Background
Pulmonary arterial hypertension (PAH) is a group of vascular diseases that are characterized by a progressive increase in pulmonary vascular resistance and pulmonary arterial pressure with secondary vascular and right ventricular remodeling. PAH leads to right ventricular dysfunction, heart failure syndrome, and finally, premature death [
1]. PAH is characterized hemodynamically by the presence of pre-capillary pulmonary hypertension as defined by a pulmonary arterial wedge ≤15 mmHg and a pulmonary vascular resistance >3 Wood units in the absence of other causes of pre-capillary pulmonary hypertension. The current PAH Group 1 classifications include idiopathic, heritable, drug- and toxin-induced, and associated with connective tissue disease, human immunodeficiency virus infection, portal hypertension, congenital heart disease, and schistosomiasis [
2]. Despite current therapeutic options, PAH remains a catastrophic disease that is related to an unacceptable high mortality [
3]. It is clinically silent in early stages; symptoms develop late in the course of the disease. Although early diagnosis is associated with improved long-term survival, at present, most patients are diagnosed at a very advanced stage of PAH indicating that the early screening for PAH is crucial [
4]. In this regard, genetic testing is an effective strategy for the early diagnosis and management of PAH. The availability of molecular diagnosis has opened up a new field in patient care, which includes genetic counseling for severe diseases; however, the major predisposing gene for PAH, BMPR2, has a highly variable penetrance within families [
5]. Physicians who manage PAH should understand the heritable PAH phenotypes and the genes that are potentially responsible for PAH. They also should know the potential benefits, and issues surrounding genetic counseling and testing for patients with PAH.
The main genetic cause of familial PAH is caused by mutations in the gene bone morphogenic protein receptor type 2 (
BMPR2) [
5]. Although mutations in specific protein domains have been studied [
6], over 300 different
BMPR2 mutations along the gene have been related to the diagnosis and prognosis of PAH [
5,
7]. Recent studies have suggested that a BMPR2 mutation promotes cell division and prevents cell death, resulting in an overgrowth of cells in the small arteries throughout the lungs [
8]. As a result, these arteries narrow in diameter, which increases the resistance to blood flow and, consequently, increases the end-diastolic volume and causes adverse right ventricular remodeling. In addition, new evidence suggests that, in spite of a similar afterload, the right ventricular function is more severely affected in
BMPR2-mutation carriers that in noncarriers [
9]. Although mutations in the
BMPR2 gene are the single most common causal factor for hereditary cases of PAH, other pathogenic mutations have been observed in approximately 25% of idiopathic PAH and the occurrence of novel mutations in patients with PAH who have a family history can be 20–30% [
5]. Recent progress in the development of next-generation sequencing has facilitated the evaluation of several novel mutations that strongly correlate with PAH [
10]. The current genetic testing panels for PAH-associated autosomal dominant genes available mainly test for BMPR2-related genes; novel and not frequently represented genes are not included.
In this systematic review, we present a classification of the evidence of genetic mutations for PAH that are reported in the literature. These results can be used to suggest a comprehensive gene panel that is implicated in PAH for screening purposes. Additionally, this systematic classification avoids biases or overrepresentation of well-known genes, it is retrospective, depends on primary sources, and considers information from diverse cohorts and unrepresented minority populations of PAH patients. We also provide a functional analysis of the involved genes whose results might provide interesting insights into etiology and progression of the disease. To our knowledge, this is the first systematic review of genetic mutations in PAH.
Discussion
Heterozygous BMPR2 mutations account for approximately 75% of hereditable PAH and up to 25% of presumably sporadic PAH cases [
5]. The most important registries around the world have identified an incidence of less than 5% for hereditable PAH with a penetrance range between 20 and 80%. The REVEAL registry recognized an incidence of 2.7% [
24], while the French registry identified 3.9% [
25]. However, the incidence in other registries from China [
26] and Brazil [
27] is unknown. Recent guidelines of the European Cardiology Society [
2] recommend analyses for genetic alterations in
BMPR2,
BMPR1B,
EIF2AK4,
CAV1,
KCNK3, and
ENG where
BMPR2 has a key role in the etiological factor of the disease. Currently, genetic testing is a suggestion, but not a recommendation because of the level of evidence that supports the use of genetic testing in the diagnostic approach. In addition, genetic counseling is an issue that must be analyzed from an ethical perspective to allow the patient to make appropriate decisions.
Through PubMed and Internet searches, we found 12 available kits to identify genetic alterations related to PAH. Some of these kits are also applied to diverse cardiovascular disorders, such as dilated cardiomyopathy, arteriovenous malformations, and congenital defects. On average, these kits considered around eight genes, which were compared with the 21 genes identified (see column Kits in Table
2). Although one kit considered 11 genes of the 21 genes, most kits considered on average only 5 genes (
BMPR2,
ACVRL1,
ENG,
CAV1, and
SMAD4). None of the kits included genes that have been associated with PAH, such as
AGTR1,
TBX4,
EDN1,
EDNRA,
NOS2,
SERPINE,
SIRT3,
THBS1,
TOPBP1, or
TRCP6. For example,
AGTR1 has been related to later age PAH at diagnosis [
28],
TBX4 has been related to childhood-onset of PAH [
29], and
EDN1,
EDNRA, and
NOS2 have been related to susceptibility to develop the disease and its clinical course as well as susceptibility to systemic sclerosis-related PAH [
16,
30‐
32].
TOPBP1 and
TRCP6 have been linked to cellular protection and proliferation, respectively [
5,
33‐
35].
SERPINE1 has been linked with a risk of venous thromboembolism [
36], while
SIRT3 was related to mitochondrial dysfunction [
37].
THBS1 has been identified [
38], but not studied functionally. The identification of an important number of genetic alterations offers the potential to expand our knowledge about PAH. This provides a better understanding of the onset and progression of the disease, as well as the development of biomarkers for early diagnosis and stratification and potential therapeutic strategies, improving the clinical scenario for patients [
5].
The map of mutations (Fig.
3) suggests that mutations specific for PAH (continuous line) are widely distributed in a few genes:
ACVRL1,
BMPR2,
EIF2AK4,
ENG,
KCNA5, and
SMAD4. Nevertheless, there are many other mutations not yet found in PAH patients that have been found in other diseases. It is sensible to believe that mutations that affect other PAH-related disease genes are likely to have an effect in PAH. Consequently, most of the genes are affected along the whole coding sequence, such as
AGTR1,
CAV1,
EDN1,
NOTCH3,
SMAD9,
TBX4, and
TRPC6. For all these genes, testing specific mutations or sequencing selected regions does not seem to be a good strategy, instead, a more broad strategy, such as sequencing, would be needed. Figure
3 also shows that
ACVRL1,
ENG,
SMAD4,
SMAD9, and
TRPC6 have some mutations in the 5’UTR suggesting that coding mutations are not the only mutations that affect PAH. This concur with recent reports that found mutation in the promoter of
BMPR2 [
39]. These mutations may alter the binding of transcription factors or the transcriptional machinery affecting the gene levels instead of the function of the produced protein. Therefore it is important to test this types of alterations.
Around 300 different mutations in
BMPR2 have been studied and found to be related to a PAH diagnosis [
6,
40]. BMPR2 is a member of the TGFβ receptors superfamily, which consists in an extracellular and a transmembrane motif and kinase domains. Recent research showed that mutations in BMPR2 promote cell division and prevent cell death, resulting in an overgrowth of cells in small arteries throughout the lungs [
41]. As reviewed here, mutations in other genes also contribute. In this context, our functional analysis showed that the mutations were, collectively, related to other biological processes, diseases, and comorbidities, including hypoxia, pulmonary hypertension, hemangiomatosis, and abnormality of the vasculature of the conjunctiva (Fig.
4). Other functional associations found were related for example to stroke, or telangiectasia. It should be determined if these vascular disorders are directly related to PAH.
Mutations associated with the genes in the TGFβ signaling pathway, such as
ALK2 (
ACVR1),
ALK1 (
ACVRL1), and
CAV1, are also very important. ALK1 and ALK2 function to activate other proteins and genes in the TGFβ and BMPR2 pathways [
42].
CAV1 has been extensively reported with many confirmed mutations specific for PAH [
43,
44]. This gene codes a protein that contains 178 amino acids in its α-isoform. The expression of this protein is implicated in caveolae formation, which are plasma membrane structures that are specialized microdomains known as lipidic rafts. Caveolae are rich in superficial receptors that interact with cell membranes. These receptors are essential in order to activate the signaling pathways. The TGFβ and the nitric oxide signaling pathway are targeted by the plasmalemmal caveolae in endothelial cells; these pathways are involved in PAH development. For instance, CAV1 can modify the signal of TGFβ at the plasmatic membrane level. Thus, mutations in
CAV1 could be related to
BMPR2 mutations in PAH [
43,
45]. During our analysis, we appreciated how a huge number of these genes and genes that were associated with the TGFβ signaling pathway were related to the vasoconstriction processes. It is also possible to appreciate how some of these genes are related to the synthesis of prostaglandins (Fig.
4a); these genes relate to vasoconstriction and increase tissue permeability. In this regard, many different drugs have been developed for vasodilatation of the pulmonary vasculature. One interesting example is sildenafil, which inhibits phosphodiesterase and enhances the vasodilatory effects of nitric oxide in PAH to promote relaxation of the vascular smooth muscle and increase blood flow. This drug produces a relatively selective reduction in pulmonary artery pressure without adverse systemic hemodynamic effects and is one of the most widely used drugs in treating PAH [
46,
47].
Finally, genes related to redox homeostasis and calcium handling were also classified. In the analysis, genes with specific mutations for PAH, such as
CAV1,
AGTR1,
EDN1, and
KCNA5, were related to the regulation of homeostasis and ion controls. As a consequence, one of the pathways shown in the analysis was for the regulation of cytosolic calcium; many of the altered genes in PAH code for the transmembrane proteins that are responsible for calcium transport. As shown in Fig.
4b, the regulation of homeostasis and ion control within the cell are all controlled by the same genes. It is important to mention that these genes, such as
EDN1,
EDNRA, and
SMAD9, are also associated with the nitric oxide biosynthesis process. For example, EDN1 and EDNRA are that are common in homeostasis control in the cell or in nitric oxide biosynthesis processes. Besides, these genes are associated with oxidative processes; for example, EDNRA, EDN1, and CAV1 are related to the synthesis of different oxidoreductases that control oxidative stress within cells. One of the most novel genes associated with oxidative stress is
SIRTUIN3, which protein is involved in the post-translational modification of several proteins complexes involved in the antioxidant cellular and mitochondrial system [
48]. Surprisingly, this gene does not appear in the results of our analysis in any of the different pathways. However, the importance of the mutations in this gene in PAH has been clearly documented [
37].
The results of our systematic review propose that other genes whose prevalence or incidence have not widely studied may be important in the pathogenesis of PAH. In particular, the pathway analysis and the biological function of genes support our view. For research, it may be sensible to assess all genes. For clinical purposes, or if cost is an issue, a series of panels may be used; the first including well-known and more likely mutated genes like BMPR2, ACVRL1, ENG, EDN1, SMAD9; then, if no mutations are found, SMAD4, KCNA5, CAV1, BMPR1B, EIF2AK4, and KCNK3 may follow; and so on with the remaining genes.
The limitations of this study relate to the PAH classification, which has recently been modified; several reports could be potentially included in our analysis that used previous classifications of PAH. Based on the results of our systematic approach, we explored the relative risk associated with mutations and the occurrence of PAH. However, because of the heterogeneity of reports, populations, and some case reports, the associations were sometimes difficult and confusing to determine. Few studies reported associations between genetic alterations and the manifestation of PAH. We were also limited by the abstracts annotations provided by third party tools like PubTator [
11] where, overall, we observed accurate annotations but also some mistakes and time delays in the annotations.