Mitochondria are the centers of energy metabolism in eukaryotes, and about 95% of the ATP is synthesized by mitochondria in human cells. Mitochondrial DNA (mtDNA) is present in the mitochondria of eukaryotic cells, and mtDNA mutations or abnormalities can cause mitochondrial diseases.
The human nuclear genome includes 24 chromosomes. The cytoplasmic genome is the mitochondrial genome, which is also known as the human chromosome 25 or M chromosome.
The total length of human mtDNA sequence is 16 569bp, which is not combined with histone, and is a naked double-stranded closed-loop DNA. Double chains are heavy chain (H) and light chain (L), respectively. Heavy chain is rich in guanine (G), while light chain is rich in cytosine (C). The ratio of G to C in H is 2.377, and the ratio of G to C in L is 0.4207. H is located in the outer ring, while L is located in the inner ring.
mtDNA has coding region and non-coding region. The coding region is conservative and encodes 37 genes, including 13 kinds of proteins, 2 kinds of rRNA and 22 kinds of tRNA. 13 genes encode proteins associated with mitochondrial oxidative phosphorylation (OXPHOS), all of which have AUG (methionine) as the initiation codon. 2 rRNA (16S, 12S) and 22 tRNA can meet the needs of all codons in the translation of mitochondrial protein. The H chain encodes 12 kinds of polypeptide chains, 2 rRNA and 14 tRNA. L chain codes 1 polypeptide chain and 8 tRNA. The mtDNA gene is very compact and has a high utilization rate. With the exception of the D-loop region, the intergenic region is only 87 bp. mtDNA has no introns, so mutations in any region of mtDNA may lead to changes in the function of mitochondrial oxidative phosphorylation. The mtDNA has two non-coding regions, one is the D-loop region and the other is the L-chain replication initiation region. The D ring region is composed of 1 122bp, and most of them are tandem repeat sequences (TRS). The D ring region is related to the replication and transcription of mtDNA, because it is rich in A=T pair and easy to melt. The D loop region is the origin of replication of the heavy chain (OH). The replication of the light chain begins at about 2/3 of the completion of the H chain, and the origin of replication of light chain is located at about 1/3 of the mtDNA. The mtDNA replication is not only in the S phase, but throughout the entire cell cycle. In addition to OH, the D-loop region also contains the transcriptional promoters of light and heavy chains (PL and PH) and four highly conserved sequences (213 ~ 235 bp, 299 ~ 315 bp, 346 ~ 363 bp and 16 147 ~ 16 172 bp). Transcription is initiated by two promoters, PL and PH, located in the D-loop region, respectively, with the light and heavy chains being transcribed in opposite directions.
Mitochondria can independently replicate, transcribe and translate 13 kinds of mitochondrial proteins by using mtDNA and independent genetic information expression systems, showing autonomy. About 99% of the 1441 proteins involved in mitochondrial function are encoded by the nuclear genome, and only 1% are encoded by the mitochondrial genome. So that most of the proteins required maintaining mitochondrial structure and function are encoded by the nuclear genome, showing semi-autonomous.
The mutation rate of mtDNA is 10~20 times higher than the nDNA mutation rate. The average mtDNA for any two individuals varies from 4 out of 1,000 base pairs up to 3%. The mutation rate of nDNA is only 0.1%. High mutation rate is the cause of large differences in mtDNA sequences between individuals and groups. mtDNA mutations can be removed by autophagy of lysosomes, so although mutations in mtDNA are common, mitochondrial genetic diseases are uncommon.
UGA codes for a stop codon in the nuclear gene, but encodes tryptophan (Trp) in the mitochondrial gene. AUA encodes isoleucine (ILE) in the nuclear gene, but encodes methionine (Met) in the mitochondrial gene. AGA and AGG encode arginine (Arg) in the nuclear gene and termination codon in mitochondria. Therefore, the mitochondrial genetic code has 4 termination codes (UAA, UAG, AGA and AGG).
Maternal inheritance refers to the phenomenon that maternal traits determine the offspring, that is, the mother passes her mtDNA to her son and daughter, but only her daughter can pass the mtDNA to the next generation. This is because when the sperm and egg are bound, the sperm has only the head into the fertilized egg, while the mitochondria are located at the tail of the sperm. So almost all of the cytoplasm in the fertilized egg comes from the egg.
There are about 100,000 mitochondria in female oocytes, but fewer than 100 mitochondria are passed on to offspring by the time the egg matures. This process of mitigating the number of mitochondria from 100,000 to fewer than 100 is called the genetic bottleneck. The bottleneck effect causes significant mtDNA heterogeneity among offspring individuals, and even identical twins can exhibit different mtDNA heterogeneities. Children with more mutated mtDNA will get the disease, and less mutated mtDNA will not become ill or less severe.
The minimum number of mutant mtDNA that can cause dysfunction of the tissue organ is called threshold. Different tissues and organs have different degrees of dependence on ATP, and the threshold values also show significant differences. The central nervous system has the highest degree of dependence on ATP, and the lower the threshold is the more likely to be affected, followed by skeletal muscle, heart, pancreas, kidney and liver. The threshold of the same tissue at different developmental stages is also different. mtDNA mutation in skeletal muscle cells does not show any pathological symptoms in neonates, but with the growth of individuals, the mutation of mtDNA reduces the ATP production. When it does not meet the needs of skeletal muscle for energy, it shows myopathy.
If mtDNA molecules in the same tissue or cell are identical, it is called homoplasmy. If mtDNA mutations cause two or more mtDNA (wild-type and mutant co-existence) existing in the same cell or the same tissue, known as heteroplasmy. Symptoms are more severe in homozygous individuals of mutation mtDNA. Whether the heteroplasmy individuals of mutation mtDNA showing symptoms or not often depend on the proportion of mutated mtDNA.
When the cell divides, the mutant and wild-type mtDNA are randomly separated, allowing the subcells to have different proportions of mutant mtDNA molecules. In the process of continuous division, the proportion of mutated mtDNA and wild-type mtDNA in heterogeneous cells will drift and develop towards homogeneity. The exuberant splinter cells (such as blood cells) tend to have a tendency to exclude mutation mtDNA. After numerous splits, cells are gradually becoming only homogenous cells of wild type mtDNA. In the less vigorous cells (such as muscle cells), the mutation mtDNA has the advantage of replication. With the gradual accumulation of mutation mtDNA, the homogenous cells with only mutant mtDNA are formed. As a result of the drift, the phenotype also changes.
The multidirectional mutation of mtDNA has an addition effect on the phenotype. For example, the more types of mutation mtDNA, the more severe the condition of Leber's hereditary optic neuropathy.
There were 772 types of mtDNA mutation, of which 621 are caused by base substitutions, and 151 are caused by deletion, insertion, inversion and rearrangement.
The mRNA gene has base substitutions, most of which are missense mutations, and cause changes in the encoded amino acid. For example, patients with Leber hereditary optic neuropathy (LHON), the G of mtDNA 11 778th site has been transformed into A (G11 778A), making 340th amino acid of NADH dehydrogenase subunit 4 (ND4) from arginine to histidine. Both eyes were simultaneously affected, if not at the same time, and then the other eye was blinded within two months of blindness in one eye. The neurodegeneration of optic nerve and retinal neuron is the main pathological feature of LHON, as well as the degeneration of peripheral nerve, heart block and hypotonia. The average age of onset is 27~34 years old.
tRNA is the tool for the transport of amino acids in protein synthesis, but the tRNA base substitutions result in structural abnormalities.
I. Mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS)
Approximately 80% of patients are due to the conversion of A to G (A3243G) at position 3243 of the tRNA gene. The mutation makes the structure of tRNALeu change, causing abnormal transcription and inducing disease. The characteristic pathological changes are a large number of abnormal mitochondrial aggregations in the arterioles and capillary wall of the brain and skeletal muscle. Abnormal mitochondria cannot metabolize pyruvate, resulting in the production of large amount of lactic acid. The accumulation of lactic acid in blood and body fluids leads to decreased pH and buffer capacity, resulting in lactic acidosis. The patient began to have recurrent shock, myopathy, ataxia, myoclonus, dementia and deafness before the age of 40.
II. Myoclonic epilepsyassociated with ragged-red fibers (MERRF)
80% to 90% of MERRF patients are caused by A8344G base substitution resulting in tRNALys mutation. tRNALys mutation causes severe defects in mitochondrial translation, resulting in multiple defects in the respiratory chain enzyme complex. Generally in childhood, the clinical manifestations of multiple system disorders, including myoclonic epilepsy, ataxia, myopathy, mild dementia, deafness and so on. A large number of massive abnormal mitochondria are commonly found in muscle cells of patients, often described as ragged red fibers (RRFs), which can be dyed red with special dyes (Gomori Trichrome).
mtDNA has two rRNA genes that encode 12SrRNA and 16SrRNA, respectively. rRNA is an important component of mitochondrial ribosome. Since the “natural target” of aminoglycoside antibiotics is the bacterial ribosome, and the structure of mitochondrial ribosome is similar to the bacterial ribosome. Changes in the structure of 12S rRNA may cause the mitochondrial ribosome of the cochlear cells to be attacked by aminoglycoside antibiotics, resulting in deafness. The molecular mechanism of aminoglcoside antibiotics induced deafness (AAID) caused by streptomycin, gentamicin, kanamycin and other aminoglycoside antibiotics is presumed to be the A1555G mutation of mtDNA 12SrRNA gene. This mutation amplifies the “pocket” of aminoglycoside antibiotics bound to the ribosome and makes the aminoglycoside antibiotics bind more closely, thereby attacking the cochlea.
The study found that base substitutions in the regulatory sequence of the D-loop region in mtDNA are also associated with mitochondrial genetic disease. For example, T16 189C can lead to type 2 diabetes.
It has been found that more than 100 deletions lead to KSS disease. Most of the mtDNA deletions are caused by somatic mutations and only 5% are caused by maternal inheritance. The common deletion is a 5 kb mtDNA fragment from 8 469 to 13 447, which is located in ATPase8 and ND5 genes, causing the deletion of ATPase8, ATPase6, COIII, ND3, ND4, ND4L, ND5 and multiple tRNA genes. The patients were characterized by external ophthalmoplegia, blepharoptosis, myopathy of limbs, dyskinesia, retinitis pigmentosa, sensory nerve injury and hearing loss, lactic acidosis and cardiac conduction dysfunction. Usually, the disease develops rapidly before the age of 20.
It is due to deletion of 5 kb mtDNA from 8 469 to 13 447bp. In 1992, Ballinger studied a family with diabetes accompanied by deafness, and the results showed that 10.4 kb large fragments deletion in mtDNA.
It's also called paralysis agitans. There was mtDNA deletion in the brain tissue, especially in substantia nigra. Patients could detect long 5 kb deletion in mtDNA. The breakpoints are located in ATPase 8 and ND5 genes, respectively, resulting in dysfunction of the mitochondrial respiratory chain, which could lead to neuronal dysfunction. PD is a kind of motor disorder of senile disease with symptoms of tremor, slow motion, and often error and so on. It is generally believed that the deletion of mtDNA is caused by somatic mutation.
mtDNA copy number mutation indicates that the mtDNA copy number is significantly lower than normal. This mutation is rare, only found in some cases of fatal infant respiratory disorder, lactic acidosis, or liver and kidney failure.
In 1992, Corral-Debrinski et al. studied the brain of 7 patients aged from 24 to 94, and found that the deletion of mtDNA fragment was positively correlated with age, especially for individuals over 80 years old. In 1992, Soong et al. showed that the gray matter region of the brain contained the major mtDNA deletion, and the deletion preferentially occurred in the neuron, but less in glial cells. In 1999, Michikawa et al. examined the fibroblasts of 14 elderly people over 65 years old, of which 8 had T414G base substitutions.
Mitochondria are the source of free radicals in cells. More than 95% of the oxygen free radicals in the body come from the respiratory chain of mitochondria. Under normal conditions, oxygen free radicals can be removed by Mn2+-SOD in mitochondria. When body aging and degenerative diseases occur, Mn2+-SOD activity decreases and oxygen free radicals accumulate in mitochondria, resulting in mtDNA damage and accelerated aging. Aerobic exercise can increase the supply of ATP, increase the autophagy of lysosomes, remove damaged mtDNA in time and delay senescence.
In mitochondria, only 1% of the proteins are encoded by the mitochondrial genome, about 99% of the proteins are encoded by nuclear DNA. Therefore, nuclear DNA abnormalities also cause the corresponding mitochondrial diseases (101 species).
The pathogenic genes OPA1, OPA3 and OPA8 are located in 3q29, 19q13.32 and 16q21 ~ q22, respectively. The products of these three genes are all located in the mitochondria. The point mutation of these genes can lead to the decrease of ATP synthesis in mitochondria, and their clinical manifestations are very similar to that of Leber hereditary optic neuropathy (LHON).
PD (OMIM 556500) is usually caused by deletion of mtDNA fragments. However, the pathogenicity gene of some adolescents with Parkinson's disease (OMIM 605909) is PINK1, which is located in 1p36.12. The occurrence of Parkinson's disease may also be the result of the combination of mtDNA and nuclear DNA. It is suggested that the occurrence of Parkinson's disease may also be the result of the combination of mtDNA and nuclear DNA.