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Published on: July 12, 2022
A method for measuring mitochondrial DNA copy number in pediatric populations
Simran Maggo1, Liam Y North2, Aime Ozuna2
1Department of Pathology and Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA, United States.
Insights
Quantifying mitochondrial DNA copy number (mtDNAcn) is crucial for disease research. A new qPCR assay using buccal swabs, instead of blood, offers a less invasive and effective method for pediatric populations.
Area of Science:
- Mitochondrial biology
- Genetics
- Pediatric research
Background:
- Mitochondria are vital organelles regulating homeostasis during stress.
- Mitochondrial DNA copy number (mtDNAcn) variation is a biomarker for disease.
- Current blood-based quantification methods are challenging in infants and young children.
Purpose of the Study:
- To validate a novel qPCR assay for mtDNAcn quantification using buccal swabs.
- To assess the assay's performance in pediatric specimens, specifically infants.
- To establish a less invasive method for pediatric mtDNAcn analysis.
Main Methods:
- DNA extraction from buccal swabs (Isohelix SK-2S).
- Quantitative PCR (qPCR) targeting mitochondrial genes (ND1, ND6) and nuclear genes (BECN1, NEB).
- Comparison of qPCR results with low-pass whole genome sequencing (lpWGS) data.
Main Results:
- The buccal swab qPCR assay demonstrated 100% concordance with lpWGS.
- The method is effective for specimens from infants under 12 months.
- Successful quantification of mtDNAcn from non-invasive buccal swabs.
Conclusions:
- A validated qPCR assay using buccal swabs overcomes challenges of blood collection in pediatric populations.
- This method facilitates easier and less distressing mtDNAcn measurement in infants and young children.
- Enables potential development of clinical assays for mitochondrial dysfunction in pediatric stress.
Abstract:
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 mtDNA to nuclear DNA. 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 it's performance in specimens from infants (age = <12 months). Utilizing qPCR to amplify ∼200 bp 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.
