Bone morphogenetic protein 9, and its genetic variants contribute to susceptibility of idiopathic pulmonary arterial hypertension

Characteristics of study population

The characteristics of 836 patients with IPAH and 900 control subjects included in this study were shown in Table 1. There were no significant differences in the distribution of age, gender, smoking and drinking status, as well as BMI between the IPAH cases and controls (P > 0.05 for all). These results indicate the comparability of this case-control study design.

The relationships of the BMP9 SNPs in IPAH risk

The genotypic distribution of the five selected tagSNPs of the BMP9 gene among the IPAH cases and controls was shown in Table 2. Genotype frequencies of five tagSNPs in controls were all in agreement with HWE (P > 0.05). Among the five SNPs, we found minor alleles of rs3740297 (OR: 0.72, 95% CI: 0.59-0.87, P=7.77×10^-5) and rs7923671 (OR: 0.76, 95% CI: 0.62-0.93, P=0.009) were significantly associated with decreased risk of IPAH. For rs3740297, the adjusted OR for the carriers with the CT genotype was 0.76 (95% CI: 0.64-0.91, P=0.003) and for those with the TT genotype was 0.51 (95% CI: 0.32-0.82, P=0.005), compared with the CC genotype. While for rs7923671, the adjusted OR for the carriers with the AG genotype was 0.78 (95% CI: 0.61-1.00, P=0.051) and for those with the AA genotype was 0.46 (95% CI: 0.23-0.95, P=0.034), compared with the GG genotype.

Effect of BMP9 rs3740297 and rs7923671 on plasma level of BMP9

As shown in Figures 1 and 2, the effect of BMP9 rs3740297 and rs7923671 on plasma level of BMP9 was presented. The plasma level of BMP9 in IPAH cases was significantly lower than that in controls (P<0.001). Next, we examined whether any of the two polymorphisms were correlated with plasma level of BMP9. We found minor alleles of rs3740297 and rs7923671 were significantly associated with increased plasma level of BMP9 in both IPAH cases and controls (P<0.001).

Effect of BMP9 rs3740297 and rs7923671 on transcriptional activity

HaploRegv4.1 and miRNASNP V2 were used to annotate the functional elements for BMP9 rs3740297 and rs7923671. We found that rs7923671 was located at a locus with multiple transcription factor (Myc, Pax-2, Pou5f1, P300) binding site, and rs3740297 was located at the 3’ UTR region of the BMP9 gene and its C>T transition could cause a binding site loss of miR-149 (Figure 3B), which suggest their potential allele-specific regulatory effect. To verify our hypothesis, we first measured the expression level of miR-149 in plasma of and PASMC cells, respectively. As shown in Figure 4, the expression level of miR-149 was not influenced by the genotypes of SNP rs3740297, as well as the disease status of IPAH (P >0.05). Meanwhile, the PASMC cells transfected with vectors containing the C allele of rs3740297 showed identical expression level of miR-149, compared to those containing the T allele (Figure 5A, P >0.05).

Then, we conducted the dual-luciferase reporter assay to validate the bioinformatics analysis findings. As shown in Figure 3, the PASMC cell lines transfected with vectors containing the A allele of rs7923671 showed higher relative luciferase activity compared to that containing the G allele (P<0.001, Figure 3A), which confirmed its allele-specific regulatory effect. For rs3740297, PASMC cell lines transfected with rs3740297 -C allele construct, miR-149 mimic, and antagomir miR-149 showed more sensitive change of the relative luciferase activity (Figure 3C, P < 0.001), which revealed its allelic binding effect to miR-149.

On the basis of results of the luciferase reporter assays, we further evaluated the regulatory role of rs3740297 on the expression of BMP9 in PASMC cell lines. As shown in Figure 5B, PASMC cell lines transfected with rs3740297 -C allele construct, miR-149 mimic, and antagomir miR-149 showed more sensitive change of the relative expression level of BMP9 (P < 0.001), while PASMC cell lines transfected with rs3740297 -T allele construct, miR-149 mimic, and antagomir miR-149, the relative expression level of BMP9 was not materially changed (P >0.05). Taking together, we can conclude that miR-149 could down-regulate the BMP9 expression with the existence of rs3740297 -C allele in PASMC cell line.

Study population

We totally recruited 836 IPAH cases, and 900 frequency-matched healthy controls by age and gender. All patients were primarily diagnosed by pulmonary angiography and right heart catheterization. The diagnosis of IPAH was made by at least 2 experienced PAH experts according to WHO criteria that a mean pulmonary arterial pressure equal to or exceeding 25 mmHg associated with normal pulmonary capillary wedge pressure. Patients with heritable, and associated PAH were not included in the present study. Controls were enrolled from the normal physical examination during the same period. Each participant was interviewed face to face to collect the demographic information. After that, each subject was asked to donate 10 mL of peripheral venous blood using sodium ethylene diamine tetra -acetic acid (EDTA) tubes. After centrifugation at 3000 × g and 4 °C for 15 minutes, the supernatants and cell sediment were frozen at -80 °C, respectively. All patients provided informed consent for participation and recruitment protocols were approved by the institutional review boards of Zhongshan Hospital.

TagSNP selection, DNA extraction and genotyping

The tagSNPs covering the BMP9 gene and its 5kb flanking region were selected from 1000 genome CHB data (phase 3, minor allele frequency ≥ 5%, pairwise r2 ≥ 0.8) using the Haploview 4.2 software. Finally, five SNPs (rs3740297, rs7923671, rs73299055, rs3781226, rs4922508) were selected as the candidates. Genomic DNA was extracted from the stored cell sediment of the blood samples using a DNA Blood Mini Kit (Qiagen, Valencia, CA, USA) according to the manufacturer’s protocol. The genotyping for the SNPs was performed using the TaqMan allelic discrimination assay on an ABI 7900 system (Applied Biosystems Inc, Foster City, CA, USA). A random 10% samples for each of the five SNPs were repeatedly genotyped and the concordance rate was 100%.

Enzyme-linked Immunosorbent Assay (ELISA) of BMP9

The plasma level of BMP9 in 100 randomly selected IPAH cases and 100 randomly selected controls was measured using a Sandwich Enzyme-linked Immunosorbent assay (ELISA) kit (R&D Systems, Minneapolis, MN) according to the manufacturer’s protocol, respectively. The inter-assay coefficient of variation was 8%, and the intra-assay coefficient of variation was 5%. All the assays were replicated in three times independently.

Luciferase reporter assay

Human pulmonary artery smooth muscle cell (PASMC) line was purchased from the Chinese Academy of Sciences Cell Bank (Shanghai, China), were cultured in Dulbecco’s Minimum Essential Medium (DMEM) medium. The plasmids were constructed by subcloning the wild types of rs3740297 and rs7923671 into the pGL-3 promoter vectors (Promega, Madison, WI, USA), respectively. Then, the mutant plasmids were created using the Quick Change Mutagenesis Kit (Stratagene, La Jolla, CA, USA). All constructs were confirmed by sequencing. All plasmids were transfected into PASMC cells using Lipofectamine 3000 reagent (Invitrogen). For rs3740297, mimic control, antagomir control, miR-149 mimic or antagomir miR-149 (GenePharma, Shanghai) were transfected in different groups, respectively. Finally, firefly and Renilla luciferase activity was measured on Synergy H1 microplate reader (BioTek Instruments, Winooski, VT, USA) using Dual-luciferase Reporter Assay System (Promega, Madison, WI, USA). Luciferase activity was normalized to Renilla activity to correct for differences in transfection efficiency. All the experiments were performed in triplicate.

Real-Time polymerase chain reaction analyses of miR-149 and the BMP9 gene

Total RNA was extracted from plasma and PASMC cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). Then cDNA was synthesized with M-MLV reverse transcriptase (Invitrogen). Real-time reverse transcription-polymerase chain reaction (RT-PCR) with SYBR Green assay (TaKaRa Biotechnology, Dalian, China) was performed to examine expression level of miR-149 and the BMP9 gene. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. The assay was conducted using the ABI 7300 system (Applied Biosystems). All reactions were done in triplicate and the expression level was calculated according to the equation 2^{-Δ Δ Ct}.

Statistical analysis

All analyses were conducted with SAS 9.3 software (SAS Institute, Inc., Cary, NC, USA), and a P value of < 0.05 for two-side was considered statistically significant. Differences in the distribution of selected demographic variables were evaluated by Pearson’s χ ² test. For quantitative variables, statistical significance was determined using unpaired t-test or one-way ANOVA. Hardy-Weinberg equilibrium (HWE) for each SNP among controls was tested using a goodness of-fit χ ² -test. The associations of each SNP with IPAH susceptibility were estimated by unconditional logistic regression analyses with odds ratios (ORs) and 95% confidence intervals (CIs), adjusted for age, gender, smoking and drinking status, and body mass index (BMI).