3.1 Laboratory examination and diagnosis

The proband was diagnosed as PAH based on clinical assessment, chest radiography, electrocardiography, echocardiography, and genetic testing. Patient clinical characteristics and parameters are shown in Table 1.

TABLE 1. Patient clinical characteristics and parameters Examination item Test value Reference value Clinical Age, years 55 Sex, M(F) F Arterial blood gas analysis PH 7.425 7.35–7.45 PO2 (mmHg) 93.1 80.0–100.0 PCO2 (mmHg) 40.0 35.0–45.0 Base excess (mmol/L) 1.2 −3.0–3.0 Alveolar-arterial oxygen tension difference (mmHg) 14.2↓ 15.0–20.0 Standard bicarbonate (mmol/L) 25.3↑ 21.3–24.8 Carbon dioxide (mmol/L) 23.1↓ 24.0–32.0 P50 (mmHg) 24.4↓ 24.8–27.8 Oxyhemoglobin (%) 96.2↑ 90.0–95.0 Laboratory parameters Cardiolipin antibody IgG (GPLU/ml) 11.9↑ 0–10.0 Immunoglobulin G (g/L) 18.4↑ 7.00–16.00 Complement C4 (g/L) 0.41↑ 0.10–0.40 Rheumatoid factor (IU/ml) 56.54↑ 0.00–30.00 Antinuclear antibody Positive Negative Anti-SSA/Ro−60KD antibody Positive(++) Negative Anti-SSA/Ro−52KD antibody Positive(++) Negative HLA-B27 Positive Negative 3.2 Gene detection

Sanger sequencing identified a novel heterozygous c.1481C>T (p. Ala494Val) mutation in the BMPR2 gene (Figure 1), whereas the mutation was not found in the controls.

Genome sequencing revealed a heterozygous c.1481C>T mutation in the BMPR2 gene, which causes amino acid p. Ala494Val. No additional mutation was found

3.3 Bioinformatics analysis

The predictions of pathogenicity tended to be pathogenic, predictive values of REVEL and CADD were 0.872 and 34, respectively. The conservation analysis indicated that the Pro residue at 494 in the BMPR2 protein was highly conserved across humans, rhesus, mice, dogs, elephants, chickens, x_tropicalis, zebrafish, and lamprey (Figure 2).

Phylogenetic conservation analysis. Evolution conservation analysis revealed that p.A494 in the BMPR2 domain was extremely evolutionarily conserved

Secondary structure was predicted to be strand by I-TASSER server with high confidence sore for this variant, solvent accessibility predicted that both normal and mutant amino acids at this position are buried in protein; the accessibility to solvent of each of these amino acids is 4 (Figure 3).

Predicted secondary structure and solvent accessibility predicted by I-TASSER server. Secondary structures of normal and mutant amino acid are predicted to be helix at position 494 with the respective confidence score of 8 and 9, the confidence ranges 0–9 wherein a higher score indicates a prediction with higher confidence. The solvent accessibility of the sequence is predicted as buried amino acid (range 0–9 wherein a higher value means higher accessibility)

The results of protein function prediction and secondary structure simulation are shown in Figure 4.

Bioinformatics analysis. The results of protein function prediction, Pfam, reveals the domain contained in the protein sequence (A). Secondary structure simulation showed the relative position of α, β, and random curl (B)

Furthermore, utilizing the STRING database, interactions between BMPR2 and other proteins showed potential implications on bone morphology development. BMP7, BMP2, ACVR1, GDF2, BMP4, BMP6, SMAD4, SMAD6, SMAD9, and SMAD5 are the ten functional partners expected to interact with BMPR2 (Figure 5).

Protein-protein interaction network of bone morphogenetic protein type II receptor (BMPR2). Predicted functional partners are as follows: BMP7: bone morphogenetic protein 7 (431 aa); BMP2: bone morphogenetic protein 2, induces cartilage and bone formation (396 aa); ACVR1: activin receptor type-1 (509 aa); GDF2: growth/differentiation factor 2 (429 aa); BMP4: bone morphogenetic protein 4, induces cartilage and bone formation (408 aa); BMP6: bone morphogenetic protein 6, induces cartilage and bone formation (513 aa); SMAD4: mothers against decapentaplegic homolog 4 (552 aa); SMAD6: mothers against decapentaplegic homolog 6 (496 aa); SMAD9: mothers against decapentaplegic homolog 9 (467 aa); and SMAD5: mothers against decapentaplegic homolog 5 (465 aa)