Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Sep 30;11(10):1158.
doi: 10.3390/genes11101158.

Customized Massive Parallel Sequencing Panel for Diagnosis of Pulmonary Arterial Hypertension

Jair Antonio Tenorio Castaño  1   2   3 , Ignacio Hernández-Gonzalez  4 , Natalia Gallego  1   2   3 , Carmen Pérez-Olivares  5 , Nuria Ochoa Parra  5 , Pedro Arias  1   2   3 , Elena Granda  1   2   3 , Gonzalo Gómez Acebo  1   2   3 , Mauro Lago-Docampo  6   7   8 , Julian Palomino-Doza  9   10 , Manuel López Meseguer  11 , María Jesús Del Cerro  12 , Spanish Pah Consortium  1   2   3   5 , Diana Valverde  6   7   8 , Pablo Lapunzina  1   2   3 , Pilar Escribano-Subías  5   9
Affiliations

Customized Massive Parallel Sequencing Panel for Diagnosis of Pulmonary Arterial Hypertension

Jair Antonio Tenorio Castaño et al. Genes (Basel). .

Abstract

Pulmonary arterial hypertension is a very infrequent disease, with a variable etiology and clinical expressivity, making sometimes the clinical diagnosis a challenge. Current classification based on clinical features does not reflect the underlying molecular profiling of these groups. The advance in massive parallel sequencing in PAH has allowed for the describing of several new causative and susceptibility genes related to PAH, improving overall patient diagnosis. In order to address the molecular diagnosis of patients with PAH we designed, validated, and routinely applied a custom panel including 21 genes. Three hundred patients from the National Spanish PAH Registry (REHAP) were included in the analysis. A custom script was developed to annotate and filter the variants. Variant classification was performed according to the ACMG guidelines. Pathogenic and likely pathogenic variants have been found in 15% of the patients with 12% of variants of unknown significance (VUS). We have found variants in patients with connective tissue disease (CTD) and congenital heart disease (CHD). In addition, in a small proportion of patients (1.75%), we observed a possible digenic mode of inheritance. These results stand out the importance of the genetic testing of patients with associated forms of PAH (i.e., CHD and CTD) additionally to the classical IPAH and HPAH forms. Molecular confirmation of the clinical presumptive diagnosis is required in cases with a high clinical overlapping to carry out proper management and follow up of the individuals with the disease.

Keywords: NGS; and genetics; digenic inheritance; massive parallel sequencing; pulmonary arterial hypertension.

PubMed Disclaimer

Conflict of interest statement

The authors declare that there is no conflict of interest regarding the publication of this article. IHG and NG contributed equally to this work.

Figures

Figure 1
Figure 1
Filtering pipeline for NGS panel HAP v1.2. A set of filters applied for variant prioritization. Only variants does not fulfil the criteria for variant quality were validated with Sanger sequencing [25].
Figure 2
Figure 2
Frequency of gene mutation detected after the analysis of 21 genes. (A) A total of 14% of pathogenic and likely pathogenic variants were identified, with a 15% of variants of unknown significance (VUS) and in approximately 65% of individuals no candidate gene variants were identified. After quality check, 6% of the analyzed samples were discarded due to low quality. (B) The gene with the highest pathogenic variants detected was BMPR2, followed by EIF2AK4, because many patients with PVOD were also included in the analysis.
Figure 3
Figure 3
Pedigree and in silico analysis of the families with more than one variant detected. (A) Genealogy of the families in which segregation analysis was performed (when available). (B) Table of in sillico analysis of the candidate variants, including the ACMG classification as well as the reference in which the variant was described. n/a: not applicable, P: pathogenic, VUS: Variant of unknown significance. α: In silico analysis was performed by dbNSFP counting the ones that predict a deleterious effect over the total available.
Figure 4
Figure 4
Variants detected in ABCC8. (A) Schematic representation of SUR1 (encoded by ABCC8) protein with its 17 transmembrane domains, and the variants detected in our cohort (red) and in Bohnen et al series (green). (B) Distribution of variants detected in ABCC8 in our cohort based on the type of variant.
Figure 5
Figure 5
Survival analysis between carriers’ vs. non-carriers. Kaplan-Meier analysis did not reflect statistical differences between individuals with PAH and pathogenic or likely pathogenic variants and individuals without any pathogenic, likely pathogenic variant detected.
See this image and copyright information in PMC

References

    1. Escribano-Subias P., Blanco I., Lopez-Meseguer M., Lopez-Guarch C.J., Roman A., Morales P., Castillo-Palma M.J., Segovia J., Gomez-Sanchez M.A., Barbera J.A., et al. Survival in pulmonary hypertension in Spain: Insights from the Spanish registry. Eur. Respir. J. 2012;40:596–603. doi: 10.1183/09031936.00101211. - DOI - PubMed
    1. Simonneau G., Montani D., Celermajer D.S., Denton C.P., Gatzoulis M.A., Krowka M., Williams P.G., Souza R. Haemodynamic definitions and updated clinical classification of pulmonary hypertension. Eur. Respir. J. 2019;53 doi: 10.1183/13993003.01913-2018. - DOI - PMC - PubMed
    1. Galie N., Humbert M., Vachiery J.L., Gibbs S., Lang I., Torbicki A., Simonneau G., Peacock A., Vonk Noordegraaf A., Beghetti M., et al. 2015 ESC/ERS guidelines for the diagnosis and treatment of pulmonary hypertension. Rev. Esp. Cardiol. 2016;69:177. doi: 10.1016/j.recesp.2016.01.002. - DOI - PubMed
    1. Thomson J.R., Machado R.D., Pauciulo M.W., Morgan N.V., Humbert M., Elliott G.C., Ward K., Yacoub M., Mikhail G., Rogers P., et al. Sporadic primary pulmonary hypertension is associated with germline mutations of the gene encoding BMPR-II, a receptor member of the TGF-beta family. J. Med. Genet. 2000;37:741–745. doi: 10.1136/jmg.37.10.741. - DOI - PMC - PubMed
    1. Navas P., Tenorio J., Quezada C.A., Barrios E., Gordo G., Arias P., Lopez Meseguer M., Santos-Lozano A., Palomino Doza J., Lapunzina P., et al. Molecular analysis of BMPR2, TBX4, and KCNK3 and genotype-phenotype correlations in Spanish patients and families with idiopathic and hereditary pulmonary arterial hypertension. Rev. Esp. Cardiol. 2016;69:1011–1019. doi: 10.1016/j.recesp.2016.03.031. - DOI - PubMed

Publication types

LinkOut - more resources