Genetic Variations And Serum Biomarkers Of Persistent Pulmonary Hypertension Of The Newborn

Persistent pulmonary hypertension of the newborn (PPHN) is a clinical syndrome characterized by persistent elevation of pulmonary vascular resistance after birth with right to left shunting of blood across the ductus arteriosus and/or foramen ovale, resulting in hypoxemic respiratory failure.Recent studies of pulmonary arterial hypertension (PAH) in adults have identified genetic basis of the disease. Genes involved in the transforming growth factor-β(TGF-β) super family such as BMPR2, ALK1 have been implicated in familial/idiopathic and congenital heart disease associated pulmonary arterial hypertension. Other studies have identified genetic variants of CPS1、ABCA3、CRHR1 and epigenetic changes to be associated with PPHN.On the other hand, vasoactive substances including nitric oxide, endothelin-1, angiotensin-Ⅱ and vascular endothelial growth factor contribute to progressive changes in pulmonary vasoreactivity and related genes are NOS3, EDN1, ACE and VEGFA. Previous studies have shown that mutations or polymorphisms of these genes may affect the serum proteins’ concentrations.The etiology of PPHN is considered to be multiple including chronic intrauterine hypoxia, birth asphyxia, lung parenchymal diseases and pulmonary hypoplasia. Genetic basis have raised more and more concern. So, this study was designed to test the hypothesis that genetic variants of NOS3, EDN1, ACE and VEGFA may impact the risk of PPHN and to explore the possible relationship of gene variants and serum proteins’ concentrations.Part Ⅰ.Relationship of NOS3, EDN1,ACE, VEGFA single nucleotide polymorphisms and risk of PPHNObjectives:To evaluate the relationship of genetic variations of NOS3, EDN1, ACE, VEGFA and risk of PPHN among neonates with respiratory distress.Methods:Neonates≥34 weeks with respiratory distress at birth were recruited between 2013 and 2014. They were classified as PPHN or non-PPHN according to clinical manifestations and cardiac echocardiogram. Coding regions of EDNl, NOS3, VEGFA and ACE were screened by next-generation sequencing (NGS) on the Ion Torrent Personal Genome Machine platform.Results:A total of 83 neonates were enrolled (PPHN= 55; non-PPHN= 27).30 of them were performed NGS (PPHN= 20; non-PPHN= 10). No pathogenic mutations were identified but 49 single nucleotide polymorphisms (SNPs). Among them, the allele and genotype frequencies of 3 SNPs (rs5370、rs2070699、rs1800543) of EDN1 differ significantly between PPHN and non-PPHN subjects. After validation in all the 80 neonates, the result for rs2070699 remains significant (P=0.002). Higher frequency of rs2070699 T allele was observed in PPHN group.Conclusions:rs2070699 polymorphism of EDN1 may play a significant role in neonates’susceptibility to PPHN and carriers of T allele are at higher risk of PPHN when hypoxia happens.Part II. Analysis of Serum Levels of ET-1、VEGF、ACE、NO in PPHN, Non-PPHN and Normal NeonatesObjectives:To investigate serum levels of ET-1、VEGF、ACE、NO in PPHN, non-PPHN and normal neonates and to identify useful serum markers that involved in the pathogenesis of PPHN. Additionally, try to reveal association of EDN1 genotypes with serum ET-1 levels.Methods:Serum ET-1, VEGF, ACE levels were determined by ELISA. Nitric oxide metabolites (NOx) were determined by colorimetric method. Compare the concentrations in different groups and find the relationship with severity of PPHN. Compare serum ET-1 levels in neonates with different EDN1 genotypes.Results:ET-1 levels [(2.8±2.5)、(1.9±1.3)、(0.7±0.9)pg/mL] were significantly (p=0.00) higher while VEGF levels [(472.3±360.9)、(783.1±312.9)、(826.2±528.3) pg/mL] were significantly (p=0.02) lower in PPHN group than the other two groups. The difference in serum NOx and ACE concentrations between groups were not significantly. The EDN1 GG genotype associated with significantly (p=0.039) lower ET-1 levels when compared to GT/TT genotypes.Conclusions:There were significantly changes of serum ET-1 and VEGF in neonates with PPHN.T allele of EDN1 rs2070699 polymorphism may increase the risk of PPHN by affecting the synthesis of ET-1.