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RationaleDespite the increasing frequency ofTBX4-associated pulmonary arterial hypertension (PAH), genotype-phenotype associations are lacking and may provide important insights.MethodsWe assembled a multi-center cohort of 137 patients harboring monoallelicTBX4variants 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 PAH patients withBMPR2causal variants (n = 162) or no identified variants in PAH-associated genes (n = 741) genotyped via the NIHR BioResource - Rare Diseases (NBR).ResultsFunctional assessment ofTBX4missense variants led to the novel finding of gain-of-function effects associated with older age at diagnosis of lung disease compared to loss-of-function (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 ofTBX4variants were diagnosed at a younger age (p < 0.001) and had worse baseline lung function (FEV1, FVC) (p = 0.009) compared to theBMPR2and no identified causal variant groups.ConclusionsWe 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.

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

Introduction PAH is a severe complication of CTD, with remarkable morbidity and mortality. SSc is most commonly associated with PAH, but it can be present in other CTD. Despite major advances in PAH therapy, survival in CTD-PAH remains poor. Furthermore, the molecular and genetic basis of PAH in CTD are not well established. Purpose This study aimed to screen for genetic defects in a cohort of patients with CTD-PAH. Methods Since November 2011, genetic testing is offered to all patients with idiopathic, hereditable and associated forms of PAH, and PVOD included in a national registry of PAH. A PAH-specific panel of 35 genes was designed. Results During enrolling, 79 patients were recruited: 59 SSc, 11 SLE and 9 other. 69 female, mean age 55,6±1,9 years, mean PVR 8,6±0,5 WU and mean DLCO 47,5±2%. Disease-associated variants were observed in 9 patients: 4 pathogenic/likely pathogenic in 4 different genes (TBX4, ABCC8, KCNA5 and GDF2/BMP9) and 5 VUS in 4 genes (ABCC8, NOTCH3, TOPBP1 and CTCFL). Clinical characteristics of patients with pathogenic/likely pathogenic variants and variant analyses are shown in Tables 1 and 2. Patient 1 is a Caucasian female with mixed CTD, diagnosed with PAH at 58 years of age. She has a frameshift pathogenic variant in TBX4. Pulmonary function test (PFT) ruled out interstitial lung disease (ILD), but a reduction in DLCO was observed (61% of predicted). Two patients carry variants in ABCC8. Patient 2 is a Caucasian female with SSc, diagnosed with PAH at 27 years of age. She carries a splicing variant in ABCC8, classified as likely pathogenic. Her mother was diagnosed with PAH associated with a repaired ASD at 61 years of age. In the genetic testing, no variants were observed in PAH genes. DLCO was 71% of predicted, without signs of ILD. Patient 3 is a Caucasian male, with clinical suspicion of PVOD associated with SSc and HIV infection. PAH was diagnosed at 57 years of age. He presented a missense variant in ABCC8, located in a gating regulatory region, and classified as VUS. Her sister was also diagnosed with PVOD associated with SSc at 48 years of age. No blood or tissue samples are available. In patient 3, DLCO was 22% of predicted value. CT scan showed the typical triad of PVOD. Patient 4 is a Latin American female with SLE, diagnosed with PAH at 25 years of age. She presented a pathogenic nonsense variant in GDF2/BMP9. Patient 5 is a Caucasian female with SSc, diagnosed with PAH at 70 years of age. She presented a pathogenic variant in KCNA5. Patients 6–9 have SSc-PAH and carry VUS in NOTCH1, CTCFL, CTCFL and TOPBP1, respectively. Conclusions We demonstrate the practical diagnostic utility of genetic testing with a panel in CTD-PAH. The discovery of rare variants in these patients forces us to take a comprehensive approach and accurate genetic counseling. Further research is still necessary to confirm these findings and help to provide a personalized medicine approach to these patients. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): This project was founded by project “Bases Genético Moleculares de la Medicina de Precisiόn en la Hipertensiόn Arterial Pulmonar”. Funder: Instituto Carlos III. Ministerio de Economía y Competitividad.

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.

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. Instituto de Salud Carlos III (España) | Ref. FISPI18/01233 Xunta de Galicia | Ref. ED481A-2018/304

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. Instituto de Salud Carlos III | Ref. RD06/0003/0012 Instituto de Salud Carlos III | Ref. PI18/01233 Xunta de Galicia | Ref. ED431G-2019/06 Xunta de Galicia | Ref. ED431C 2018/54 Xunta de Galicia | Ref. ED481A-2018/304 Ministerio de Economía y Competitividad (España) | Ref. RYC-2015-18241

Background: Pulmonary arterial hypertension is a disease wlith an extremely important genetic basis. Micro RNAs (miRNAs) are non-coding RNAs that range in size 17-25 nucleotides and regulate a large variety of biological functions. The aim of our study was to look for miRNAs with potential role in the pathogenesis of PAH. Methods: Plasma samples from 25 patients with idiopathic PAH and 10 healthy controls were included. Serch and subsequent sequencing of the miRNAs were performed Results: More than 500 miRNAs were detected of wich 189 were deregulated between PAH-control groups. After filtering based on a false discovery rate of 1% and a fold change greater than 1.5, 26 miRNAs were selected. Og these, it should be noted that all of those who were under-expressed (hsa-let-7a-5p, hsa-let-7c-5p, hsa-let-7b-5p, hsa-let-7f-5p, hsa-miR-454-3p) have as one of their main tatgets the endothelin gen (EDN1) Conclusions: The expresion of miRNAs is diferent between PAH patients and general population. miRNAs related to EDN1 gene are under-expressed in PAH patients. This could lead to increased expresion on endothelin 1, a key agent in the development of PAH

Resumen Introduccion y objetivos La hipertension arterial pulmonar asociada a lupus eritematoso sistemico (HAP-LES) es una rara enfermedad de baja incidencia. En este estudio se caracterizaron molecularmente los genes y modificadores geneticos relacionados con la HAP en pacientes con HAP-LES. Pacientes y metodos 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 patogenicas en los genes BMPR2 y ENG. Tras el analisis in silico, las mutaciones patogenicas 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 presento polimorfismos en los genes BMPR2, ACVRL1 y ENG. Se identificaron diversas variaciones en los modificadores geneticos en las 3 pacientes analizadas. La presencia de estos modificadores, junto con las mutaciones patogenicas, podrian dar lugar a un fenotipo mas severo en los pacientes con HAP. Conclusiones Presentamos, por primera vez, pacientes con HAP-LES portadores de mutaciones patogenicas en los principales genes relacionados con la HAP y con alteraciones en los modificadores geneticos.

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.

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. This project was founded by Project "Bases Gene´tico Moleculares de la Medicina de Precisio´n en la Hipertensio´n Arterial Pulmonar". Funder: Instituto Carlos III. Ministerio de Economı´a y Competitividad. https://www.isciii.es/Paginas/Inicio.aspx Award number: PI 18/01233 Grant Recipient: P E-S

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.Three patients diagnosed with PAH-SLE and 100 control individuals were analysed after signing an informed consent.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.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.

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‐β1 in thyroid tissue from patients with GD This study was supported by the grants CO‐0115‐14 from Actelion Pharmaceuticals and has received financial support from the Xunta de Galicia (Centro singular de investigación de Galicia accreditation 2016‐2019) and the European Union (European Regional Development Fund—ERDF) SI