Genetics of PAH

To date, there are at least eight genes associated with HPAH and IPAH (table 2). From these genes, bone morphogenetic protein receptor 2 (BMPR2), is the major gene associated with HPAH and IPAH. Today over 300 BMPR2 mutations have been identified in patients with PAH. BMPR2 mutations are estimated to account for approximately 75% of patients with HPAH and up to 25% of IPAH cases.3 The penetrance of the disease associated with BMPR2 mutations has originally been considered low. These observations have been questioned as disease manifestations can take 75 years and phenotypical variability exists. Supporting evidence for higher penetrance was established through the Vanderbilt Pulmonary Hypertension Registry where overall penetrance was 27% (female 42% and the male 14%).4 Over 40% penetrance and high proportion of mutation positive individuals among patients with HPAH demonstrates that PAH is a genetic disease comparable with other forms of genetic diseases, i.e., cardiomyopathies and channelopathies. BMPR2 mutations are associated with childhood-onset PAH (table 2).5 ,6

PAH associated with hereditary haemorrhagic telangiectasia (HHT) has been identified in multiple studies to associate with pathogenic mutations in the ACVRL1 gene and in some occasions with Endoglin (ENG), a type III TGF-β receptor.7 ACVRL1 mutations can also be identified in patients with PAH with no features of HHT. SMAD9 mutations have been suggested as a genetic cause of PAH although existing evidence is mainly based on experimental data derived from functional in vitro and animal studies.8 Two missense mutations in another bone morphogenetic protein (BMP) signaling associated gene, BMPR1B, were identified in children with IPAH.9 An exome sequencing analysis of a three-generation family with multiple family members presenting with PAH revealed CAV1 as a novel gene associated with PAH. Another exome sequencing study on a family with multiple affected individuals revealed a missense mutation in a potassium channel gene KCNK3.10 A recent report suggested that mutations in the NOTCH3 gene could also cause childhood-onset IPAH.11 A recent breakthrough in pulmonary veno-occlusive disease (PVOD) has revealed that mutations in the EIF2AK4 gene play a major role in hereditary forms of PVOD.12 Genetic testing for mutations in EIF2AK4 gene should be considered for children with suspicion of PVOD. To date the evidence for causality in human IPAH and HPAH is considered significant for the following genes: ACVRL1, BMPR2, CAV1, ENG and KCNK3, whereas the role of BMPR1B, NOTCH3 and SMAD9 is still unconfirmed (table 2).

There is evidence for genotype-phenotype correlations in PAH. Multiple studies from different registries have implicated that carriers of a BMPR2 mutation, irrespective of family history, develop PAH at a younger age than mutation negative individuals.13–16 Furthermore, patients with the hereditary form of PAH developed higher mean pulmonary arterial pressure (PAP), lower cardiac index, higher pulmonary vascular resistance (PVR), and were more likely to be treated with parenteral prostanoid therapy or lung transplant when compared with patients with idiopathic disease. Both children and adults with PAH in association with a BMPR2 mutation are less likely to respond to vasodilators and are unlikely to benefit from treatment with calcium channel blockers. Symptomatic patients with HPAH carrying ACVRL1 mutations, most without HHT, have an earlier age of disease onset and show more rapid disease progression than patients with BMPR2 mutations.16 A recent study on 54 children with IPAH and HPAH revealed that 25 individuals (46%) were positive for either BMPR2 (18 patients; 33%) or ACVRL1 (7 patients; 13%) mutations.6 In this study the 5-year survival was significantly lower in patients tested positive for BMPR2 and ACVRL1 mutations. The study outcome correlated well with previous studies showing earlier onset and more severe phenotype in mutation positive patients. These studies suggest that patients with PAH carrying the BMPR2 or ACVRL1 mutation should be considered for more aggressive treatment strategies.

PAH is a rare complication in many genetic disorders such as Down syndrome (trisomy 21) where PAH can be caused by the high incidence of left-to-right shunt, upper airway obstructions and sleep apnoea. Other disorders where PAH has been described are congenital heart disease (CHD), 22q11 deletion syndrome (DiGeorge), VACTERL syndrome, CHARGE syndrome, Scimitar syndrome, Noonan syndrome, chromosomal anomalies associated with congenital diaphragmatic hernia, Cantu syndrome, autoimmune polyendocrine syndrome, mitochondrial disorders including mitochondrial encephalopathy lactic acidosis and stroke-like episodes, Gaucher disease, and glycogen storage diseases (GSDI and GSDIII). Establishing the underlying molecular genetic defect in the majority of these syndromes can be beneficial in determining the exact diagnosis and estimating prognosis, clinical follow-up as well as treatment strategies. This recommendation does not focus on the molecular genetics of these syndromes nor does it recommend testing of PAH-associated genes in these patients.

Genetic testing in PAH

Considering the possible hereditary nature of PAH, identification of a child with idiopathic PAH will initiate concerns of disease susceptibility in other family members, including the parents. Clinical evaluation of patient and family members will not necessarily establish the aetiology of IPAH and cannot define their risk to develop PAH later in life. The latter, like in other hereditary diseases, causes significant distress for the family. Identification of the genetic background enables effective family member testing and risk stratification among relatives. Considering the relatively high penetrance, especially among women, identification of the causative mutation allows effective clinical monitoring of those individuals at increased risk for the disease. Similarly, negative testing in family members following the molecular diagnosis for an index patient reduces stress burden in family members and allows discontinuation of future clinical monitoring in these individuals. This approach could therefore enable the establishment of cost-effective monitoring and follow-up programmes.2 Follow-up strategies for mutation carrier relatives have to be evaluated individually based on the age and sex of the family member, and existing family history and disease characteristics.

Genetic counselling is recommended for families with children suffering from a genetic disorder. Genetic counselling has to elucidate to the patient and family the rationale for genetic testing and describe the expected yield of genetic testing. Counselling also includes addressing the complex issues of incomplete penetrance and the possibility of finding variants that currently are classified as variants of unknown significance. All variant classification scenarios in genetic testing have to be clear for the referring physician.17 Different end points may affect the future surveillance of family members. It is also recommended to discuss concerns about genetic discrimination, and psychosocial issues of guilt and blame that accompany genetically based diseases. Although there have been concerns that predictive genetic testing in clinically unaffected children and adults could provoke anxiety and distress, current studies suggest instead that it can provide reassurance, lessen uncertainty and promote a sense of control.18 Genetic counselling should be provided by a healthcare professional with experience in genetics and PAH. Determining the best age for testing the asymptomatic children has to be evaluated separately in every family based on the family history and family’s requests after counselling. An algorithm for genetic testing of a child with IPAH or HPAH has been described in figure 1.

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Figure 1

Algorithm for applying genetic testing for a child with IPAH or HPAH, and family members. (–) negative test result and (+) positive test result with identification of pathogenic or likely pathogenic mutation. If NGS-based comprehensive analysis of PAH genes is not available consider gene-specific testing for BMPR2 and ACVRL1 genes as first-line diagnostics. *If genetic test reveals a mutation classified as likely pathogenic consider new genetic counselling in 5 years to confirm classification and validity of applied follow-up strategy. # Follow-up strategies for mutation carrier relatives have to be evaluated individually based on the age and sex of the family member, and existing family history and disease characteristics. HPAH, hereditary pulmonary arterial hypertension; IPAH, idiopathic pulmonary arterial hypertension; NGS, next generation sequencing; PAH, pulmonary arterial hypertension.

An important part of the counselling process is to establish an inclusive family history and determine the clinical features of the index patient. This allows comprehensive description of the disease phenotype and will facilitate interpretation of genetic test results. Any family history of HHT-like symptoms could point towards a mutation in the ACVRL1 or ENG gene, whereas at the same time a negative family history of HHT, does not rule out the possibility of disease-causing mutation in the ACVRL1 gene. On the contrary, suspicion of PVOD, especially if recessive inheritance is possible, suggests mutations in the EIF2AK4 gene. Although BMPR2 and ACVRL1 are currently the major disease-causing genes in PAH, any patient with IPAH or HPAH could harbour a mutation in any of the other known PAH genes. As the number of disease-causing genes is expanding there is increasing rationale to select more effective high-throughput next generation sequencing (NGS) strategies instead of gene-specific direct sequencing (Sanger sequencing). NGS-based applications are already enabling more cost-effective, comprehensive and faster analysis of causative genes in diagnostic setting. As the field and technologies are evolving rapidly there is increasing pressure for the referring physician to select optimal diagnostic tools to ensure high-end test quality and interpretation of test results. NGS strategies are already enabling the analysis of all nine genes mentioned in this statement with the same costs as single gene analysis using conventional technologies. Multigene strategies will provide simultaneous full analysis of BMPR2 and ACVRL1 and can include the analysis of other genes causative for PAH as well as potential new candidates. Adapting this strategy will allow for increasing diagnostic power but may also accumulate variant data impacting future diagnostics. Today's genetic diagnostics is increasingly based on public databases, which accumulate variant information from diagnostics and research. It is becoming a standard procedure in modern genetic diagnostics to publish variant data and classifications in public databases such as ClinVar (http://www.ncbi.nlm.nih.gov/clinvar/).

The use of biomarkers in paediatric PAH

Existing and potential biomarkers in PAH relate to heart failure, inflammation, haemostasis, vascular remodeling and endothelial cell-smooth muscle cell dysfunctions. In current PAH guidelines, brain natriuretic peptide (BNP) and N-terminal fragment of pro-BNP (NT-proBNP) have been recommended.19 Other prognostic markers such as growth differentiation factor-15, osteopontin and red cell distribution width, are considered emerging biomarkers of PAH. Anaemia, hypocapnia, elevated uric acid and C reactive protein levels are unspecific markers of disease severity. Certain chemokines, matrix metalloproteases and apelin have been linked to the vascular pathology in PAH and are considered as potential biomarkers. Circulating endothelial cells (CECs) and endothelial progenitor cells (EPCs) have received much attention as markers of disease activity but with controversial findings. Circulating microparticles and microRNAs have also been suggested. The majority of the studies in this field have been done with adults and the role of most candidate biomarkers in paediatric PAH is unknown. As in adults, the strongest evidence is on the use of BNP and NT-proBNP in monitoring disease severity in paediatric PAH. Biomarkers in children with PAH are likely to be impacted by additional factors, more so than in adult patients with PAH. These factors include degree of physical activity, age and/or developmental stage, gender differences, puberty and nutritional status. Although several promising markers have been identified over the past few years, the development of more specific markers, standardisation and prospective validation are warranted, also in paediatric PAH.

Natriuretic peptides and other plasma protein biomarkers

There is solid evidence in adult and paediatric patients with PAH that natriuretic peptides have prognostic value.20–22 In these studies, elevated natriuretic peptides correlated with poor haemodynamics and increased mortality. The natriuretic peptides atrial natriuretic peptide (ANP) and BNP are synthesised in the atrial and the ventricular myocardium. These peptides regulate natriuresis, diuresis and vasomotor tone. Atrial natriuretic peptide and BNP are elevated in association with pressure overload in order to reduce the workload of the heart and therefore have been found to be elevated in PAH. BNP is produced as a 108 amino acid prohormone, which is cleaved and secreted as a biological active sequence (BNP-32) and an inactive NT-proBNP peptide. BNP-32 has a short half-life of around 20 min and NT-proBNP a longer half-life of several hours, but both fragments are cleared by the kidney and influenced by renal function. A recent study elucidates that NT-proBNP levels correlated with haemodynamic parameters and outcome in PAH regardless of renal function.23 Large variability of normal NT-proBNP levels at different ages during childhood has to be acknowledged when applied in paediatric PAH.24

Cardiac troponin T has also been shown as a significant biomarker in PAH correlating independently with mortality.2 Evidence in paediatric population is lacking. Another study on children with CHD and PAH revealed elevated serum levels of soluble intracellular adhesion molecule 1.3 In addition growth differentiation factor-15, galectin-3, endothelins and C reactive protein are promising as biomarkers in adult PAH, at least with respect to inflammation. A large number of other inflammatory proteins have also been suggested as candidates for biomarkers in PAH. As in genetics, the need for a comprehensive approach targeting large number of PAH-specific analytes is useful to optimise use of protein biomarkers in everyday clinical practice.

Uric acid

Uric acid is the final degradation product of purines and an endogenous free radical scavenger that is elevated following tissue ischaemia and hypoxia. Raised levels have been described in chronic obstructive pulmonary disease,8 chronic heart failure9 and cyanotic CHD.10 This was underlined by reductions of uric acid levels in response to chronic vasodilator therapy. Potential confounding factors, however, include renal dysfunction and diuretic use and hence uric acid levels must be interpreted in the context of these factors. The role of uric acid in monitoring PAH severity and treatment responses may be useful although further studies are needed.11 ,25

Circulating endothelial cells

Circulating endothelial cells (CECs) have been established as non-invasive markers for vascular damage, remodeling and dysfunction. CECs have been measured in children with IPAH and PAH associated with CHD, before and after treatment.26 Rising CEC counts predicted clinical deterioration, suggesting that CECs may be an important tool to evaluate prognosis.26^, 27 Another study reported elevated levels of CECs associated with reversible APAH-CHD in children.27 These studies26^, 27 involved analysis of soluble biomarkers, and determined that markers of angiogenic cytokines, inflammation or endothelial microparticles were not able to predict PAH reversibility. These studies suggest that circulating CECs could be a potential biomarker in the assessment of paediatric PAH. On the other hand, some studies have failed to establish any correlation between CECs and PAH.6 ,28 The lack of standards for cell isolation and characterisation methods as well as differences in the pathological mechanisms of the investigated patients may contribute to the existing discrepancies.

Conclusions

Emerging evidence is accumulating supporting the use of genetic testing and blood biomarkers in paediatric PAH to individualise diagnostics,32 prognostics and therapy.33 In addition, genetic testing supports efficient risk stratification in family members. As the number of genes and biomarkers associated with PAH is accumulating, we need more comprehensive diagnostic approaches in both fields. In addition to new diagnostic technologies, advanced experimental approaches and patient registries including multicentre approaches are needed, to strengthen current evidence and develop future recommendations.