The Mitochondrial DNA copy number acts as a biomarker in the pathological state

Zhang Tong1，2，Zhao Guoqiang1，2，Liu Yanan1，2，Wei Fengxiang*1，2

  • The Genetics Laboratory, Longgang District Maternity and Child Healthcare Hospital, Shenzhen 518000, China;

2.School of Clinical Medical，Jiamusi University, Jiamusi 154007, China.)

Abstract：

Mitochondria are vital organelles in cells. Mitochondrial DNA is the only genetic material that exists outside the nucleus, and there are hundreds to thousands of copies in each cell in a tissue-specific way. When mitochondrial DNA is subjected to oxidative damage and carcinogen attack, it causes overproduction of reactive oxygen species, damage to some large molecules, insufficient energy supply and production of essential molecules such as heme and nucleotides, and abnormal mitochondrial signaling function. It can easily lead to mutations in mitochondrial DNA, which can lead to abnormalities in energy metabolism and oxidative stress. This is also one of the important reasons leading to mitochondrial dysfunction. At present, the change of mitochondrial DNA copy number is very important in various pathological conditions in the body such as diabetes, cardiovascular disease, mental disease, cancer, and aging, and reflects the biomarkers of various pathological conditions in the human body. Therefore, the regulation and detection of mitochondrial DNA copy number is an important aspect of mitochondrial genetics and biogenesis. And it is helpful for targeted disease prevention, early diagnosis and individualized treatment. It is of great significance for applications in clinical medicine, genetics, and life sciences. Nowadays, digital PCR technology can be used to identify diseases in the early stage of disease progression. Continue to track the existence, expansion and disappearance of pathogenic microorganisms and genetic variations in cancer, infectious diseases, and other human diseases. Nucleic acid detection with high resolution and low target level has a significant impact in the field of diagnosis, providing great advantages for diagnosis and preventive medicine. In short, changes in plasma mitochondrial DNA content have been associated with a variety of disease types, suggesting that the mitochondrial genome may be a key contributor to the occurrence of some diseases. It is necessary to further understand the relationship between the content of mitochondrial DNA in plasma and the clinicopathological characteristics of the disease and targeted treatment. Using a non-invasive and dynamic method to assess disease is a very promising method for cancer diagnosis and prognosis.

Keywords: mitochondrial DNA copy number；mitochondrial dysfunction；hereditary diseases；digital PCR；

  1. Mitochondrial DNA copy number

Mitochondrial are semi-autonomous organelles with their own genetic material and genetic system, and participate in a variety of cellular functions such as energy production, metabolic regulation, apoptosis, calcium homeostasis, cell proliferation and movement, and the generation of free radicals [1].  Human mitochondrial DNA is composed of a 16569 base pair circular double-stranded DNA molecule, whose genome is divided into non-coding region and coding region dominated by D-loop region. The molecular coding respiratory chain includes 13 peptides, 22 transport RNA and 2 ribosomal RNA[2].  And there are hundreds to thousands of copies in each cell in a tissue-specific manner [1].  

Mitochondrial DNA copy number (mtDNA-cn) represents the number of mitochondria in each cell and the number of mitochondrial genome in each mitochondria, and is an indirect biomarker reflecting mitochondrial function [3].  In recent years, the copy number of mitochondrial DNA has changed in various pathological states of human body, including endocrine system diseases [4], cardiovascular diseases [5,6], neurodegenerative diseases [7], mental diseases [8,9], various cancers [10,11] and the aging process [12], etc.  It is also one of the important causes of mitochondrial dysfunction.  In the case of mitochondrial dysfunction, it can cause excessive production of reactive oxygen species (ROS), damage of macromolecules, insufficient energy supply, production of essential molecules such as heme and nucleotide, and abnormal mitochondrial signaling function [13].  Therefore, mitochondrial DNA copy number may be considered as a biomarker reflecting various pathological states in the human body [1]. 

  • Regulation of mitochondrial DNA copy number

2.1 Regulation model

At present, the model of mitochondrial DNA copy number regulation includes: ATP requirement determines the amount of mitochondrial DNA. Nucleotide availability regulation, mitochondrial DNA replication regulation, and initiation of mitochondrial DNA replication may be necessary steps to maintain mitochondrial DNA quality. Mitochondrial DNA copy number is regulated by multiple origin of replication and mitochondrial transcription factor A (TFAM) can affect the mitochondrial DNA copy number by changing the replication mechanism.  Threshold hypothesis of mitochondrial DNA copy number control: different thresholds work together to push the mitochondrial DNA copy number to the middle range and regulate the content of mitochondrial DNA in each cell [14].

2.2 Mitochondrial oxidative stress

The supply and consumption of oxygen in the body is a dynamic equilibrium process. Therefore, under normal circumstances, the generation and consumption of ROS in the body are in a dynamic equilibrium state.  When there is a large increase in mitochondrial reactive oxygen species caused by various reasons, the increase of oxidative stress may lead to a decrease in the level of mitochondrial DNA copy number, and mitochondrial oxidative stress plays an important role in the process of disease and aging through irreversible damage to mitochondrial DNA.  Although active oxygen is necessary for normal signal transduction, but in a lot of mitochondrial ROS increase associated with mitochondrial dysfunction, defects due to histones, DNA repair ability is limited, and mitochondrial DNA near the location of the mitochondrial ROS produced, mitochondrial DNA will occur, including mitochondrial DNA copy number reduce various types of damage,  At the same time, it can lead to insufficient energy supply and production of essential molecules (such as heme and nucleotide), weaken the activity of the mitochondrial Electronic delivery chain (ETC), and abnormal mitochondrial signaling function, leading to the gradual failure of cellular mechanisms, thus promoting various clinically relevant diseases and aging processes.  It destroys the balance of apoptosis, mitochondrial bioenergetics and biogenesis [16].  

2.2.3 Mitochondrial-associated proteins

The decrease in mitochondrial DNA copy number in vitro is associated with down-regulation of mitochondrial transcription and decreased levels of proteins involved in oxidative phosphorylation, especially mitochondrial NADH dehydrogenase subunit 1(ND1), Cytochrome B (Cyt B) and Cytochrome C oxidase subunit I (COX-I) [18].  Among them, single stranded DNA binding protein (SSB), TFAM, DNA polymerase γ (DNA Pol γ) and Twinkle helicase are all mitochondrial specific DNA effectors and play important roles in nucleoid DNA polymerase β (DNA Pol β) has been detected in mitochondrial protein extracts from mammalian tissues and cells, suggesting that Pol β gene knockout leads to mitochondrial dysfunction including reduced membrane potential and mitochondrial content [17].  

  • Detection method of mitochondrial DNA copy number

At present, the mitochondrial DNA copy number level can be measured from DNA extracted from peripheral blood or other tissues by simple and convenient methods. When the rare target nucleic acid is detected in complex clinical samples, a novel, sensitive, accurate and multivariate absolute quantitative strategy is mainly adopted—-digital PCR technology, also known as single-molecule PCR.  Traditional digital PCR techniques have cumbersome dilution steps, so digital PCR introduces a variety of digital PCR sample dispersion methods including droplet, micowell capillary force drive, channel and print.  Moreover, the appropriate use of these sample dispersion methods provides reaction zones of hundreds of millions to millions of nanoliters or even picoliters, which can carry out sensitive and accurate absolute quantification of a variety of target nucleic acids [18].  

Among them, droplet digital PCR (ddPCR) technology uses the method of separating different DNA templates to obtain better amplification efficiency and realize the simultaneous amplification of different samples, so that the target nucleic acid in the samples can be accurately quantified.  The absolute copy number was quantified by determining the ratio of positive mitochondrial probe copy number to nuclear probe copy number.  This new PCR detection technique provides a simple workflow and is capable of producing highly stable DNA molecular segmentation [19].  

Compared with other methods for mitochondrial DNA copy number detection, digital PCR no longer requires standard dilution curve and reaction efficiency [18], but still provides a highly accurate and precise detection method.  At present, the digital PCR technique can identify the disease in the early stages of the disease progression, track cancer, infectious diseases and other pathogenic microorganisms, the existence of genetic variation in human disease, extension, and disappear, through high resolution and low target nucleic acid detection have a significant impact in the field of diagnosis, provides great advantages for diagnosis and preventive medicine [20].  

  • Changes in mitochondrial DNA copy number in different states of the organism

4.1 Endocrine system diseases

Diabetes is one of the most common diseases of the endocrine system.  At present, metabolic syndrome represented by abdominal obesity, hypertension, insulin resistance, dyslipidemia and other metabolic disorders has been proved to have a potential intermediary role in the risk of type 2 diabetes, which reduces the mitochondrial DNA copy number and increases the risk of type 2 diabetes.  Deletion of its mitochondrial DNA has been proved to be associated with the occurrence of insulin resistance and obesity, and precedes the development of type 2 diabetes [4].  The main cause of diabetes is the loss of mitochondrial DNA and the excessive production of ROS are susceptible to the influence of oxidative sugar metabolism and oxidative stress, resulting in qualitative and quantitative changes and abnormal mitochondrial dynamics [21].  Moreover, the quantitative status of mitochondrial DNA is considered to be an early genetic marker for type 2 diabetes and insulin resistance syndrome.  

4.2 Cardiovascular disease

It has been reported [3] that mitochondrial DNA copy number is associated with cardiovascular disease (CVD) and may have potential clinical application value in improving the risk classification of cardiovascular disease.  Currently, the mechanism by which mitochondrial DNA copy number affects disease is not clear, but one of the mechanisms may be the differential expression of specific genes that may affect human health and disease by altering cellular signaling pathways through changes in nuclear DNA (nDNA) methylation at specific sites [5]

Because defective mitochondrial DNA can lead to mitochondrial dysfunction, processes that promote inflammation and cellular aging disrupt energy homeostasis, leading to atherosclerosis.  Therefore, mitochondrial dysfunction is considered to be a core component of several chronic diseases, including cardiovascular disease, and may be associated with atherosclerosis, playing an important role in the occurrence and progression of cardiovascular disease.  Atherosclerosis is the main pathological lesion of cardiovascular diseases, and most coronary heart disease (CHD) occurs in the context of coronary atherosclerosis. The etiology of stroke is more diverse, including the confluence of atherosclerosis, cardiac embolism and small vessel disease [3].  Therefore, mitochondrial DNA copy number is involved in the atherosclerotic process that triggers coronary heart disease.  It has been reported  that[6] the reduction of mitochondrial DNA copy number in patients with CHD is considered as an indicator of increased oxidative stress cell level and suggests that mitochondrial DNA copy number can be used as a biomarker to quantify the severity of CHD.  

4.3 Neurodegenerative disease

Blood-derived mitochondrial DNA copy number is related to gene expression in multiple tissues including nuclear genes required for mitochondrial DNA replication, and can predict the occurrence of neurodegenerative diseases [7].  Some important processes in the pathophysiology of neurodegeneration are damage to mitochondrial energy generation pathways, oxidative phosphorylation, increased production of reactive oxygen species, and apoptosis.  Alzheimer’s Disease (AD) is a neurodegenerative Disease that mainly leads to dementia, and its preclinical stage can reflect impaired mitochondrial biogenesis by detecting low levels of mitochondrial DNA in cerebrospinal fluid.  It also helps to distinguish other neurodegenerative diseases including Creutzfeldt-Jakob disease [22].  Therefore, mitochondrial DNA in CSF is considered to be an interesting biomarker [23].  

4.4 Mental sickness

Mental illness may be a set of symptoms reflecting a separation from reality caused by chronic oxidative stress or exposure to drug toxicity.  It has been reported [8] that the mitochondrial DNA copy number decreases with the increase of the severity of psychosis, and the mitochondrial DNA copy number of leukocytes decreases in patients with schizophrenia and type I bipolar disorder.  

Schizophrenia is a complex neuropsychiatric disease with significant genetic susceptibility.  In some patients with schizophrenia, mitochondrial dysfunction can be explained by genomic defects such as mitochondrial DNA copy number variation, which is considered to be a sensitive indicator of cellular oxidative stress [9].  The mitochondrial DNA copy number decreased significantly in schizophrenia patients, suggesting that mitochondrial dysfunction is one of the reasons for the elevated oxidative stress in schizophrenia patients.  Due to the high energy requirements of neuronal function, changes in mitochondrial DNA copy number and consequent impairment of mitochondrial physiology significantly affect the pathogenesis of schizophrenia.  Therefore, mitochondrial DNA copy number may serve as a proxy marker for mitochondrial function in schizophrenia.  

4.5 Cancer

Cancer is one of the world’s highest rates of morbidity and mortality.  Mitochondrial DNA content is considered a molecular diagnostic tool that can help identify genetic abnormalities in human tumors.  According to a report from the Cancer Genome Atlas Project investigating copy number changes in mitochondrial DNA in 22 tumor types, mitochondrial DNA encodes proteins that only target mitochondrial electron transport chains and ATP synthase[10]. Therefore, the fluctuation of mitochondrial DNA copy number is related to the expression of respiratory genes, immune response genes and cell cycle genes, and may affect the transcription level of these genes.

Changes in mitochondrial DNA have been reported in a variety of cancer literature. The distribution of mitochondrial DNA content in cancer cells and changes in mitochondrial DNA may play an important role in the development of cancer, and may be a crucial factor in the progression of cancer.  The mitochondrial DNA of bladder cancer, breast cancer and kidney cancer has a tendency of deletion relative to adjacent normal tissues [10], and it was found in the report on the comprehensive molecular characteristics of human cancer mitochondrial genome:  increased mitochondrial DNA copy numbers were detected in cancer samples from patients with chronic lymphocytic leukemia, lung squamous cell carcinoma, and pancreatic cancer, but decreased in samples from patients with renal clear cell carcinoma, hepatocellular carcinoma, and myeloid proliferative tumors.  Moreover, different cancer subtypes from the same tissue sometimes exhibit different mitochondrial DNA copy number distributions.  For example, the mitochondrial DNA copy number of the renal chromatism cell group was significantly higher than that of the renal clear cell group and the renal papilla group [11].  This may be related to the general insufficiency of mitochondrial quality control and the resulting increase in steady-state mitochondrial DNA copy number, such as renal eosinophils [24].  

Mutations in the coding and regulatory regions of mitochondrial DNA have been found in many cancers.  In conclusion, changes in plasma mitochondrial DNA content have been associated with multiple cancer types, suggesting that mitochondrial genome may be a key contributing factor to cancer.  Further understanding of the relationship between plasma mitochondrial DNA content and the clinicopathological characteristics and targeted treatment of cancer can provide a non-invasive and dynamic method for disease assessment, which is a very promising method for cancer diagnosis and prognosis.

4.6 Aging

Mitochondria are involved in the regulation of biological aging and can be reflected by DNA phenotype and leukocyte telomere length.  According to reports [12],  Mitochondrial copy number and several aging biomarkers—-DNA methylation age, DNA methylation PhenoAge, DNA methylation damage and leukocyte telomere length, and higher mitochondrial DNA copy number may be associated with higher DNA phenotypes and increased risk of age-related disease and mortality.  At present, further elucidation of the breadth of mitochondrial interactions with aging biomarkers in human aging could help identify differences in risk of functional decline, disease onset and death between individuals.  

  • Conclusion and future perspective

In conclusion, mitochondrial are the vital organelles in cells, and mitochondrial DNA is the only genetic material that exists outside the nucleus.  Mitochondrial DNA mutations lead to abnormal energy metabolism and oxidative stress, which may lead to cellular metabolic disorders, mitochondrial dysfunction, and ultimately cell necrosis or apoptosis, resulting in serious clinical diseases and cell aging.  Therefore, in a relatively stable environment, the detection and regulation of human mitochondrial DNA copy number is of great significance to the study of detection methods, therapeutic targets, prognosis and rehabilitation of clinically related diseases and its application in the fields of medicine and life sciences. 

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