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Molecular genetic framework underlying pulmonary arterial hypertension


Pulmonary arterial hypertension (PAH) is a rare, progressive disorder typified by occlusion of the pulmonary arterioles owing to endothelial dysfunction and uncontrolled proliferation of pulmonary artery smooth muscle cells and fibroblasts. Vascular occlusion can lead to increased pressure in the pulmonary arteries, often resulting in right ventricular failure with shortness of breath and syncope. Since the identification of BMPR2, which encodes a receptor in the transforming growth factor-β superfamily, the development of high-throughput sequencing approaches to identify novel causal genes has substantially advanced our understanding of the molecular genetics of PAH. In the past 6 years, additional pathways involved in PAH susceptibility have been described through the identification of deleterious genetic variants in potassium channels (KCNK3 and ABCC8) and transcription factors (TBX4 and SOX17), among others. Although familial PAH most often has an autosomal-dominant pattern of inheritance, cases of incomplete penetrance and evidence of genetic heterogeneity support a model of PAH as a Mendelian disorder with complex disease features. In this Review, we outline the latest advances in the detection of rare and common genetic variants underlying PAH susceptibility and disease progression. These findings have clinical implications for lung vascular function and can help to identify mechanistic pathways amenable to pharmacological intervention.

Key points

  • Heterozygous germline mutations in BMPR2 represent the central susceptibility factor in the precipitation and progression of pulmonary arterial hypertension (PAH).

  • Causal rare disease alleles have been identified in both bone morphogenetic protein (BMP) signalling and non-BMP pathways, confirming locus heterogeneity in PAH.

  • Next-generation sequencing has been instrumental in expanding the genetic architecture of PAH by broadening the mutation spectrum in known genes and identifying novel genetic risk alleles.

  • Childhood-onset PAH is associated with greater morbidity and mortality than adult-onset disease and has a distinctive genetic signature.

  • PAH is a Mendelian disorder with complex disease traits, indicating a role for modifying common variation in disease development.

  • Elucidating the genetic architecture of PAH provides unprecedented potential for the development of novel, precision medicine options in disease management.


Pulmonary arterial hypertension (PAH) is a rare vascular disorder with an estimated annual incidence of 1–2 cases per million individuals1. Until 2019, PAH was defined clinically as mean pulmonary arterial pressure >25 mmHg at rest with normal left atrial pressure, but this definition has since been revised to mean pulmonary arterial pressure >20 mmHg, normal left atrial pressure and pulmonary vascular resistance ≥3 Wood units2. Consistent features among patients with PAH include exertional weakness or syncope, dyspnoea and right ventricular hypertrophy in the absence of any underlying cardiac or pulmonary disease3. PAH was first described in 1891 in the case report of a patient with right ventricular failure in which the autopsy revealed no obvious reason for the observed pathology, referred to as ‘sclerosis of the pulmonary arteries’4. The term ‘primary pulmonary hypertension’ was subsequently employed in 1951 to set apart the elevated pulmonary arterial pressure observed in a cohort of 39 patients from other patients with a discernible cause of right ventricular failure5. The current terminology, adopted and adapted since 2004, takes into account the genetic contribution to disease and includes various categories of PAH, including idiopathic or sporadic cases (IPAH), heritable cases (HPAH; describing patients with a family history or identified germline mutation) and associated forms (APAH; attributable to specific risk factors, such as anorexigens, liver disease, congenital heart disease and connective tissue diseases)6,7.

PAH typically manifests in the third to fourth decade of life but can also be diagnosed in children, in whom prognosis is more severe8. Clinical features of disease in HPAH and IPAH are largely indistinguishable clinically, although patients with HPAH have an earlier age of onset with worse haemodynamic features upon initial diagnosis9. In the absence of contemporary ameliorative treatment, PAH is frequently fatal10. Histopathological examination of PAH indicates the presence of occluded pulmonary arterioles resulting from the proliferation of pulmonary artery endothelial cells (PAECs), pulmonary artery smooth muscle cells (PASMCs) and fibroblasts, which leads to right heart hypertrophy and eventual cardiac failure11.

Familial segregation has been reported in approximately 6–10% of all patients with PAH, wherein the disease segregates as a monogenic, autosomal-dominant trait with incomplete penetrance, ranging from 14% in males to 42% in females12. PAH is observed much more frequently in females, with a ratio of occurrence in women to men ranging between 2:1 and 4:1, which reflects the distinctions across different patient populations and PAH subtypes13. Both intrafamilial and interfamilial variability of phenotypic expression and differential age of onset have also been also observed. Taken together, these clinical findings indicate that the pathophysiology of PAH is complex, wherein the development and progression of this condition can be influenced by both environmental and genetic modifying factors. In this Review, we discuss the genetic aetiology of PAH and the factors that might influence screening and treatment strategies in the future.

TGFβ signalling in PAH

In 1997, the linkage interval for PAH was established on the chromosomal locus 2q33–34, leading to an international positional cloning approach to determine the causal gene14. Subsequent systematic analyses of the genes contained within the minimal linkage interval in two independent, multigenerational familial cohorts resulted in the identification of heterozygous germline mutations in the gene encoding bone morphogenetic protein receptor type 2 (BMPR2), a member of the transforming growth factor-β (TGFβ) superfamily of signalling molecules15,16. Subsequent interrogation of BMPR2 in patients with IPAH provided independent validation of a pathogenic variation in a subset of patients17,18. The mature BMPR2 polypeptide comprises 1,038 amino acids encompassing an extracellular signal peptide, a ligand-binding domain, a single-pass transmembrane domain, an intracellular catalytic kinase domain and an atypically long cytoplasmic tail19. Canonical bone morphogenetic protein (BMP) signal transduction is initiated at the plasma membrane upon ligand binding with the formation of a heteromeric complex of BMPR2 and a cognate type I receptor, namely activin receptor-like 1 (ACVRL1; also known as ALK1), activin receptor type 1 (ACVR1; also known as ALK2), BMPR1A (also known as ALK3) or BMPR1B (also known as ALK6). BMP signalling is cell-specific; for example, signalling in endothelial cells is triggered by the BMP9 ligand and relayed via a BMPR2–ACVRL1–endoglin receptor complex20,21. Next, the constitutively active BMPR2 transphosphorylates its type I receptor partner, which, in turn, activates a BMP-specific series of cytoplasmic intermediaries, namely mothers against decapentaplegic homologue 1 (SMAD1), SMAD5 and SMAD8. In conjunction with the nuclear chaperone SMAD4, this macromolecular complex translocates to the nucleus, where it combines with a series of co-activators and co-repressors to regulate the transcription of a limited set of target genes22.

BMPR2 mutations in PAH

Since the identification of BMPR2, numerous screening studies have been undertaken in patient cohorts of various sizes and diverse ethnicities, which have together established that mutations in BMPR2 account for 53–86% of patients with a family history of PAH and 14–35% of patients with IPAH23. Indeed, BMPR2 mutations of likely pathogenicity have been detected in >800 patients with PAH, of which 486 involve distinct, non-recurrent variants. These data indicate that the spectrum of genetic variants is similar in both idiopathic and familial forms of PAH and that these mutations fall into the major categories. Specifically, 25% of PAH-specific variations in BMPR2 are missense mutations that result in amino acid substitutions (Table 1). By contrast, the vast majority of variants are predicted to lead to premature protein truncation owing to nonsense mutations (27%), frameshift mutations arising from small-nucleotide insertions or deletions (23%), gene rearrangements (14%) or splice-site mutations (10%). Given that truncating mutations spare the terminal exon 13, mutant transcripts are most probably degraded via the nonsense-mediated decay pathway. Recurrent mutations have been observed in approximately 40% of patients with PAH-specific BMPR2 variations, most likely as a result of small insertions and deletions at sites of low genomic complexity, such as mononucleotide runs, or as a consequence of spontaneous cytosine deamination23. The highly recurrent mutation c.1471C>T (p.R491W), which affects a critical residue within the catalytic domain, is most likely to be a result of the latter mechanism. Indeed, a study involving a genetically isolated Finnish population indicated that detected BMPR2 mutations in PAH arose on distinct haplotypes, thereby precluding the presence of a founder mutation24.

Table 1 Genetic and molecular features of the 16 genes underlying PAH

Whereas missense mutations are widely distributed across BMPR2 exons, the majority are localized within key functional domains, specifically the ligand-binding domain encoded by exons 2–3 and the highly conserved catalytic kinase region functionally delineated by exons 6–9 and 11 (ref.23). Of note, the majority of studies to identify genetic variants in BMPR2 have historically been confined to the exonic space. The limited analyses of non-coding regions have identified substitutions within the BMPR2 5′ UTR, including the recurrent variant c.–669G>A, which has been predicted to abrogate specificity for an SP3 transcription factor binding site25,26. However, the high frequency at which this variant is present in control populations indicates that further investigation is required to establish its pathogenicity. Together, this extended data set consolidates BMPR2 haploinsufficiency as the primary molecular mechanism underlying hereditary PAH18.

PAH-associated congenital heart disease

Congenital heart disease is an established complication of PAH. In 2004, an analysis of BMPR2 variation was performed on a combined panel of 40 adults and 66 children with APAH encompassing a range of congenital heart conditions including patent ductus arteriosus and atrial and ventricular septal defects27. Direct screening of protein-coding exons and flanking intronic sequences led to the detection of six missense mutations, representing 6% of the total cohort. Three defects within the BMPR2 extracellular domain (c.125A>G, p.Q42R; c.304A>G, p.T102A; and c.319T>C, p.S107P) were observed in adults with complete type C atrioventricular canal defects. Of the patients with mutation-positive, childhood-onset APAH, two presented with an atrial septal defect and patent ductus arteriosus, whereas one had an aortopulmonary window and a ventricular septal defect27. In contrast to the adult-onset form of the disease, BMPR2 mutations in patients with childhood-onset APAH were not clustered within a specific exon or functional domain of the protein (c.140G>A, p.G47N; c.556A>G, p.M186V; and c.1509A>C, p.E503D). Despite the limitations of small sample size and potential bias of ascertainment for PAH, this study is important in that these mutations are unique to PAH-associated congenital heart disease and are potentially under-represented because the authors did not screen for gene rearrangements or promoter variants. These genetic data are reinforced by a study in a mouse model that revealed the need for BMP signalling in the developing embryonic heart28.

Rare TGFβ family alleles in PAH

Hereditary haemorrhagic telangiectasia (HHT) is an inherited vascular condition clinically characterized by arteriovenous malformations and subcutaneous telangiectasia. HHT and PAH infrequently co-present in families, suggesting a common molecular aetiology. HHT has autosomal-dominant transmission and is caused by defects in the type I receptor ACVRL1 and the type III accessory receptor endoglin, which are members of the TGFβ signalling family and thus represent good candidate genes for PAH. Analyses of patients with both PAH and HHT confirmed the existence of a pathogenic variant in ACVRL1 and, at lower frequency, in ENG29. Since then, ACVRL1 mutations have been reported in patients with evidence of PAH without concomitant HHT, typically in early-onset disease30,31. The vast majority of ACVRL1 variants are missense mutations, of which approximately 89% are contained within the vital catalytic domain, indicating a high likelihood of pathogenicity23.

Subsequent interrogation of the BMP-specific pathway genes SMAD1, SMAD4, SMAD5 and SMAD9 in European and Asian populations led to the identification of novel independent variants that were absent from ethnically matched healthy individuals32,33. Functional analyses of these variants by in vitro luciferase SMAD-responsive element reporter assays confirmed that mutations in SMAD9 (encoding SMAD8) markedly impaired SMAD transcriptional activity, further validated by downregulation of the BMP target gene ID1 (ref.33). By contrast, defects in SMAD1 and SMAD4 caused less severe suppression of transcriptional activation and have consequently been defined as variants of unknown significance, albeit with the caveat that SMAD-independent pathways were not explored33. Taken together with the discovery of a novel homozygous c.76C>T (p.Q26*) nonsense mutation in GDF2 (which encodes the ligand BMP9) in a Hispanic boy aged 5 years34, these genetic findings underline the critical role of the wider TGFβ signalling network in the precipitation of PAH.

Expanding the genetic architecture of PAH

Whereas classic gene identification studies have proven invaluable in identifying causal factors underlying the development of many diseases, technological advances have revolutionized the way in which the genetics and genomics of PAH have been analysed. Next-generation sequencing technologies, in particular whole-exome sequencing and whole-genome sequencing, have been powerful tools for the detection of rare, highly penetrant gene variants in PAH and have thus established PAH as a genetically heterogeneous condition through analysis of families negative for mutations in known TGFβ pathway genes. Employing a direct approach of variant filtering based on a model of autosomal-dominant inheritance has led to the isolation of rare familial genetic variants in the genes encoding caveolin 1 (CAV1) and the potassium channel subfamily K member 3 (KCNK3), both of which have been validated as causative genes by replication in patients with IPAH35,36.

Caveolin 1

Implicated causal factors for PAH fall within cellular pathways pertinent to PAH pathology, particularly CAV1. Caveolin 1 is critical in forming the flask-shaped invaginations required for the regulation of BMP-driven, SMAD-dependent signalling in lung mesenchymal cells and endothelial cells37,38. Variants in CAV1 were initially implicated in PAH pathogenesis by exome sequencing of four patients with PAH within a three-generational family who were negative for the established variants in the TGFβ family35. Further examination of the remaining affected members confirmed segregation of the likely truncating mutation in exon 3 (c.474delA, p.L159Sfs*22). Replication of CAV1 variants was achieved through the identification of an adjacent de novo mutation in a patient with IPAH (c.473delC, p.P158Hfs*23)35. A separate study has further confirmed a third CAV1 frameshift mutation (c.471delC, p.D157fs) in an adult patient with PAH39. Although reported as a novel mutation, on the basis of the standardized mutation nomenclature from the Human Genome Variation Society40, this variant is actually identical to the previously described c.473delC mutation35. Assuming that these two patients are unrelated, the three mutations identified thus far are harboured in the terminal exon of CAV1 and the gene product would, as such, not be predicted to undergo transcript degradation via the nonsense-mediated decay pathway. Indeed, functional analysis of a PAH-specific frameshift mutation in CAV1 has indicated demonstrable impairment of caveolae assembly owing to retention of the mutant protein in the endoplasmic reticulum, with concomitant sequestration of the wild-type protein form41. Moreover, the c.474delA CAV1 mutation leads to hyperphosphorylation of SMAD1, SMAD5 and SMAD8, consequently resulting in a reduction of the anti-proliferative function of caveolin 1, thereby supporting SMAD gain of function as the underlying molecular mechanism of disease in patients with this CAV1 variant42. Although uncommon in human PAH, a causal role for CAV1 is consolidated by the observation that caveolin 1-deficient mice develop clinical features of pulmonary vascular disease analogous to PAH43,44,45.

Potassium channel dysregulation

Membrane potential is essential for contraction of PASMCs, and the dysregulation of potassium channels has been implicated in the pathogenesis of PAH. Transcript levels of KCNA5, which encodes the voltage-gated potassium channel subfamily A member 5, are reduced in PASMCs from patients with PAH compared with controls46, suggesting that the delayed-rectifier voltage-gated potassium current in PASMCs from patients with PAH is likely to be attenuated47. Of note, potassium channel expression is regulated by BMP signalling, providing a putative association with established molecular defects in BMPR2 (ref.48). Defects in KCNA5 have also been identified in patients with PAH49,50. However, in the absence of any independent replication studies or comprehensive examination of functional consequences, this gene remains an unvalidated causal factor. In 2013, Ma et al. reported in three independent families with PAH the segregation of KCNK3 mutations leading to amino acid substitutions36. Moreover, this finding was supported by the discovery of additional deleterious missense variants in KCNK3 in three patients with IPAH by the direct sequencing of 230 individuals. Electrophysiological analyses to examine the functional effect of these mutations showed a variable loss of function of the mutant channels that probably cause resting membrane potential depolarization36,51. Of note, the observed reduction in current across the potassium channel was reversed after treatment with a phospholipase inhibitor. The subsequent identification of a homozygous mutation underlying an aggressive form of HPAH has served to further consolidate a causal role for KCNK3 in this disorder52. Taken together, the detection of germline mutations in KCNK3 with deleterious functional effects reinforces a pathological role for potassium channel dysregulation in both familial and idiopathic forms of PAH.

Autosomal-recessive PAH subtypes

Under the present clinical classification of pulmonary hypertension, pulmonary veno-occlusive disease (PVOD) and pulmonary capillary haemangiomatosis (PCH) fall within PAH subgroup 1.6, namely PAH with overt features of venous or capillary involvement2. Typically, PVOD and PCH have histological characteristics that are indicative of pulmonary venous and capillary proliferation with a diffuse presentation53,54. Patients with PVOD or PCH respond poorly to available vasodilatating agents, and these drugs might provoke life-threatening pulmonary oedema54,55. Occasionally, pulmonary arterial involvement is also observed53,56. In contrast to PAH, both PVOD and PCH are inherited in an autosomal-recessive manner. To determine the genetic basis of PVOD, exome sequencing was performed to identify homozygous and/or compound heterozygous variations57. Mutations in EIF2AK4, which encodes eIF2α kinase GCN2, were identified in all of the analysed kindred (n = 5) and in 25% of those with sporadic disease (n = 20). Concurrently, Best et al. independently detected biallelic EIF2AK4 mutations in heritable and sporadic PCH58. GCN2 is essential for the activation of the integrated stress-response pathway, which is triggered by environmental stress via the phosphorylation of the α-subunit of eukaryotic translation initiation factor 2 (ref.59). The clinical and histological overlap between PAH, PVOD and PCH prompted the assessment of potential pathogenic EIF2AK4 defects in patients diagnosed with PAH. Analysis of a HPAH and IPAH patient cohort (n = 66) who did not have mutations in any of the major causative genes for PAH led to the identification of a novel homozygous EIF2AK4 mutation (c.257+4A>C) in two sisters with severe disease, indicating that variations in this gene constitute a rare molecular trigger for PAH60. Further validation for a pathogenic role of EIF2AK4 mutations in patients clinically diagnosed with PAH was provided by a study that identified 9 patients (out of 864) with biallelic EIF2AK4 mutations61. Notably, these individuals had clinical characteristics distinct from patients with PAH who did not have EIF2AK4 mutations, namely a younger age at PAH diagnosis (29 (interquartile range 23–38) years versus 51 (interquartile range 37–65) years) and reduced survival61. Taken together, these data emphasize the importance of an accurate genetic diagnosis for the efficient management and treatment of patients with PAH.

Sample size and gene-variant detection

The locus heterogeneity present in PAH has reduced the statistical power of genetic studies to detect causal genes, which is further confounded by small or clinically heterogeneous patient cohorts and the limited effect size of underlying variants. To advance our understanding of the increasingly complex aetiology of Mendelian disease, genetic studies are now contingent on the development of large, multicentre consortia such as the National Institute for Health Research BioResource for Rare Diseases. This resource is focused on novel gene-variant identification in conjunction with prospective phenotypic analyses through the generation of a unique biobank of patients and families with rare diseases, such as PAH62,63,64. To evaluate existing loci and expand our understanding of the genetics of PAH, a large cohort of 1,038 patients with PAH from the United Kingdom and Western Europe was recruited for whole-genome sequencing studies65. The cohort predominantly comprised patients with IPAH (87%; n = 908) of European descent (89%; n = 806). Initial screening of the established PAH gene variants confirmed BMPR2 mutation as the major risk factor, with a mutation frequency of 12% in patients with IPAH and 76% in patients with HPAH65. The distribution of variation across the other PAH risk factors indicated that variants in ACVRL1, ENG, SMAD9, KCNK3 and TBX4 had a modest contribution to the disease, with variants in each gene accounting for ~1% of cases. Of note, no pathogenic coding variation was identified in CAV1, SMAD1 or SMAD4, further reiterating the need for larger PAH sample sets in such studies65.

Genome-wide gene burden tests, employed to identify novel gene variants through a comparison of variant frequencies between patients with PAH and control individuals, detected statistically significant enrichment of rare deleterious variants in ATP13A3, AQP1 and SOX17 (ref.65). In keeping with previous studies23, the heritability ascribed to each novel gene variant was relatively small, accounting for 0.5–1.0% of the overall cohort. The majority of reported mutations were missense and nonsense variants, with ATP13A3 additionally harbouring three frameshift and two splice-site defects, probably reflecting loss of protein function. AQP1 and SOX17 mutation carriers had a significantly younger age at diagnosis, putatively suggesting early-onset PAH; however, this observation requires validation in larger patient cohorts. The identified variants in AQP1 and SOX17 were further demonstrated to segregate with the phenotype in a limited set of HPAH pedigrees65. Importantly, this study also provided further evidence of a prominent role for GDF2 as a key PAH risk factor, with the identification of one frameshift mutation and seven missense mutations, all of which are predicted with high confidence scores to be deleterious. Subsequently, the causal role of GDF2 mutations was confirmed in a prospective study that employed a gene panel66 and in a whole-exome sequencing study in Chinese patients with PAH, in which GDF2 mutations were detected in 6.7% of 331 patients with IPAH67. Moreover, preliminary evidence suggests a role for two putative loss-of-function mutations of BMP10 in disease susceptibility, further implicating the endothelial cell as the primary site of PAH initiation66.

Functional effect of novel gene defects

Probable cation-transporting ATPase 13A3 (ATP13A3), a member of the P5B subfamily of ATPases involved in ion channel transport, is highly expressed in major vascular cell types and represents a strong candidate for involvement in PAH pathogenesis65. Many of the ATP13A3 mutations identified in PAH cluster within the catalytic phosphorylation domain, indicating a likely important effect on protein function. However, the cellular function and transported substrate of ATP13A3 remain poorly understood. PAH is considered a neoplastic growth disorder and, of note, ATP13A3 promotes cell proliferation in multiple cancers68,69. Indeed, preliminary analyses have demonstrated reduced proliferation and increased apoptosis of endothelial cells following ATP13A3 knockdown with small interfering RNA65, consistent with reports that PAH is initiated by endothelial dysfunction-mediated survival of apoptotic-resistant cells, which subsequently results in the uncontrolled growth of pre-proliferative cells at key sites of disease70,71.

Expression of AQP1, which encodes the plasma membrane water channel aquaporin 1, is integral to the maintenance of vascular tone72. Animal studies indicate that abrogation of aquaporin 1 in PASMCs results in an attenuation of clinical features in hypoxia-induced PAH, providing the potential for targeted development of therapeutic options73. Aqp1-null mice have impaired endothelial cell migration and angiogenesis74. By contrast, elevated aquaporin 1 levels promote PASMC proliferation and migration through upregulation of the expression of the β-catenin targets MYC proto-oncogene protein and cyclin D1 (ref.75).

The majority of variants detected in SOX17 are also predicted to affect β-catenin function through either loss of the β-catenin binding region in SOX17 or abrogation of OCT4 (also known as POU domain, class 5, transcription factor 1)-mediated β-catenin interaction76,77. The endothelial transcription factor SOX17 depends on β-catenin binding to activate the transcription of target genes. Given that missense variations within the conserved high-mobility group box of SOX17 are anticipated to affect DNA binding of the transcription factor, all identified variants are likely to result in loss-of-function defects65. A whole-exome sequencing study involving 256 patients with PAH strongly implicated SOX17 mutations as a major risk factor in PAH-associated congenital heart disease and also provided independent validation for their role in IPAH78. Likely pathogenic variants were detected in 0.7% of patients with IPAH and 3.2% of patients with PAH-associated congenital heart disease, with the vast majority clustered within the high-mobility group box. A similar study in a cohort of Japanese patients with HPAH or IPAH identified three additional SOX17 variants79. Of interest, gene-based case–control analyses also revealed an over-representation of rare deleterious variations in several putative SOX17 target genes, indicating a key role for this pathway in PAH aetiology78. The SOX protein family have critical functions during human development and SOX17 is fundamental in angiogenic processes, including arteriovenous differentiation and development of the lung microvasculature80,81,82. In conjunction with Notch signalling, SOX17 is involved in the regulation of tip-cell and stalk-cell specification of endothelial cells, but the exact mechanisms by which SOX17 mutations can precipitate PAH remain to be determined81,83,84.

GDF2 encodes BMP9, a major ligand of the BMPR2–ACVRL1 receptor complex and a potent inhibitor of endothelial cell migration and growth85. BMP9 is processed by the serine endoprotease furin to its fully activated form, which is secreted as a mature prodomain-bound protein86. Mapping of identified amino acid substitutions onto the crystal structure of the BMP9 homodimer revealed a grouping of PAH-associated variants on the interface of the prodomain and growth factor domain65,86,87. Consistent with the hypothesis that PAH-specific variants reduce ligand stability, analysis of BMP9 levels in HEK293T cells demonstrated significantly reduced secretion of mature ligands in the supernatants of cells transfected with mutant BMP9 constructs compared with cells transfected with wild-type BMP9 (ref.65). In support of these findings, plasma levels of BMP9 were also reduced in patients with GDF2 mutations67.

The importance of these newly identified gene variants in the pathology of PAH is yet to be fully defined. These data provide a compelling framework of PAH as a genetically heterogeneous disorder comprising low-frequency mutations of high impact in seemingly disparate genes. Taken together, these findings provide important insights into the distinct mutation spectrum across different clinical categories for each causal gene, thereby facilitating focused diagnostic protocols (Table 2).

Table 2 Clinical and demographic features associated with reported PAH mutations

Genetics of childhood-onset PAH

The clinical features of childhood-onset and adult PAH have conspicuous differences. Specifically, poor survival and resistance to conventional therapies in paediatric patients have long indicated a distinct genetic aetiology to the disease in adult patients39. Candidate-gene analyses in limited sample sets have established BMPR2 variants as the major risk factor in familial and idiopathic early-onset PAH, with ACVRL1 variants also having a prominent role in conferring susceptibility31,32,88,89,90. In addition, rare PAH-associated alleles have been identified in ENG, KCNK3, SMAD9 and BMPR1B30,39,89,91. Of note, the mutant allele frequency in these genes is comparable between adult and paediatric patients.

To obviate the restrictions of previous investigations and more comprehensively assess the mutation spectrum in childhood disease, whole-exome sequencing was performed in a cohort of 155 patients with childhood-onset PAH and 257 patients with adult-onset PAH39. The analysis revealed a substantial enrichment of likely disease-causing alleles in TBX4 (encoding the T-box transcription factor TBX4) among the childhood-onset IPAH sample set (10 of 130 patients) compared with the adult-onset IPAH cohort (0 of 178 patients; P < 0.0001). TBX4 mutations have previously been identified as a prominent risk factor in small patella syndrome, a rare developmental bone dysplasia characterized by small or absent kneecaps92, and heterozygous loss-of-function TBX4 mutations have been described in childhood-onset PAH, with or without associated small patella syndrome90,93,94. These data, generated from the largest study of its kind, provide compelling evidence that, after BMPR2 mutations, variants in TBX4 confer the highest degree of genetic risk in childhood-onset PAH (9.2% versus 7.7%)39. TBX4 has an important role in the regulation of embryogenesis, in particular the formation of the hindlimb95. Additionally, the gene is expressed in the lung and trachea mesenchyme, and in the atria of the heart96. TBX4 mutations associated with early-onset PAH predominantly cluster in the critical T-box domain and typically result in damaging amino acid substitutions or premature truncation of the mutant transcript. Moreover, PAH has a significantly earlier age of onset in TBX4 carriers (7.9 ± 9.0 (0.6–33.0) years) compared with individuals with BMPR2 heterozygous mutations (28.2 ± 15.4 (1.5–72.0) years; P < 0.0001), suggestive of a specific genotype–phenotype correlation39.

A follow-up report in a cohort of patients with childhood-onset (n = 99) and adult-onset (n = 134) PAH who were negative for BMPR2 or ACVRL1 mutations identified a de novo missense variant in ABCC8 (encoding the ATP binding cassette subfamily C member 8 (ABCC8); also known as sulfonylurea receptor 1) in a patient with childhood-onset IPAH97. The mutation (c.2873G>A, p.R958H) was predicted to be deleterious, occurring at a highly conserved site within a critical domain of the encoded protein, which is a regulatory subunit of the ATP-sensitive potassium channel97. Screening of ABCC8 mutations in an extended cohort identified likely deleterious, heterozygous, missense mutations distributed across the length of the gene in paediatric patients with IPAH, familial PAH or congenital heart disease-associated PAH. Expression of the identified ABCC8 variants in a fibroblast-like cell line decreased the function of the ATP-sensitive potassium channel, further indicating that a subset of PAH might be mechanistically described as a potassium channelopathy97. Of interest, treatment of these cells with the pharmacological agent diazoxide, a selective ABCC8 activator, resulted in the recovery of channel function to normal levels97.

In addition to being an important determinant of early-onset PAH, TBX4 and ABCC8 mutations are also key to the pathogenesis of the adult form of the disease39,97. Indeed, in an extensive study of adult-onset IPAH, heterozygous TBX4 defects were observed in 14 patients (accounting for 1.3% of the total cohort), constituting the joint-second most substantive risk factor for PAH development after BMPR2 abnormalities65. These data serve to underscore the central importance of extensive study populations to investigate the composition of deleterious genetic variants in rare diseases with marked genetic heterogeneity.

Common variation in PAH

The first genome-wide association study (GWAS) in PAH was conducted in 2013 by Germain et al. in a French patient cohort comprising unrelated patients with IPAH or familial PAH without identifiable BMPR2 or ACVRL1 mutations98. Using a discovery data set of ~400,000 single-nucleotide polymorphisms genotyped across 340 patients and 1,068 healthy individuals, an association was identified with two single-nucleotide polymorphisms located 52 kb downstream of CBLN2 on chromosome 18q22.3. This signal was independently replicated in a US-based validation cohort of 284 patients with IPAH or HPAH and 456 controls98. Across the total sample set, the risk allele was associated with a twofold increased risk of disease with an odds ratio of 1.97 (95% CI 1.59–2.45; P = 7.47 × 10−10). CBLN2 encodes the precursor of cerebellin 2, which is predominantly expressed in the brain. Analysis of CBLN2 expression in the whole lung revealed significantly higher mRNA levels in explants of patients with PAH and in cultured pulmonary arterial cells; CBLN2 was predominantly expressed in PAECs and poorly expressed in PASMCs. Treatment of PASMCs with cerebellin 2 led to a significant reduction in cell proliferation in a dose-dependent manner, supporting a potential role for this protein in the physiopathology of PAH98.

A GWAS has also been conducted in a Japanese cohort of 44 patients with IPAH or HPAH and 2,993 control individuals99. Replication analysis in 21 patients and 991 control individuals led to the validation of two novel susceptibility single-nucleotide polymorphisms located midway between PDE1A (encoding calcium/calmodulin-dependent 3′,5′-cyclic nucleotide phosphodiesterase 1A (PDE1A)) and DNAJC10 (encoding DnaJ homologue subfamily C member 10) on chromosome 2q32.1 (ref.99). Odds ratios obtained from the validation study were 5.18 (95% CI 1.86–14.42) and 5.16 (95% CI 1.86–14.37) for rs71427857 and rs13023449, respectively. Based on immunohistochemical studies demonstrating increased immunoreactivity for PDE1A in distal pulmonary arteries from patients with IPAH, the authors suggest that PDE1A could be a novel therapeutic target for PAH99. However, owing to the small sample size of this GWAS, these findings will require further validation in a larger cohort with different ethnic groups.

In 2019, a comprehensive GWAS involving >2,000 patients of European ancestry with PAH was conducted100. The study design, which involved two independent discovery data sets followed by a meta-analysis to cross-validate loci attaining genome-wide significance, was unique in that it utilized whole-genome sequencing data to obtain genotypes for one cohort. The cohort comprised patients from the United Kingdom, the United States and Western Europe, including patients from the previously published French-led GWAS98. A combined association comparison of 2,085 patients with PAH against 9,659 population-matched control individuals demonstrated two regions of genome-wide significance across the HLA–DPA1/DPB1 locus (P = 7.65 × 10−20) and upstream of SOX17 (ref.100). Of note, the previously reported associations between CBNL2 and PDE1ADNAJC10 were not replicated in this study, indicating that these might represent population-specific signals. An investigation of putative correlation between the HLA–DPA and DPB1 risk variant and patient survival indicated a significantly improved prognosis with two copies of the risk allele. The SOX17 risk variants were located within an enhancer region, and functional evaluation of PAECs supported a role of these variants in the regulation of gene expression, with PAH risk alleles demonstrating reduced enhancer activity compared with non-risk alleles100. Taken together with the report of rare, deleterious SOX17 variants underlying PAH65, this finding suggests that both common and rare variants might predispose to PAH.

Clinical implications

The assessment of the genetic architecture of PAH has facilitated the directed mutation analysis of established causal genes, which provides molecular confirmation of the diagnosis. Moreover, numerous studies have identified exonic regions that are likely to harbour recurrent mutations, defects clustering within conserved domains and areas of functional importance, thereby expediting the accurate assessment of deleterious variants in contrast to variants of unknown significance. Importantly, predictions of pathogenicity must be evaluated in the context of the guidelines provided by the American College of Medical Genetics and Genomics for interpretation of sequence variants101. Within families, the precise determination of causal mutations provides an opportunity for informed genetic counselling, carrier testing and preimplantation genetic diagnosis in at-risk individuals102,103.

Aligned with these opportunities, the integration of genetic clinics into routine patient care within specialist PAH centres will be critical to support the development of mutation screening programmes and, as a progressive goal, the identification of early biomarkers of disease. For patients with a documented family history of PAH, clinical genetic testing is of exceptional importance to facilitate early monitoring and treatment intervention of relatives harbouring known gene defects. However, given the established role of heritable risk factors in PAH, we would also advocate genetic screening of all patients with IPAH to support risk calculations for family members and successive generations, consistent with international guidelines104. In the case of BMPR2, genetic data indicate that genotype–phenotype correlations differ between mutation carriers and non-carriers, and these correlations influence features of disease, such as age of onset and severity, that have the potential to direct specific treatment strategies. Furthermore, these molecular data permit differential diagnosis between subtypes of pulmonary hypertension, for example PAH and PVOD, thus informing efficacious patient management53. Ultimately, the identification of specific genetic defects in a given patient might provide options for personalized medicine.

Notably, the elucidation of disease genes has provided key insights into the pathways central to the maintenance of the pulmonary vascular bed and enabled the identification of molecular targets for the development of novel treatment strategies (Fig. 1). Proof-of-concept studies for restoration of BMPR2 function as a therapeutic approach in PAH holds promise, as has been demonstrated in patient-derived cells and in animal models105,106,107,108,109. These studies have been reviewed in detail previously110. To date, trials in patients based on genetically isolated targets have been focused on use of the calcineurin inhibitor tacrolimus, which, among other effects, activates BMP signalling to reverse vascular occlusion in rat models and seems to be well tolerated in patients with PAH111. Although preliminary in vitro findings support the potential restoration of potassium channel activity after pharmacological intervention in loss-of-function KCNK3 and ABCC8 cell models, further preclinical testing is required to evaluate the feasibility of manipulating these targets for patient benefit36,97,112. The endothelial cell-specific ligand BMP9, a known susceptibility factor for PAH, has been found to reverse cellular characteristics of disease in three established experimental models, including a murine Bmpr2-knock-in version of the recurrent human mutation p.R899* (ref.113). Critically, the fundamental understanding of the BMP9, ACVRL1 and BMP receptor signalling nexus has been integral to the progression of research from laboratory testing to clinical application, with a protein-engineered variant of BMP9 now in preclinical development. Based on this proof-of-principle notion, the novel genetic findings and wealth of genomic information that are now accessible lend promise to the progressive development of precision medicine treatments targeted at multiple pathways114.

Fig. 1: Major pathways and gene variants in heritable pulmonary arterial hypertension.

Several gene variants associated with heritable pulmonary arterial hypertension (HPAH) converge within the bone morphogenetic protein 9 (BMP9) and BMP10 signalling pathways, including BMPR2, ACVRL1 (also known as ALK1), ENG, SMAD4 and SMAD9. Caveolin 1 (encoded by CAV1) can also participate in BMP signalling. KCNK3 encodes the K2P3.1 potassium channel and ABCC8 encodes a subunit of the ATP-sensitive potassium (KATP) channel. The ATP13A3 gene product is an ATPase that resides on the endosomal compartment. SOX17 and TBX4 encode transcription factors critical for organogenesis. Biallelic mutations in EIF2AK4, which encodes an important kinase in the integrated stress response, are implicated in the pathogenesis of pulmonary veno-occlusive disease (PVOD) and pulmonary capillary haemangiomatosis (PCH). ACVRL1, activin receptor-like 1; ATP13A3, probable cation-transporting ATPase 13A3; BRE, BMP-responsive element; BMPR2, bone morphogenetic protein receptor type 2; GCN2, eIF2α kinase GCN2; SMAD, mothers against decapentaplegic homologue.

Future perspectives

To date, 16 genetic risk factors for PAH have been identified. For the majority of genes in which familial inheritance has been assessed, transmission occurs in an autosomal-dominant manner with incomplete penetrance. This method of transmission is particularly relevant in the case of BMPR2 variants, which suggests that although germline mutations represent a substantial genetic risk, additional environmental and genetic factors might be required to precipitate manifest disease. Indeed, anorexigens confer an established environmental risk to PAH development2. The identification of PAH-associated variants through GWAS analysis has demonstrated the likely existence of genetic modifiers98,99,100. Therefore, the genetic background of a mutation carrier in concert with environmental insults might explain whether the condition is potentiated. Taken together, these observations indicate that protein function needs to fall below a certain level for PAH to occur.

Missing heritability in PAH remains a vexed question. Although large data sets have been analysed for exonic and flanking variation with rigour, inadequate systematic examination of copy-number variations across implicated susceptibility loci remains. Indeed, large deletions and duplications are estimated to comprise approximately 14% of the mutation spectrum in BMPR2 alone (Table 1). Furthermore, the functional genomics of non-coding variations remains to be robustly analysed to best interrogate the contribution of impaired regulatory elements to disease precipitation, for example, via a combination of transcriptomics and chromatin-conformation capture technologies. For the genetic architecture of a rare, heterogeneous disorder to be fully annotated, a global initiative is required. Specifically, international patient ascertainment, data collection and amalgamation onto a joint platform is central to identification of novel rare disease alleles. The formation of the International Consortium for Genetic Studies in PAH aims to provide the necessary statistical power to advance discovery of the complete genetic architecture of PAH towards smaller effect sizes.

Critically, the functional analysis of detected variants is a requisite for the reliable determination of pathogenicity (Table 1). To investigate the effect of gene defects in the poorly characterized genes, an important first step would be the assessment of mRNA levels compared with wild-type controls to establish the stability and abundance of mutant transcripts. Similarly, the analysis of protein stability by western blotting and determination of subcellular localization by immunofluorescence is desirable. Where truncating or splice-site mutations are suspected, experiments aimed at examining nonsense-mediated decay might be of value. Further to this, in vitro assays will need to be specifically tailored to the known or predicted protein function on a gene-specific basis. Specific examples include electrophysiological studies to examine channel function36,97 and ligand-binding assays115 or luciferase assays to evaluate signalling activity32,33. Employing a relevant cell type, for example PAECs and patient-derived cells, is an important factor in the accurate interpretation of results in the context of disease. Long-term analyses might include the use of model systems to demonstrate in vivo effects of identified variants.

In addition to genomics-focused strategies to understanding the pathogenesis of PAH, additional insights can be leveraged through a more holistic approach by combining multilayered omics studies with comprehensive clinical phenotyping. In 2014, the Redefining Pulmonary Hypertension through Pulmonary Vascular Disease Phenomics (PVDOMICS) initiative was launched in the United States, with the aim of supporting clinical research with a molecular-based classification of pulmonary vascular disease. Over the next few years, the PVDOMICS consortium plan to integrate existing genomic data with patient transcriptomic, proteomic, metabolomic, coagulomic and cell-biomic profiles, which can be influenced by environmental and genetic factors116,117. In combination with improvements in tissue functioning and imaging, these studies will help to precisely categorize features of PAH and pulmonary vascular disease, providing a foundation to facilitate the development of precision medicine and personalized therapeutic interventions.


The genetics of PAH has evolved rapidly since the introduction of massively parallel sequencing technologies that facilitate the evaluation of genomic variation across multiple genes concurrently. Active collaboration between molecular biologists and dedicated clinicians has facilitated the identification of novel causal genes and pathways, highlighting important genotype–phenotype correlations that will inform diagnosis and improve clinical management. Dysregulation of BMP signalling remains the predominant risk factor for the development of PAH, and the seminal identification of BMPR2 haploinsufficiency as the primary molecular mechanism of disease is now evolving into the development of targeted therapeutic options. Continued gene discovery alongside mutation screening of newly identified genes will likely support the transition of molecular testing into PAH clinics. Nonetheless, there remains a substantial gap with regard to providing a definitive molecular genetic diagnosis to all patients. The continued efforts of global consortia are, therefore, central to fully resolving the molecular mechanisms of thisdevastating disease.


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L.S. is supported by the Wellcome Trust Institutional Strategic Support Fund (204809/Z/16/Z) awarded to St George’s, University of London, UK. N.W.M. is supported by a British Heart Foundation Personal Chair Award.

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Correspondence to Nicholas W. Morrell.

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Southgate, L., Machado, R.D., Gräf, S. et al. Molecular genetic framework underlying pulmonary arterial hypertension. Nat Rev Cardiol 17, 85–95 (2020).

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