Methods

Fifty-seven unrelated patients aged ≤15 years at diagnosis with PAH were selected from 19 hospitals throughout Japan and China ( Figure 1 ). There was no duplication in this selection process. Some of these patients have been described in previous reports. Written informed consent was obtained from guardians of all study subjects in accordance with the Declaration of Helsinki. We assessed each patient by clinical history, physical examination, current therapy, 6-minute walking distance, and a review of medical records through March 2011.

Patient disposition. BMPR1A = bone morphogenetic protein receptor type 1A; BMPR1B = bone morphogenetic protein receptor type 1B. ENG = endoglin.

Diagnosis of IPAH and HPAH was made through clinical evaluation, chest x-ray, electrocardiography, echocardiography, and cardiac catheterization on the basis of current international consensus criteria (mean pulmonary artery pressure >25 mm Hg at rest or >30 mm Hg during exercise). Patients with PAH associated with other diseases such as portal hypertension, congenital heart disease, or persistent pulmonary hypertension of the newborn were systematically excluded from this study by trained cardiologists using a diagnostic algorithm. A significant acute response to nitric oxide and/or epoprostenol was defined by the following 3 criteria: (1) a >20% decrease in mean pulmonary artery pressure, (2) no change or an increase in cardiac index, and (3) no change or a decrease in the ratio of pulmonary vascular resistance to systemic vascular resistance.

The BMPR2 and ALK1 coding regions and exon-intron boundaries were amplified from patients’ genomic deoxyribonucleic acid. Amplified products were purified using the QIAquick polymerase chain reaction purification method (Qiagen, Hilden, Germany) and screened using bidirectional direct sequencing with an ABI 3130xl DNA Analyzer (Applied Biosystems, Foster City, California). Fourteen BMPR2 mutations and 7 ALK1 mutations were identified by direct sequencing ( Figure 1 ). Regarding BMPR2 and ALK1 , mutation-negative samples, multiplex ligation–dependent probe amplification (MLPA) was used to detect exonic deletions or duplications in BMPR2 , ALK1 , and endoglin. MLPA was performed according to the manufacturer’s instructions using a SALSA MLPA HHT/PPH1 probe set (MRC-Holland, Amsterdam, The Netherlands). MLPA analysis revealed that 4 of 36 patients had exonic deletions in BMPR2 ( Figure 1 ). In patients with no mutations in BMPR2 or ALK1 , all coding exons and adjacent intronic regions for endoglin, SMAD1 , SMAD2 , SMAD3 , SMAD4 , SMAD5 , SMAD6 , SMAD7 , SMAD8 , BMPR1A , and BMPR1B were amplified using a polymerase chain reaction. Polymerase chain reaction–amplified products were purified and directly sequenced like BMPR2 and ALK1 . One SMAD8 nonsense mutation and 2 BMPR1B missense mutations were detected, as described previously. They were excluded from this analysis because the number of cases representing each mutation was too small. No mutations were identified in endoglin, SMAD1 , SMAD2 , SMAD3 , SMAD4 , SMAD5 , SMAD6 , SMAD7 , or BMPR1A in 29 patients who had no mutations in BMPR2 , ALK1 , SMAD8 , or BMPR1B ( Figure 1 ). When a mutation was detected, we confirmed that it was not present in >200 healthy controls by direct sequencing.

Clinical features are expressed as mean ± SD, median (interquartile range), or number of patients (percentage), as appropriate. Univariate comparisons between ALK1 mutation carriers, BMPR2 mutation carriers, and mutation noncarriers were done using 1-way analysis of variance or the median test for continuous measures and the chi-square test for categorical measures. Death rate is presented as the number per person-year, and the difference between gene mutations was evaluated using a Poisson regression model.

A Kaplan-Meier overall survival curve was constructed to demonstrate the overall survival difference among mutation groups and compared using the log-rank test. Cox proportional-hazards regression modeling was performed to examine the relation between mutations and all-cause death. Similar survival analyses were performed by gender.

A p values <0.05 was considered significant. Statistical analyses were performed using JMP for Windows version 9 (SAS Institute Inc., Cary, North Carolina).

Results

From January 1, 1995, to March 31, 2011, 54 pediatric patients with IPAH or HPAH, corresponding to 24 male and 30 female patients, were included in this analysis. Eighteen (33%) were BMPR2 mutation carriers, 7 (13%) were ALK1 mutation carriers, and the rest were without these mutations ( Figure 1 , Table 1 ). The average age at diagnosis was 8.5 years, and there was no significant difference in ages among the 3 groups. Familial cases in ALK1 mutation carriers were largest in these groups. No significant differences were seen in brain natriuretic peptide or 6-minute walking distance. Cardiac catheterization was performed in 44 of 54 patients ( Figure 1 , Table 2 ). Forty-one of 54 patients were subjected to acute vasodilator response testing, 15 of whom were responders. There were no significant differences among the 3 groups in terms of hemodynamic characteristics or response to vasoreactivity testing ( Table 2 ).

There were no significant differences in current therapy among the 3 groups ( Table 3 ). As shown in Figure 2 , intravenous epoprostenol strongly improved functional class in all groups. Sildenafil, vasodilators except sildenafil or tadalafil and bosentan or ambrisentan, and oxygen also improved functional class moderately. The difference of each drug effect among each group was not significant (data not shown).

Functional class improvement through treatment in ALK1 mutation carriers (a) , BMPR2 mutation carriers (b) , mutation noncarriers (c) , and all patients (d) . Four of 7 ALK1 mutation carriers, 10 of 18 BMPR2 mutation carriers, and 16 of 29 mutation noncarriers could be investigated for the effects of the treatment. Changes in functional class were assessed by each attending physician retrospectively in March 2011. BMPR1A = bone morphogenetic protein receptor type 1A; BMPR1B = bone morphogenetic protein receptor type 1B; IV = intravenous.

Eight of 18 BMPR2 mutation carriers died, and the causes of death were all directly related to PAH. Two of 7 ALK1 mutation carriers died of PAH. Three of 29 mutation noncarrriers died, and the causes of death were related to PAH. The rate of death was worst in BMPR2 mutation carriers ( Table 4 ). Overall 5- and 10-year survival of all patients was 76% and 72%, respectively ( Figure 3 ). As shown in Figure 4 , male patients’ outcomes tended to be worse than female patients’ outcomes. In Figure 5 , overall mortality was worst in BMPR2 mutation carriers. Five- and 10-year survival was lower in BMPR2 mutation carriers than mutation noncarriers (55% and 37% vs 90% and 90%, respectively). ALK1 mutation carriers also had a tendency to have worse outcomes than mutation noncarriers (5- and 10-year survival rates were 64%). A multivariate Cox proportional-hazards model for time to death indicated that BMPR2 mutation carriers had significantly worse outcome than mutation noncarriers ( Table 5 ). The difference in outcomes between ALK1 mutation carriers and mutation noncarriers was marginally significant. No significant differences were observed in time to death among the 3 subgroups of BMPR2 mutation carriers ( Figure 6 ).

Kaplan-Meier curves for survival of all patients. Five- and 10-year survival probabilities were 76% and 72%, respectively.

Time to death in all patients according to gender (log-rank test, p = 0.08).

Survival of BMPR2 and ALK1 mutation carriers and mutation noncarriers with PAH (log-rank test, p = 0.0005).

Influence of BMPR2 mutation types on clinical outcomes of patients with PAH (log-rank test, p = 0.670).

Some BMPR2 and ALK1 mutations have been previously reported by other groups, and 7 BMPR2 mutation cases and 5 ALK1 mutation cases have been previously published by our colleagues ( Tables 6 and 7 ). Of 18 BMPR2 mutations, 6 were nonsense mutations, 3 were missense mutations, 2 were small deletions resulting in frame shifts, and 3 were splice-site mutations. MLPA analysis detected 4 exonic deletions ( Table 6 ). We identified 5 BMPR2 mutations that were not previously reported. All ALK1 mutations were missense mutations ( Table 7 ). One patient previously diagnosed with HHT subsequently developed features of PAH at the age of 11 years. We identified 2 ALK1 mutations that were not previously reported.

Table 6

Details of BMPR2 mutations

Proband	Mutation Location	Mutation Category	Domain	Nucleotide Change	Amino Acid Change	Reference
1	Exon 1	Nonsense	ECD	c.38 G>A	W13X	This study
2	Intron 2	Splice-site	ECD	c.248-3 T>G	—	This study
3	Exon 3	Nonsense	ECD	c.339 C>G	Y113X	Fujiwara et al
4	Exon 3	Nonsense	ECD	c.339 C>G	Y113X	Fujiwara et al
5	Exon 3	Missense	ECD	c.367 T>C	C123R	Machado et al
						Fujiwara et al
6	Exon 3	Deletion	ECD	c.247-? _420+? del	—	Machado et al
7	Exons 2-3	Deletion	ECD	c.76-? _420+? del	—	Machado et al
8	Exons 2-3	Deletion	ECD	c.76-? _420+? del	—	Machado et al
9	Intron 4	Splice-site	ECD	c.529+2 T>C	—	This study
10	Exon 6	Nonsense	KD	c.631 C>T	R211X	Thomson et al
11	Exon 6	Missense	KD	c.727 G>C	E243Q	This study
12	Intron 8	Splice-site	KD	c.1128+1 G>T	—	Pfarr et al
13	Exon 9	Nonsense	KD	c.1207 C>T	Q403X	Uehera et al
						Fujiwara et al
14	Exons 8-9	Deletion	KD	c.967-? _1275+? del	—	Aldred et al
15	Exon 10	Frameshift	KD	c.1376_1377 del GA	R459fsX10	Sankelo et al
16	Exon 10	Nonsense	KD	c.1397 G>A	W466X	Koehler et al
17	Exon 11	Missense	KD	c.1472 G>A	R491Q	Deng et al
18	Exon 12	Frameshift	CD	c.2289 del C	T762fsX9	This study

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