The mitochondrion is a multifunctional organelle that modulates multiple systems critical for homeostasis during pathophysiological stress. Variation in mitochondrial DNA (mtDNA) copy number (mtDNAcn), a key mitochondrial change associated with chronic stress, is an emerging biomarker for disease pathology and progression. mtDNAcn can be quantified from whole blood samples using qPCR to determine the ratio of nuclear DNA to mtDNA. However, the collection of blood samples in pediatric populations, particularly in infants and young children, can be technically challenging, yield much smaller volume samples, and can be distressing for the patients and their caregivers. Therefore, we have validated a mtDNAcn assay utilizing DNA from simple buccal swabs (Isohelix SK-2S) and report here its performance in specimens from infants (age = <12 months). Utilizing qPCR to amplify ~200bp regions from two mitochondrial (ND1, ND6) and two nuclear (BECN1, NEB) genes, we demonstrated absolute (100%) concordance with results from low-pass whole genome sequencing (lpWGS). We believe that this method overcomes key obstacles to measuring mtDNAcn in pediatric populations and creates the possibility for development of clinical assays to measure mitochondrial change during pathophysiological stress.

The authors have declared no competing interest.

This research project was supported by the California Initiative to Advance Precision Medicine ACES Program (OPR20139) and The JPB Research Network on Toxic Stress: A project of the Center on the Developing Child at Harvard University.

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The details of the IRB/oversight body that provided approval or exemption for the research described are given below:

IRB of Children's Hospital Los Angeles gave ethical approval for this work. Specifically, the IRB approved the following study cohorts presented in this work include Adverse Childhood Experiences (ACEs) program (Scalable Measurement and Clinical Deployment of Mitochondrial Biomarkers of Toxic Stress IRB CHLA2100174) and the Your Baby: Healthy Development and Resiliency (IRB CHLA1800547). Participants' or their legal representatives' provided written consent to take part in the aforementioned studies.

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