Biological and translational attributes of mitochondrial DNA copy number: Laboratory perspective to clinical relevance

Table 1 Key genes regulating mtDNA copy number

Gene	Role in mtDNA regulation	Associated disorders	Ref.
PGC-1α	Master regulator of mitochondrial biogenesis	Neurodegeneration, cancer, metabolic syndrome	[3,5,6]
TFAM	Maintains mtDNA integrity and replication	Mitochondrial diseases, aging	[5,9]
POLγ	Essential for mtDNA replication	MELAS, Kearns-Sayre syndrome, cancer	[88-90]
NRFs (1 and 2)	Coordinate expression of mitochondrial genes	Neurodegeneration, metabolic disorders	[3,5]

mtDNA: Mitochondrial DNA; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; TFAM: Mitochondrial transcription factor A; POLγ: Polymerase gamma; NRFs: Nuclear respiratory factors; MELAS: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes.

Table 2 Methods for assessing mitochondrial DNA copy number

Method	Steps involved	Applications	Advantages	Limitations	Ref.
qPCR	DNA extraction → primer design → amplification → analysis	Widely used in clinical and research diagnostics	High sensitivity, high throughput, cost-effective, rapid	Susceptible to bias in low-quality DNA, issues with heterogeneous samples, requires careful primer design	[15,24-26]
NGS	DNA extraction → library preparation → sequencing → bioinformatics analysis	Genome-wide studies, detects mtDNA mutations alongside copy number analysis	Genome-wide analysis, accurate quantification, detects mtDNA heteroplasmy	Requires advanced bioinformatics, high cost, computational complexity	[21,28-32]
Southern blot hybridization	DNA extraction → gel electrophoresis → hybridization → quantification	Historically the gold standard, reliable for mtDNA integrity assessment	High reliability, detects large-scale deletions	Time-intensive, requires large DNA quantities, semi-quantitative	[22]
FISH	Sample preparation → probe hybridization → microscopy	Single-cell resolution studies, spatial visualization of mtDNA	Single-cell resolution, visualizes mtDNA distribution	Labor-intensive, provides only rough mtDNA estimates, technically demanding	[23]

qPCR: Quantitative polymerase chain reaction; NGS: Next-generation sequencing; FISH: Fluorescent in situ hybridization; mtDNA: Mitochondrial DNA.

Table 3 Clinical relevance of mitochondrial DNA copy number in diseases

Disease category	Role of mtDNAcn	Key observations	Diagnostic/prognostic value
Neurodegenerative disorders[64-70]	Biomarker for disease progression and severity	Reduced mtDNAcn in AD brains; increased mtDNAcn in peripheral blood of AD patients	Correlates with tau pathology in CSF; potential for non-invasive diagnosis using blood mtDNA levels
Cancer[15,62,100-102]	Indicator of tumor aggressiveness and treatment response	Elevated mtDNAcn associated with tumor proliferation; decreased mtDNAcn linked to poor prognosis	Distinguishes between cancerous and non-cancerous tissues; early-stage cancers show higher mtDNAcns, while advanced stages may show depletion
Metabolic disorders[4,15,80-83]	Reflects mitochondrial dysfunction	mtDNAcn dysregulated in diabetes and other metabolic syndromes, indicating stress or compensation mechanisms	Biomarker for mitochondrial stress in diabetes; changes in mtDNAcn can indicate early disease onset or progression
Aging[47-49,55,56]	Associated with age-related cellular dysfunction	Decline in mtDNAcn in various tissues (e.g., blood, muscle) with age; some tissues exhibit increased mtDNA	Low mtDNAcn linked with poor health outcomes in aging populations, including cognitive and physical decline
Inherited mitochondrial disorders[82-93]	Indicates heteroplasmy levels and disease severity	Variations in mtDNAcn linked to phenotypes like MELAS, Pearson’s syndrome, and Leber’s hereditary optic neuropathy	High mtDNAcn linked to milder phenotypes; can guide prognosis and therapy for conditions like Kearns-Sayre syndrome and mitochondrial encephalopathy

mtDNAcn: Mitochondrial DNA copy number; AD: Alzheimer’s disease; MELAS: Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes; CSF: Cerebrospinal fluid.

Table 4 Role of mtDNA copy number in various stages of cancer development and progression

Aspect	Role of mtDNAcn	Mechanism/impact	Examples
Cancer risk	Altered mtDNAcn (increase or decrease) may predispose individuals to cancer development	Imbalance in ROS production	Decreased mtDNAcn linked to breast cancer risk
		Compromised cellular energy metabolism	Increased mtDNAcn linked to lung cancer risk
Tumor initiation	Changes in mtDNAcn can affect mitochondrial biogenesis and metabolic reprogramming	Promotes a shift to aerobic glycolysis (Warburg effect)	Low mtDNAcn observed in colorectal cancer tissues
		Increases ROS, leading to genomic instability
Tumor progression	Dynamic changes in mtDNAcn support adaptation to tumor microenvironment	High mtDNAcn enables oxidative metabolism in hypoxic conditions	Elevated mtDNAcn associated with metastatic breast cancer
		Supports invasive and metastatic properties
Therapeutic resistance	Altered mtDNAcn contributes to drug resistance	High mtDNAcn enhances oxidative phosphorylation, reducing sensitivity to certain chemotherapies	Increased mtDNAcn linked to resistance in lung cancer treatments
Prognostic biomarker	mtDNAcn alterations can predict cancer outcomes	Low mtDNAcn correlates with poor prognosis in many cancers	Reduced mtDNAcn in gastric cancer linked to poor survival
		High mtDNAcn may predict aggressive tumor behavior
Immune evasion	Changes in mtDNAcn influence immune responses within the tumor microenvironment	mtDNA release into the cytoplasm activates inflammatory pathways	mtDNA-derived DAMPs in melanoma
		Alters immune surveillance mechanisms
Angiogenesis	mtDNAcn modulates energy demand and oxidative stress, indirectly affecting vascular growth	High mtDNAcn supports angiogenic signaling	Increased angiogenesis in glioblastoma with altered mtDNAcn
Metastasis	Altered mtDNAcn facilitates energy supply for metastatic spread	Provides metabolic flexibility for survival in secondary sites	Elevated mtDNAcn in metastatic colorectal cancer

mtDNAcn: Mitochondrial DNA copy number; ROS: Reactive oxygen species; DAMPs: Damage-associated molecular patterns.

Table 5 Comparison of techniques for modulating mtDNA copy number therapeutic strategy

Therapeutic strategy	Mechanism	Applications	Advantages	Challenges
AMPK activation	Enhances mitochondrial biogenesis via PGC-1α activation	Neurodegenerative diseases, aging	Promotes energy balance	Off-target effects, limited clinical trials
Genome editing tools	Targets mtDNA mutations or modulates copy number	Mitochondrial diseases, cancer therapy	Precision targeting	Ethical concerns, technical challenges
Autophagy induction	Removes damaged mitochondria	Improves mitochondrial quality	Enhances cellular health	Excessive clearance may have long-term side effects
Small molecules and vitamins	Increases mtDNAcn	Metabolic and neurodegenerative disorders	Cost-effective	Limited understanding of long-term effects

AMPK; Adenosine monophosphate-activated protein kinase; PGC-1α: Peroxisome proliferator-activated receptor gamma coactivator 1-alpha; mtDNA: Mitochondrial DNA; mtDNAcn: Mitochondrial DNA copy number.

Table 6 Broad spectrum of therapeutic applications of mtDNA copy number

Application	Role of mtDNAcn assessment	Therapeutic implications	Examples
Cancer prognostication	mtDNAcn serves as a biomarker for predicting cancer outcomes	Guides risk stratification and treatment intensity	Low mtDNAcn linked to poor prognosis in gastric and colorectal cancers
		Identifies patients with aggressive disease
Therapeutic targeting	Abnormal mtDNAcn highlights mitochondrial vulnerabilities	Enables development of drugs targeting mitochondrial pathways (e.g., OXPHOS inhibitors)	mtDNAcn modulation as a target in ovarian and pancreatic cancer therapies
Monitoring treatment response	Changes in mtDNAcn reflect tumor response to therapy	Serves as a real-time marker to monitor chemotherapy, radiotherapy, or immunotherapy efficacy	mtDNAcn alterations used to monitor cisplatin therapy in ovarian cancer
Personalized medicine	mtDNAcn variations help tailor therapies based on mitochondrial function	Facilitates selection of specific treatment modalities (e.g., glycolysis inhibitors vs OXPHOS inhibitors)	mtDNAcn guiding metabolic therapy choices in lung and breast cancer
Radiotherapy sensitization	Altered mtDNAcn may increase sensitivity or resistance to radiotherapy	Identifies patients who might benefit from combined mitochondrial and radiotherapy interventions	Elevated mtDNAcn linked to radio-resistance in glioblastoma
Metabolic modulation	mtDNAcn assessment reveals metabolic dependencies of tumors	Guides therapies targeting cancer metabolism (e.g., ketogenic diets, mitochondrial uncouplers)	Low mtDNAcn tumors treated with glycolysis inhibitors
Early disease detection	mtDNAcn alterations in blood or tissue serve as a non-invasive biomarker for early cancer detection	Allows early initiation of treatment, potentially improving outcomes	Reduced mtDNAcn detected in circulating cell-free DNA in lung and breast cancers
Combination therapies	mtDNAcn dynamics predict synergy between mitochondrial-targeted drugs and conventional therapies	Combines metabolic modulators with standard chemotherapy or immunotherapy for enhanced efficacy	mtDNAcn-directed combination strategies in melanoma treatment
Toxicity management	mtDNAcn levels predict susceptibility to mitochondrial toxicity from certain drugs	Assists in preemptive dose adjustments or alternative drug selection to avoid adverse effects	Monitoring mtDNAcn to prevent cardiotoxicity from anthracyclines
Rare mitochondrial disorders	mtDNAcn assessment aids in the diagnosis and management of mitochondrial diseases with cancer overlap	Develops therapies that normalize mtDNAcn or enhance mitochondrial biogenesis	mtDNAcn restoration therapies in mitochondrial depletion syndromes

mtDNAcn: Mitochondrial DNA copy number; OXPHOS: Oxidative phosphorylation.