Identification of genetic factors underlying persistent pulmonary hypertension of newborns in a cohort of Chinese neonates

Persistent pulmonary hypertension of the newborn (PPHN) is caused by a failure in the normal circulatory transition at birth and is characterized by elevated pulmonary vascular resistance (PVR), which leads to right-to-left shunting and hypoxemia. The incidence of PPHN ranges from 2 to 6 per 1000 live births and the mortality rate is 10–20% [1]. PPHN can be idiopathic or may be caused by multiple pulmonary diseases including perinatal asphyxia, meconium aspiration syndrome (MAS), respiratory distress syndrome (RDS), pulmonary dysplasia and congenital diaphragmatic hernia [2].

To date, only a few genetic polymorphisms have been identified in infants with PPHN. Pearson et al. first found that a T1405 N variant of carbamoyl phosphate synthetase I (CPS1) exhibited a different distribution between infants with PPHN and the general population [3]. A homozygous missense variant (L326R) in the ABCA3 gene was identified in a newborn with severe hypoxemic respiratory failure and refractory pulmonary hypertension [4]. Single nucleotide polymorphisms (SNPs) in the corticotropin-releasing hormone receptor 1 (CRHR1) and corticotropin-releasing hormone-binding protein (CRHBP) genes were significantly associated with PPHN [5]. Most recently, rs2070699 in endothelin 1 (EDN1) was found to increase the risk of PPHN with respiratory distress [6].

PPHN is a subgroup of pulmonary arterial hypertension (PAH), which is a complex disorder characterized by elevation of PVR and failure of right heart function. It is a common complication of many clinical diseases. PAH is classified into five types according to the pathogenesis of the disease (WHO classification) and PPHN is a specific group [7]. Pathogenic variants among several genes have been reported in PAH patients exhibiting both adulthood onset and childhood onset. Bone morphogenic protein receptor type 2 (BMPR2), a member of the transforming growth factor beta (TGF-β) superfamily, is associated with 70% of familial pulmonary arterial hypertension (FPAH)/heritable pulmonary arterial hypertension (HPAH) cases and 20% of idiopathic pulmonary hypertension (IPAH) cases [8]. SMAD9 [9], CAV1 [10], KCNK3 [11] are also known PAH genes listed in the Online Mendelian Inheritance in Man (OMIM) database. Hereditary hemorrhagic telangiectasia (HHT) gene variants in the activin receptor-like kinase 1 (ACVRL1) [12] and endoglin (ENG) [13] have been identified in PAH patients. To date, no causal genes for PPHN have been reported, and the genetic etiology remains unclear. We suggest that the genetic pathogenesis of PPHN may share some similarities with PAH in adults and children.

Therefore, in the present study, we applied clinical exome sequencing to investigate the genetic etiology of PPHN in 115 Chinese patients. We aimed to identify causal variants in reported PPHN/PAH-related genes and genetic risk polymorphisms for PPHN patients.

PPHN is a severe clinical problem and accounts for ~ 6% of our NICU patients. The role of genetics in the pathogenesis of PPHN remains elusive. The present study investigated the genetic contributions to the pathogenesis of PPHN in 115 Chinese PPHN patients using exome sequencing. Among all cases, 41 were preterm infants and 74 were late preterm and term infants. We identified three patients with P/LP variants in TBX4 and BMPR2 and six patients with VUSs in BMPR2 and 4 other reported PAH-related genes. CPS1, NOTCH3 and SMAD9 were identified as important risk genes for late preterm and term PPHN through case-control analysis.

PPHN has generally been recognized to occur among late preterm and term infants, but studies have reported an increasing rate of PPHN in preterm infants [25]. In this study, most of the infants with PPHN were late preterm and term infants (≥ 34 weeks gestation), and preterm infants also accounted for 35.7% of the patients. Among the 9 patients with genetic findings, only 1 patient with c.596A > T (p.D199V) in BMPR2 was born before 34 weeks gestation (32 + 3 weeks). The genetic diagnosis rates were different in the two groups (8/74 in the ≥34-week gestation group vs 1/41 in the < 34-week gestation group). Our findings indicated that preterm complications play major roles in preterm infants with PPHN, while genetic factors have a greater effect on late preterm and term infants.

In terms of the genetic background of PPHN, previous studies have not found the disease-causing gene for PPHN patients thus far, and only polymorphisms in 5 genes have been reported to be associated with PPHN. PAH has been widely studied in both adults and children, and 20 genes have been associated with the development of the disease (Additional file 1: Table S1). The genetic etiology of PPHN in newborns is complex and unclear and may share some similarities with PAH in adults and children. In this study, we identified several variants in PAH-related genes, which verified that PAH and PPHN potentially exhibit a common genetic pathogenesis. We also found three disease-causing genes in the three other patients. However, these genes were not associated with the development of pulmonary hypertension. Further studies are needed to investigate other potential disease-causing genes related to PPHN.

Among the genes identified in this study, several genetic variants in the BMP/TGF–β/SMAD pathways were identified, including three P/LP variants in TBX4 and BMPR2 and one VUS in TGFB1 related to severe clinical phenotypes in four patients. BMP/TGF–β/SMAD signaling (especially BMPR2) has been reported to be involved in the regulation of the proliferation and apoptosis of pulmonary arterial smooth muscle cells (PASMCs) and pulmonary arterial endothelial cells (PAECs) [26]. In a previous study of PAH, BMPR2 variants were more commonly found in females than males (3.6:1 ratio in adult-onset PAH cases and 1.7:1 ratio in pediatric-onset PAH cases) [27]. The sex ratio was similar (3:1, female:male ratio) in our study among the 4 BMPR2 variant-carrying PPHN patients. TBX4 is a member of the T-box genes that is important for the development of airway branching and the regulation of lung fibrosis. TBX4 variants have been reported in childhood-onset PAH patients [28] and might contribute to PAH by decreasing the activation of the BMP/TGF–β/SMAD signaling pathways [29]. TGFB1 (transforming growth factor β1) is a member of the TGF-β superfamily, whose members are important modulators of cell growth, inflammation and apoptosis. TGFB1 can suppress the proliferation and migration of endothelial and smooth muscle cells and thereby inhibit vascular remodeling. Variants in TGFB1 might affect its function and lead to pulmonary hypertension [30]. Both the TGF-β and BMP signaling pathways ultimately converge on SMADs. One rare SMAD9 variant, A196V, located in the linker domain of the protein was identified in one patient (Fig. 2b). The linker region of SMAD9 is rendered shorter than those of other SMADs, which suppresses its transcriptional activity and ability to activate BMP signaling, while facilitating interaction with other molecules [31]. In addition, two ion channel genes, which might also play important roles in PAH, were identified in our patients. The Kv1.5 channel gene (KCNA5) is a pore-forming α-subunit that forms a voltage-gated K⁺ channel in PASMCs. Downregulation of KCNA5 causes membrane depolarization and increases the cytosolic Ca²⁺ concentration, resulting in pulmonary vasoconstriction, and pulmonary vascular remodeling [32]. A novel D549Y variant in KCNA5 was identified in one girl. The residue is located in the C-linker region following transmembrane domain segment 6. Another novel variant, F443I in TRPC6, was identified in another patient. TRPC6 is an important member of the TRPC channels of the transient receptor potential (TRP) superfamily expressed in the lungs and PASMCs [33]. A SNP in the promoter region of TRPC6 has been demonstrated to increase the risk of IPAH by recruiting NF-κB [34].

Furthermore, we performed SNP association and gene-level analyses in 25 PPHN/PAH-disease related genes among 74 late preterm and term PPHN cases and 115 controls with matched clinical characteristics to further investigate the genetic etiology of PPHN. We identified 3 SNPs in CPS1 and 1 SNP in NOTCH3 associated with PPHN. The CPS1 SNPs rs192759073 and rs2229589 are synonymous variants, and rs1047883 is a missense variant. The heterozygous rs192759073 T allele was identified in 3 female PPHN patients and none of the controls. For rs1047883 and rs2229589, homozygous SNPs were found in 19 PPHN cases and 20 controls and heterozygous SNPs were found in 41 PPHN cases and 58 controls. The synonymous SNP rs1044008 in NOTCH3 was detected in three PPHN patients (heterozygous). These SNPs are reported to be associated with PPHN for the first time in this study. CPS1 (carbamoyl phosphate synthase 1) encodes one of the key enzymes located in mitochondria involved in the urea cycle. A functional deficiency in the CPS1 enzyme can affect the catalysis of the first step of the urea cycle and the generation of nitric oxide, which plays a critical role in regulating pulmonary vascular resistance [35]. Sixteen polymorphisms, including three in coding regions (rs1047891, rs2287599 and rs41272667) of CPS1 [3, 36], have been reported to be associated with PPHN. However, rs1047891 and rs2287599 were not significant in our cohort, and rs41272667 (close to the noncoding region) was not included in our study. The reason for this difference may be that the genetic risk factors for PPHN differ in different ethnic populations. NOTCH3 belongs to the Notch signaling pathway, which plays an important role in the regulation of cellular proliferation, differentiation and apoptosis. Heterozygous variants in NOTCH3 might affect cell proliferation and NOTCH3-HES5 signaling resulting in PAH [37]. Gene-level analysis also identified CPS1 and SMAD9 as genetic risk factors for PPHN.

There are several limitations to our study. We used clinical exome sequencing (with 16 PPHN/PAH disease-related genes included in the panel) for genetic testing, and the other 9 genes need to be further studied. Genetic risk polymorphisms are usually identified in noncoding regions, which cannot be detected using exome sequencing panels. However, exome sequencing provides more information for variants spread throughout genes than candidate SNP genotyping has provided in previous studies. Additionally, we could not study the association between nitric oxide metabolites and PPHN since the plasma concentrations of nitric oxide metabolites were not measured/recorded for all patients.

In summary, this study identified 9 rare variants in PAH-related genes (TBX4, BMPR2, SMAD9, TGFB1, KCNA5 and TRPC6) in 9 PPHN patients in a Chinese cohort. We also revealed an association between the CPS1, NOTCH3 and SMAD9 genes and late preterm and term PPHN infants. Our findings have important clinical implications for the earlier detection of PPHN in preterm infants and provide more insight into the pathogenesis of PPHN and potential strategies for disease treatment.