Diagnosis of mitochondrial diseases

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

Due to the fact that myotochondrial diseases in some cases can be caused by damage to the nuclear genome, transmission of the disease will correspond to the Mendelian laws of inheritance. In the same cases, when the development of the disease is caused by mitochondrial DNA mutations, inheritance will correspond to the mitochondrial type, that is, to be transmitted along the maternal line. Finally, when pathology develops with simultaneous damage to the genes of nuclear and mitochondrial genomes, inheritance will be complex and will be determined by various factors. In this connection, genealogical analysis based on a formal characteristic (the nature of genealogy inheritance) reveals a variety of inheritance types: autosomal dominant, autosomal recessive, X-linked, mitochondrial.

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

In the case of recessive, X-linked type of inheritance (for example, the neonatal form of glutar aciduria II type or the deficiency of the subunit E1 of the pyruvic complex, Menkes disease, etc.), the boys are more often affected, and mothers are carriers of mutations and transmit them to their sons. Maternal inheritance differs from X-linked by defeat of persons of both sexes. In these cases, when analyzing the pedigree, it is necessary to analyze the incidence in men, since it will not appear in women. The genealogy does not trace the transmission of the disease along the father-son line, since the father can only give the son to the Y chromosome.

In the development of the disease, due to damage to the mitochondrial genome (for example, a number of respiratory chain diseases, Leber's optical neuropathies, MELAS, MERF, NARP, etc.), maternal inheritance can be traced, as the mitochondrial child inherits from the mother, and she can transmit them as boys , and girls. Thus, people of both sexes are equally affected. In this regard, the pedigree should be followed by the transmission of the disease along the mother's line.

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

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

However, in contrast to the Mendelian 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 hereditary transmission of the disease is of great importance for the medical-genetic prognosis and requires an in-depth analysis of clinical signs with knowledge of the mechanisms of mitochondrial pathology formation and types of its inheritance.

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

Exchange disorders observed in mitochondrial diseases are, in most cases, progressive. Initial symptoms are often mild, subsequently progressing and can lead to significant disabling disorders. Rare forms of pathology, such as benign infantile myopathy and some forms of Leber's optical neuropathy, can be favorable and reverse.

At laboratory research pay attention to characteristic attributes of mitochondrial diseases:

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

The diagnostic value of these indicators is higher for food load than for fasting. In practice, a diagnostic test has proven itself: the determination of lactate in the blood on the background of glucose loading, which makes it possible to more clearly identify the inconsistency of the respiratory chain with an additional load of glucose.

For laboratory diagnosis of mitochondrial dysfunction and its specific nosological forms, routine, routine biochemical methods of investigation are not enough, special tests are necessary. It is especially convenient to analyze enzyme activity in skeletal muscle biopsy specimens 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 of morphological and molecular genetic studies

Morphological studies in the diagnosis of mitochondrial pathology are of particular importance. Due to the great informative importance, it is often necessary to perform a biopsy of the muscle tissue and histochemical examination of the obtained biopsy specimens. Important information can be obtained by simultaneous examination of the material by light and electron microscopy.

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

Other morphological features of mitochondrial pathology include:

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

In patients with mitochondrial diseases with light microscopy of muscle tissue, nonspecific morphological features can be identified: local necrosis of muscle fibers, accumulation of sarcoplasmic masses, presence of lysis of subarachromolemal areas of sarcoplasm, basophilia of sarcoplasm, increased number of muscle nuclei, activation of regeneration processes, etc.

The study of the role of the phenomenon of "torn" red fibers showed its importance for the diagnosis of such conditions as MELAS, MERRF, Cairns-Seir syndrome, chronic progressive ophthalmoplegia and others associated with mtDNA mutations. This phenomenon can develop in other diseases: Duchenne myodystrophy, dermatomyositis, myotonic dystrophy, medication intake (clofibrate), and other pathological conditions. Thus, along with primary mitochondrial diseases, the phenomenon of RRF may accompany secondary mitochondrial dysfunctions.

At present, histochemical and electron microscopic examination of muscle tissue has been widely used to detect signs of mitochondrial insufficiency. In some cases, they help diagnosis, especially with a normal morphological picture of the muscle tissue according to light microscopy.

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

In some patients, it is possible to detect cytochemical abnormalities in leukocytes.

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

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

Criteria for diagnosis

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

• Clinical:
  • established diagnoses: syndromes MERRF, MELAS, NARP, MNGIE, Pearson, Leber's neuropathy, Leah's disease, Alpers;
  • Presence 2 or a combination of the following features:
    • multisystemic damage pathognomonic for respiratory chain diseases;
    • progressing 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 - the detection of the phenomenon of RRF in more than 2% of muscle tissue.
• Enzymatic:
  • cytochrome C-oxidase-negative fibers;
  • a decrease in the activity of enzymes of the respiratory chain complex (<20% of the norm in the tissue, <30% in cells or several tissues).
• Functional - decrease in ATP synthesis in fibroblasts more than 3 standard deviations.
• Molecular-genetic - pathogenetically significant mutations of nuclear or mtDNA.

Additional diagnostic criteria (second group).

• Clinical - nonspecific symptoms that occur in diseases of the respiratory chain (stillbirth, decreased motor activity of the fetus, early neonatal death, motor disorders, developmental disorders, muscular tonus disorder in the neonatal period).
• Histological - a small percentage of the phenomenon of RRF, subarachromolemal accumulation of mitochondria or their anomalies.
• Enzymatic - a low activity of the enzymes of the respiratory complex (20-30% of the norm in tissues, 30-40% - in cells or cell lines).
• Functional - reducing the synthesis of ATP in fibroblasts by 2-3 standard deviations or lack of growth of fibroblasts in a medium with galactose.
• Molecular-genetic - the detection of mutations of nuclear or mtDNA with a presumed pathogenetic link.
• Metabolic - the detection of one or more metabolites, indicating a violation of cellular bioenergetics.

[1], [2], [3], [4]