Clinical Implications of the Genetic Background in Pediatric Pulmonary Arterial Hypertension: Data from the Spanish REHIPED Registry

Abstract

Background: Pulmonary arterial hypertension (PAH) is a severe and rare disease with an important genetic background. The influence of genetic testing in the clinical classification of pediatric PAH is not well known and genetics could influence management and prognosis.

Objectives: The aim of this work was to identify the molecular fingerprint of PH children in the REgistro de pacientes con HIpertensión Pulmonar PEDiátrica (REHIPED), and to investigate if genetics could have an impact in clinical reclassification and prognosis.

Methods: We included pediatric patients with a genetic analysis from REHIPED. From 2011 onward, successive genetic techniques have been carried out. Before genetic diagnosis, patients were classified according to their clinical and hemodynamic data in five groups. After genetic analysis, the patients were reclassified. The impact of genetics in survival free of lung transplantation was estimated by Kaplan-Meier curves.

Results: Ninety-eight patients were included for the analysis. Before the genetic diagnoses, there were idiopathic PAH forms in 53.1%, PAH associated with congenital heart disease in 30.6%, pulmonary veno-occlusive disease-PVOD-in 6.1%, familial PAH in 5.1%, and associated forms with multisystemic disorders-MSD-in 5.1% of the patients. Pathogenic or likely pathogenic variants were found in 44 patients (44.9%). After a genetic analysis, 28.6% of the cohort was "reclassified", with the groups of heritable PAH, heritable PVOD, TBX4, and MSD increasing up to 18.4%, 8.2%, 4.1%, and 12.2%, respectively. The MSD forms had the worst survival rates, followed by PVOD.

Conclusions: Genetic testing changed the clinical classification of a significant proportion of patients. This reclassification showed relevant prognostic implications.

Keywords: genetics; heritable pulmonary arterial hypertension; pediatric pulmonary hypertension; pulmonary veno-occlusive disease.

Figure 1

Workflow for variant prioritization. Schematic representation of the pipeline for tertiary/prioritization analysis of panel (A) and WES (B) samples. Custom NGS panels (HAPv1.2, NGS v2, and NGS v3) were designed with NimbleDesign (Roche, Indianapolis, IN, USA). Fragmentation and library preparation was performed with a SeqCap EZ Choice Enrichment Kit (Roche, Indianapolis, IN, USA), and sequencing was performed with the Illumina MiSeq platform (Illumina, San Diego, CA, USA). Genes included in HAPv1.2.: ACVRL1, GDF2, BMPR1B, BMPR2, CAV1, EIF2AK4, ENG, KCNA5, KCNK3, NOTCH3, SMAD1, SMAD4, SMAD5, SMAD9, TBX4, TOPBP1, SARS2, CPS1, ABCC8, CBLN2, and MMACHC; HAPv2: those included in HAPv1.2. and KLF2, NOTCH1, FOXF1, COX5A, CCDC80, HRG, VCAN, NRG1, CTCFL, APOH, MYSM1, FCER2, and CYP1A1; HAPv3: those included in HAPv2 and AQP1, ATP13A3 and SOX17. Library preparation for whole exome sequencing was carried out by Agilent SureSelect TM (v 6.0), and all exon kits were followed by sequencing in a NovaSeq Sequencer (Illumina, USA). The exomes were analyzed by VarSeq (Golden Helix, Bozeman, MT, USA) to detect both single nucleotide variants (SNVs) and copy number variants. A three-step prioritization algorithm was performed. † Only variants who have had rare allele frequency (≤0.01) in control population databases such as gnomAD exomes (v2.1.1), gnomAD genomes (v2.1.1), Kaviar (version 160204-Public), Bravo, were kept. ‡ BAM review was performed through Alamut and IGV; § Splicing analysis through SpliceSiteFinder-like, MaxEntScan, GeneSplicer, NNSPLICE.

Figure 2

Flow-chart of included patients and the definitive diagnosis after genetic testing. Patients in the REHIPED registry with genetic analysis and classified as a group 2 or group 3 pulmonary hypertension were excluded. FOXF1 (Forkhead Box F1 gene), GBE1 (1,4-Alpha-Glucan Branching Enzyme 1 gene), MECP2 (Methyl-CpG Binding Protein 2 gene), MMACHC (Methylmalonic aciduria and homocystinuria type C protein), MSD (multisystemic disorders), PAH (pulmonary arterial hypertension), PAH-CHD (pulmonary arterial hypertension associated with congenital heart disease), PH (pulmonary hypertension), PVOD (pulmonary veno-occlusive disease), TBX4 (T-Box Transcription Factor 4), VUS (variant of unknown significance), VHL (Von-Hippel-Lindau gene).

Figure 3

Sankey diagram demonstrating the reclassification of patients after genetic analysis. The left column shows the clinical classification prior to genetic analysis. The right column reflects the clinical classification after reclassification. PAH (pulmonary arterial hypertension); PAH-CHD (pulmonary arterial hypertension associated with congenital heart disease); PVOD (pulmonary veno-occlusive disease). The size of each section represents the number of subjects in each classification; for example, the number of subjects classified with IPAH was lower after reclassification according to genetic testing results.

Figure 4

Kaplan–Meier curves of free survival of death or lung transplantation comparing the different types of pediatric PAH after genetic testing (A) and comparing exclusively the specific genetic background (B). DLD (development lung disorders), PAH (pulmonary arterial hypertension); PAH-CHD (pulmonary arterial hypertension associated with congenital heart disease); PVOD (pulmonary veno-occlusive disease).