Data source and study population

This is a multi-centre ethnicity-based study that investigates PAH gene mutations in the Han Chinese. Twenty PAH patients were enrolled into this study. Informed consent was obtained from all participants and their family members for collection of blood samples and genetic analysis. The Institutional Review Board (IRB) of Kaohsiung Veterans General Hospital approved this study (IRB number: KSVGH21-CT1-21).

Whole exome sequencing, alignment, variant calling and annotation

Genomic DNA was isolated from peripheral blood leukocytes of PAH patients. Polymerase chain reaction (PCR) was used to amplify the exons and flanking intronic bases of the five genes, including BMPR2 (13 exons), ALK-1 (10 exons), CAV1 (3 exons), ENG (14 exons), and KCNK3 (2 exons). The primers used for PCR were designed using reference sequences deposited in the GenBank database. Standard DNA sequencing reactions were performed using the fluorescence-labelled dideoxy chain termination method with the BigDye Terminator ABI Prism Kit and the ABI PRISM™ 3700 DNA Analyser (Applied Biosystems, Foster City, CA, USA), according to the manufacturer’s instructions.

Gene mutation and expression analysis

The DAVID database (https://david.ncifcrf.gov) was used to functionally annotate mutated genes of PAH patients in this study [12, 13]. BMPR2, ALK1, KCNK3, CAV1, and ENG were entered into the gene list, with ‘Official_Gene_Symbol’ as the selected identifier, and human/homo sapiens as the selected species; background population was set as homo sapiens. Associated diseases, functional categories, gene ontology, and protein interaction were analysed using the Functional Annotation Tool in the DAVID database. The aforementioned genes were also entered into the GEO profiles database, where search results on mutations and expressions related to PAH were manually reviewed. Pulmonary hypertension was also used as a key word for the ClinVar database [14]; genetic locus mutations were downloaded from the website in text format, which were transformed to Excel format for further comparisons with gene mutations in our study.

Statistical analyses

The SPSS version 22 (IBM, Chicago, IL, USA) was used for data analysis. Percentile values were used to express categorical data, and were analysed using the chi-square test. Mean (μ) and standard deviation values were used for continuous variables using the Student’s unpaired t-tests. A p-value of < 0.05 (–Log P value > 1.3) was considered to be statistically significant.

Patient characteristics

All patients’ basic characteristics are listed in Table 1. There was one variant at ‘G410DfsX1’ in BMPR2, as well as three locus variants in ALK1, at ‘ex7 L300P,’ ‘ex4 S110PfsX40,’ and ‘ex7 E295Afs96X.’ In addition, one variant at ‘c.-2C>A (IVS1–2 C>A)’ in CAV1, and one at ‘ex8 D366H’ in ENG were also found. There were 11 patients without gene mutations, whereas patient 15 (P15) had gene mutations at both ex7 L300P in ALK1 and D366H in ENG. Table 2 shows the comparison between gene subgroups, including gender, age, body height, and body weight. Out of the 20 patients, there were three (15%) males and 17 (85%) females, with a mean age of 48.6 ± 11.1. There was a 25-year-old male in the BMPR2 group, who was considerably younger than patients in other groups.

DAVID database analysis

The DAVID database was used for functional annotation, and the output data are shown in Figure 1. Mutated genes in this study, including BMPR2, ALK1, KCNK3, CAV1, and ENG, are displayed in the gene list. Commonly associated diseases for these mutations included pulmonary hypertension (-Log P value: 1.9), liver cirrhosis, and associated hepatopulmonary syndrome (–Log P value: 2.8) (Fig. 1A). Mutated genes and their associated proteins included transforming growth factor beta receptor 1 (TGF-β1) (–Log P value: 2.0), bone morphogenetic protein 7 (BMP7) (Log P value: 2.6), and activin A receptor type 1 (ACVR1) (–Log P value: 2.7), as shown in Figure 1B.

Gene cluster and ontologies were analysed, and the results are shown in Figure 2. All mutated genes in this study were found in the gene cluster of ‘disease mutation’ (–Log P value: 2.72) (Figure 2A). Moreover, gene ontologies of input genes included ‘negative regulation of nitric-oxide synthase activity’ (–Log P value: 2.85), ‘negative regulation of endothelial cell proliferation’ (–Log P value: 2.28), ‘positive regulation of BMP signalling pathway’ (–Log P value: 2.26), ‘positive regulation of pathway-restricted Smad protein phosphorylation’ (–Log P value: 2.07), ‘vasculogenesis’ (–Log P value: 2.00), ‘negative regulation of TGF-β receptor signalling pathway’ (–Log P value: 1.96), ‘regulation of cell proliferation’ (–Log P value: 1.48), and ‘BMP binding’ (–Log P value: 2.74), as shown in Figure 2B.

GEO profile database analysis

GEO profile database associated with gene mutations and expressions was shown in Figure 3. The data discussed in Figure 3A were deposited into the NCBI’s Gene Expression Omnibus (Edgar et al., 2002), which are accessible through GEO Series accession numbers. BMPR2 mutation (GDS5610) is associated with PAH expression (Figure 3A) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE67492). BMP2 (GDS5610/39998_at) expression is associated with IPAH (Figure 3B) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE2559), and changes in CVA1 (212097_at) expression could lead to IPAH (Figure 3C) (https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE22356).

Locations of gene mutations and family pedigrees

The locations of gene mutations and further details are illustrated in Table 3, and family pedigrees of volunteer PAH patients are shown in Figure 4. For example, gene mutation location of patient 11 (P11) was at IVS1–2 C>A in the KCNK3ex1 fragment (Figure 4A); patient 12 (P12) had a gene mutation located at D366 (D, H) in the ENGex8 fragment (Figure 4B); patient 13 (P13) showed mutation at G410DfsX1 in the BMPR2ex9 fragment (Figure 4C).

Gene mutation for PAH was associated with age and gender difference

Compared with PAH patients without BMPR2 mutations, those with BMPR2 mutations were younger, with a mean age of 35.4 years [8]. Previous study indicated a mean age of 42 years for BMPR2 non-carriers [8]. Patients with gene mutations other than BMPR2 in our study were older than previously reported (49.7–62.8 years in ALK1, KCNK3, CAV1, and ENG subgroups). Our study also indicated that patients with BMPR2 mutations were younger than patients with other gene mutations.

The occurrence of BMPR2 mutations in sporadic PAH cases without a family history can be attributed to low penetrance of BMPR2 mutations (20%–30%) and de novo mutations [15]. The estimated penetrance in male and female carriers is 14% and 42%, respectively [15, 16]. It has been shown that female is the single most important determinant for the penetrance of BMPR2 mutations in PAH [15, 16]; male patients were significantly more likely to have BMPR2 mutations than female patients [17]. Similarly, in our study, only one male was found to have the BMPR2 mutation.

PAH occurs predominantly in females; the sex ratio of female-to-male is 2.4:1. It has been suggested that oestrogen and its metabolism may be associated with the pathogenesis of PAH [16, 18, 19]. However, mortality rate of PAH is higher in males as compared with that of females, particularly in male BMPR2 mutation carriers [20]. The Registry to Evaluate Early and Long-Term Pulmonary Arterial Hypertension Disease Management (REVEAL) showed a higher female predominance of PAH irrespective of BMPR2 status, and found that the female-to-male ratio for PAH was 3.9:1 in races other than whites, black and Hispanic [21]. Our study results agreed with previous evidence that suggested female dominance in IPAH or HPAH (Table 1) [17].

Gene ontologies from the Han Chinese patients correlated with PAH mechanism

Mutated genes in our study, including BMPR2, ALK1, KCNK3, CVA1, and ENG were considered to have evidence of mutation in patients with PAH [22]. Among which, BMPR2, ALK1 and ENG were clearly recognized for their biological functions in PAH [22]. BMPR2 is particularly highly expressed on the cell surface of pulmonary vascular endothelium [23], and BMP9 functions as a circulating vascular quiescence factor to counterbalance cell apoptosis and excessive proliferation in endothelial cells [24]. In BMPR2 mutated pulmonary artery smooth muscle cells, there was a loss of antiproliferative effects from BMPs, resulting in excessive Smad1/5 signalling, which led to hyper-proliferative cells [24]. The BMPR2 protein forms a complex with ALK1; ENG plays a co-receptor to form a complex on the membrane and signal specifically in response to the circulating BMP ligands [25]. CAV1 is highly expressed in endothelial cells, and is an important constituent protein of caveolae. BMP receptors are localized in caveolae, and loss of CAV1 inhibits BMPR2 membrane localization and signalling [26, 27]. KCNK3 encodes a potassium channel that generates the membrane potential needed to regulate pulmonary vascular tone [28]. BMPR2, composed of an extracellular motif and transmembrane kinase domains, and is a part of the TGF-β receptor superfamily [29]; mutated genes associated with the TGF-β signalling pathway include ALK1, CVA1, and ENG [30]. All of the genes analyzed in our study encode for membrane or transmembrane proteins [22].

The TGF-β pathway processes angiogenesis via two distinct signalling pathways, including the ALK5-Smad2/3 pathway and the ALK1-Smad1/5/8 pathway [31]. Endoglin counterbalances the stabilizing role of ALK5 to stabilise the vessel and inhibit endothelial cell overproliferation [32]. However, BMPR2 or ACVRL1 mutation disrupt the SMAD1/5/8 pathway and BMP signalling. This inhibits SMC apoptosis, which leads to SMC proliferation, vascular remodelling, and ultimately results in PAH [33]. Our study (Figure 2) reported gene ontologies, including ‘positive regulation if BMP signaling pathway’ and ‘negative regulation of TGF-β signaling pathway,’ which confirmed the imbalance between the ALK5-Smad2/3 pathway and ALK1-Smad1/5/8 pathways in in these gene mutations, resulting in PAH. Gene ontology of input genes in our study also suggested that in addition to their roles in BMP and TGF-β pathways, they also function in pathways associated with nitric oxide signalling and regulation, which coincides with the pathobiology of PAH; the results of this study was supported by a previous study [22].

New pathogenic mutations for IPAH might exist in the Han Chinese population

There are differences in genetic, physiological, and anatomic factors between races, which affected the structure and function of the right ventricle [34]. However, studies focused on the Han Chinese are limited. The REVEAL trial enrolled only 3.3% Asians, and it showed a higher PAH prevalence in Hispanic patients as compared with that of earlier registries [21]. A Chinese registry reported a lower 1-year survival rate for Chinese PAH patients [35]. Early molecular genetics studies can strengthen clinical diagnosis and assist decision-making in adopting effective treatment [36].

Locus variants in our study included ‘G410DfsX1’ in BMPR2, ‘ex7 L300P,’ ‘ex4 S110PfsX40,’ and ‘ex7 E295Afs96X’ in ALK1, ‘c.-2C>A (IVS1–2 C>A)’ in CAV1, and ‘ex8 D366H’ in ENG, which were not found in the ClinVar database associated with PAH. Further studies are needed to determine whether these new mutations are associated with sporadic primary PAH or HAPH in the Han Chinese. This study also revealed similar gender and age distribution with previous large studies, which implies that other genetic mutations or environmental factors may contribute to the poor survival of Han Chinese PAH patients [35]. Further studies on offspring of these PAH patients may be needed to confirm the association between these new mutations in Han Chinese patients with PAH.

Study limitations

The sample size of this study was small, and not all family members of enrolled PAH patients received whole genome sequencing. Although the characteristics of patients included in this study were similar to those of previous large studies. Further investigations are needed for PAH patients from the Han Chinese population.

Study strengths

This is a multi-centre ethnicity-based study to determine gene mutations in PAH patients. Current trials are focused mostly on white or Hispanic patients; studies of PAH patients in the Han Chinese population are rare. This study focused on the Han Chinese and used databases, including DAVID, ClinVar, and GEO profiles to analyse the relationship between mutated genes and their functional ontologies. This study offers valuable genetic data for the Han Chinese population.