. The results showed 11 novel changes in ABCC8 and 1 in SMAD1 present in PAH patients. After in silico and in vitro biochemical analyses, we classified 2 as pathogenic (c.3288_3289del and c.3394G > A), 6 as likely pathogenic (c.211C > T, c.1429G > A, c.1643C > T, c.2422C > A, c.2694 + 1G > A, c.3976G > A and SMAD1 c.27delinsGTAAAG) and 3 as Variants of Uncertain Significance (c.298G > A, c.2176G > A and c.3238G > A). In all, we show that coupling in silico tools with in vitro biochemical studies can improve the classification of genetic variants.
Subject(s)
Exons , Genetic Markers , Mutation, Missense , Pulmonary Arterial Hypertension/epidemiology , Pulmonary Arterial Hypertension/genetics , RNA Splicing , Sulfonylurea Receptors/genetics , Adult , Female , Humans , Incidence , Male , Pulmonary Arterial Hypertension/pathology , Spain/epidemiology , Young AdultABSTRACT
BACKGROUND: The knowledge of hereditary predisposition has changed our understanding of Pulmonary Arterial Hypertension. Genetic testing has been widely extended and the application of Pulmonary Arterial Hypertension specific gene panels has allowed its inclusion in the diagnostic workup and increase the diagnostic ratio compared to the traditional sequencing techniques. This is particularly important in the differential diagnosis between Pulmonary Arterial Hypertension and Pulmonary Venoocclusive Disease. METHODS: Since November 2011, genetic testing is offered to all patients with idiopathic, hereditable and associated forms of Pulmonary Arterial Hypertension or Pulmonary Venoocclusive Disease included in the Spanish Registry of Pulmonary Arterial Hypertension. Herein, we present the clinical phenotype and prognosis of all Pulmonary Arterial Hypertension patients with disease-associated variants in TBX4. RESULTS: Out of 579 adults and 45 children, we found in eight patients from seven families, disease-causing associated variants in TBX4. All adult patients had a moderate-severe reduction in diffusion capacity. However, we observed a wide spectrum of clinical presentations, including Pulmonary Venoocclusive Disease suspicion, interstitial lung disease, pulmonary vascular abnormalities and congenital heart disease. CONCLUSIONS: Genetic testing is now essential for a correct diagnosis work-up in Pulmonary Arterial Hypertension. TBX4-associated Pulmonary Arterial Hypertension has marked clinical heterogeneity. In this regard, a genetic study is extremely useful to obtain an accurate diagnosis and provide appropriate management.
Subject(s)
Familial Primary Pulmonary Hypertension/genetics , Genetic Variation , T-Box Domain Proteins/genetics , Adolescent , Adult , Child , Child, Preschool , Codon, Nonsense , Diagnosis, Differential , Familial Primary Pulmonary Hypertension/diagnosis , Familial Primary Pulmonary Hypertension/diagnostic imaging , Female , Gene Deletion , Genetic Predisposition to Disease , Humans , Male , Middle Aged , Mutation, Missense , Pedigree , Phenotype , Polymorphism, Single Nucleotide , Prognosis , Pulmonary Veno-Occlusive Disease/diagnosis , Pulmonary Veno-Occlusive Disease/geneticsABSTRACT
Introducción y objetivos: La hipertensión arterial pulmonar asociada a lupus eritematoso sistémico (HAP-LES) es una rara enfermedad de baja incidencia. En este estudio se caracterizaron molecularmente los genes y modificadores genéticos relacionados con la HAP en pacientes con HAP-LES. Pacientes y métodos: Se analizaron 3 pacientes diagnosticados de HAP-LES y 100 individuos control, previa firma del consentimiento informado. Resultados: Dos de las 3 pacientes con HAP-LES analizadas resultaron ser portadoras de mutaciones patogénicas en los genes BMPR2 y ENG. Tras el análisis in silico, las mutaciones patogénicas se buscaron en individuos control y en diferentes bases de datos, siendo este resultado negativo, por lo que fueron analizadas funcionalmente. La tercera paciente tan solo presentó polimorfismos en los genes BMPR2, ACVRL1 y ENG. Se identificaron diversas variaciones en los modificadores genéticos en las 3 pacientes analizadas. La presencia de estos modificadores, junto con las mutaciones patogénicas, podrían dar lugar a un fenotipo más severo en los pacientes con HAP. Conclusiones: Presentamos, por primera vez, pacientes con HAP-LES portadores de mutaciones patogénicas en los principales genes relacionados con la HAP y con alteraciones en los modificadores genéticos
Background and objective: Pulmonary arterial hypertension associated with systemic lupus erythematosus (PAH-SLE) is a rare disease with a low incidence rate. In this study, PAH related genes and genetic modifiers were characterised molecularly in patients with PAH-SLE. Patients and methods: Three patients diagnosed with PAH-SLE and 100 control individuals were analysed after signing an informed consent. Results: Two out of the three analysed patients with PAH-SLE were carriers of pathogenic mutations in the genes BMPR2 and ENG. After an in silico analysis, pathogenic mutations were searched for in control individuals and different databases, with negative results, and they were thus functionally analysed. The third patients only showed polymorphisms in the genes BMPR2, ACVRL1 and ENG. Several genetic variants and genetic modifiers were identified in the three analysed patients. These modifiers, along with the pathogenic mutations, could lead to a more severe clinical course in patients with PAH. Conclusions: We present, for the first time, patients with PAH-SLE carrying pathogenic mutations in the main genes related to PAH and alterations in the genetic modifiers
Subject(s)
Humans , Female , Adult , Middle Aged , Hypertension/genetics , Hypertension, Pulmonary/genetics , Lupus Erythematosus, Systemic/pathology , Hypertension, Pulmonary/pathology , Mutation/genetics , Genes, ModifierABSTRACT
BACKGROUND AND OBJECTIVE: Pulmonary arterial hypertension associated with systemic lupus erythematosus (PAH-SLE) is a rare disease with a low incidence rate. In this study, PAH related genes and genetic modifiers were characterised molecularly in patients with PAH-SLE. PATIENTS AND METHODS: Three patients diagnosed with PAH-SLE and 100 control individuals were analysed after signing an informed consent. RESULTS: Two out of the three analysed patients with PAH-SLE were carriers of pathogenic mutations in the genes BMPR2 and ENG. After an in silico analysis, pathogenic mutations were searched for in control individuals and different databases, with negative results, and they were thus functionally analysed. The third patients only showed polymorphisms in the genes BMPR2, ACVRL1 and ENG. Several genetic variants and genetic modifiers were identified in the three analysed patients. These modifiers, along with the pathogenic mutations, could lead to a more severe clinical course in patients with PAH. CONCLUSIONS: We present, for the first time, patients with PAH-SLE carrying pathogenic mutations in the main genes related to PAH and alterations in the genetic modifiers.
Subject(s)
Activin Receptors, Type II/genetics , Bone Morphogenetic Protein Receptors, Type II/genetics , Endoglin/genetics , Hypertension, Pulmonary/genetics , Lupus Erythematosus, Systemic/complications , Mutation/genetics , Adult , Case-Control Studies , Female , Genetic Variation , Humans , Hypertension, Pulmonary/complications , Middle Aged , Polymorphism, Genetic , Serotonin Plasma Membrane Transport Proteins/genetics , TRPC6 Cation Channel/geneticsABSTRACT
In this study, we analysed the possible influence of the c.419-43delT BMPR2 variant in patients with Graves' disease (GD), in a molecular basis, focusing our efforts on possible alterations in the mRNA processing and synthesis. The molecular assessment of this variant in patients with GD would shed light on the association between the BMPR2 gene and the disease. The variant was detected in 18%, 55% and 10% of patients with pulmonary arterial hypertension, GD and in general population, respectively. Patients with GD fold change showed increased BMPR2 expression when matched against the controls, with a mean of 4.21 ± 1.73 (P = 0.001); BMPR2 was overexpressed in the analysed cell cycle stages. Fold change analysis of variant carriers and non-carriers showed slight overexpression and differences between phases, but none of them were statistically significant. BMPR2 expression was confirmed in the lymphoblastoid cell lines (LCLs) with a molecular weight of 115 kD, and no differences between variant carriers and non-carriers were detected. To conclude, the BMPR2 variant c.419-19delT appears in high frequency in patients with GD, and independently of its presence, BMPR2 is overexpressed in the LCLs from the GD patients tested. This increase could be paired with the described decreased expression of transforming growth factor-ß1 in thyroid tissue from patients with GD.