Identification of a novel mutation in the BMPR2 gene in a pulmonary arterial hypertension patient using next‐generation sequencing

Abstract Background Pulmonary arterial hypertension (PAH) is a hemodynamic state that is characterized by pulmonary vasoconstriction and vascular remodeling, leading to a continuous increase in mean pulmonary arterial pressure, and eventually right heart failure. Mutations of the bone morphogenetic protein type II receptor (BMPR2) gene are the most common genetic cause of PAH. Methods A 52‐year‐old woman was admitted to Shaoxing People's Hospital after suffering from a cough for 2 months. In our hospital, the proband got a thorough medical examination and was diagnosed with PAH following genetic testing. Results Genetic test showed that the proband carried a novel heterozygous c.1481C>T (p.Ala494Val) mutation in the BMPR2 gene. The new mutation was initially discovered as a potential pathogenic variant by bioinformatics research, but it needed to be functionally verified. Conclusions The novel mutation may be related to the development of the PAH. In addition to general examinations, clinicians must thoroughly examine molecular genetics to provide an accurate diagnosis in the clinic, particularly for rare disorders.


| INTRODUC TI ON
Pulmonary arterial hypertension (PAH) is a hemodynamic state that is characterized by pulmonary vasoconstriction and vascular remodeling, leading to a continuous increase in mean pulmonary arterial pressure, and eventually right heart failure. 1 In the present study, we report a 52-year-old woman diagnosed with PAH carried a novel heterozygous c.1481C>T (p. Ala494Val) mutation in the BMPR2 gene. Detailed clinical data and the phenotypegenotype associated with the disease were delineated.

| Subjects
All procedures conducted in this study involving human participants were conducted in accordance with the Declaration of Helsinki and following the ethical standards of the Ethical Committee of Shaoxing People's Hospital. Informed consent was obtained from all participants in the study. The healthy people acted as controls.
The proband was a 52-year-old woman admitted to our hospital after suffering from a cough for two months. She was given a series of clinical and laboratory tests in Shaoxing people's hospital, including brain magnetic resonance imaging (MRI), magnetic resonance spectroscopy (MRS), next-generation sequencing (NGS), and so on.

| Samples collection
Peripheral blood samples of all participants were collected for the extraction of genomic DNA using a genomic DNA kit (TIANGEN BIOTECH, Beijing, China, DP304) according to the manufacturer's instructions.

| Genetics test
Genomic DNA was sequenced by next-generation sequencing (NGS), which refer to the related studies. [5][6][7][8] The NGS was performed by Agilent SureSelect Human All Exon V6 kits and Illumina NovaSeq 6000 sequencing platform. The paired-end reads (PE150) were aligned to a Genome Reference Consortium Human Genome Build 37 (GRCh37)-derived alignment set including decoy sequences using the Burrows-Wheeler Aligner (BWA). Single nucleotide variants (SNVs), small insertions and deletions (indels), and copy number variants were called with GATK Best Practices. The sequencing data for all samples underwent standard quality control checks. It must be achieved that the average coverage depth is more than 100 X, 90.00% of the target region sequencing depth is greater than 20X, and Q30 is not less than 90%. The pathogenicity of the variants was estimated using the American College of Medical Genetics and Genomics (ACMG) guidelines. Suspected pathogenic variation was verified by Sanger sequencing with specific primers (forward primer: 5′ -GAGCATGTTCCGTAATCC-3′ and reverse primer: 5′ -TTGTTGGGTCTCAGTTTC-3′). This part was conducted by the Joingenome Diagnostics Co., Ltd.

| Molecular evolution analysis
The modified MYBPC3 protein was also subjected to bioinformatics analysis utilizing accessible software tools. The software Clustal X1.83 was used to analyze evolution conservation among different paralogs and orthologs. Sequences were obtained from https:// www.ncbi.nlm.nih.gov/. In addition, the iterative threading assembly refinement (I-TASSER) server was used to predict the tertiary structure. STRING database (version 11.0) was used to predict protein-protein interactions of MYBPC3 protein, and the minimum required interaction score and number were set to 0.700 and 10, respectively. Pfam (https://pfam.xfam.org/) and PredictProtein (http://www.predi ctpro tein.org/) were used to determine aligned protein regions, protein secondary structure, and prediction of mutation function.

| 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.

| 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.

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

| DISCUSS ION
Bone morphogenetic proteins (BMPs) are secreted ligands of the transforming growth factorβ (TGFβ) family that control embryonic patterning, as well as tissue development and homeostasis. 9 Mutations in the BMPR2 gene that induce loss of function are the most common cause of PAH. 10 BMPR2-associated PAH is an autosomal dominant disease and considered a rare disease, with an estimated incidence of 1-2 per million cases. 11,12 To date, more than 298 BMPR2 mutations responsible for 55%-70% of heritable PAH (HPAH) and 11%-40% of idiopathic PAH (IPAH) have been reported. 13 Despite the impact of BMPR2 as the main genetic factor for PAH, currently, the specific mechanism of pathogenesis of BMPR2 in PAH is not fully understood.
Normally, BMP signaling activates SMAD proteins that counteract the effects of the TGF-beta signaling pathway. There is a delicate balance of SMAD signaling among the TGF-beta receptors.

| CON CLUS IONS
In conclusion, a unique heterozygous c.1481C>T (p. Ala494Val) mutation in the BMPR2 gene was discovered in a patient with pulmonary arterial hypertension, which appears to be linked to BMP malfunction. However, more research is needed to fully understand and elucidate the underlying mechanism.
F I G U R E 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) F I G U R E 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)

ACK N OWLED G M ENTS
The authors thank the Shaoxing Bureau of Science and Technology (no. 2017B70023), for financial support.

CO N FLI C T O F I NTE R E S T
None to declare.

DATA AVA I L A B I L I T Y S TAT E M E N T
Data sharing is not applicable to this article as no new data were created or analyzed in this study.