Genetic Variants Associated with Persistent Pulmonary Hypertension of Newborn: A Systematic Review

Authors: Srinivasan Mani, Seth I. Berger

Abstract

Introduction

Persistent pulmonary hypertension of newborn (PPHN) occurs due to the impairment in the expected fall in pulmonary vascular resistance during the fetal to neonatal circulatory transition, with a prevalence of 1.9 per 1,000 live births and a significant mortality rate of 4–33%. We aimed to systematically review the genetic variants associated with PPHN in term and late preterm infants without a known genetic syndrome.

Methods

In February 2025, the MEDLINE Ovid, Scopus, and Cochrane databases were searched for eligible studies without publication date restriction. Our review included cohort studies, case-control studies, and case series that examined the association of PPHN and genetic variants in term and late preterm infants. We extracted data regarding the methodology, participant characteristics, and outcome measures.

Results

We included nine studies (7 case-control studies and 2 cohort studies) that enrolled 1,494 participants. The risk of bias assessment using the Quality of Genetic Association Studies tool showed that 100% of the studies were of moderate or good quality. Our review found reports of positive associations between specific genetic variants in genes such as CPS1, CRHR1, NOTCH3, EDN1, EPAS1, WWC2, ABCA3, RFX3, EP300, GNA11, PKLR, SLC2A1, BMPR2, and EGLN1. One study reported no association between an *ACE *gene variant and PPHN.

Discussion

Studies of common genetic variants associated with an increased risk of PPHN in term and late preterm infants are limited, based on small cohorts and frequently focused on small sets of candidate genes, yielding inconsistent results across studies.

Introduction

Persistent pulmonary hypertension of newborn (PPHN) occurs due to the impairment in the expected fall in pulmonary vascular resistance during the fetal to neonatal circulatory transition at birth [1]. The resulting elevation in the pulmonary artery and the right heart pressures leads to intracardiac, extracardiac, and intrapulmonary right-to-left shunts, causing hypoxemia, systemic hypoperfusion, and metabolic acidosis. PPHN has a prevalence of 1.9 per 1,000 live births, with a significant mortality rate of 4–33% [2]. The leading causes of PPHN are infection, meconium aspiration syndrome, idiopathic respiratory distress syndrome, and congenital diaphragmatic hernia [3]. The significance of the nearly 42% genetic contribution to pediatric-onset pulmonary artery hypertension (PAH) has been recognized recently [4]. Pediatric-onset PAH is associated with both inherited and de novo genetic variants in known PAH risk genes such as BMPR2, ABCC8, ACVRL1, ATP13A3, BMPR1B, CAV1, EIF2AK4, ENG, GDF2, KCNA5, KCNK3, KDR, NOTCH1, SMAD1, SMAD4, SMAD9, SOX17, and TBX4. Infants with specific genetic syndromes, such as Down syndrome and small patella syndrome, have a higher predilection for pulmonary hypertension.

The association of PPHN and variants in genes related to the urea cycle, nitric oxide pathway, glucocorticoids, endothelin system, transforming growth factor beta superfamily, and potassium channel family has been reported [5–8]. A genetic study using exome sequencing in the high-altitude Tibetan population identified 166 PPHN-associated genetic variants with significant involvement of hypoxia-related genes [9]. The association of PPHN and variants involving hypoxia-related genes was validated in the low-altitude dwelling Han population, confirming the relationship independent of residence at high altitudes. Chen et al. [10] found pathogenic and likely pathogenic rare variants associated with PPHN in genes such as FOXF1, KMT2D, NKX2-1, CHD7, and CACNA1E. Galambos et al. [11] studied the rare variants of the *TBX4 *gene in neonates with PPHN and found specific copy number and rare variants. The genes that harbor rare variants associated with syndromes presenting with PPHN are relatively better characterized. In contrast, the role of common genetic variants associated with PPHN is unclear. The methodologies used to identify genetic risk factors vary across studies, including genome-wide association studies using microarrays, exome sequencing, targeted gene panels, and genome sequencing [12]. The relative significance of the known genetic variants associated with PPHN and their generalizability across populations is unclear. We aimed to systematically review the genetic variants associated with PPHN and identify their relative contribution to the susceptibility and severity of the disease in term and late preterm infants without a known genetic syndrome.

Methods

This systematic review was conducted based on the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) reporting guidelines [13]. A review protocol was developed that included the objectives, eligibility criteria, information sources, and search strategy. The protocol was registered in the International Prospective Register of Systematic Reviews (PROSPERO) (ID: CRD420251009500). The protocol can be accessed at https://www.crd.york.ac.uk/PROSPERO/view/CRD420251009500.

Eligibility Criteria

The review included cohort studies, case-control studies, and case series examining the association of PPHN and genetic variants in preterm infants. The following PECO framework was adapted.

Population: we included infants born at gestational age ≥34 weeks admitted to the NICU. We excluded all patients with known genetic syndromes, congenital abnormalities, congenital heart disease, or who were not undergoing active treatment.

Exposure/risk the presence of specific genetic variants known to be associated with PPHN.

Comparison: the absence of specific genetic variants known to be associated with PPHN.

Outcome: diagnosis of PPHN confirmed by echocardiography.

Information Sources

In February 2025, a Boolean search strategy without publication date restriction was developed to find eligible studies. The following electronic databases were MEDLINE Ovid (1946 to February 2025), Scopus (1974 to February 2025), PubMed (1966 to February 2025), and Cochrane (data inception to February 2025). The reference lists of related articles were searched for additional eligible studies.

Search Strategy

The search strategy for PubMed is included in online supplementary Material 1 (for all online suppl. material, see https://doi.org/10.1159/000550289). This strategy was adapted to suit MEDLINE Ovid, Scopus, and Cochrane.

Selection Process

One reviewer (S.M.) reviewed the abstracts and included the studies based on the inclusion criteria following standard methods. The second reviewer (S.B.) verified the eligibility of the studies included in the review before data extraction. The Covidence, a web-based collaboration software platform, was used to screen titles and abstracts, select articles for full review, and organize the systematic review [14].

Data Extraction

One reviewer (S.M.) assessed the methodological quality of each study and extracted data from the eligible studies. The senior reviewer (S.B.) independently verified the accuracy of the data extracted from the selected studies. A list of all the extracted data items is included in online supplementary Material 2.

Risk of Bias (Quality) Assessment

The quality of the genetic studies (Q-Genie) tool was used to assess the risk of biases among included studies [15]. Two reviewers (S.M. and S.B.) conducted quality assessments separately. The studies were assessed for quality based on eleven criteria, each with a minimum score of 1 and a maximum score of 7. The studies with control groups with scores ≤35 were considered as poor quality, >35 and ≤45 as moderate quality, and >45 as good quality. For studies without control groups, the Q-Genie scores ≤32 were considered poor quality; >32 and ≤40 as moderate quality; and >40 as good quality.

Results

Study Selection

A comprehensive literature search identified 2,295 references across three databases (Scopus, MEDLINE, CENTRAL). Five hundred sixty-five duplicate references were removed, 1,730 studies were screened, and 1,662 studies were excluded based on the inclusion criteria. The full texts of 68 studies were retrieved and thoroughly assessed for inclusion. From full-text analysis, 59 studies were excluded, and nine studies were included for review [5–9, 16–19]. The PRISMA flowchart detailing the study selection is shown in Figure 1. The studies excluded from the review, along with the reasons for exclusion, are included in online supplementary Table 1 (for all online suppl. material, see https://doi.org/10.1159/000550289).

Study Characteristics

Nine studies included in the review enrolled 1,494 participants, of whom 585 had PPHN confirmed by echocardiography. We included seven case-control studies and two cohort studies. Four included studies (44%) were conducted in the USA, three in China, and two in Thailand. Fifty-six percent of the studies (5/9) recruited infants after 2013, and 67% of the studies were published after 2015. The average gestational age of the infants included across the eleven studies was 38.4 weeks ± 2 weeks. The average birth weight of the infants across the included studies was 3,235 g ± 603 g. On average, 62% of the infants included in the selected studies were males. The characteristics of the individual studies are presented in Table 1.

Risk of Bias in Studies

The risk of bias in the included studies was assessed using the Quality of Genetic Association Studies (Q-Genie) tool. This tool included 11 assessment items, (1) rationale for the study, (2) selection and definition of the outcome of interest, (3) selection and comparability of the comparison group, (4) technical classification of the exposure, (5) nontechnical classification of the exposure, (6) other sources of bias, (7) sample size and power, (8) a priori planning of analyses, (9) statistical methods and control for confounding, (10) testing of assumptions and inferences for genetic analyses, and (11) appropriateness of inferences drawn from results. The risk of bias assessment revealed that 67% of studies were of moderate quality, while 33% were of good quality. The Q-Genie scores of the individual studies are shown in online supplementary Table 2.

Results of Individual Studies

Byers et al. [6] studied 32 genetic variants in 12 candidate genes. They found that genetic variants in *CRHBP *(corticotropin-releasing hormone-binding protein) and CRHR1 (corticotropin-releasing hormone receptor I) were associated with PPHN. After Bonferroni correction, the CRHR1 rs4458044 variant was identified as a significant variant associated with PPHN. De Jesus et al. [16] studied the ACE gene variant of an insertion (I) or a deletion (D) of a 287-bp within intron 16 on chromosome 17. He found that the DD genotype was not associated with an increased risk of PPHN or its severity. Kaluarachchi et al. [17] studied the genetic variants in urea cycle enzymes and showed a significant association between rs41272673, rs4399666, and rs2287599 in the *CPS1 *(carbamoyl phosphate synthase 1) gene and PPHN. Liu et al. [8] analyzed the genetic variants in known PPHN-associated genes and reported that rs192759073, rs1047883, and rs2229589 in CPS1, as well as rs1044008 in NOTCH3, are markers for PPHN risk. Mei et al. [7] observed a significant association between the EDN1 gene variant rs2070699 and PPHN.

Nakwan et al. [18] studied 285 variants in candidate genes and found that the rs17034984 variant in the EPAS1 (endothelial PAS domain protein 1) gene was significantly associated with PPHN [9]. The same author analyzed 659,184 genetic variants and found 14 variants associated with PPHN [10]. They are *WWC2 *(WW and C2 domain containing 2) rs149768622, rs78232913 in uncharacterized genes LOC102723906, LOC105377599, rs147741304 in the intergenic region between the *KRT82 *and KRT75 genes, *CADM4 *(cell adhesion molecule 4) rs112876885, rs56956847 in the intergenic region between *GRM2 *and *RGS7 *genes, GPM6A (glycoprotein M6A) rs191330351, *CIT *(citron rho-interacting serine/threonine kinase); *MIR1178 *(microRNA 1178) rs56074206, *RIMBP2 *(RIMS binding protein 2) rs7134357, LOC105374510 rs145547469, LOC105375193 rs10239513, *PTPRN2 *(protein tyrosine phosphatase receptor type N2) rs11764121, CDK14 (cyclin dependent kinase 14) rs144344757, LCORL (ligand-dependent nuclear receptor corepressor-like) rs187729743, rs79741635 in the intergenic region that lies between the CLDN12 and *CKD14 *genes. Pearson et al. [5] observed that the variant T1405N in the *CPS *gene had a skewed distribution in their study population [1]. Wang et al. [9] found 166 PPHN-related variants, of which 49% were related to 43 specific hypoxia-related genes. In their two-stage study, the authors used a validation cohort comprising of Han population living in low altitudes and showed that 413 gene variants found in 127 hypoxia-related genes were associated with PPHN [8]. The full list of genes and the genetic variants reported by Wang et al. [9] is included in the online supplementary Table 3.

Results Synthesis

The included studies employed various methodologies, including targeted evaluations of single variants (2 studies), a limited panel of variants from 1 to 50 genes (3 studies), genome-wide polymorphism evaluation using microarrays (2 studies), and gene sequencing with exome (2 studies). Sample sizes ranged from 65 in a study evaluating a single variant to 387 in a genome-wide association study. While no two studies reported the same associated variants, study populations varied across studies, including those in predominantly white populations, Chinese populations, and Thai populations. Genetic variants in the carbamoyl phosphate synthase 1 gene were observed to be associated with PPHN in three of the nine included studies. However, the three studies reported different variants in their respective studies. One of these studies tested only a single variant, while another evaluated 48 different variants across six urea cycle genes. In two studies, genetic variants related to hypoxia response genes and vasoconstriction/vascular smooth muscle were found to be associated with PPHN. Among the included studies, only one reported a lack of association between PPHN and the genetic variant studied, leaving 89% of studies to report a positive association between specific genetic variants and PPHN in their respective study populations. The significant genetic variants associated with PPHN identified in the reviewed studies and their plausible link to PPHN are listed in Table 2. A gene network created using STRING v12.0 is shown in Figure 2 highlighting the complex interactions between the 15 genes identified through this review [20]. The intended quantitative synthesis of the pooled data could not be undertaken because of the differences in the variants and genes reported in the included studies. The data required for the preplanned subgroup analysis were not reported uniformly across the reported studies.

Discussion

Our systematic review, which included 1,494 newborn infants from nine studies, investigated and synthesized the evidence relating to the genetic contribution of PPHN in term and late preterm infants without a known congenital heart disease or genetic syndrome. We specifically excluded studies investigating the genetic association of PPHN in infants with known alveolar capillary dysplasia with misalignment of pulmonary veins (ACDMPV) and congenital diaphragmatic hernia (online suppl. Table 1). We excluded reports of individual cases of genetic variants for monogenic syndromes associated with PPHN to reduce bias in the evidence synthesis. Our review identified reports of positive associations between specific genetic variants in genes such as CPS1, CRHR1, NOTCH3, EDN1, EPAS1 (HIF-2alpha), ABCA3, RFX3, EP300, GNA11, PKLR, SLC2A1, BMPR2, EGLN1, and WWC2. In the following section, we will briefly describe the biological role of genes identified in our review and their plausible relationship with PPHN. We also describe the genetic variants that showed significant association with PPHN by using the information available from the National Center for Biotechnology Information databases, such as Gene, SNP, and ClinVar [21].

*CPS1 *genetic variants are the most frequently reported association. Carbamoyl phosphate synthase 1 is a rate-limiting enzyme in the urea cycle. Arginine is a critical intermediate in the urea cycle, which is involved in the production of nitric oxide, which plays a central role in reducing pulmonary vascular resistance during the circulatory transition in the immediate post-natal life [22, 23]. The variants in the CPS1 gene reported to be associated with PPHN based on our review are predominantly common variants except c.195C>T and c.1708-61G>A. They are classified as benign intronic, synonymous, or missense variants. The clinical significance of these variants is unknown and has not been reported in ClinVar [24].

Corticotropin-releasing hormone receptor 1 is a transmembrane receptor belonging to the secretin family of G-protein-coupled receptors in the pituitary [6, 25]. It interacts with corticotropin to regulate the secretion of ACTH by the anterior pituitary. ACTH, in turn, regulates cortisol production, a key effector hormone involved in the hypothalamic-pituitary-adrenal axis. The hypothalamic-pituitary-adrenal axis plays a vital role in fetal lung development, maturation, and the circulatory transition at birth [26]. The variant c.34-10649G>C at 45796361 (GRCh38.p14) is classified as a benign intronic variant. This is a common variant with a minor allele frequency of 0.2118.

Endothelin 1 (EDN1) gene codes for endothelin, a vasoconstrictor. The endothelin receptors are drug targets for the treatment of PAH. The rs2070699 associated with PPHN corresponds to c.233+30G>T at 12292539 (GRCh38). This is classified as a benign germline intron variant with G as the major allele and T as the minor allele, with a global frequency of 0.35623. The endothelial PAS domain protein 1(EPAS1) gene encodes a protein by the same name. This transcription factor induces the oxygen-regulated genes and regulates the expression of vascular endothelial growth factor. The rs17034984 variant corresponds to c.27-20925 C>T at 46325948 (GRCh38.p14). The clinical significance of this intron variant is not reported in ClinVar because it is considered benign based on population frequency.

The *NOTCH3 *gene on chromosome 19 encodes the Notch receptor 3, which is involved in the Notch cellular signaling pathway. The receptor function plays a vital role in the function and survival of smooth muscle cells in blood vessels. The NOTCH3 gene has been implicated in diseases such as cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) and lateral meningocele syndrome [27]. The tubulin tyrosine ligase-like 3 (TTLL3) gene codes for a protein that plays a key role in axoneme assembly, microtubule cytoskeleton, and ciliary function [28]. The WW and C2 domain containing 2 (WWC2) gene encodes a protein that regulates the Hippo signaling pathway [29]. This gene has been implicated in long QT syndrome.

The ATP-binding cassette subfamily A member 3 (ABCA3) gene, located on chromosome 16, encodes a transporter protein involved in surfactant synthesis. Dysfunction of this gene results in the respiratory distress syndrome phenotype, characterized by surfactant deficiency and severe hypoxia. The hypoxia-related pulmonary vasoconstriction could be a mechanistic pathway linking this gene to PPHN. Genes such as the EP300 lysine acetyltransferase (EP300) and the egl-9 family hypoxia inducible factor 1 (EGLN1) are involved in hypoxia-inducible factor pathways. The variants reported in these genes could affect an infant’s normal response to hypoxia, leading to abnormal pulmonary vascular tone that evolves into PPHN. The G-protein subunit alpha 11 (GNA11) gene is involved in G-protein-coupled receptor signaling in the remodeling of the pulmonary vasculature. The bone morphogenetic protein receptor type 2 (BMPR2) gene encodes a receptor in the bone morphogenetic pathway, involved in lung embryogenesis, and plays an active role in pulmonary vascular remodeling.

Welch and Chung [4] reviewed the genetics and genomics of pediatric pulmonary arterial hypertension (PAH) broadly and reported differences between pediatric and adult PAH in the genes involved in etiopathogenesis and in the genetic contribution to the overall burden of this disease. The review found that developmental genes such as *TBX4 *and *SOX17 *are involved, with significant contributions from de novo variants. The genetic factors associated with PPHN, in particular, have not been systematically reviewed before this study. Our review specifically aimed to identify the role of genetic underpinnings in PPHN cases without a known monogenic syndrome or copy number variants. The discussion on genetic syndromes with a high risk of pulmonary hypertension, such as Down, CHARGE, VACTERL, DiGeorge, Scimitar, and Noonan syndromes, falls beyond the scope of our review and is found elsewhere [30]. In summary, the genetic variants associated with PPHN, as discussed in this review, were related to genes involved in the urea cycle, the hypothalamic-pituitary axis, the oxygen-sensing mechanism, and regulatory genes that control gene expression during lung development and pulmonary vascular remodeling. Most of the reviewed studies reported common variants, single-nucleotide variants with a minor allele frequency greater than 0.05, that were classified as benign or likely benign [31].

PPHN is a rare disorder (1.9/1,000 births) affecting newborn infants [2]. So, the studies we reviewed had smaller sample sizes than the expected sample sizes required for well-powered genome-wide association studies to detect small effects [32]. This is a notable limitation of the evidence synthesized in our review. All the included studies were single institutional in nature. The racial and ethnic diversity within each study was minimal, limiting the generalization of the results to other populations. The genetic methodology used to study the genetic variability differed across the studies. Only 22% of our included studies performed exome sequencing and 22% performed genome-wide polymorphism analysis, whereas the remainder performed candidate gene analysis. The candidate gene panel differed between the studies in terms of the genes of interest. The reporting of the variants was not uniform. In 2015, the American College of Medical Genetics and Genomics (ACMG) in collaboration with the Association for Molecular Pathologists (AMP) proposed guidelines for standardizing the classification and terminology of genetic variants associated with Mendelian disorders [33]. The term “variant” was recommended to replace gene mutation and polymorphism and was divided into a 5-tier pathogenic, likely pathogenic, uncertain significance, likely benign, and benign with 28 specific criteria (16 pathogenic and 12 benign) for classification. This classification typically applies to rare and high-impact variants and is less often applied to association studies where the marker variant has a smaller impact or may be in linkage disequilibrium with a causative variant. Thus, only a few recently published studies in our review used this classification system while reporting their findings, and the reported variants would typically be classified as “benign” due to being common population polymorphisms. Although the ACMG variant classification system is widely adopted, it aims to classify gene variants as causal or not. This approach limits its practical application for gene variants in the context of linkage disequilibrium, polygenic risk, and disease predisposition [34].

Understanding the limitations of the current literature in finding the genetic factors associated with PPHN gives us opportunities to design better studies with an adequate sample size. National and international research collaborations are required to have a diverse population of neonates with PPHN. Exome or genome sequencing should be preferred over the candidate gene approach, with select genetic variants [35]. These large datasets generated by broad genomic research could provide evidence for the development of precise Next-generation sequencing based gene panels for clinical decision-making, thereby enhancing diagnosis, classification, and management options for PPHN. Future studies should report the severity of PPHN based on objectively defined echocardiographic criteria, therapy modalities, and treatment response, especially to inhaled nitric oxide (iNO). Adding other “omics” technologies such as transcriptomics, epigenomics, and metabolomics could be valuable for understanding the impact of the genetic variants on the disease process and the response to conventional therapy. Alternatively, reverse phenotyping or a genotype-first research approach could be used, leveraging large genomic databases, to identify clinical phenotypes with severe disease presentation, suboptimal response to iNO, and a need for extracorporeal membrane oxygenation.

Conclusion

Common genetic variants in specific genes, such as CPS1, CRHR1, EDN1, and EPAS1, could be associated with an increased risk of PPHN in term and late preterm infants. However, the current literature is underpowered to assess the generalizability of these associations definitively. Future research in this field needs robust study designs with a large, diverse population to understand the genetic risk factors of PPHN. Identifying specific genetic variants associated with an increased risk of PPHN could help us develop novel biomarkers and drug targets for infants with PPHN, especially for those with suboptimal response to conventional treatment.

Acknowledgment

We thank Abby L. Adamczyk, MLIS, AHIP, Graduate Medical Education Librarian at Scott Memorial Library, Thomas Jefferson University, Philadelphia, for her guidance in formulating the search strategy.

Statement of Ethics

This is a systematic review of published data, so ethics approval and consent to participate are not applicable.

Conflict of Interest Statement

The authors have no conflicts of interest to declare.

Funding Sources

This study was not supported by any sponsor or funder.

Author Contributions

S.M. conceptualized the study, designed the study, acquired the data, interpreted the data, drafted the initial manuscript, and revised the manuscript. S.I.B. designed the study, interpreted the data, reviewed, and helped revise the manuscript. All the authors approved the final manuscript as submitted and agreed to be accountable for all aspects of the work.