The results of the study indicate that the proportion of genetic alterations in children with various forms of PAH is higher than previously thought and is related to several different genes of the TGFß pathway. Mutations of the BMPR2, ACVRL1, and ENG gene, respectively, occurred in 8 out of 29 (27.5%) of I/HPAH patients and even in 2 of 11 patients (18.2%) with CHD-APAH. Children carrying a mutation had a significantly lower PVR than non carriers. Two BMPR2 and one ACVRL1 mutation detected in this study have not been described before. Genetic analysis in children with various forms of PAH may be of clinical relevance and shows the complexity of the genetic background in childhood.
Prevalence of gene alterations in children with I/HPAH
The prevalence of the here identified mutations in 27.5% of I/HPAH patients in the BMPR2, ACVRL1 and ENG genes is similar as previously reported. In 22% of 18 I/HPAH-children Harrison et al. found mutations in these 3 genes . Rosenzweig et al. found in 10% of patients BMPR2 mutations . In contrast to these 3 genes sequence alterations of the SMAD genes (SMAD1, SMAD5 and SMAD9) seem to be a rare event in adult or paediatric PAH patients (<2%) with unclear functional significance . In this study we identified no mutation in any of the SMAD genes, but 3 rare polymorphisms in SMAD9 in 3 I/HPAH patients.
Genetic findings in children with CHD-APAH and classification
The findings of this study (mutations in 18.2%) confirm that gene defects play also a role in CHD-APAH-patients. However, BMPR2 mutations have been a rare finding in CHD-APAH and occurred in only 6% of cases of a mixed cohort of adults and children . In our CHD-APAH-patient we found a deletion of the first exon of the BMPR2 gene which has been previously observed only in I/HPAH [32, 33]. Although such deletions are predicted to cause an altered translation, their penetrance is variable and clinically unaffected mutation carriers have also been reported  and have been seen in the sister of our index patient. HPAH and CHD-APAH are simultaneously present in this family. Thus, the classification in HPAH and CHD-APAH according to the guidelines [1, 34] seems to be difficult in this case since both forms can occur in one patient.
Mutations of ACVRL1/ENG and HHT in childhood
This study shows that ACVRL1 and ENG mutations can cause the development of severe PAH in childhood, without any symptoms or familial history of HHT. Wide intrafamilial phenotypic variations were previously observed in carriers of a mutation at position 484 . Some of the mutation carriers in these families showed symptoms of HHT, others of pulmonary hypertension and one a combination of both phenotypes , as was seen in our family with altered amino acid at this position (p.R484Q). The novel mutation p.I317T identified in patient A3385 was also present in three unaffected family members. Neither the index patient nor the mutation carriers in this family showed any clinical signs of HHT and occurrence of HHT was also not reported in the family history.
In our cohort we have detected two missense mutations in the ENG gene: one in a patient with IPAH (A15836, p.G214S), and one in a patient with CHD-APAH (D3783). ENG mutations are quite common in HHT patients and both mutations have previously been described in patients with HHT only [29–31]. In our patients neither the index patient nor any of the mutation positive family members showed any clinical signs of HHT.
The ENG mutations identified in a CHD-APAH patient and in an IPAH patient without phenotypic appearance of HHT sheds new light on the variability of the phenotype caused by defects in TGFß pathway genes. Both mutations lead to an exchange from a highly conserved glycine residue to a serine residue but are located on different sites of the protein: p.G214S at the N-terminal and p.G545S at the C-terminal part of the endoglin/CD105 domain. They are both considered to be damaging predicted by PolyPhen .
Beside the here identified genetic defects in children with I/HPAH and CHD-APAH there are most likely further not yet identified genetic factors, in some families possibly inherited in a recessive mode of inheritance as previously suggested .
Clinical relevance of genetic alterations
We compared hemodynamic parameters between the 10 mutation carriers and 30 non carriers. In our study, patients with mutation had a significantly lower PVR than patients without mutation. Due to the small number of patients we cannot exclude that this difference could have been accidentally occurred particularly with respect to the not significantly different PVRI. Rosenzweig et al.  found no significant differences in PVRI between mutation carriers and non carriers as well. These comparisons are anyhow difficult with respect to the genetic heterogeneity in this population.
I/HPAH vs. CHD-APAH
In this study we did not find significant differences in hemodynamic parameters or age at onset between I/HPAH and CHD-APAH patients. This is in contrast to data from Barst et al.  and Hill et al.  who both found a significantly higher PVRI and an older age at onset of the disease in I/HPAH patients. Again this might be due to the relatively small numbers of patients. Further studies with larger cohorts are needed to investigate the differences between I/HPAH and CHD-APAH.
In conclusion, mutations in different TGFß genes occurred in 8 out of 29 I/HPAH patients and 2 out of 11 CHD-APAH-patients and may influence the clinical status of the disease. Therefore, genetic analysis in children with PAH may be of clinical relevance and shows the complexity of the genetic background.