Diagnosis of mitochondrial diseases

Evaluation of the nature of inheritance and manifestation of clinical signs of mitochondrial diseases

Since myotochondrial diseases in some cases can be caused by damage to the nuclear genome, the transmission of the disease will correspond to the Mendelian laws of inheritance. In those cases where the development of the disease is caused by mutations in mitochondrial DNA, the inheritance will correspond to the mitochondrial type, that is, it will be transmitted through the maternal line. Finally, when the pathology develops with simultaneous damage to the genes of the nuclear and mitochondrial genomes, the inheritance will be complex and determined by various factors. In this regard, in genealogical analysis by a formal feature (the nature of inheritance according to the pedigree), it is possible to state the most different types of inheritance: autosomal dominant, autosomal recessive, X-linked, mitochondrial.

The development of such myotochondrial diseases as pyruvic acid metabolism disorder or beta-oxidation of fatty acids, Krebs cycle, is associated with mutations of nuclear genome genes. These pathologies are characterized by autosomal recessive inheritance, when the parents are carriers of mutations (heterozygotes), and the child is a carrier of both inherited mutations received from the father and mother (homozygote). The parents are usually outwardly healthy, and a similar disease or its microsigns should be sought in the brothers and sisters of the sick child (proband sibs) and in relatives on both the mother's and father's side (cousins).

In the case of a recessive, X-linked inheritance type (e.g. neonatal glutaric aciduria type II or deficiency of the E1 subunit of the pyruvic complex, Menkes disease, etc.), boys are more often affected, and mothers are carriers of mutations and pass them on to their sons. Maternal inheritance differs from X-linked inheritance in that both sexes are affected. In these cases, when analyzing the pedigree, it is necessary to analyze the incidence of the disease in men, since it will not manifest itself in women. The pedigree does not show the transmission of the disease along the father-son line, since the father can only pass on the Y chromosome to his son.

When a disease develops due to damage to the mitochondrial genome (for example, a number of respiratory chain diseases, Leber's optic neuropathy, MELAS, MERF, NARP syndromes, etc.), maternal inheritance is traced, since the child inherits mitochondria from the mother, and she can pass them on to both boys and girls. Thus, both sexes are affected equally. In this regard, the transmission of the disease along the mother's line should be traced in the pedigree.

When analyzing a family tree and searching for symptoms of mitochondrial diseases in relatives, it should be remembered that the severity of the disease (expressiveness of the sign) can vary widely, which may be due to the different number of damaged mitochondria, the nature of their damage, the different distribution of mutations in cells, etc. Thus, it is not always possible to detect obvious signs of diseases. In some cases, either individual, erased symptoms or signs are detected that can be detected during their targeted search.

The development of mitochondrial pathology may be associated with damage to large areas of mitochondria, the so-called microdeletions (for example, Kearns-Sayre syndrome, Pearson syndrome, some forms of diabetes mellitus with deafness, progressive external ophthalmoplegia, etc.). In these cases, characteristic symptoms are often not found in relatives, since the development of diseases is associated with the emergence of new mutations that occurred in the zygote immediately after fertilization of the egg ( de novo mutation ). The disease is sporadic. Often, along with these diseases, a number of conditions associated with multiple mutations of mitochondrial DNA are inherited in an autosomal dominant manner: for example, some forms of encephalomyopathy, myopathy with eye damage, despite the presence of mtDNA mutations (multiple deletions), have an autosomal dominant type of inheritance.

However, unlike Mendelian inheritance, the autosomal dominant type of inheritance in mitochondrial pathology is characterized by a large number of affected individuals in subsequent generations.

Finally, some mitochondrial diseases, often associated with depletion of mtDNA mitochondria or their absence in cells, can be inherited in an autosomal recessive manner. These include congenital forms of myopathy, cardiomyopathy, neurodistress syndrome, lactic acidosis, liver damage, etc.

The study of the nature of the hereditary transmission of the disease is important for medical and genetic prognosis and requires a deep analysis of clinical signs with knowledge of the mechanisms of formation of mitochondrial pathology and the types of its inheritance.

The manifestation of clinical manifestations varies widely from the first days of life to adulthood. When analyzing this indicator, it is necessary to take into account nosological forms, since each of them has a certain age of debut.

Metabolic disorders observed in mitochondrial diseases are progressive in the vast majority of cases. Initial symptoms are often mild, then progress and can lead to significant disabling disorders. Rare forms of pathology, such as benign infantile myopathy and some forms of Leber's optic neuropathy, can be benign and subject to regression.

During laboratory testing, attention is paid to the characteristic signs of mitochondrial diseases:

• presence of acidosis;
• elevated levels of lactate and pyruvate in the blood, an increase in the lactate/pyruvate index of more than 15, especially increasing with glucose load or physical exercise;
• hyperketonemia;
• hypoglycemia;
• hyperammonemia;
• increased concentrations of acetoacetate and 3-hydroxybutyrate;
• increased 3-hydroxybutyric acid/acetoacetic acid ratio in the blood;
• increased levels of amino acids in the blood and urine (alanine, glutamine, glutamic acid, valine, leucine, isoleucine);
• elevated levels of fatty acids in the blood;
• hyperexcretion of organic acids in urine;
• decreased levels of carnitine in the blood;
• increase in myoglobin content in biological fluids;
• decreased activity of mitochondrial enzymes in myocytes and fibroblasts.

The diagnostic value of these indicators is higher with a food load than on an empty stomach. In practice, a diagnostic test has proven itself well: determining lactate in the blood against the background of a glucose load, which allows for a more distinct detection of the failure of the respiratory chain to an additional glucose load.

For laboratory diagnostics of mitochondrial dysfunction and its specific nosological forms, conventional, routine biochemical methods of research are not enough; it is necessary to conduct special tests. It is especially convenient to analyze the activity of enzymes in skeletal muscle biopsies than in other tissues. It is possible to determine the activity of respiratory chain enzymes, especially citrate synthetase, succinate dehydrogenase and cytochrome C oxidase.

Data from morphological and molecular genetic studies

Morphological studies are of particular importance in the diagnosis of mitochondrial pathology. Due to their great informative value, muscle tissue biopsy and histochemical examination of the obtained biopsies are often required. Important information can be obtained by simultaneously examining the material using light and electron microscopy.

One of the important markers of mitochondrial diseases is the phenomenon of "ragged" red fibers [the RRF phenomenon (ragged red fibres)], established in 1963. It is associated with the formation of genetically altered abnormal mitochondria along the edge of the muscle fiber due to proliferation and focal accumulation. This phenomenon is detected by light microscopy using a special Gomori stain, but in recent years various mitochondrial markers and various immunological methods have been used for this purpose.

Other morphological signs of mitochondrial pathology include:

• a sharp increase in the size of mitochondria;
• accumulation of glycogen, lipids and calcium conglomerates in the subsarcolemma;
• decreased activity of mitochondrial enzymes;
• disrupted distribution of granules of activity of enzymes succinate dehydrogenase (SDH), NADH oxidoreductase, cytochrome C oxidase, etc.

In patients with mitochondrial diseases, light microscopy of muscle tissue can reveal non-specific morphological signs: local necrosis of muscle fibers, accumulation of sarcoplasmic masses, the presence of lysis of subsarcolemmal areas of sarcoplasm, basophilia of sarcoplasm, an increased number of muscle nuclei, activation of regeneration processes, etc.

The study of the role of the phenomenon of "ragged" red fibers showed its importance for the diagnosis of such conditions as MELAS, MERRF, Kearns-Sayre syndromes, chronic progressive ophthalmoplegia and others associated with mtDNA mutations. This phenomenon can develop in other diseases: Duchenne muscular dystrophy, dermatomyositis, myotonic dystrophy, taking medications (clofibrate) and other pathological conditions. Thus, along with primary mitochondrial diseases, the RRF phenomenon can accompany secondary mitochondrial dysfunctions.

Currently, histochemical and electron microscopic examination of muscle tissue to detect signs of mitochondrial insufficiency has become widespread. In some cases, they help in diagnosis, especially with normal morphological picture of muscle tissue according to light microscopy data.

Electron microscopic signs - detection of mitochondrial proliferation, disruption of their shape and size, disorganization and enlargement of cristae, accumulation of abnormal mitochondria under the sarcolemma, accumulation of lipid and abnormal paracrystalline (mainly consisting of protein) or osmophilic inclusions localized between the inner and outer membranes or within the cristae, spherical clusters, often located in the matrix (consisting mainly of triglycerides), etc.

In some patients, cytochemical abnormalities in leukocytes can be detected.

The complex of biochemical and morphological studies is supplemented by modern methods of molecular diagnostics (detection of nuclear or mitochondrial mutations), which are performed in specialized DNA diagnostic laboratories. In mitochondrial diseases, various types of mutations are detected: point mutations, deletions, duplications, quantitative DNA anomalies, etc.

In the absence of mutations in mtDNA, if mitochondrial pathology is suspected, a nuclear DNA study is performed.

Diagnostic criteria

There are 2 groups of diagnostic criteria for mitochondrial diseases. Main diagnostic criteria (first group).

• Clinical:
  • established diagnoses: MERRF, MELAS, NARP, MNGIE, Pearson syndromes, Leber neuropathy, Leigh and Alpers diseases;
  • the presence of 2 or a combination of the following signs:
    • multisystemic lesion, pathognomonic for respiratory chain diseases;
    • progressive course with episodes of exacerbation or the presence of mitochondrial mutations in the family;
    • exclusion of metabolic and other diseases by conducting appropriate tests.
• Histological - detection of the RRF phenomenon in more than 2% of muscle tissue.
• Enzymatic:
  • cytochrome c oxidase-negative fibers;
  • decreased activity of enzymes of the respiratory chain complex (<20% of normal in tissue, <30% in cells or several tissues).
• Functional - a decrease in ATP synthesis in fibroblasts by more than 3 standard deviations.
• Molecular genetic - pathogenetically significant mutations of nuclear or mtDNA.

Additional diagnostic criteria (second group).

• Clinical - non-specific symptoms that occur in diseases of the respiratory chain (stillbirth, decreased motor activity of the fetus, early neonatal death, movement disorders, developmental disorders, impaired muscle tone in the neonatal period).
• Histological - small percentage of RRF phenomenon, subsarcolemmal accumulation of mitochondria or their anomalies.
• Enzymatic - low activity of respiratory complex enzymes (20-30% of the norm in tissue, 30-40% in cells or cell lines).
• Functional - a decrease in ATP synthesis in fibroblasts by 2-3 standard deviations or the absence of fibroblast growth in a medium with galactose.
• Molecular genetic - detection of mutations in nuclear or mtDNA with a putative pathogenetic link.
• Metabolic - detection of one or more metabolites indicating a disturbance in cellular bioenergetics.

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