Structural and functional alterations of nitric oxide synthase 3 due to missense variants associate with high-altitude pulmonary edema through dynamic study

Abstract

The human endothelial nitric oxide synthase (NOS3) is 28 Kbp at 7q36.1 and encodes protein comprising of 1280 amino acids. Being a major source of nitric oxide, the enzyme is crucial to the vascular homeostasis and thereby to be an important pharmaceutical target. We hence have been investigating this molecule in a high-altitude disorder namely, high-altitude pulmonary edema (HAPE). We performed a genome-wide association study (GWAS) in a case-control design of sojourners that included healthy controls and HAPE patients (n = 200) each. Four NOS3 missense SNPs i.e. rs1799983 (E298D), rs3918232 (V827M), rs3918201 (R885M) and rs3918234 (Q982L), were associated significantly with HAPE (P-value < 0.05). Furthermore, extensive in silico analyses were performed to predict the detrimental effect of the four variant types and their three most relevant co-factors namely, heme, flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) that are accountable for amendment of protein stability leading to structural de-construction. Subsequently, we validated the findings in a larger sample size of the two study groups. HAPE patients had a higher frequency of the four variants and significantly decreased levels of circulating nitric oxide (NO) (P-value < 0.001). The in silico and human subjects findings complement each other. This study explored the impact of HAPE-associated NOS3 variants with its protein structure stability and holds promise to be current and future drug targets.

Communicated by Ramaswamy H. Sarma

Keywords:

• Hypobaric hypoxia
• high-altitude pulmonary edema
• GWAS
• NOS3
• missense SNPs
• MD simulations

Acknowledgements

We thank the Director of CSIR-Institute of Genomics and Integrative Biology, Delhi for the infrastructure. The cooperation of Dr. Neha Chanana and Ms. Tsering Palmo in reviewing the manuscript is appreciated.

Disclosure statement

No potential conflict of interest was reported by the authors.

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Additional information

Funding

This

work is majorly supported by CSIR, India (grant numbers MLP1401 and BSC0123) and in part by ICMR (fellowship to KH under grant No 5/4/1-6/14-NCD-II; and EMS to QP).

Notes on contributors

Hema Kanipakam

KH contributed to the extensive in silico investigations, writing and correcting the manuscript. KS contributed the wet-lab experiments and findings, the related write-up and corrections. TT and GM contributed to human samples and reviewed the manuscript. QP conceived, design and supervised the project, contributed to human sample collection, analyses, writing and correcting the manuscript.

Kavita Sharma

KH contributed to the extensive in silico investigations, writing and correcting the manuscript. KS contributed the wet-lab experiments and findings, the related write-up and corrections. TT and GM contributed to human samples and reviewed the manuscript. QP conceived, design and supervised the project, contributed to human sample collection, analyses, writing and correcting the manuscript.

Tashi Thinlas

KH contributed to the extensive in silico investigations, writing and correcting the manuscript. KS contributed the wet-lab experiments and findings, the related write-up and corrections. TT and GM contributed to human samples and reviewed the manuscript. QP conceived, design and supervised the project, contributed to human sample collection, analyses, writing and correcting the manuscript.

Ghulam Mohammad

KH contributed to the extensive in silico investigations, writing and correcting the manuscript. KS contributed the wet-lab experiments and findings, the related write-up and corrections. TT and GM contributed to human samples and reviewed the manuscript. QP conceived, design and supervised the project, contributed to human sample collection, analyses, writing and correcting the manuscript.

M. A. Qadar Pasha

KH contributed to the extensive in silico investigations, writing and correcting the manuscript. KS contributed the wet-lab experiments and findings, the related write-up and corrections. TT and GM contributed to human samples and reviewed the manuscript. QP conceived, design and supervised the project, contributed to human sample collection, analyses, writing and correcting the manuscript.