Health Worlds

[Abstract] In recent years the emergence of mitochondrial disease increased significantly, especially common in multiple neuromuscular and multi-system disease patients. The molecular mechanism of complex and sometimes exist in the nuclear DNA mutations and mitochondrial DNA (mtDNA). mtDNA mutations are usually maternally inherited, and sometimes adjust the mtDNA from the biosynthesis of genetic mutations in nuclear genes of the chromosome. mtDNA mutations produce clinical manifestations and severity of even in the same family will have a significant difference. Some clinical syndromes have been found with some of the special mutation, which the relationship between phenotype and genotype has been
Clear, but most patients do not comply with these symptoms have been identified as a syndrome, the mutation is rare or newly issued. Most of the patients exist mutant and wild-type mtDNA ("heterogeneity"), some mtDNA mutations are homogeneous. The clinical diagnosis of mtDNA, including detection of common mutations is usually to PCR-based detection technologies and large fragments (> 500 bp) rearrangement of the Southern blot or PCR. For the samples tested negative, but also the need for other methods for mtDNA deletion to further testing. Prenatal diagnosis is currently difficult to find this type of disease.
[Keywords] mitochondria; DNA; diagnosis1 OverThe past decade, a significant increase in mitochondrial disease, but there is no one specific DNA or protein level in t


he diagnostic methods. Clinicians may have heard of some diseases related with mitochondria, including diabetes, cancer, stroke, myocardial disease, dementia, migraine headaches and other diseases, even aging is also related to the doctor's diagnostic tests on mitochondrial strong interest. However, due to the diversity of its clinical manifestations, the lack of detection of the gold standard, the lack of doctors and laboratory personnel, and many other good reasons for collaboration, making diagnosis of mitochondrial disease becomes very complex. Common doctor will send blood samples to a molecular diagnostic laboratory demands the removal of mitochondrial disease, which is an impossible task. This article will seek to provide guidance to laboratory diagnosis of mitochondrial disease based on the performance of knowledge, some of the clinical diagnosis of molecular diagnostic methods can apply the new standards, and lead to disease manifestations and molecular genetic diagnostic specificity of mitochondrial complex aspects of knowledge.2 Overof mitochondrial geneticsAlmost all mitochondria of human cells exist, all the energy of the pump cell function. There are more than 1,000 kinds of mitochondrial proteins, most of them by the nuclear-encoded, only 13 proteins encoded by the mitochondria. Nuclear encoded mitochondrial proteins and other non-encoded protein, as encoded by the genes on the chromosomes. mRNA is transported to the cytoplasm of the ribosome, protein transport through the complex mechanism of the mitochondrial membrane into the mitochondria. These nuclear encoded proteins, including pyruvate dehydrogenase complex (PDHC) and the citric acid cycle enzymes, membrane transport proteins, electron transfer chain, transport and assembly factor. Electron transport chain will eventually break down the equivalent of food energy into ATP chemical energy of the system, including 87 kinds of proteins, in addition to 13 kinds of proteins encoded by nuclear outside all nuclear encoded mitochondrial protein mutation will lead to energy deficiency, which causes congenital mitochondrial energy metabolism (AKA mitochondrial disease) and other diseases.
As with most metabolic diseases, nuclear-encoded mitochondrial disease by autosomal recessive inheritance. For example SURFI, it is the cytochrome C oxidase (COX; electron transport chain complex IV) in the assembly of proteins, the defects often lead to Leigh disease (later). Research has also found other genetic methods, such as X linked and autosomal dominant inheritance. Amplification of trinucleotide repeats resulting Friedrech ataxia is a relatively common mitochondrial disease due to play a role in iron balance mutations of mitochondrial proteins. Nuclear encoded mitochondrial proteins caused by mutations in the important and complex disease, but not the focus of this article.
This article focuses on the mitochondrial DNA (mtDNA) mutations occur. mtDNA length of 16.6kb (nuclear genome length of 3.0 billion kb), even more than the number of only 1 kind of protein coding nuclear genes is also small. MtDNA contains such a small 37 genes, encoding 13 proteins, 22 tRNA, 2 rRNA. 13 proteins encoded by mtDNA are all subunits of the electron transport chain, so mtDNA is very important for energy metabolism. Mitochondria ribosomal protein can be translated. Since the first report since 15 years ago, mtDNA point mutations have been over 180 missing and the same number of human diseases with a variety of reports (, closing ). mtDNA and the mtDNA disease in a number of pages to be reed and addressed.
Because mtDNA only comes from the oocyte, so many of mtDNA mutations in primary came from the maternally inherited. Many nuclear regulatory proteins encoded by mtDNA exist and influence each other, so these mutations can lead to secondary mtDNA mutations, the genetic model can often stained recessive or dominant inheritance. Because of these mutations lead to nuclear-encoded mtDNA mutation is common in clinical diagnostic tests, this paper will also discuss its related diseases.
Since both are nuclear and mitochondrial protein encoded by mitochondrial DNA, in which mutations in any one genome may lead to mitochondrial disease. a small number of mtDNA coding genes, to some extent make up for its high copy number, high mutation rate and the ability relative lack of repair mechanisms that may cause problems. Is not yet clear mtDNA mutations in nuclear genes and the role of mitochondrial disease. Most mutations in nuclear genes encoding proteins of mitochondrial disease caused by serious, and in early childhood and early onset. On the contrary, mtDNA mutations lead to disease conditions light, late onset, but some mtDNA-related cases of severe early-onset reports. Therefore, although studies suggest that nuclear DNA mutations in childhood diseases, dominant, mtDNA mutations in adult dominant disease, clinical practice shows that the two genomes of mitochondria in patients of any age are equally important in the pathogenesis.
As before, mitochondrial disease and many clinical manifestations, and if a patient just the symptoms of mitochondrial disease and has been unanimously confirmed to be directly related to the mutation of the disease, DNA testing, but for most patients in terms of it is often difficult to determine the nuclear gene mutation in the end is still in the mtDNA, so screening for the mutation will be very useful.3, the characteristics of mitochondrial genetics3.1 maternal mode of inheritance of genetic and other
Asexual reproduction by mtDNA from mother to child. Paternal inheritance and recombination in humans is very rare. Therefore, in addition to a new mutation in mtDNA sequences in children with their mothers, brothers and sisters, half brothers and sisters, family, aunt, uncle, grandmother is the same. Genetic relationship of these and more distant maternal relatives showed a familial clinical typically carry mtDNA mutations, which is maternally inherited pattern. There are other characteristics of the mtDNA, such as heterogeneity, maternally inherited mutations come within the family and family members between the clinical presentation, age of onset and severity of the disease that has significant variability. Because all the children of a woman inherited the same mtDNA sequence, then usually by maternally inherited, siblings of children's future will be very high risk of recurrence, close to 100%, but their clinical performance may be significantly different.
MtDNA mutations occur in the blood usually the mother and other maternal relatives were found, but the number of mtDNA mutations in some or all of the maternal relatives in the incidence of mutations may be lower than the threshold amount, it may not have clinical signs or symptoms rarely. Also found that the blood did not, and can be isolated from muscle mtDNA mutations. It is assumed that they occurred in the early embryo, it does not appear in their relatives.
mtDNA deletion and deletion / duplication of the genetic mechanism is complex. A single mtDNA deletion usually is sporadic, not inherited from your mother will not be passed on to her children, but there are exceptions to the report. However, the lack of repetition is usually related to maternal inheritance, so the family may have a high risk for relapse.
As mentioned earlier, all the regulatory proteins of mtDNA, including the polymerase enzyme topology, replication components and mtDNA repair enzymes encoded by nuclear genes. Therefore, mutations in these genes can be secondary effects of primary mtDNA, mtDNA generally result in loss or reduction of mtDNA copy number ("failure"), the genetic mechanism could be autosomal recessive or dominant inheritance.3.2 The high copy number and heterogeneity
Each usually has two copies of nuclear genes, and mtDNA copy number up to several hundred to tens of thousands per cell. Therefore, mutations in nuclear genes into homozygous (100% mutation) and heterozygous (50% mutation), while the mtDNA mutation rate may be 0% to 100% in any one of proportion. When two (sometimes more species) mtDNA sequences exist in the same mitochondria, cells, tissues or individuals, known as heterogeneity. Clinical diagnosis, while there wild-type (normal) and mutant (disease) of the mtDNA sequence, generally considered to be heterogeneous, but sometimes also appear heterogeneous benign polymorphism, and its mtDNA are associated with two different related diseases. MtDNA mutations in patients generally are based on the form of heterogeneity may be due to homogeneity (100%) mutation is lethal. There are exceptions, such as in Leber hereditary optic neuropathy (LHOH) three kinds of mtDNA mutations may also appear.
Because selection pressure and other factors, the proportion of mutant and normal mtDNA (mutation load) in each individual the proportion of the different organizations with different, such as the A3243G and other mtDNA mutations in the number of different organizations are different, such as T8993G or C mutation in various the same organizations, which may be the exception. Therefore, blood or amniotic fluid in the mtDNA mutation load of mtDNA mutations in other organizations predict load of little significance, especially in some mutant load and clinical phenotypes related organizations, such as brain, bone marrow muscle, heart and so on.3.3 The bottleneck effect
Oocytes with 10 million copies of mtDNA, in the process of egg formation, the number of molecules can be transferred to the next generation of very small, estimated that only one or a few mtDNA genome, known as the bottleneck, so the children of the same mother, the degree of heterogeneity there may be very different. This mechanism within the family led to the diversity of clinical manifestations of mitochondrial disease mainly, but not all reasons, such as heterogeneity LHOH significant mutation in the family because of the different performance of the important nuclear modifier genes rather than mtDNA. For individuals, the proportion of mutant mtDNA in cell division or organization of the mitotic period possible after the change in mtDNA replication, leading to different phenotypes appear with time change.3.4 Threshold
MtDNA of wild-type cells can compensate for the reduction, until the emergence of exceeding the threshold, or involving other features of apoptosis, when cells in the affected tissue when sufficient amount of clinical disease appears. Threshold depends on the mutation characteristics and cell types. Neurons and muscle cells with high energy requirements, and therefore high mutation load tolerance is low, its incidence is lower than the threshold, such as connective tissue of other organizations that require low power.4 Mitochondrial Medicine4.1 Summary
Normal function of cells produced highly dependent on mitochondrial ATP, in particular neurons and muscle cells. These organizations demanding performance on mitochondrial function in even a brief lack of oxygen, or ischemia can lead to stroke and myocardial infarction. Therefore, selective mitochondrial disease affecting the nervous and muscle, often referred to as "mitochondrial myopathy." Mitochondrial disease can also be found in various types of endocrine glands and kidney (especially tubular), even can affect the bone marrow, liver and exocrine glands.
Previously described characteristics of mitochondrial genetics led to inconsistent clinical manifestations of mitochondrial disease, which each carry mtDNA in the maternal relatives in the system or organization involved, disease extent (from fatal to asymptomatic) and age (from birth to late adult ), and many have a significant diversity. Lung and skin, bones, tendons and other tissues of the rare low energy requirements involved, but also by neuromuscular disease, gastroesophageal reflux, respiratory muscle weakness, abnormalities of central pulmonary ventilation. High-energy requirements of tissue dysfunction, including growth hormone deficiency, and renal tubular acidosis can lead to disease, mitochondrial disease also need to pay attention to symptoms may occur intermittently, there may be infection or increased energy demand such as excessive exercise or fasting, etc. the case of energy supply to reduce induced.4.2 with the mtDNA-related syndromes
Most clinical experience has shown that mitochondrial disease is not fully consistent with a particular syndrome, but the association between genotype and phenotype is not strong, such a syndrome may not occur in patients with mutations associated with the disease. MELAS A3243G mutation is usually a result of mtDNA may be caused by large deletions; Kearns-Sayre syndrome (KSS) is usually related with the large fragment of mtDNA deletion, but can also be A3243G mutation. Although the mutation does not appear related to the known, when one of the following syndrome, or strongly suggest the existence of mitochondrial disease, more genetic information to guide treatment and advice.4.2.1 and A1555G deafness phenotype related 12S rRNA gene is a point mutation. This gene and other genes in mtDNA point mutations have been reported in deaf individuals. Mitochondrial gene pool record with 13 different deafness-related mtDNA mutations.4.2.2 The cyclic vomiting syndrome (CVS) CVS is the exacerbation of the performance of nausea, vomiting and lethargy, asymptomatic in the interim period. This situation is typical common migraine or familial autonomic dysfunction and other diseases. Many cases also occur neuromuscular symptoms, some associated with maternal inheritance of migraine and other familial autonomic dysfunction related to performance. Many cases appear disease-related changes in control region sequences, but lack the known common mutation in the clinical diagnosis difficult. Have reported a case of large fragments of mtDNA appears rearrangement.4.2.3 Kearns-Sayre syndrome (KSS) KSS showed chronic progressive external ophthalmoplegia (CPOE, muscle weakness, eye movements), atypical retinitis pigmentosa, muscle weakness, usually before the age of onset at 20, may be associated with one of the following symptoms: cardiac conduction defects, cerebellar ataxia, or CSF protein more than 100mg/dl. Some patients will appear dementia or endocrine diseases. The syndrome mainly by the lack of a single large fragment of mtDNA caused by common is 5kb long absence. The possible reasons include mtDNA deletion / duplication and A3243G point mutation, etc.
In addition, KSS / CPEO phenotype may also be associated with mtDNA mutations in the nuclear genes, including adenine transfer factor -1 (ANT1), Twinkle (mtDNA topoisomerase imaginary), and polymerase (POLG), which species complex mtDNA deletions can be found by autosomal dominant or recessive inheritance.4.2.4 Leigh disease, also known as subacute necrotizing encephalopathy, showing cranial nerve abnormalities, respiratory dysfunction, and associated with the basal ganglia, cerebellum or brain stem of the high-intensity signals ataxia. And other similar mitochondrial disease, Leigh disease is usually not progressive or fatal disease only when the infection worsened. Typical of its genetic diversity, there are two kinds of genomic mutations. On the common due to mitochondrial T8993G / C mutation (usually more than 95%), and some genes on the nuclear DNA defects, including complex I (NDVFV1 mutation), complex IV (such as SURF1 mutation) and PDHC and so on.4.2.5 Leber hereditary optic neuropathy (LHON) showed adolescence or early adulthood central visual loss occurs. Many cases have dystocia history. And most of the difference is that mtDNA mutations, LHON mutations usually appear homogeneous (100% mutation), a common G11778A, G3406A, and T14484C. Explicit the disease is low, the possibility of male and female blind 3 to 4 times.4.2.6 mitochondrial encephalopathy, lactic acidosis and stroke-like episodes (MELAS) manifested as reversible stroke-like episodes, usually starting from 5 to 15 years old disease, from infants to adults may be at any time in the beginning the disease, the majority of cases caused by the A3243G mutation, there are some other points by the T3271C mutation, and rearrangement of large fragment of mtDNA or nuclear gene deletion (with the exception mtDNA) cause. The disease generally does not appear coarse red fibers and abnormal electron transport chain activities.
A3243G mutation in the leucine tRNA gene in one of the two, is a mutation hot spot. Mutations affect tRNA function, thereby affecting protein synthesis in mitochondria. Not only is the cause of MELAS A3243G also been associated with maternally inherited diabetes, deafness, cognitive impairment, migraine headaches and a series of diseases.4.2.7 epileptic myoclonus and rough red fiber disease (MERRF), and clonus showed progressive dementia, usually from the beginning of childhood or adult onset. Most cases of the disease gene is located on the lysine A8344G point mutation. Some patients and other parts of the neck associated with multiple symmetric lipomas.
Rough red fiber refers to collections of mitochondrial staining in the Gomori under the red, many mitochondrial diseases, there will be different this performance, is generally not seen in children.4.2.8 Gastrointestinal neural mitochondrial encephalopathy (MNGLE) usually adult-onset, mainly to the performance of the gastrointestinal tract, including diarrhea, pseudo-obstruction (very severe constipation.) Muscle could be seen in a variety of mtDNA deletion and failure, can occur thymidine phosphorylase (nuclear encoded) in autosomal recessive mutation.4.2.9 mtDNA wasting syndrome in infancy is usually the first disease, is caused by nuclear genes encoding mtDNA genes necessary for the regular staining recessive mutations, involving polymerase, thymidine kinase 2 (TK2) and deoxyguanosine glucoside kinase (dGk) and so on mtDNA replication and deoxyguanosine to maintain the necessary gene pool. The performance of the general myopathy caused by the TK2 gene mutations, liver performance by the DGK gene mutation, and some mtDNA mutations failure patients do not appear above, that may also involve other genes to be studied further elucidated.
failure of molecular diagnosis of mtDNA muscles based (not blood) in the proportion of mtDNA relative to nuclear DNA was significantly lower than normal controls.4.2.10 neurodegenerative diseases, ataxia and retinitis pigmentosa (NARP) T8993G or T8993C mutation of its rate can reach 70% -95% of children below the Leigh disease. Leigh disease have been reported, NARP and some other less serious cases also appear in a matriarchal family. T8993G or C mutations typically affect only the energy stored into the form of complex CATP ATP synthase, which does not affect protein synthesis in mitochondria.
Ataxia means unsteady gait. Retinitis pigmentosa is a retinal disease is currently the general performance of night blindness.4.2.11 Peason disease in an infant stage of iron promyelocytic refractory anemia, hematopoietic system also includes a number of abnormalities and pancreatic function disorders. The reason that there is a single large fragment of the mtDNA deletion or deletion / rearrangement. Survivors may appear KSS. KSS / CPEO and Pearson disease, a single mtDNA deletion was considered related to multiple clinical phenotypes, which are involved and other neuromuscular system.Chinica chimica Acta, 2005,354. (Translated by Huang Jie)



Friends Links:Automation Control Blog
Automation Products Order Numbers