Next Article in Journal
The HSP90 Inhibitor, AUY-922, Protects and Repairs Human Lung Microvascular Endothelial Cells from Hydrochloric Acid-Induced Endothelial Barrier Dysfunction
Next Article in Special Issue
Expanding the Evidence of a Semi-Dominant Inheritance in GDF2 Associated with Pulmonary Arterial Hypertension
Previous Article in Journal
The Role of Prostaglandins in Different Types of Cancer
Font Type:
Arial Georgia Verdana
Font Size:
Aa Aa Aa
Line Spacing:
Column Width:
Brief Report

Novel Genetic and Molecular Pathways in Pulmonary Arterial Hypertension Associated with Connective Tissue Disease

Ignacio Hernandez-Gonzalez
Jair Tenorio-Castano
Nuria Ochoa-Parra
Natalia Gallego
Carmen Pérez-Olivares
Mauro Lago-Docampo
Julian Palomino Doza
Diana Valverde
Pablo Lapunzina
2,3,4 and
Pilar Escribano-Subias
Department of Cardiology, Hospital Universitario Río Hortega, 47012 Valladolid, Spain
Institute of Medical and Molecular Genetics (INGEMM)-IdiPAZ, Hospital Universitario La Paz-UAM, Paseo de La Castellana, 261, 28046 Madrid, Spain
CIBERER, Centro de Investigación Biomédica en Red de Enfermedades Raras, ISCIII, Melchor Fernández Almagro Street, 3, 28029 Madrid, Spain
ITHACA, European Reference Network on Rare Congenital Malformations and Rare Intellectual Disability, Hospital Universitario La Paz, 28046 Madrid, Spain
Unidad Multidisciplinar de Hipertensión Pulmonar, Servicio de Cardiología, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain
CINBIO, Universidade de Vigo, 36310 Vigo, Spain
Instituto de Investigación Sanitaria Galicia Sur (IIS Galicia Sur), SERGAS-UVIGO, 36312 Vigo, Spain
Unidad de Miocardiopatías Familiares, Servicio de Cardiología, Hospital Universitario 12 de Octubre, 28041 Madrid, Spain
CIBERCV, Centro de Investigación Biomédica en Red de Enfermedades Cardiovasculares, ISCIII, 28029 Madrid, Spain
Author to whom correspondence should be addressed.
Cells 2021, 10(6), 1488;
Submission received: 6 May 2021 / Revised: 5 June 2021 / Accepted: 7 June 2021 / Published: 13 June 2021
(This article belongs to the Special Issue BMP Signaling and Beyond: Breaking the Cell Code of PAH)


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.

1. Introduction

Pulmonary Arterial Hypertension (PAH) is a feared complication of Connective Tissue Diseases (CTD), with remarkable morbidity and mortality [1]. Systemic sclerosis (SSc) is most commonly associated with PAH, but it can be present in other CTD such as Systemic Lupus Erythematous (SLE) or Mixed CTD (MCTD) [2]. CTD-associated PAH (CTD-PAH) is present in up to 12% of patients with SSc and it is one of the leading disease-related causes of death [3]. Moreover, CTD-PAH represents 15–30% of cases in PAH registries [4,5]. Despite major advances in PAH therapy, survival in CTD-PAH remains poor, with a three-year survival of 40–50% [4].
To date, 12 genes have been associated with PAH with a high level of evidence, and 5 have been associated with a low level of evidence [6]. Furthermore, high-throughput sequencing (HTS) technologies have led to the identification of novel associated genes [7]. The main gene involved in PAH encodes the bone morphogenic protein receptor type 2 (BMPR2), a receptor belonging to the transforming growth factor beta (TGF-β) superfamily [8]. Other genes have also been identified: potassium channel genes (KCNK3, KCNA5, ABCC8), T-box transcription factor 4 (TBX4), and other genes in the TGF-β/BMP signaling pathway (BMP9/GDF2, SMAD1, SMAD4, SMAD9, BMPR1B) [6]. Previous studies have demonstrated that rare coding mutations are present in ~80% of familial forms and ~20% of sporadic cases [8].
Currently, the molecular and genetic basis of PAH in CTD has not been fully addressed. Previous studies have suggested that genetic factors may play a significant role in the development of Pulmonary Vascular Disease (PVD) in other conditions, such as congenital heart disease [9]. However, the role of genetic abnormalities in CTD-associated PVD remains unclear.
This study aimed to screen for genetic defects in a cohort of patients with CTD-PAH.

2. Materials and Methods

2.1. Study Patients

Since November 2011, genetic testing has been offered to all patients with idiopathic, hereditable and associated forms of PAH, and Pulmonary Venooclusive Disease (PVOD), included in the Spanish Registry of Pulmonary Arterial Hypertension (REHAP). A full list of REHAP centers and investigators is provided in the Supporting Information (See Table S1).
Pulmonary Arterial Hypertension was defined according to the 2015 ERS/ESC Guidelines for the Diagnosis and Treatment of Pulmonary Hypertension [10]. Routine diagnostic workup included medical history, physical examination, 6-min walking test (6MWT), echocardiogram, multidetector computed tomography (MDCT), ventilation/perfusion lung scan, pulmonary function tests (PFT), and screening of connective tissue disease, HIV infection and portal hypertension. Right Heart Catheterism (RHC) at diagnosis includes Right Atrium Pressure, Mean Pulmonary Artery Pressure, Pulmonary Wedge Pressure, Cardiac Output, Cardiac Index and Pulmonary Vascular Resistance. Pulmonary vasoreactivity testing was performed in Idiopathic PAH (IPAH), Hereditable PAH (HPAH) and drug-induced PAH. Routine diagnostic workup included medical history, physical examination, 6-min Walking Test (6MWT), echocardiogram, Multidetector Computed Tomography (MDCT), ventilation/perfusion lung scan, pulmonary function tests (PFT), and screening of Connective Tissue Disease, HIV infection and Portal Hypertension. PFT included the diffusing capacity for carbon monoxide (DLCO), which was considered moderately reduced when DLCO 43–62% of predicted values and severely reduced when DLCO < 43% of predicted values [11]. Therapeutic management is left to the discretion of individual physicians.
All patients or legal tutors included in the analysis gave their written informed consent and the project was approved by the ethical committee for scientific research of the participant centers. We obtained written parental consent from the parents or guardians of minors included in this study.

2.2. Molecular Analysis

A PAH-specific HTS panel of 35 genes was designed, including all PAH-associated genes at that date with a variable level of evidence [6]. Review, classification and interpretation of variants were carried out according to the American College of Medical Genetics and Genomics guidelines [12]. The ethical principles of the European Board of Medical Genetics and the 2015 ERS/ESC guidelines for the diagnosis and treatment of pulmonary hypertension offer accurate information on the range of options available to make informed decisions, and allow equal access to genetic counseling and testing [10]. Pre- and post-test genetic counseling was provided. In the pre-test visit, family history information was collected, but only probands were studied. When a positive result was observed, a genetic study was offered to first degree relatives where available. Cascade or co-segregation genetic tests were also performed. When an unaffected carrier was identified, a complete diagnostic was performed, including electrocardiogram, echocardiogram, N-terminal pro-brain natriuretic peptide (NT-proBNP) and 6 Minute Walking Test. This evaluation is periodically repeated. When a sustained suspicion of early-stage PAH was observed, RHC was performed to rule out the condition.

3. Results

During patients’ enrolling, 79 CTD-PAH patients were recruited: 59 SSc, 11 Systemic Lupus Erythematous (SLE) and 9 other CTD (Figure 1). Baseline characteristics are shown in Table 1. Sixty-nine patients were female, mean age was 55.6 ± 1.9 years, mean pulmonary vascular resistance (PVR) was 8.6 ± 0.5 wood units (WU) and mean diffusing capacity of the lung for carbon monoxide (DLCO) was 47.5 ± 2% of predicted value.
Disease-associated variants were observed in nine patients. Four of them were classified as pathogenic or likely pathogenic in four different genes (TBX4, ABCC8, KCNA5 and GDF2/BMP9), and five as variants of unknown significance (VUS) in four genes (ABCC8, NOTCH3, TOPBP1 and CTCFL). Clinical characteristics of patients with pathogenic or likely pathogenic variants and variant analyses are shown in Table 2 and Table 3, respectively.
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: (NM_018488.3): c.1112dupC:p.(Pro372Serfs*14). A Pulmonary function test (PFT) at diagnosis ruled out interstitial lung disease (ILD), but a reduction in DLCO was observed (61% of predicted value). Small Patella Syndrome was also ruled out. Up-front oral combination therapy was prescribed. After six years of follow-up, she has a low-risk profile under double oral combination therapy.
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: (NM_000352.6): c.2694+1G>A, classified as likely pathogenic. Her mother was diagnosed with PAH, associated with a repaired atrial septal defect, at 61 years of age. In the genetic testing, no variants were observed in ABCC8 or other PAH genes. ILD was also ruled out in patient 2. However, a mild reduction in diffusion capacity was observed at diagnosis (DLCO 71% of predicted value). Monotherapy was initiated. During follow-up, goal-oriented PAH therapy was applied, and risk profile was assessed periodically. Eighteen years after diagnosis, she presents a low-risk profile under triple combination therapy, including systemic prostanoids. In this time, diffusion capacity progressively worsened (current DLCO 45% of predicted value), 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 (NM_000352.6):c.298G>A p.(Glu100Lys), located in a gating regulatory region, and classified as VUS. His sister was also diagnosed with PVOD associated with SSc at 48 years of age. Referred to the lung transplant unit, she died on the waiting list. No blood or tissue samples are available for histological or genetic analysis. In patient 3, DLCO at diagnosis was 22% of predicted value. MDCT showed the typical PVOD triad, consisting of ground grass opacification, interlobular septal thickening or mediastinal lymphadenopathy. He also had respiratory insufficiency when resting and a significant drop in oxygen desaturation during exercise. Although referred to the lung transplant unit, he was not eligible due to advanced age and comorbidities. The clinical course was progressive, and he died 4.5 years after the diagnosis.
Patient 4 is a Latin American female with SLE, diagnosed with PAH at 25 years of age. She presented a nonsense variant in GDF2/BMP9: (NM_016204.4): c.642G>A: (p.Trp214*) which causes the appearance of a premature stop codon, classified as pathogenic. Three years after diagnosis, she presented a low-risk profile under dual oral therapy.
Patient 5 is a Caucasian female with SSc, diagnosed with PAH at 70 years of age. She presented a pathogenic variant in KCNA5: (NM_002234.3):c.1685delC(p.Phe563fs*21). During follow-up, goal-oriented PAH therapy was applied. She died 8.5 years after diagnosis due to progressive heart failure.
Another patient with SSc carries a VUS in NOTCH1. Three patients show variants in novel PAH-related genes. Patients 9 and 10 had a previous diagnosis of SSc and carry a VUS in CTCFL. Patient 11 was diagnosed with SSc-PAH and carries a VUS in TOPBP1.

4. Discussion

The pathobiology of PAH-CTD remains incompletely understood. On the one hand, it is speculated that this complication may be triggered by immune dysregulation present in CTD [2,13,14]. On the other hand, an imbalance in the TGF-β/BMP axis might also contribute to CTD-PAH development [15,16]. However, despite progress in our knowledge of CTD-PVD, neither genetic theory nor inflammatory theory have been proven. In hereditable and idiopathic forms of PAH, BMPR2 haploinsufficiency is the most common inherited molecular mechanism [8,17]. However, the penetrance of the disease phenotype is incomplete and additional stimuli are necessary [18]. Female sex is the single most important factor influencing the development of PAH in mutation carriers [19]. Other factors might be genetic (a second variant in another gene), epigenetic or environmental [17,20]. Furthermore, inflammatory cells and their mediator also contribute to pulmonary vascular remodeling in idiopathic forms [21]. Whether the proinflammatory state in CTD is a trigger in genetically susceptible individuals remains unclear [22,23,24,25]. One might speculate that the simultaneous occurrence of genetic and inflammatory factors might explain PAH in CTD [6]. Nevertheless, the genetic basis of CTD-PAH has not been well elucidated to date.
In our CTD-PAH cohort, four patients (5.1%) carried a pathogenic or likely pathogenic variant in a PAH-related gene. Furthermore, VUS were observed in another five patients (6.3%). Functional assays must be performed in order to confirm or discard the possible role of these variants in protein function, and whether this can be related to the phenotype. Some previous studies failed to observe variants in PAH-related genes throughout this population [26]. The most likely explanation for this is that only one or a small number of genes were included. Furthermore, current guidelines for the management of PAH do not recommend genetic testing in associated forms, and most previous studies have excluded them [10]. However, a recent study by Zhu et al. studied 722 CTD-PAH patients [7]. Rare coding variants were observed in 5.26% of the cohort. However, phenotype and clinical information are not provided. For our study, a PAH-specific HTS panel was designed (35 genes), and associated forms of PAH and PVOD were also included. As a result, we also obtained a significant number of variant carriers.
Our study highlights different molecular pathways involved in CTD-PAH. Half of the pathogenic or likely pathogenic variants were located in potassium channel genes (KCNA5, ABCC8). Only one gene was included in the TGF β pathway (GDF2/BMP9) and no variants were observed in BMPR2. Another pathogenic variant was present in TBX, whose mutations express an ever-expanding phenotype. With this in mind, one might speculate that screening genetic variants may be a practical non-invasive tool to identify high risk CTD patients. Furthermore, it might be especially useful in rare forms of CTD-PAH: early-onset SSc or CTD other than SSc. However, the presence of a genetic defect does not seem to influence clinical course or prognosis.
TGF-β signaling pathway expression and activity is reduced in both idiopathic and hereditable PAH, regardless of the presence of germline mutations [27]. Restoration of the BMPR2 axis is a promising therapy to prevent or treat PAH, by restoring the balance between proliferative and anti-proligerative pathways [13,28,29] Recently, PULSAR trial has demonstrated the benefit of sotatercept in PAH, including a high proportion of CTD-PAH patients. BMP9/GDF2 is a ligand of the BMP signaling pathway, recently identified as a PAH gene [30]. Furthermore, the application of recombinant BMP9 reversed pulmonary hypertension in animal models [31]. Restoring BMPR2 expression is also a promising treatment target [13,32,33]. The presence of germline mutations in BMP pathway genes suggests that this mechanism might contribute to PVD in CTD patients.
TBX4 is involved in the regulation of embryonic developmental processes and its haploinsufficiency was classically associated with Small Patella Syndrome. Pathogenic variants in this gene are a common cause of hereditable PAH in infants and children [34,35]. Recently, our group has demonstrated that PAH associated with TBX4 variants shows a wide spectrum of clinical presentations, with overlapping forms of PAH [36]. A severely reduced DLCO was a common finding. However, TBX4 mutations are not an established cause of CTD-PAH. Thus, the relationship between TBX4 variants and connective tissue diseases remains unclear. Further investigation is necessary to address this issue.
In our cohort, three patients carry a variant in a potassium channel gene. There is growing evidence supporting the role of potassium channel dysfunction in PAH [37]. KCNK3 and KCNA5 are considered to play a predominant role in pulmonary vascular tone [38]. ABCC8 codifies for SUR1, a subunit of the ATP-sensitive potassium channel that is mostly present in the β pancreatic cells. This explains why pathogenic variants in ABCC8 have been widely related to diabetes mellitus and congenital hyperinsulinism. However, how ABCC8 can cause PAH remains unclear. Previous studies done by our group and others have observed rare coding mutations in ABCC8 in idiopathic, familial and CHD-associated PAH [39,40]. However, the role of potassium channels in PAH associated with CTD is not well researched. Further investigation might explain the importance of resting membrane potential in the development of pulmonary vasculopathy in this population.

5. Conclusions

These findings suggest that genetic testing might be a useful tool in screening or initial diagnosis work-up of CTD-PAH. The discovery of rare variants in these patients forces us to take a comprehensive approach and provide accurate genetic counseling. Further research is still necessary to confirm these findings and help to provide a personalized medicine approach to these patients.

Supplementary Materials

The following are available online at, Table S1: Spanish Registry of Pulmonary Arterial Hypertension Centers and Investigators.

Author Contributions

Conceptualization, P.E.-S. and P.L.; methodology, P.E.-S. and P.L.; software, N.O.-P.; validation, J.T.-C., N.G., D.V., P.E.-S. and P.L.; formal analysis, I.H.-G., N.G. and J.T.-C.; investigation, I.H.-G., J.T.-C., N.O.-P., C.P.-O., P.E.-S. and P.L.; resources, P.E.-S. and P.L.; data curation, I.H.-G. and J.T.-C.; writing—original draft preparation, I.H.-G.; writing—review and editing, M.L.-D., J.T.-C., D.V., J.P.D., P.E.-S. and P.L.; visualization, P.E.-S. and P.L.; supervision, P.E.-S. and P.L.; project administration, P.E.-S. and P.L.; funding acquisition, J.T.-C., P.E.-S. and P.L. All authors have read and agreed to the published version of the manuscript.


This research was funded by project “Bases Genético Moleculares de la Medicina de Precisión en la Hipertensión Arterial Pulmonar”. Funder: Instituto de Salud Carlos III. Ministerio de Economía y Competitividad. Grant number: PI 18/01233.

Institutional Review Board Statement

The study was conducted according to the guidelines of the Declaration of Helsinki, and approved by the ethical committee for scientific research of the participant centers. A full list of REHAP centers and investigators is provided in the Supporting Information.

Informed Consent Statement

Written informed consent was obtained from all subjects involved in the study.

Data Availability Statement

All relevant data are within the manuscript and its Supporting Information files.


This project was supported by the Spanish Pulmonary Arterial Hypertension Registry (REHAP), Registry of Pediatric Pulmonary Hypertension Patients (REHIPED), Xunta de Galicia and Centro de Investigación Biomédica en Red de Enfermedades Cardiovascular (CIBERCV). In Addition: we would like to thank our patients for making this study possible.

Conflicts of Interest

The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.


  1. Rubio-Rivas, M.; Corbella, X.; Guillen-Del-Castillo, A.; Tolosa Vilella, C.; Colunga Arguelles, D.; Argibay, A.; Vargas Hitos, J.A.; Todoli Parra, J.A.; Gonzalez-Echavarri, C.; Ortego-Centeno, N.; et al. Spanish scleroderma risk score (RESCLESCORE) to predict 15-year all-cause mortality in scleroderma patients at the time of diagnosis based on the RESCLE cohort: Derivation and internal validation. Autoimmun. Rev. 2020, 19, 102507. [Google Scholar] [CrossRef] [PubMed]
  2. Sung, Y.K.; Chung, L. Connective tissue disease-associated pulmonary arterial hypertension. Rheum. Dis. Clin. N. Am. 2015, 41, 295–313. [Google Scholar] [CrossRef] [PubMed]
  3. Morrisroe, K.; Stevens, W.; Sahhar, J.; Rabusa, C.; Nikpour, M.; Proudman, S.; Australian Scleroderma Interest, G. Epidemiology and disease characteristics of systemic sclerosis-related pulmonary arterial hypertension: Results from a real-life screening programme. Arthritis Res. Ther. 2017, 19, 42. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  4. Quezada Loaiza, C.A.; Velazquez Martin, M.T.; Jimenez Lopez-Guarch, C.; Ruiz Cano, M.J.; Navas Tejedor, P.; Carreira, P.E.; Flox Camacho, A.; de Pablo Gafas, A.; Delgado Jimenez, J.F.; Gomez Sanchez, M.A.; et al. Trends in Pulmonary Hypertension Over a Period of 30 Years: Experience From a Single Referral Centre. Rev. Esp. Cardiol. 2017, 70, 915–923. [Google Scholar] [CrossRef] [PubMed]
  5. Escribano-Subias, P.; Blanco, I.; Lopez-Meseguer, M.; Lopez-Guarch, C.J.; Roman, A.; Morales, P.; Castillo-Palma, M.J.; Segovia, J.; Gomez-Sanchez, M.A.; Barbera, J.A.; et al. Survival in pulmonary hypertension in Spain: Insights from the Spanish registry. Eur. Respir. J. 2012, 40, 596–603. [Google Scholar] [CrossRef] [PubMed]
  6. Morrell, N.W.; Aldred, M.A.; Chung, W.K.; Elliott, C.G.; Nichols, W.C.; Soubrier, F.; Trembath, R.C.; and Loyd, J.E. Genetics and genomics of pulmonary arterial hypertension. Eur. Respir J. 2019, 53, 1801899. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  7. Zhu, N.; Pauciulo, M.W.; Welch, C.L.; Lutz, K.A.; Coleman, A.W.; Gonzaga-Jauregui, C.; Wang, J.; Grimes, J.M.; Martin, L.J.; He, H.; et al. Novel risk genes and mechanisms implicated by exome sequencing of 2572 individuals with pulmonary arterial hypertension. Genome Med. 2019, 11, 69. [Google Scholar] [CrossRef] [Green Version]
  8. Girerd, B.; Montani, D.; Jais, X.; Eyries, M.; Yaici, A.; Sztrymf, B.; Savale, L.; Parent, F.; Coulet, F.; Godinas, L.; et al. Genetic counselling in a national referral centre for pulmonary hypertension. Eur. Respir. J. 2016, 47, 541–552. [Google Scholar] [CrossRef] [Green Version]
  9. Liu, D.; Liu, Q.Q.; Guan, L.H.; Jiang, X.; Zhou, D.X.; Beghetti, M.; Qu, J.M.; Jing, Z.C. BMPR2 mutation is a potential predisposing genetic risk factor for congenital heart disease associated pulmonary vascular disease. Int. J. Cardiol. 2016, 211, 132–136. [Google Scholar] [CrossRef]
  10. Galie, N.; Humbert, M.; Vachiery, J.L.; Gibbs, S.; Lang, I.; Torbicki, A.; Simonneau, G.; Peacock, A.; Vonk Noordegraaf, A.; Beghetti, M.; et al. 2015 ESC/ERS Guidelines for the diagnosis and treatment of pulmonary hypertension: The Joint Task Force for the Diagnosis and Treatment of Pulmonary Hypertension of the European Society of Cardiology (ESC) and the European Respiratory Society (ERS): Endorsed by: Association for European Paediatric and Congenital Cardiology (AEPC), International Society for Heart and Lung Transplantation (ISHLT). Eur. Respir. J. 2015, 46, 903–975. [Google Scholar]
  11. van der Bruggen, C.E.; Spruijt, O.A.; Nossent, E.J.; Trip, P.; Marcus, J.T.; de Man, F.S.; Jan Bogaard, H.; Vonk Noordegraaf, A. Treatment response in patients with idiopathic pulmonary arterial hypertension and a severely reduced diffusion capacity. Pulm Circ. 2017, 7, 137–144. [Google Scholar] [CrossRef] [PubMed]
  12. Richards, S.; Aziz, N.; Bale, S.; Bick, D.; Das, S.; Gastier-Foster, J.; Grody, W.W.; Hegde, M.; Lyon, E.; Spector, E.; et al. Standards and guidelines for the interpretation of sequence variants: A joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology. Genet. Med. 2015, 17, 405–424. [Google Scholar] [CrossRef] [PubMed]
  13. Spiekerkoetter, E.; Kawut, S.M.; de Jesus Perez, V.A. New and Emerging Therapies for Pulmonary Arterial Hypertension. Annu. Rev. Med. 2019, 70, 45–59. [Google Scholar] [CrossRef] [PubMed]
  14. Odler, B.; Foris, V.; Gungl, A.; Muller, V.; Hassoun, P.M.; Kwapiszewska, G.; Olschewski, H.; and Kovacs, G. Biomarkers for Pulmonary Vascular Remodeling in Systemic Sclerosis: A Pathophysiological Approach. Front. Physiol. 2018, 9, 587. [Google Scholar] [CrossRef]
  15. Wang, Y.; and Kahaleh, B. Epigenetic repression of bone morphogenetic protein receptor II expression in scleroderma. J. Cell Mol. Med. 2013, 17, 1291–1299. [Google Scholar] [CrossRef] [PubMed]
  16. Gilbane, A.J.; Derrett-Smith, E.; Trinder, S.L.; Good, R.B.; Pearce, A.; Denton, C.P.; Holmes, A.M. Impaired bone morphogenetic protein receptor II signaling in a transforming growth factor-beta-dependent mouse model of pulmonary hypertension and in systemic sclerosis. Am. J. Respi.r Crit. Care Med. 2015, 191, 665–677. [Google Scholar] [CrossRef]
  17. Castano, J.A.T.; Hernandez-Gonzalez, I.; Gallego, N.; Perez-Olivares, C.; Ochoa Parra, N.; Arias, P.; Granda, E.; Acebo, G.G.; Lago-Docampo, M.; Palomino-Doza, J.; et al. Customized Massive Parallel Sequencing Panel for Diagnosis of Pulmonary Arterial Hypertension. Genes 2020, 11, 1158. [Google Scholar] [CrossRef]
  18. Loyd, J.E.; Primm, R.K.; Newman, J.H. Familial primary pulmonary hypertension: Clinical patterns. Am. Rev. Respir. Dis. 1984, 129, 194–197. [Google Scholar] [PubMed]
  19. Larkin, E.K.; Newman, J.H.; Austin, E.D.; Hemnes, A.R.; Wheeler, L.; Robbins, I.M.; West, J.D.; Phillips III, J.A.; Hamid, R.; Loyd, J.E. Longitudinal analysis casts doubt on the presence of genetic anticipation in heritable pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 2012, 186, 892–896. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  20. Eichstaedt, C.A.; Song, J.; Benjamin, N.; Harutyunova, S.; Fischer, C.; Grunig, E.; Hinderhofer, K. EIF2AK4 mutation as “second hit” in hereditary pulmonary arterial hypertension. Respir. Res. 2016, 17, 141. [Google Scholar] [CrossRef] [Green Version]
  21. Rabinovitch, M.; Guignabert, C.; Humbert, M.; Nicolls, M.R. Inflammation and immunity in the pathogenesis of pulmonary arterial hypertension. Circ. Res. 2014, 115, 165–175. [Google Scholar] [CrossRef]
  22. Thompson, A.A.R.; Lawrie, A. Targeting Vascular Remodeling to Treat Pulmonary Arterial Hypertension. Trends Mol. Med. 2017, 23, 31–45. [Google Scholar] [CrossRef]
  23. Dorfmuller, P.; Zarka, V.; Durand-Gasselin, I.; Monti, G.; Balabanian, K.; Garcia, G.; Capron, F.; Coulomb-Lhermine, A.; Marfaing-Koka, A.; Simonneau, G.; et al. Chemokine RANTES in severe pulmonary arterial hypertension. Am. J. Respir. Crit. Care Med. 2002, 165, 534–539. [Google Scholar] [CrossRef]
  24. Cracowski, J.L.; Chabot, F.; Labarere, J.; Faure, P.; Degano, B.; Schwebel, C.; Chaouat, A.; Reynaud-Gaubert, M.; Cracowski, C.; Sitbon, O.; et al. Proinflammatory cytokine levels are linked to death in pulmonary arterial hypertension. Eur. Respir. J. 2014, 43, 915–917. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  25. George, P.M.; Oliver, E.; Dorfmuller, P.; Dubois, O.D.; Reed, D.M.; Kirkby, N.S.; Mohamed, N.A.; Perros, F.; Antigny, F.; Fadel, E.; et al. Evidence for the involvement of type I interferon in pulmonary arterial hypertension. Circ. Res. 2014, 114, 677–688. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  26. Koumakis, E.; Wipff, J.; Dieude, P.; Ruiz, B.; Bouaziz, M.; Revillod, L.; Guedj, M.; Distler, J.H.; Matucci-Cerinic, M.; Humbert, M.; et al. TGFbeta receptor gene variants in systemic sclerosis-related pulmonary arterial hypertension: Results from a multicentre EUSTAR study of European Caucasian patients. Ann. Rheum. Dis. 2012, 71, 1900–1903. [Google Scholar] [CrossRef] [Green Version]
  27. Dewachter, L.; Adnot, S.; Guignabert, C.; Tu, L.; Marcos, E.; Fadel, E.; Humbert, M.; Dartevelle, P.; Simonneau, G.; Naeije, R.; et al. Bone morphogenetic protein signalling in heritable versus idiopathic pulmonary hypertension. Eur. Respir. J. 2009, 34, 1100–1110. [Google Scholar] [CrossRef] [Green Version]
  28. Guignabert, C.; Bailly, S.; Humbert, M. Restoring BMPRII functions in pulmonary arterial hypertension: Opportunities, challenges and limitations. Expert Opin. Ther. Targets 2017, 21, 181–190. [Google Scholar] [CrossRef] [PubMed]
  29. Humbert, M.; McLaughlin, V.; Gibbs, J.S.R.; Gomberg-Maitland, M.; Hoeper, M.M.; Preston, I.R.; Souza, R.; Waxman, A.; Escribano Subias, P.; Feldman, J.; et al. Sotatercept for the Treatment of Pulmonary Arterial Hypertension. N Engl J. Med. 2021, 384, 1204–1215. [Google Scholar] [CrossRef] [PubMed]
  30. Wang, X.J.; Lian, T.Y.; Jiang, X.; Liu, S.F.; Li, S.Q.; Jiang, R.; Wu, W.H.; Ye, J.; Cheng, C.Y.; Du, Y.; et al. Germline BMP9 mutation causes idiopathic pulmonary arterial hypertension. Eur. Respir. J. 2019, 53, 1801609. [Google Scholar] [CrossRef]
  31. Long, L.; Ormiston, M.L.; Yang, X.; Southwood, M.; Graf, S.; Machado, R.D.; Mueller, M.; Kinzel, B.; Yung, L.M.; Wilkinson, J.M.; et al. Selective enhancement of endothelial BMPR-II with BMP9 reverses pulmonary arterial hypertension. Nat. Med. 2015, 21, 777–785. [Google Scholar] [CrossRef] [Green Version]
  32. Spiekerkoetter, E.; Sung, Y.K.; Sudheendra, D.; Bill, M.; Aldred, M.A.; van de Veerdonk, M.C.; Vonk Noordegraaf, A.; Long-Boyle, J.; Dash, R.; Yang, P.C.; et al. Low-Dose FK506 (Tacrolimus) in End-Stage Pulmonary Arterial Hypertension. Am. J. Respir. Crit. Care Med. 2015, 192, 254–257. [Google Scholar] [CrossRef] [PubMed] [Green Version]
  33. Spiekerkoetter, E.; Sung, Y.K.; Sudheendra, D.; Scott, V.; Del Rosario, P.; Bill, M.; Haddad, F.; Long-Boyle, J.; Hedlin, H.; Zamanian, R.T. Randomised placebo-controlled safety and tolerability trial of FK506 (tacrolimus) for pulmonary arterial hypertension. Eur. Respir. J. 2017, 50. [Google Scholar] [CrossRef] [Green Version]
  34. Kerstjens-Frederikse, W.S.; Bongers, E.M.; Roofthooft, M.T.; Leter, E.M.; Douwes, J.M.; Van Dijk, A.; Vonk-Noordegraaf, A.; Dijk-Bos, K.K.; Hoefsloot, L.H.; Hoendermis, E.S.; et al. TBX4 mutations (small patella syndrome) are associated with childhood-onset pulmonary arterial hypertension. J. Med. Genet. 2013, 50, 500–506. [Google Scholar] [CrossRef] [Green Version]
  35. Haarman, M.G.; Kerstjens-Frederikse, W.S.; Vissia-Kazemier, T.R.; Breeman, K.T.N.; Timens, W.; Vos, Y.J.; Roofthooft, M.T.R.; Hillege, H.L.; Berger, R.M.F. The Genetic Epidemiology of Pediatric Pulmonary Arterial Hypertension. J. Pediatr. 2020, 225, 65–73. [Google Scholar] [CrossRef]
  36. Hernandez-Gonzalez, I.; Tenorio, J.; Palomino-Doza, J.; Martinez Menaca, A.; Morales Ruiz, R.; Lago-Docampo, M.; Valverde Gomez, M.; Gomez Roman, J.; Enguita Valls, A.B.; Perez-Olivares, C.; et al. Clinical heterogeneity of Pulmonary Arterial Hypertension associated with variants in TBX4. PLoS ONE 2020, 15, e0232216. [Google Scholar] [CrossRef]
  37. Le Ribeuz, H.; Capuano, V.; Girerd, B.; Humbert, M.; Montani, D.; Antigny, F. Implication of Potassium Channels in the Pathophysiology of Pulmonary Arterial Hypertension. Biomolecules 2020, 10, 1261. [Google Scholar] [CrossRef] [PubMed]
  38. Ma, L.; Roman-Campos, D.; Austin, E.D.; Eyries, M.; Sampson, K.S.; Soubrier, F.; Germain, M.; Trégouët, D.A.; Borczuk, A.; Rosenzweig, E.B.; et al. A novel channelopathy in pulmonary arterial hypertension. N. Engl. J. Med. 2013, 369, 351–361. [Google Scholar] [CrossRef] [Green Version]
  39. Lago-Docampo, M.; Tenorio, J.; Hernandez-Gonzalez, I.; Perez-Olivares, C.; Escribano-Subias, P.; Pousada, G.; Baloira, A.; Arenas, M.; Lapunzina, P.; Valverde, D. Characterization of rare ABCC8 variants identified in Spanish pulmonary arterial hypertension patients. Sci Rep. 2020, 10, 15135. [Google Scholar] [CrossRef] [PubMed]
  40. Bohnen, M.S.; Ma, L.; Zhu, N.; Qi, H.; McClenaghan, C.; Gonzaga-Jauregui, C.; Dewey, F.E.; Overton, J.D.; Reid, J.G.; Shuldiner, A.R.; et al. Loss-of-Function ABCC8 Mutations in Pulmonary Arterial Hypertension. Circ. Genom Precis Med. 2018, 11, e002087. [Google Scholar] [CrossRef] [PubMed] [Green Version]
Figure 1. Patient selection flow chart. PAH: Pulmonary Arterial Hypertension; CTD-PAH: Pulmonary Arterial Hypertension Associated with Connective Tissue Disease.
Figure 1. Patient selection flow chart. PAH: Pulmonary Arterial Hypertension; CTD-PAH: Pulmonary Arterial Hypertension Associated with Connective Tissue Disease.
Cells 10 01488 g001
Table 1. Baseline characteristics of 79 CTD patients.
Table 1. Baseline characteristics of 79 CTD patients.
Female69 (87.3%)
Age (years)55.6 ± 1.9
Right Heart Catheterism
RAP (mmHg)8.6 ± 0.6
mPAP (mmHg)42 ± 1.4
PCWP (mmHg)9.8 ± 0.4
CO (l/min)4.3 ± 0.1
CI (l/min/m2)2.6 ± 0.1
PVR (UW)8.6 ± 0.5
SvO2 (%)65.8 ± 1.5
Pulmonary Function Test
FEV1 (% predicted)80.5 ± 2.2
FVC (% predicted)81.7 ± 2.2
TLC (% predicted)88.5 ± 2.6
DLCO (% predicted)47.5 ± 2
6MWT (m)340 ± 16
Exercise O2 Sat (%)87 ± 1.3
Functional Class
I2 (2.5%)
II27 (34.2%)
III43 (54.4%)
IV7 (8.9%)
Data are median mean ± SD, or n (%)
DLCO: Diffusing Capacity of the Lung for Carbon Monoxide; VFC: Vital Forced Capacity; FE1V: Forced Expiratory Volume in 1 s; TLC: Total Lung Capacity; RAP: Right Atrium Pressure; mPAP: mean Pulmonary Artery Pressure; PCWP: Pulmonary Capillary Wedge Pressure; CO: Cardiac Output; CI: Cardiac Index; PVR: Pulmonary Vascular Resistance; WU: Wood Units; 6MWT: Six Minute Walk Test; m: meters; SvO2: Mixed venous oxygen saturation; O2: oxygen.
Table 2. Clinical characteristics of patients with pathogenic or likely pathogenic variants.
Table 2. Clinical characteristics of patients with pathogenic or likely pathogenic variants.
Patient 1Patient 2Patient 4Patient 5
Age PAH diagnosis58262570
Age CTD diagnosis562822NA
CTD manifestationsArthritisRaynaud, digital ulcersDiscoid lupus, enteritis, serositis, poliarthritisNA
CTD serologyNAANA, ACAANA, anti-DNA, anti-SmNA
CTD treatmentNoneCorticosteoids, AZACorticosteoids, MTX, cyclophosmamide, hydroxychloroquineNone
mPAP (mmHg)37713045
PCWP (mmHg)4368
CI (l/min/m2)
PVR (WU)12.610.23.754.5
FEV1 (% predicted)831028675
TLC or FVC (% predicted)1098710381
DLCO (% predicted)6174
6MWT (m)389463NA180
Final statusAliveAliveAliveDeath
Follow-up (years)6183.58.5
DLCO: Diffusing Capacity of the Lung for Carbon Monoxide; VFC: Vital Forced Capacity; FE1V: Forced Expiratory Volume in 1 s; mPAP: mean Pulmonary Artery Pressure; PCWP: Pulmonary Capillary Wedge Pressure; CO: Cardiac Output; CI: Cardiac Index; WU: Wood Units; PVOD: Pulmonary Venooclusive Disease; SSc: Systemic Sclerosis, SLE: Systemic Erythematous Lupus, CTD: Connective Tissue Disease; HRCT: High-Resolution Computed Tomography; FC: Functional Class; Ex: Exercise; Sat: Saturation; AZA: azathioprine; MTX: methotrexate; NA: not available.
Table 3. Variant analysis.
Table 3. Variant analysis.
PatientGeneGenomic Coordinate (hg19)cDNA and Protein LocationExon/IntronMutation TypePopulation Frequency Pathogenicity Predictors ACMG Prediction §Reference
1TBX4chr17:59560351dupNM_018488.2:c.1112dupC:p. (Pro372Serfs*14)8frameshift03/3PPMID: 32348326
2ABCC8chr11:17432062C>TNM_000352.4:c.2694+1G>AIVS21splicing0.0000039792/2LPLago-Docampo et al.
3ABCC8chr11:17491762G>ANM_000352.6:c.298G>A p. (Glu100Lys)3missense0.000071622/9VUSLago-Docampo et al.
4GDF2chr10:48414226C>TNM_016204.3:c.642G>A:p. (Trp214*)2nonsense03/3PTenorio et al.
5KCNA5chr12:5154998delNM_002234.3:c.1685delC (p.Phe563fs*21)1frameshift02/2PTenorio et al.
6NOTCH3chr19:15278219C>TNM_000435.2:c.5203G>A:p. (Glu1735Lys)29missense0.000004098/9VUSTenorio et al.
7CTCFLchr20:56078510G>ANM_001269041.1:c.1822G>A (p.Glu608Lys)9missense01/9VUSThis study
8CTCFLchr20:56093935A>CNM_001269041.1:c.938A>G (p.Tyr313Cys)4missense04/9VUSThis study
9TOPBP1chr3:133371445T>GNM_007027.3:c.951T>G (p.Ile317Met)8missense0.00002994/9VUSThis study
gnomAD exomes, gnomAD genomes, Kaviar, 1000G phase III, ESP. Number of in silico tools that predict pathogenic effect over the total analysed from dbNSFP (MutationTaster, MutationAssessor, FATHMM, FATHMM-MKL, MetaSVM, MetalR, Provean, LRT, SIFT). § ACMG prediction: P: Pathogenic, LP: Likely Pathogenic, VUS: Variant of Unknown Significance.
Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Share and Cite

MDPI and ACS Style

Hernandez-Gonzalez, I.; Tenorio-Castano, J.; Ochoa-Parra, N.; Gallego, N.; Pérez-Olivares, C.; Lago-Docampo, M.; Palomino Doza, J.; Valverde, D.; Lapunzina, P.; Escribano-Subias, P. Novel Genetic and Molecular Pathways in Pulmonary Arterial Hypertension Associated with Connective Tissue Disease. Cells 2021, 10, 1488.

AMA Style

Hernandez-Gonzalez I, Tenorio-Castano J, Ochoa-Parra N, Gallego N, Pérez-Olivares C, Lago-Docampo M, Palomino Doza J, Valverde D, Lapunzina P, Escribano-Subias P. Novel Genetic and Molecular Pathways in Pulmonary Arterial Hypertension Associated with Connective Tissue Disease. Cells. 2021; 10(6):1488.

Chicago/Turabian Style

Hernandez-Gonzalez, Ignacio, Jair Tenorio-Castano, Nuria Ochoa-Parra, Natalia Gallego, Carmen Pérez-Olivares, Mauro Lago-Docampo, Julian Palomino Doza, Diana Valverde, Pablo Lapunzina, and Pilar Escribano-Subias. 2021. "Novel Genetic and Molecular Pathways in Pulmonary Arterial Hypertension Associated with Connective Tissue Disease" Cells 10, no. 6: 1488.

Note that from the first issue of 2016, this journal uses article numbers instead of page numbers. See further details here.

Article Metrics

Back to TopTop