Basic Study
Copyright ©The Author(s) 2022. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Transl Med. Aug 6, 2022; 10(2): 14-28
Published online Aug 6, 2022. doi: 10.5528/wjtm.v10.i2.14
Cell-free mitochondrial DNA quantification in ischemic stroke patients for non-invasive and real-time monitoring of disease status
Nusrath Fathima, Sandhya Manorenj, Sandeep Kumar Vishwakarma, Aleem Ahmed Khan
Nusrath Fathima, Sandeep Kumar Vishwakarma, Aleem Ahmed Khan, Central Laboratory for Stem Cell Research and Translational Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Hyderabad 500058, Telangana, India
Sandhya Manorenj, Department of Neurology, Princess Esra Hospital, Deccan College of Medical Sciences, Hyderabad 500058, Telangana, India
Author contributions: Fathima N performed lab experiments, collected the data, performed the statistical analysis, and wrote and formatted the manuscript; Manorenj S provided samples and clinical inputs for the study; Khan AA and Vishwakarma SK provided inputs, designed the study, helped in data analysis and presentation, and wrote and edited the manuscript.
Institutional review board statement: All study procedures were carried out with the approval of the Institutional Review Board of Deccan College of Medical Sciences, Hyderabad, Telangana, India.
Institutional animal care and use committee statement: All animal experiments conformed to the internationally accepted principles for the care and use of laboratory animals.
Conflict-of-interest statement: All authors declare that they have no conflict of interest.
Data sharing statement: No additional data are available.
ARRIVE guidelines statement: All authors have read the ARRIVE guidelines, and the manuscript was prepared and revised according to the ARRIVE guidelines.
Open-Access: This article is an open-access article that was selected by an in-house editor and fully peer-reviewed by external reviewers. It is distributed in accordance with the Creative Commons Attribution NonCommercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited and the use is non-commercial. See: https://creativecommons.org/Licenses/by-nc/4.0/
Corresponding author: Aleem Ahmed Khan, PhD, Senior Scientist, Central Laboratory for Stem Cell Research and Translational Medicine, Centre for Liver Research and Diagnostics, Deccan College of Medical Sciences, Kanchanbagh, Hyderabad 500058, Telangana, India. aleem_a_khan@rediffmail.com
Received: April 2, 2022
Peer-review started: April 2, 2022
First decision: May 31, 2022
Revised: June 14, 2022
Accepted: July 16, 2022
Article in press: July 16, 2022
Published online: August 6, 2022
Processing time: 122 Days and 16 Hours
Abstract
BACKGROUND

Acute ischemic stroke (AIS) is one of the major causes of the continuous increasing rate of global mortality due to the lack of timely diagnosis, prognosis, and management. This study provides a primitive platform for non-invasive and cost-effective diagnosis and prognosis of patients with AIS using circulating cell-free mitochondrial DNA (cf-mtDNA) quantification and validation.

AIM

To evaluate the role of cf-mtDNA as s non-invasive, and affordable tool for real-time monitoring and prognosticating AIS patients at disease onset and during treatment.

METHODS

This study enrolled 88 participants including 44 patients with AIS and 44 healthy controls with almost similar mean age group at stroke onset, and at 24 h and 72 h of treatment. Peripheral blood samples were collected from each study participant and plasma was separated using centrifugation. The cf-mtDNA concentration was quantified using nanodrop reading and validated through real-time quantitative polymerase chain reaction (RT-qPCR) of NADH-ubiquinone oxidoreductase chain 1 (ND1) relative transcript expression levels.

RESULTS

Comparative analysis of cf-mtDNA concentration in patients at disease onset showed significantly increased levels compared to control individuals for both nanodrop reading, as well as ND1 relative expression levels (P < 0.0001). Intergroup analysis of cf-mtDNA concentration using nanodrop showed significantly reduced levels in patients at 72 h of treatment compared to onset (P < 0.01). However, RT-qPCR analysis showed a significant reduction at 24 h and 72 h of treatment compared to the disease onset (P < 0.001). The sensitivity and specificity were relatively higher for RT-qPCR than nanodrop-based cf-mtDNA quantification. Correlation analysis of both cf-mtDNA concentration as well as ND1 relative expression with National Institute of Health Stroke Scale score at baseline showed a positive trend.

CONCLUSION

In summary, quantitative estimation of highly pure cf-mtDNA provides a simple, highly sensitive and specific, non-invasive, and affordable approach for real-time monitoring and prognosticating AIS patients at onset and during treatment.

Keywords: Cell-free mitochondrial DNA; NADH-ubiquinone oxidoreductase chain 1; Ischemic stroke; Circulating biomarker; National Institute of Health Stroke Scale score; Stroke assessment; Severity and outcome

Core Tip: Several blood biomarkers have been studied to determine the severity and outcome of ischemic stroke with limited applications. Hence, we need to establish molecular markers which can provide more comprehensive information on the stroke pathophysiology and treatment response. Dynamic quantification of plasma cell-free DNA appears to be a valid and reliable option. Hence, we compared the real-time expression of cell-free mitochondrial DNA (NADH-ubiquinone oxidoreductase chain 1 gene) in ischemic stroke patients with healthy controls and studies its prognostic value during the treatment. This study could aid in the development of clinical values for assessing real-time, non-invasive mode of ischemic stroke status in the future.