DDAH1 SNP rs480414 that protects against the development of pulmonary hypertension in bronchopulmonary dysplasia results in lower nitric oxide production in neonatal cord blood-derived lymphoblastoid cell lines

Avante D. Milton, Hanadi Almazrouea, Yi Jina, Gloria Zenderb, Jennifer K. Trittmanna
Abigail Wexner Research Institute at Nationwide Children’s Hospital. Ohio State University College of Medicine.
United States

Journal of Neonatal and Perinatal Medicine
J Neonatal Perinatal Med 2022; 15: 113-121
DOI: 10.3233/NPM-210710

Abstract
Background: Bronchopulmonary dysplasia (BPD) is chronic lung disease of prematurity and pulmonary hypertension (PH) is a major contributor to morbidity and mortality in BPD patients. Nitric oxide (NO) is a vasodilator and apoptotic mediator made by NO synthase (NOS). NOS is inhibited by asymmetric dimethylarginine (ADMA), and dimethylarginine dimethylaminohydrolase (DDAH) hydrolyzes ADMA. Previously, in a BPD patient cohort, we identified single nucleotide polymorphism (SNP) DDAH1 rs480414 (G > A) that was protective against developing PH. This study aims to determine functional consequences of the DDAH1 SNP in lymphoblastoid cell lines (LCLs) derived from neonatal cord blood. We tested the hypothesis that DDAH1 SNP (AA) results in DDAH1 gain of function, leading to greater NO-mediated apoptosis compared to DDAH1 wild-type (GG) in LCLs.
Methods: LCLs were analyzed by Western blot (DDAH1, cleaved and total caspase-3 and -8, and β-actin), and RT-PCR (DDAH1, iNOS). Cell media assayed for nitrites with chemiluminescence NO analyzer, and conversion of ADMA to L-citrulline was measured by spectrophotometry.
Results: LCLs with DDAH1 SNP had similar levels of DDAH1 protein and mRNA expression, as well as DDAH activity, compared to DDAH1 WT LCLs. There were also no changes in cleaved caspase-3 and -8 protein levels. LCLs with DDAH1 SNP had similar iNOS mRNA expression. Nitrite levels in media were lower for DDAH1 SNP LCLs compared to DDAH1 WT LCLs (p < 0.05).
Conclusion: Contrary to our hypothesis, we found that NO production was lower in DDAH1 SNP LCLs, indicative of a loss of function phenotype.

Category
Class III. Pulmonary Hypertension Associated with Lung Disease
Genetic Factors Associated with Pulmonary Vascular Disease

Age Focus: Pediatric Pulmonary Vascular Disease

Fresh or Filed Publication: Filed (PHiled). Greater than 1-2 years since publication

Article Access
Free PDF File or Full Text Article Available Through PubMed or DOI: Yes

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