The concept of mitochondrial diseases. Encephalomyopathic mitochondrial DNA depletion syndrome. DNA depletion syndrome

02.07.2020

There are a large number of chronic diseases, one of the pathogenetic links of which is secondary mitochondrial deficiency. Their list is far from complete and is expanding to this day.

All these disorders are polymorphic, may have varying degrees of severity and be of interest to medical specialists in various fields - neuropathologists, cardiologists, neonatologists, nephrologists, surgeons, urologists, otorhinolaryngologists, pulmonologists, etc.

According to our data, at least a third of all disabled children in the symptom complex of their diseases have signs of a polysystemic disorder of cellular energy. It should be noted that in recent years the number of children with diseases accompanied by a high probability of tissue hypoxia has significantly increased.

Studies recently conducted at the Moscow Research Institute of Pediatrics and Pediatric Surgery in children admitted to a genetic clinic with undifferentiated disorders of physical and neuropsychic development showed that half of them had disturbances in cellular energy exchange. For the first time, employees of this institute discovered the presence of mitochondrial disorders in such pathologies in children: connective tissue diseases (Marfan and Ehlers-Danlos syndromes), tuberous sclerosis, a number of non-endocrine syndromes accompanied by growth retardation (osteochondrodysplasia, Aarskog syndrome, Silver-Russell syndrome, etc.), the influence of mitochondrial deficiency on the course of a number of cardiological, hereditary, surgical and other diseases was revealed. Together with the staff of the Smolensk Medical Academy, a decompensating mitochondrial insufficiency was described in type 1 diabetes mellitus in children with a disease period of more than 5 years.

Of particular note are polysystemic mitochondrial dysfunctions caused by ecopathogenic factors. Among the latter are both well-known (for example, carbon monoxide, cyanides, heavy metal salts) and relatively recently described (primarily side effects of a number of drugs - azidothymidine, valproates, aminoglycosides, and some others). In addition, the same group includes mitochondrial dysfunctions caused by a number of nutritional disorders (primarily a deficiency of B vitamins).

Finally, it should be mentioned separately that, according to many researchers, an increase in the number of mitochondrial dysfunctions is, if not the main, then one of the most important mechanisms of aging. At the International Symposium on Mitochondrial Pathology, held in Venice in 2001, the discovery of specific mitochondrial DNA mutations that appear with aging was reported. These mutations are not found in young patients, and in the elderly they are determined in various cells of the body with a frequency of over 50%.

Pathogenesis.

A decrease in oxygen delivery to the nerve cell under conditions of acute ischemia leads to a number of regulatory functional and metabolic changes in mitochondria, among which disturbances in the state of mitochondrial enzyme complexes (MFCs) play a leading role and which lead to the suppression of aerobic energy synthesis. The general response of the body to acute oxygen deficiency is characterized by the activation of urgent regulatory compensatory mechanisms. In a neuronal cell, cascade mechanisms of intracellular signal transduction are activated, which are responsible for gene expression and the formation of adaptive traits. Such activation appears already after 2-5 minutes of oxygen starvation and proceeds against the background of a decrease in respiration associated with the suppression of MFC-1. Confirmation of the involvement of intracellular signaling systems in adaptive processes, which are necessary for the formation of genome-dependent adaptive reactions, is the activation of protein kinases - the final links of these systems, the opening of the mito-KATP channel, the enhancement of the ATP-dependent K+ transport associated with it, and increased generation of H2O2.

At this stage of adaptive reactions, the key role is assigned to the families of the so-called early genes, the products of which regulate the expression of late-acting genes. To date, it has been established that in the brain these genes include NGFI-A, c-jun, junB, c-fos, which play an important role in the processes of neuronal plasticity, learning, survival/death of neurons. When preconditioning had a protective effect and corrected the disturbances caused by severe hypoxia in hypoxia-sensitive brain structures, an increase in the expression of mRNA of all these genes, as well as mRNA of mitochondrial antioxidant genes, was observed.

A longer stay in conditions of reduced oxygen content is accompanied by a transition to a new level of regulation of oxygen homeostasis, which is characterized by economization of energy metabolism (a change in the kinetic properties of oxidative metabolism enzymes, which is accompanied by an increase in the efficiency of oxidative phosphorylation, the emergence of a new population of small mitochondria with a set of enzymes that allow them to work in this new mode). In addition, under these conditions, adaptation to hypoxia at the cellular level is closely related to the transcriptional expression of hypoxia-induced late-acting genes that are involved in the regulation of multiple cellular and systemic functions and are necessary for the formation of adaptive traits. It is known that at low oxygen concentrations, this process is primarily controlled by a specific transcription factor induced by hypoxia in all tissues (HIF-1). This factor, discovered in the early 1990s, functions as the main regulator of oxygen homeostasis and is the mechanism by which the body, in response to tissue hypoxia, controls the expression of proteins responsible for the mechanism of oxygen delivery to the cell, i.e. regulates adaptive cell responses to changes in tissue oxygenation.

Currently, more than 60 direct target genes have been identified for it. All of them contribute to the improvement of oxygen delivery (erythropoiesis, angiogenesis), metabolic adaptation (glucose transport, increased glycolytic ATP production, ion transport) and cell proliferation. HIF-1 regulated products act at different functional levels. The end result of this activation is an increase in O2 entry into the cell.

The identification and cloning of HIF-1 made it possible to establish that it is a heterodimeric redox-sensitive protein consisting of two subunits: the inducibly expressed oxygen-sensitive HIF-1b subunit and the constitutively expressed HIF-1c subunit (aryl hydrocarbon receptor nuclear translocator). -- ARNT). Heterodimerizing with the arylcarboxylic receptor (AHR), it forms a functional dioxin receptor. Other proteins of the HIF-1b family are also known: HIF-2b, HIF-3b. All of them belong to the family of basic proteins containing in the amino acid terminal part of each subunit the basic helix-loop-helix (bHLH) domain, which is characteristic of a wide variety of transcription factors and is necessary for dimerization and binding. DNA.

HIF-1b consists of 826 amino acid residues (120 kD) and contains two transcription domains at the C-terminal end. Under normoxic conditions, its synthesis occurs at a low rate and its content is minimal, since it undergoes rapid ubiquitination and degradation by proteasomes. This process depends on the interaction of the primary structure of HIF-1b and its specific oxygen-dependent degradation domain (ODDD) with the von Hippel Lindau (VHL) protein, which is widespread in tissues, a tumor growth suppressor that acts as a protein ligase. .

The molecular basis for such regulation is the O2-dependent hydroxylation of its two proline residues P402 and P564, which are part of the structure of HIF-1b, by one of three enzymes collectively known as “proteins of the prolyl hydroxylase domain (PHD)”, or “HIF-1b-prolylyl hydroxylase ”, which is necessary for the binding of HIF-1b to the VHL protein. Obligatory components of the process are also β-ketoglutarate, vitamin C and iron. Along with this, hydroxylation of the asparagine residue in the C-terminal transactivation domain (C-TAD) occurs, which leads to the suppression of the transcriptional activity of HIF-1b. After hydroxylation of the proline residues in ODDD and the asparagine residue, HIF-1b binds to the VHL protein, which makes this subunit of proteasomal degradation available.

Under conditions of a sharp oxygen deficiency, the oxygen-dependent process of hydroxylation of prolyl residues, which is characteristic of normoxia, is suppressed. Because of this, VHL cannot bind to HIF-1b, its degradation by proteasomes is limited, which makes its accumulation possible. In contrast, p300 and CBP can bind to HIF-1b, since this process does not depend on asparaginyl hydroxylation. This ensures the activation of HIF-1b, its translocation to the nucleus, dimerization with HIF-1b, leading to conformational changes, the formation of a transcriptional active complex (HRE), which triggers the activation of a wide range of HIF-1-dependent target genes and the synthesis of protective adaptive proteins in response. for hypoxia.

The above mechanisms of intracellular signal transduction occur in the cell during its adaptation to hypoxia. In the case when disadaptation sets in, a significant concentration of ROS accumulates in the cell, and the processes of its apoptotic death are activated.

Among the former are, in particular, the transfer of phosphatidylserine to the outer membrane layer and DNA fragmentation under the action of ROS and NO. In this membrane, phosphatidylserine is usually present only in the inner lipid layer. Such an asymmetric distribution of this phospholipid is due to the action of a special transport ATPase that transfers phosphatidylserine from the outer lipid layer of the plasma membrane to the inner one. This ATPase is either inactivated by the oxidized form of phosphatidylserine or simply "does not recognize" the oxidized phospholipid. That is why the oxidation of phosphatidylserine by ROS leads to its appearance in the outer layer of the plasma membrane. Apparently, there is a special receptor that detects phosphatidylserine in the outer lipid layer. It is assumed that this receptor, by binding phosphatidylserine, sends a signal of apoptosis into the cell.

Phosphatidylserine plays a key role in the so-called forced apoptosis caused by a certain type of leukocyte. A cell with phosphatidylserine in the outer layer of the cell membrane is "recognized" by these leukocytes, which initiate its apoptosis. One of the apoptogenic mechanisms used by leukocytes is that leukocytes begin to secrete proteins perforin and granzymes into the intercellular space near the target cell. Perforin makes holes in the outer membrane of the target cell. Granzymes enter the cell and trigger apoptosis in it.

Another way used by the leukocyte to force the target cell to enter apoptosis is to bombard it with superoxide produced outside the leukocyte via a special transmembrane respiratory chain of the plasma membrane. This chain oxidizes intracellular NADPH, from which electrons are transferred to flavin and then to a special cytochrome b, which can be oxidized by oxygen to release superoxide outside the leukocyte. Superoxide and other ROS formed from it oxidize the plasma membrane phosphatidylserine of the target cell, thereby enhancing the apoptotic signal sent to the cell by this phospholipid.

In addition, leukocytes include tumor necrosis factor. TNF binds to its receptor on the outer side of the plasma membrane of the target cell, which activates several parallel pathways for triggering apoptosis. In one of them, the formation of active caspase-8 from pro-caspase-8 occurs. Caspase-8 is a protease that cleaves the cytosolic Bid protein with the formation of its active form tBid (truncated Bid). tBid changes the conformation of another protein, Bax, causing the formation of a protein-permeable channel in the outer membrane of mitochondria, which leads to their exit from the intermembrane space into the cytosol.

The diversity of pathways of ROS-dependent apoptosis is illustrated in Fig. 1. The true picture, in all likelihood, is even more complex, since in addition to TNF there are other extracellular inducers of apoptosis (cytokines), each acting through its own receptor. In addition, there are anti-apoptotic systems that oppose pro-apoptotic systems. Among them are proteins of the Bcl-2 type, which inhibit the proapoptotic activity of Bax; the already mentioned caspase inhibitors (IAP); protein NFkB (nuclear factor kB) induced by TNF. NFkB includes a group of genes, among which are those encoding superoxide dismutase and other antioxidant and anti-apoptotic proteins.

All these difficulties reflect the obvious circumstance that for a cell "the decision to commit suicide" is an extreme measure when all other possibilities to prevent its erroneous actions have been exhausted.

Taking into account the above, we can imagine the following scenario of events designed to protect the body from ROS generated by mitochondria. Formed in mitochondria, ROS cause the opening of a pore and, as a consequence, the release of cytochrome C into the cytosol, which immediately activates additional antioxidant mechanisms, and then mitoptosis. If only a small part of the intracellular population of mitochondria goes into mitoptosis, the concentrations of cytochrome C and other mitochondrial proapoptotic proteins in the cytosol do not reach the values necessary to activate apoptosis. If more and more mitochondria become ROS superproducers and “open kingstones”, these concentrations increase and apoptosis of the cell containing many defective mitochondria begins. As a result, the tissue is cleared of cells whose mitochondria produce too much ROS.

Thus, we can speak of mitochondrial dysfunction as a new pathobiochemical mechanism of a wide range of neurodegenerative disorders. Currently, two types of mitochondrial dysfunction are distinguished - primary, as a result of a congenital genetic defect, and secondary, arising under the influence of various factors: hypoxia, ischemia, oxidative and nitrosative stress, and expression of pro-inflammatory cytokines. In modern medicine, the doctrine of polysystemic disorders of cellular energy metabolism, the so-called mitochondrial pathology, or mitochondrial dysfunction, occupies an increasingly important place.

Mitochondrial dysfunctions are a heterogeneous group of pathologies caused by genetic, biochemical and structural and functional defects of mitochondria with impaired cellular and tissue respiration. The classification of mitochondrial dysfunction has its own history. One of the first was a scheme based on biochemical defects in metabolism. The systematization by clinical syndromes was also not deep enough, among them the following were previously distinguished:

1) syndromes of established mitochondrial nature;
2) syndromes of presumably mitochondrial nature;
3) syndromes are consequences of mitochondrial pathology.

The first mention of a disease associated with a defect in mitochondria refers to 1962: R. Luft et al. described a case of a disease in which there was a violation of the conjugation of respiration and phosphorylation in the mitochondria of skeletal muscles in a patient with non-thyroidal hypermetabolism. In subsequent years, the clinical, biochemical and morphological aspects of mitochondrial encephalomyopathies were described. The use of modified Gomori staining played an important role in the development of this direction, with the help of which it was possible to detect fibers with altered mitochondria in skeletal muscles - the so-called ragged-red fibers (RRF).

Later, with the discovery of the mitochondrial genome and mDNA or nuclear DNA mutations, it was possible to apply the genetic principle of classification for primary, congenital mitochondrial dysfunction - first in a simplified form, then in a more complicated one. The key area of mitochondrial pathology is hereditary syndromes, which are based on mutations in the genes responsible for mitochondrial proteins (Kearns-Sayre syndromes, MELAS, MERRF, Pearson, Barth, etc.). Mitochondrial dysfunctions are manifested by a wide range of clinical symptoms. These mutations may involve tRNA, rRNA, or structural genes and may be expressed biochemically as defects in the entire electron transport chain or as defects in individual enzymes.

Throughout the 1990s, the identification of many mitochondrial defects that cause clinically very different disorders baffled clinicians regarding the diagnosis of heterogeneous and complex syndromes characterized by the following features:

- skeletal muscles: low exercise tolerance, hypotension, proximal myopathy, including facial and pharyngeal muscles, ophthalmoparesis, ptosis;
- heart: cardiac arrhythmias, hypertrophic myocardiopathy;
- CNS: optic nerve atrophy, retinopathy pigmentosa, myoclonus, dementia, stroke-like episodes, mental disorders;
- peripheral nervous system: axonal neuropathy, impaired motor activity of the gastrointestinal tract;
-- endocrine system: diabetes, hypoparathyroidism, impaired exocrine pancreatic function, short stature.

Since primary mitochondrial dysfunction manifests itself in a person with a number of different symptoms, clinicians have tried to combine some groups of the most common combinations of symptoms into syndromes:

MELAS - Mitochondrial Myopathy, Encephalopathy, Lactic Acidosis and Stroke-like episodes (mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes).
CPEO / PEO - External Ophtalmoplegia, Ophtalmoplegia plus syndrome (ophthalmoplegia associated with damage to the oculomotor muscles, ophthalmoplegia plus syndrome).
KSS - Kearns - Sayre Syndrome - retinopathy, proximal muscle weakness, cardiac arrhythmia and ataxia (retinopathy, proximal muscle weakness, arrhythmia, ataxia).
· MERRF -- Myoclonic Epilepsy associated with Ragged Red Fibres.
LHON - Leber Hereditary Optic Neuropathy (congenital neuropathy of the optic nerve).
· Leig syndrome -- infantile subacute necrotizing encephalopathy (infantile subacute necrotizing encephalopathy).
· NAPR -- Neuropathy, Ataxia and Pigmentary Retinopathy (neuropathy, ataxia and pigmentary retinopathy).

post updated 02/28/2019

Introduction(features of human mitochondria). A feature of the functioning of mitochondria is the presence of their own mitochondrial genome - circular mitochondrial DNA (mtDNA) containing 37 genes, the products of which are involved in the process of energy production in the respiratory chain of mitochondria. mtDNA is located in the inner membrane of mitochondria and consists of five conjugated enzyme complexes, which have a total of 86 subunits. They are mainly encoded by nuclear genes (nDNA), but seven subunits of the first enzyme complex (ND1, 2, 3, 4, 4L, 5, 6), one of the third (cytochrome b), three of the fourth (COI, COII, COIII) and two of the fifth (ATPase 6 and 8) are encoded by mtDNA structural genes. Thus, enzyme complexes (i.e., proteins) encoded by both nuclear (nDNA) and mitochondrial genes (mtDNA) are involved in providing diverse biochemical functions of mitochondria.

note! The main biochemical processes that are related to energy metabolism and occur in mitochondria are: tricarboxylic acid cycle (Krebs cycle), beta-oxidation of fatty acids, carnitine cycle, electron transport in the respiratory chain and oxidative phosphorylation. Any of these processes can be disturbed and cause mitochondrial insufficiency.

Cause of mitochondrial disease (hereinafter MB). The main properties of the mitochondrial genome are the cytoplasmic inheritance of genes, the absence of recombinations (i.e., the reorganization of genetic material through the exchange of individual segments, regions, DNA double helixes) and a high mutation rate. The mitochondrial genome is characterized by pronounced instability and a high rate of nucleotide substitutions, on average 10–17 times higher than the mutation rate of nuclear genes, and somatic mutations often occur in it during the life of an individual. The immediate cause of the onset and development of mitochondrial dysfunction lies in defects in the oxidative phosphorylation system, imperfection of repair mechanisms, the absence of histones, and the presence of free oxygen radicals, which are by-products of aerobic respiration.

Mutations in the mitochondrial genome are characterized by the phenomenon [ !!! ] heteroplasmy, in which (due to the specificity of mitochondrial inheritance), as a result of cell division, the distribution (which varies widely - from 1 to 99%) of mutant mtDNA between daughter cells occurs randomly and unevenly, as a result of which copies of mtDNA carrying normal and/or mutant allele. At the same time, different tissues of the body or neighboring areas of the same tissue may differ in the degree of heteroplasmy, i.e. according to the degree of presence and ratio in the cells of the body of mitochondria with both mutant and normal mtDNA (in subsequent generations, some cells may have only normal mtDNA, another part only mutant, and a third part - both types of mtDNA). The content of mitochondria with mutant mtDNA increases gradually. Due to this “lag period” (from the English “lag” - delay), future patients often reach sexual maturity (and give birth to offspring that almost always carry the same mutations in mtDNA). When the number of mutant copies of mtDNA in a cell reaches a certain concentration threshold, the energy metabolism in cells is significantly impaired and manifests itself in the form of a disease (note: a feature of hereditary MB is often the complete absence of any pathological signs at the beginning of the patient's life).

note! Heteroplasmy is characterized by the simultaneous existence of mutant and normal mtDNA in the same cell, tissue, or organ, which determines the severity, nature, and age of MB manifestation. The number of altered mtDNA can also increase with age under the influence of various factors and gradually reach a level that can cause clinical manifestations of the disease.

In accordance with the above features of the double mitochondrial genome, the type of MB inheritance can be different. Since mtDNA in the body is almost exclusively of maternal origin, when a mitochondrial mutation is transmitted to offspring, a maternal type of inheritance takes place in the pedigree - all children of a sick mother get sick. If a mutation occurs in a nuclear gene (nDNA) encoding the synthesis of a mitochondrial protein, the disease is transmitted according to classical Mendelian laws. Sometimes a mtDNA mutation (usually a deletion) occurs de novo at an early stage of ontogeny, and then the disease manifests itself as a sporadic case.

note! Currently, more than 100 point mutations and several hundred mtDNA structural rearrangements are known to be associated with characteristic neuromuscular and other mitochondrial syndromes ranging from lethal in the neonatal period of life to diseases with a late onset.

Definition. MB can be characterized as diseases caused by genetic and structural-biochemical defects of mitochondria and accompanied by a violation of tissue respiration and, as a result, a systemic defect in energy metabolism, as a result of which the most energy-dependent tissues and target organs are affected in various combinations: the brain, skeletal muscles and myocardium. (mitochondrial encephalomyopathies), pancreas, organ of vision, kidneys, liver. Clinically, violations in these organs can be realized at any age. At the same time, the heterogeneity of symptoms complicates the clinical diagnosis of these diseases. The need to exclude MB arises in the presence of multisystem manifestations that do not fit into the usual pathological process. The frequency of respiratory chain dysfunction is estimated from 1 per 5-10 thousand to 4-5 per 100 thousand newborns.

Semiotics. Neuromuscular pathology in MB is usually represented by dementia, seizures, ataxia, optic neuropathy, retinopathy, sensorineural deafness, peripheral neuropathy, and myopathy. However, about 1/3 of MB patients have normal intelligence and no neuromuscular manifestations. MB includes, in particular, Kearns-Sayre encephalocardiomyopathy (retinitis pigmentosa, external ophthalmoplegia, complete heart block); MERRF syndrome (myoclonus epilepsy, "torn" red fibers); (mitochondrial encephalomyopathy, lactic acidosis, stroke-like episodes); Pearson syndrome (encephalomyopathy, ataxia, dementia, progressive external ophthalmoplegia); NAPR syndrome (neuropathy, ataxia, retinitis pigmentosa); and some forms of ophthalmopathic myopathy. All these forms are united by a myopathic syndrome expressed to one degree or another.

note! The two main clinical signs of MB are the increase over time in the number of organs and tissues involved in the pathological process, as well as the almost inevitable damage to the central nervous system. The polymorphism of clinical manifestations, including organ damage, at first glance, physiologically and morphologically unrelated, combined with different periods of manifestation and the steady progression of disease symptoms with age, makes it possible to suspect a [genetic] mtDNA mutation.

note! In clinical practice, the ability to differentiate the clinical picture of MB from more common somatic, autoimmune, endocrine and other pathological conditions, most of which are treatable, is of great importance. It is necessary to conduct a thorough assessment of the family history, data from routine clinical and laboratory-instrumental methods of examination, before assigning specific genetic and biochemical tests to the patient, aimed at the search for mitochondrial pathology.

Diagnostics . The algorithm for diagnosing any MB should include the following steps: [ 1 ] identification of a typical clinical picture of the mitochondrial syndrome or an "inexplicable" multisystemic lesion and a hereditary history confirming the maternal type of inheritance; [ 2 ] further diagnostic search should be aimed at detecting common markers of mitochondrial dysfunction: an increase in the level of lactate/pyruvate in the blood serum and cerebrospinal fluid, a violation of carbohydrate, protein, amino acid metabolism, as well as a clinical picture involving at least three of these systems in the pathological process: CNS, cardiovascular system, muscular, endocrine, renal, organs of vision and hearing; [ 3 ] in case of clinical and confirmed laboratory and instrumental signs of mitochondrial pathology, PCR analysis of blood lymphocytes is performed for targeted search for mtDNA point mutations; a study that is considered the gold standard for diagnosing MB [cytopathies] - a biopsy of skeletal muscles with histochemical, electron microscopic, immunological and molecular genetic analyzes, characteristic changes in which will be with any MB (see below); [ 5 ] the most sensitive tests for diagnosing MB are methods for assessing the level of pathological mtDNA heteroplasmy in various organs and tissues: fluorescent PCR, cloning, denaturing high-performance liquid chromatography, sequencing, Southern blot hybridization, etc.

Histochemical study of muscle biopsy specimens of patients, including trichrome staining according to the Gomory method, demonstrates changes characteristic of MB - torn red fibers of myofibrils, which contain a large number of proliferating and damaged mitochondria, forming agglomerates along the periphery of the muscle fiber. In this case, the number of torn red fibers in the biopsy should be ≥ 2%. Enzyme-histochemical analysis shows a deficiency of cytochrome C-oxidase in 2 and 5% of myofibrils (for patients younger than 50 and older than 50 years) of their total number in biopsy specimens. Histochemical analysis of succinate dehydrogenase (SDH) activity demonstrates CDH-positive staining of myofibrils (ragged blue fibers), which, in combination with SDH-positive staining of arterial walls that supply blood to muscles, indicates a high degree of damage to myocyte mitochondria. When conducting electron microscopy of muscle biopsy specimens, pathological inclusions, structural rearrangements of mitochondria, changes in their shape, size and number are determined.

note! Despite significant progress since the discovery of mtDNA genetic mutations, most of the diagnostic methods used in clinical practice have a low degree of specificity for individual MBs. Therefore, the diagnostic criteria for a particular MB, first of all, consist of a combination of specific clinical and morphological patterns.

Principles of treatment . Therapy for MB (cytopathies) is exclusively symptomatic and is aimed at reducing the rate of progression of the disease, as well as improving the quality of life of patients. For this purpose, patients are prescribed a standard combination of drugs, including coenzyme Q10, idebenone - a synthetic analogue of CoQ10, creatine, folic acid, vitamins B2, B6, B12 and other drugs that improve redox reactions in cells (electron carrier drugs in the respiratory chain and cofactors of enzymatic reactions of energy metabolism). These compounds stimulate the synthesis of ATP molecules and reduce the activity of free radical processes in mitochondria. Meanwhile, according to a systematic review, most of the drugs with antioxidant and metabolic effects used in MB have not been evaluated in large randomized placebo-controlled trials. Therefore, it is difficult to assess the severity of their therapeutic effect and the presence of significant side effects.

Read more about MB in the following sources:

article "Neuromuscular pathology in mitochondrial diseases" L.A. Saykova, V.G. Pustozers; St. Petersburg Medical Academy of Postgraduate Education of Roszdrav (magazine "Bulletin of the St. Petersburg Medical Academy of Postgraduate Education" 2009) [read];

article "Chronic diseases of non-inflammatory genesis and mutations of the human mitochondrial genome" K.Yu. Mitrofanov, A.V. Zhelankin, M.A. Sazonova, I.A. Sobenin, A.Yu. Postnov; Skolkovo Innovation Center. Research Institute of Atherosclerosis, Moscow; GBOU Research Institute of General Pathology and Pathophysiology of the Russian Academy of Medical Sciences, Moscow; Institute of Clinical Cardiology. A.L. Myasnikova FGBU RKNPK of the Ministry of Health and Social Development of the Russian Federation (magazine "Cardiology Bulletin" No. 1, 2012) [read];

article "Mitochondrial DNA and human hereditary pathology" N.S. Prokhorova, L.A. Demidenko; Department of Medical Biology, State Institution "Crimean State Medical University named after I.I. S.I. Georgievsky", Simferopol (magazine "Tauride Medical and Biological Bulletin" No. 4, 2010) [read];

article "Mitochondrial genome and human mitochondrial diseases" I.O. Mazunin, N.V. Volodko, E.B. Starikovskaya, R.I. Sukernik; Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk (journal "Molecular Biology" No. 5, 2010) [read];

article "Prospects for Mitochondrial Medicine" by D.B. Zorov, N.K. Isaev, E.Yu. Plotnikov, D.N. Silachev, L.D. Zorova, I.B. Pevzner, M.A. Morosanova, S.S. Yankauskas, S.D. Zorov, V.A. Babenko; Moscow State University M.V. Lomonosov, Institute of Physical and Chemical Biology named after A.I. A.N. Belozersky, Research Institute of Mitoengineering, Laser Research Center, Faculty of Bioengineering and Bioinformatics; Russian National Research Medical University. N.I. Pirogov (magazine "Biochemistry" No. 9, 2013) [read];

article "Strokes in mitochondrial diseases" N.V. Pizov; Department of Nervous Diseases with courses in neurosurgery and medical genetics, SBEI HPE "Yaroslavl State Medical Academy" (journal "Neurology, Neuropsychiatry, Psychosomatics" No. 2, 2012) [read];

article "Diagnosis and prevention of nuclear-encoded mitochondrial diseases in children" E.A. Nikolaev; Research Clinical Institute of Pediatrics, Moscow (journal "Russian Bulletin of Perinatology and Pediatrics" No. 2, 2014) [read];

article "Epilepsy in children with mitochondrial diseases: features of diagnosis and treatment" Zavadenko N.N., Kholin A.A.; GBOU VPO Russian National Research Medical University. N.I. Pirogov of the Ministry of Health and Social Development of Russia, Moscow (journal "Epilepsy and paroxysmal conditions" No. 2, 2012) [read];

article "Mitochondrial pathology and problems of the pathogenesis of mental disorders" by V.S. Sukhorukov; Moscow Research Institute of Pediatrics and Pediatric Surgery of Rosmedtekhnologii (Journal of Neurology and Psychiatry, No. 6, 2008) [read];

article "Algorithm for the diagnosis of mitochondrial encephalomyopathies" S.N. Illarioshkin (Nervous Diseases magazine No. 3, 2007) [read];

article "Actual issues of treatment of mitochondrial disorders" by V.S. Sukhorukov; Federal State Budgetary Institution "Moscow Research Institute of Pediatrics and Pediatric Surgery" of the Ministry of Health of Russia (journal "Effective Pharmacotherapy. Pediatrics" No. 4, 2012 [read];

article "Leukoencephalopathy with a predominant lesion of the brainstem, spinal cord and increased lactate in MR spectroscopy (clinical observation)" V.I. Guzeva, E. A. Efet, O. M. Nikolaeva; St. Petersburg Pediatric Medical University, St. Petersburg, Russia (journal "Neurosurgery and neurology of childhood" No. 1, 2013) [read];

teaching aid for third-year students of the medical diagnostic faculty of medical universities "Hereditary mitochondrial diseases" T.S. Ugolnik, I. V. Manaenkova; Educational Institution "Gomel State Medical University", Department of Pathological Physiology, 2012 [read];

fast: Mitochondrial diseases(neurodegeneration) - to the site with 17 links to sources (articles, presentations, etc.).

© Laesus De Liro

Not so long ago, questions of mitochondrial dysfunction were of interest only to researchers and individual attending physicians. For some time now, doctors using a biomedical approach and parents of children with ASD have begun to talk about it more and more.

The mitochondrial complex is the part of the cells responsible for energy production. Mitochondrial dysfunction is seen as one of the possible causes of many manifestations of autism.

I note right away that there is simply a huge amount of data on mitochondria that need to be systematized, generalized, and a working model created. Genetics, complex chemical reactions, the movement of electrons and the permeability of cell membranes - all these issues are relevant to the problem of the efficiency of mitochondrial functioning in patients with ASD.

A large number of children with autism have similar symptoms that may be due to insufficient cell energy:

Low activity of smooth muscles. This is especially detrimental to the work of the digestive tract, which leads to reflux (reflux of stomach contents into the esophagus), dyskinesias, constipation and yeast overgrowth due to the long stay of food in the intestines.
General muscle weakness. This leads to clumsiness and poor gross motor skills, which in turn causes developmental delays.
Decreased effectiveness of body detoxification. Organs that perform detoxification, such as the liver, require a very large amount of energy. . If not, then not all toxins will be processed. As a result, the body is poisoned more and more, and potentially harmful substances that come with food and water have an unexpectedly strong effect.
Insufficient supply of energy to the nervous system. This leads to distortion of the signals in the sensory system. When nerve impulses from the brain to the muscles pass with great difficulty, this further hinders the smoothness and clarity of movements.
Decreased energy potential of brain cells. A brain deprived of sufficient energy will not be able to fully perform its functions: produce and absorb neurotransmitters, grow new cells, get rid of old ones, and transmit signals. As a result, problems with memory and concentration can be observed.

If the child exhibits the symptoms listed, then the doctor's task is to check the functioning of all body systems and decide whether laboratory studies of mitochondrial function are necessary.

Read also The influence of diet on the course of autism: where and how to look for chances for improvement

It can be assumed that not all conditions accompanying ASD are irreversible. The saturation of certain deficiencies, which include mitochondrial dysfunction, will provide the child's body with the energy that it sorely lacks.

As a result, we will be able to observe an improvement in the functioning of almost all body systems, which will increase the patient's learning ability and facilitate his integration into society.

The list of factors and substances that lead to a deterioration in the functioning of mitochondria:

infections, especially viral ones;
inflammatory process;
heat;
dehydration;
prolonged hunger;
extreme heat or cold;

paracetamol;
non-steroidal anti-inflammatory drugs;
antipsychotics;
antidepressants;
antiepileptic drugs;
anesthesia;
heavy metals;
insecticides;
cigarette smoke.

Parents of children with ASD should avoid the following:

Alcohol consumption by children
Keeping children around cigarette smoke
Eating meals with monosodium glutamate (almost all processed foods that can be found on supermarket shelves)
High fever use of paracetamol (take ibuprofen instead, which is safer)
Taking antipsychotics.

Here is the list antibiotics that impair the functioning of the mitochondrial system:

Linezolid
Rifampicin
Tetracycline
Chloramphenicol
Imipenem
Penicillin
Cephalosporins

Quinolones (ciprofloxacin, levofloxacin, ofloxacin)
Macrolides (azithromycin, clarithromycin, erythromycin)
Sulfanilamide co-trimoxazole

Mitochondrial disorders are best treated with:

Ketogenic diet (high fat, adequate protein, low carbs)
Using vitamins and nutritional supplements to help rectify the situation:

Vitamin B12 in the form of subcutaneous injections
A complex of B vitamins, such as B-50. These are all B vitamins at 50mg each
S-adenosylmethionine (SAM, ademethionine)
L-cysteine and glutathione
Coenzyme Q10
Ginkgo biloba extract
Complexes of antioxidants, which include vitamins A, C, E and minerals selenium and zinc

Doctors began to observe how mitochondrial diseases manifest themselves in the 20th century. In an effort to determine what could be causing any of the mitochondrial diseases, experts have identified more than 50 types of diseases that are associated with disorders affecting the mitochondria.

Depending on the causes, there are three main subgroups of mitochondrial diseases, namely:

Diseases caused by mutations in mitochondrial DNA. Such defects are associated with a point mutation of various elements and are inherited mainly from the mother. Also, structural dislocation can cause disease. This category of diseases includes such hereditary syndromes of Kearns-Sayre, Pearson, Leber, etc.
Diseases caused by defects at the level of nuclear DNA. Mutations lead to disruption of mitochondrial function. In addition, they can cause negative changes in the enzymes involved in the cyclic biochemical process, in particular, the provision of cells in the body with oxygen. These include Luft and Alpers syndromes, diabetic diseases, etc.
Diseases caused by defects at the level of nuclear DNA and, as a result, causing secondary deformation of mitochondrial DNA. The list of secondary changes includes liver failure and syndromes, such as the one identified by De Toni-Debre-Fanconi.

Symptoms

Over a long period of time, mutations and, as a result, mitochondrial diseases, may not manifest themselves in a minor patient. However, over time, the accumulation of unhealthy organelles increases, as a result, the first signs of a particular disease begin to appear.

Since diseases of the mitochondrial group represent a whole group of pathologies, the signs of these diseases differ significantly depending on which organs and systems of the child's body were damaged. Given the relationship between mitochondrial defects and energy function, a particular susceptibility to damage to the nervous and muscular systems can be identified.

Among the characteristic signs of the pathology of the muscular system can be recognized:

Restriction or complete absence of motor activity due to the inability to perform normal activities due to muscle weakness or, as this condition is called, myopathy.
Reduced blood pressure.
Pain syndrome or muscle spasms, accompanied by severe pain.

In children, headache, intense and recurrent vomiting, and weakness after minimal physical exertion are primarily manifested.

If we are talking about damage to the nervous system, then the following manifestations take place:

lag in psychomotor development;
inability to perform actions with which the child coped previously - developmental regression;
convulsive seizures;
periodic manifestations of apnea and tachypnea;
frequent loss of consciousness and falling into a coma;
changes in the level of acid-base balance;
change in gait.

In older children, you can notice numbness, paralysis, loss of sensation, stroke-like seizures, pathologies in the form of involuntary movements, etc.

Involvement of the sense organs is expressed in the deterioration of visual function, ptosis, cataracts, defects in the retina and visual field, hearing impairment or complete deafness of a neurosensory nature. Damage to organs in a child's body manifests itself in the form of problems with the heart, liver, kidneys, pancreas. As for diseases associated with the endocrine system, here are noted:

retardation in growth and sexual development,
reduced production of glucose by the body,
thyroid dysfunction,
other metabolic problems.

Diagnosis of mitochondrial diseases in a child

In order to diagnose the presence of mitochondrial diseases, the doctor studies the history, conducts a physical examination, examining, first of all, the child's strength and endurance. Additionally, an examination by a neuropathologist is prescribed, including an assessment of vision, reflexes, speech and cognitive abilities. With the help of specialized analyzes - muscle biopsy, MRS, and so on - confirm the suspicions. Computed and magnetic resonance imaging and DNA diagnostics are also performed with a consultation with geneticists.

Complications

The danger of mitochondrial defects depends on the type of disease. For example, when the muscular system is damaged, there is a complete paralysis and disability, including intellectual regression.

Treatment

What can you do

First aid from parents depends on what exactly the manifestations of the disease are. In any case, if there is the slightest suspicion and deviations from the norm, it is necessary to contact a specialist and find out what to do with the disease if it is present.

What does a doctor do

Regardless of the type of disease, it can be treated by administering drugs that normalize energy metabolism. Also, the child is prescribed symptomatic and specialized treatment in the manner prescribed for a particular disease. Physical exercises and physiotherapy procedures help to cure pathologies faster or normalize the patient's condition.

Prevention

Mitochondrial diseases cannot be prevented because they occur at the genetic level. The only way to somewhat minimize the risks is to lead a healthy lifestyle without bad habits.

Keywords

NEWBORN CHILDREN / MITOCHONDRIAL DISEASE / MTDNA WASTE SYNDROME TYPE 13 / ENCEPHALOMYOPATHY/ LACTAT ACIDOSIS / NEONATAL MANIFESTATION/ GENE FBXL4 / NEWBORNS / MITOCHONDRIAL DISORDER / 13 TYPE MTDNA DEPLETION SYNDROME/ ENCEPHALOMYOPATHY / LACTIC ACIDOSIS / NEONATAL MANIFESTATION / FBXL4 GENE

annotation scientific article on clinical medicine, author of scientific work - Degtyareva A.V., Stepanova E.V., Itkis Yu.S., Dorofeeva E.I., Narogan M.V.

A clinical observation of a child with early neonatal manifestation a rare hereditary disease of mitochondrial DNA depletion syndrome (mtDNA) type 13, laboratory-confirmed in Russia. Mutations in the FBXL4 gene cause disturbances in mtDNA replication and a decrease in the activity of mitochondrial respiratory chain complexes, resulting in a violation of the functional state of various organs and systems, primarily the muscular system and the brain. Antenatally, the child was diagnosed with hydronephrosis on the right, subependymal cysts of the brain, partial intestinal obstruction against the background of polyhydramnios. The condition deteriorated sharply towards the end of the first day of life. There was a clinical symptom complex of sepsis, a pronounced syndrome of depression, muscle hypotension, decompensated metabolic lactic acidosis, an increase in the concentration of mitochondrial markers in blood plasma and urine, as well as changes in the basal ganglia of the brain. Differential diagnosis was carried out with hereditary diseases proceeding according to the type of "sepsis-like" symptom complex with lactic acidosis: a group of metabolic disorders of amino acids, organic acids, defects in β-oxidation of fatty acids, diseases of the mitochondrial respiratory chain, glycogen disease. mtDNA depletion syndrome type 13 has an unfavorable prognosis, however, accurate diagnosis is extremely important for medical genetic counseling and helps prevent the rebirth of a sick child in the family.

The text of the scientific work on the topic "Clinical observation of a patient with mitochondrial DNA depletion syndrome"

Clinical observation of a patient with mitochondrial DNA depletion syndrome

A.V. Degtyareva1,3, E.V. Stepanova1, Yu.S. Itkis2, E.I. Dorofeeva1, M.V. Narogan1,3, L.V. Ushakova1, A.A. Puchkova1, V.G. Bychenko1, P.G. Tsygankova2, T.D. Krylova2, I.O. Bychkov2

1FGBU "Scientific Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakov» of the Ministry of Health of the Russian Federation, Moscow;

2FGBNU "Medical Genetic Research Center", Moscow;

3FGAOU HE First Moscow State Medical University. THEM. Sechenov of the Ministry of Health of the Russian Federation, Moscow, Russia

Clinical case of Mitochondrial DNA Depletion

A.V. Degtyareva1,3, E.V. Stepanova1, Yu.S. Itkis2, E.I. Dorofeeva1, M.V. Narogan1,3,

L.V. Ushakova1, A.A. Puchkova1, V.G. Bychenko1, P.G. Tsygankova2, T.D. Krylova2, I.O. Bychkov2

1"Research Center for Obstetrics, Gynecology and Perinatology" Ministry of Healthcare of the Russian Federation 2FSBI "Research Center for Medical Genetics"

3First Moscow State Medical University I.M. Sechenov of Ministry of Healthcare

The paper presents a clinical observation of a child with early neonatal manifestation of a rare hereditary disease - mitochondrial DNA depletion syndrome (mtDNA) type 13, laboratory-confirmed in Russia. Mutations in the FBXL4 gene cause disturbances in mtDNA replication and a decrease in the activity of mitochondrial respiratory chain complexes, resulting in a violation of the functional state of various organs and systems, primarily the muscular system and the brain. Antenatally, the child was diagnosed with hydronephrosis on the right, subependymal cysts of the brain, partial intestinal obstruction against the background of polyhydramnios. The condition deteriorated sharply towards the end of the first day of life. There was a clinical symptom complex of sepsis, a pronounced syndrome of depression, muscle hypotension, decompensated metabolic lactic acidosis, an increase in the concentration of mitochondrial markers in blood plasma and urine, as well as changes in the area of the basal ganglia of the brain. Differential diagnosis was carried out with hereditary diseases proceeding according to the type of "sepsis-like" symptom complex with lactic acidosis: a group of metabolic disorders of amino acids, organic acids, defects in fatty acid p-oxidation, diseases of the mitochondrial respiratory chain, glycogen disease. Type 13 mtDNA depletion syndrome has an unfavorable prognosis, but accurate diagnosis is extremely important for medical genetic counseling and helps prevent the rebirth of a sick child in the family.

Key words: newborns, mitochondrial disease, type 13 mtDNA depletion syndrome, encephalomyopathy, lactic acidosis, neonatal manifestation, FBXL4 gene.

For citation: Degtyareva A.V., Stepanova E.V., Itkis Yu.S., Dorofeeva E.I., Narogan M.V., Ushakova L.V., Puchkova A.A., Bychenko V.G. , Tsygankova P.G., Krylova T.D., Bychkov I.O. Clinical observation of a patient with mitochondrial DNA depletion syndrome. Rosvestn perinatol and pediatrician 2017; 62:(5): 55-62. DOI: 10.21508/1027-4065-2017-62-5-55-62

Abstract: The article reports clinical case of early neonatal manifestation of a rare genetic disease - mitochondrial DNA depletion syndrome, confirmed in laboratory in Russia. Mutations of FBXL4, which encodes an orphan mitochondrial F-box protein, involved in the maintenance of mitochondrial DNA (mtDNA), ultimately leading to disruption of mtDNA replication and decreased activity of mitochondrial respiratory chain complexes. It "s a reason of abnormalities in clinically affected tissues, most of all the muscular system and the brain. In our case hydronephrosis on the right, subependimal cysts of the brain, partial intestinal obstruction accompanied by polyhydramnios were diagnosed antenatal. Baby"s condition at birth was satisfactory and worsened dramatically towards the end of the first day of life. Clinical presentation includes sepsis-like symptom complex, neonatal depression, muscular hypotonia, persistent decompensated lactic acidosis, increase in the concentration of mitochondrial markers in blood plasma and urine, and changes in the basal ganglia of the brain. Imaging of the brain by magnetic resonance imaging (MRI) demonstrated global volume loss particularly the subcortical and periventricular white matter with significant abnormal signal in bilateral basal ganglia and brainstem with associated delayed myelination. Differential diagnosis was carried out with hereditary diseases that occur as a "sepsis-like" symptom complex, accompanied by lactic acidosis: a group of metabolic disorders of amino acids, organic acids, p-oxidation defects of fatty acids, respiratory mitochondrial chain disorders and glycogen storage disease. The diagnosis was confirmed after sequencing analysis of 62 mytochondrial genes by NGS (Next Generation Sequencing). Reported disease has an unfavorable prognosis, however, accurate diagnosis is very important for genetic counseling and helps prevent the re-birth of a sick child in the family.

Key words: newborns, mitochondrial disorder, 13 type mtDNA depletion syndrome, encephalomyopathy, lactic acidosis, neonatal manifestation, FBXL4 gene.

For citation: Degtyareva A.V., Stepanova E.V., Itkis Yu.S., Dorofeeva E.I., Narogan M.V., Ushakova L.V., Puchkova A.A., Bychenko V.G., Tsygankova P.G., Krylova T.D., Bychkov I.O. Clinical case of FBXL4-Related Encephalomyopathic Mitochondrial DNA Depletion. Ros Vestn Perinatal i Pediatr 2017; 62:(5): 55-62 (in Russ). DOI: 10.21508/1027-4065-2017-62-5-55-62

Mitochondria are complex organelles that play a key role in cell homeostasis. They are the main source of intracellular energy synthesis in the form of ATP molecules, are closely involved in the processes of calcium and free radical metabolism, and also participate in apoptosis. Tissues and organs, especially dependent on these functions, are the first to suffer in mitochondrial diseases - this affects the muscle tissue, nervous and endocrine systems most of all. Most mitochondrial diseases are progressive in nature, leading to disability and premature death. These diseases are classified as rare, with a prevalence of 1-1.5: 5,000-10,000 newborns. Mitochondrial diseases can develop at any age. About 30% of cases manifest in the neonatal period.

According to the genetic classification, mitochondrial diseases are divided into the following groups: 1) diseases caused by point mutations in mitochondrial DNA (mtDNA) - MELAS, MERRF, LHON, NARP syndromes with maternal inheritance; 2) diseases caused by single large mtDNA rearrangements - Kearns-Sayre, Pearson syndromes; 3) diseases associated with mutations in the nuclear genes of structural proteins

Correspondence address: Degtyareva Anna Vladimirovna - Doctor of Medical Sciences, Head. in clinical work of the Department of Neonatology and Pediatrics of the Scientific Center for Obstetrics, Gynecology and Perinatology named after academician V.I. Kulakova, prof. Department of Neonatology, First Moscow State Medical University named after I.M. Sechenova, ORCID 0000-0003-0822-751X Stepanova Ekaterina Vladimirovna - Resident of the Scientific Center for Obstetrics, Gynecology and Perinatology named after academician V.I. Kulakova Dorofeeva Elena Igorevna - Candidate of Medical Sciences, Head. on clinical work of the Department of Neonatal Surgery of the Department of Neonatology and Pediatrics of the Scientific Center for Obstetrics, Gynecology and Perinatology named after academician V.I. Kulakova

Narogan Marina Viktorovna - Doctor of Medical Sciences, Leading Professor scientific collaborator Department of Pathology of Newborns and Preterm Infants, Department of Neonatology and Pediatrics, Scientific Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakova, prof. Department of Neonatology, First Moscow State Medical University named after I.M. Sechenova Lyubov Vitalievna Ushakova - Candidate of Medical Sciences, Neurologist of the Scientific Consultative Pediatric Department of the Department of Neonatology and Pediatrics of the Scientific Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakova

Puchkova Anna Alexandrovna - Ph.D., head. on clinical work of the Scientific Advisory Pediatric Department of the Department of Neonatology and Pediatrics of the Scientific Center for Obstetrics, Gynecology and Perinatology named after academician V.I. Kulakova

Bychenko Vladimir Gennadievich - Candidate of Medical Sciences, Head. Department of Radiation Diagnostics of the Scientific Center for Obstetrics, Gynecology and Perinatology named after Academician V.I. Kulakova 117997 Moscow, st. Academician Oparin, 4

Itkis Yulia Sergeevna - Researcher of the Medical Genetic Research Center

Krylova Tatyana Dmitrievna - laboratory geneticist of the Medical Genetic Research Center

Bychkov Igor Olegovich - post-graduate student of the Medical Genetic Research Center 115478 Moscow, st. Moskvorechye, 1

respiratory chain of mitochondria, - Lee's syndrome, infantile encephalomyopathy inherited autosomal recessively or X-linked; 4) diseases associated with mutations in the nuclear genes of carrier proteins and assemblers of mitochondrial respiratory chain complexes - Lee's syndrome, infantile encephalomyopathies inherited autosomal recessively or X-linked; 5) diseases associated with mutations in nuclear genes responsible for mtDNA biogenesis - mtDNA depletion syndromes with autosomal recessive inheritance.

One of the biochemical markers of mitochondrial diseases is a high level of lactate in the blood. The complex of the first line of examinations with suspicion of this pathology includes the determination of the content of amino acids, acylcarnitines and organic acids in the blood and urine. Recently, the high information content of determining the concentration of fibroblast growth factor-21 (FGF-21) and growth differentiation factor-15 (GDF-15) in blood plasma has been shown, however, the effectiveness of these biomarkers for diagnosing certain groups of mitochondrial diseases is still being studied by various groups of scientists. The final diagnosis of mitochondrial disease is established on the basis of the result of molecular genetic analysis.

Currently, there are no effective treatments for mitochondrial diseases. Symptomatic therapy is based on the use of metabolic drugs such as coenzyme Q10, creatine monohydrate, riboflavin, idebenone, carnitine, thiamine, dichloroacetate, etc. Special attention should also be paid to the child's nutrition; A transition to a low-protein diet with a large amount of fat in the diet is recommended. The use of valproic acid preparations and barbiturates is contraindicated.

mtDNA depletion syndromes are a clinically and genetically heterogeneous group of diseases inherited in an autosomal recessive manner and caused by mutations in genes that maintain mtDNA biogenesis and integrity. With such disorders, there is a decrease in the number of mtDNA copies in the affected tissues without its structural damage. Clinically, three forms of diseases associated with a decrease in mtDNA copy number are distinguished: encephalomyopathic, myopathic, and hepatocerebral. There are 20 known genes whose mutations lead to mtDNA wasting syndromes: ABAT, AGK, C10ORF2 (TWINKLE), DGUOK, DNA2, FBXL4, MFN2, MGME1, MPV17, OPA1, POLG, POLG2, RNASEH1, RRM2B, SLC25A4, SUCLA2, SUCLG1, TFAM , TK2, TYMP. In the Russian Federation, in the laboratory of hereditary metabolic diseases of the Medical Genetic Research Center, 36 patients were diagnosed with

mtDNA depletion syndromes with mutations in the POLG and TWINKLE genes (encephalomyopathic and hepatocerebral forms), DGUOK and MPV17 (hepatocerebral form), which accounted for a significant proportion of all early forms of mitochondrial diseases.

mtDNA depletion syndrome type 13 (MIM http://omim.org/entry/615471 615471) is caused by mutations in the FBXL4 gene located at the 6q16.1-q16.27 locus. This disorder was first described in 2013 by P.E. Bonnen and X. Gai independently. Currently 26 clinical observations are known in the world. The FBXL4 gene encodes a protein (F-box and leucine-rich repeat 4 protein), which is one of the subunits of the ubiquitin protein ligase complex, which plays an important role in the destruction of defective proteins in the cell, including mitochondria. The exact function of this protein is unknown, but it has been shown in cell cultures that ATP synthesis is reduced in damaged mitochondria and mtDNA replication is disrupted, which ultimately leads to a decrease in its copies in tissues and disruption of the mitochondrial respiratory chain.

In most cases, type 13 mtDNA depletion syndrome manifests itself in the early neonatal period, however, observations of a later manifestation up to 24 months of age have been described. The disease is characterized by encephalopathy, hypotension, lactic acidosis, severe developmental delay, and changes in the basal ganglia on magnetic resonance imaging (MRI) of the brain. According to M. Huemer et al. , in patients with mutations in the FBXL4 gene, such phenotypic features as a narrow and long face, a protruding forehead, thick eyebrows, narrow palpebral fissures, a wide bridge of the nose, and a saddle nose are noted.

The prognosis is extremely unfavorable, most children die in the first 4 years of life. Establishing a diagnosis of a disease is of great importance for medical genetic counseling and possible prenatal diagnosis.

The purpose of this publication is to provide a clinical description of the first Russian case of a mitochondrial disease caused by mutations in the FBXL4 gene and to determine the main criteria for diagnosing mtDNA depletion syndromes in early childhood.

Patient and research methods

The girl was born and was under dynamic observation at the Scientific Center for Obstetrics, Gynecology and Perinatology named after. IN AND. Kulakov. A comprehensive clinical, laboratory and instrumental examination was carried out. Some biochemical and molecular genetic studies were carried out in the laboratory of hereditary

metabolic diseases of the Medical Genetic Research Center. Organic acids in urine were analyzed by gas chromatography with mass spectrometric detection as trimethylsilyl ethers. Sample preparation was carried out according to the method proposed by M. Lefevere. The analysis was performed on a 7890A/5975C instrument (Agilent Technologies, USA) with an HP-5MS column (30 m*0.25 mm*4 µm). The results obtained were calculated using the internal standard method. The concentration of mitochondrial markers FGF-21 and GDF-15 in blood plasma was measured using kits based on the enzyme immunoassay method from Biovendor (Czech Republic).

DNA was isolated from whole blood using Isogene kits (Russia) according to the manufacturer's protocol. Sequencing of 62 nuclear mitochondrial genes was carried out using the NGS (Next Generation Sequencing) method on the Ion Torrent PGM™ System for Next-Generation Sequencing instrument (Life Technologies, Thermo Fisher Scientific). Sample preparation of DNA samples was carried out with the Ion AmpliSeq™ Library Kit 2.0 reagent kit (primer pool design using Ampliseq technology) according to the manufacturer's protocol. Visualization of the alignment of the sequenced fragments to the reference sequence of the human genome Human.hg19 was carried out using the IGV program. The detected changes were annotated using the ANNOVAR program. The predictive functional significance of previously undescribed mutations was assessed using various open source programs (PolyPhen2, Mutation taster, SIFT). The identified variants were filtered by the frequency of occurrence in populations according to the data presented in the open databases ExAc, 1000 genomes, etc. Nucleotide substitutions other than the reference sequence were analyzed in databases of mutations and polymorphisms (HGMD, Ensemble, dbSNP). The mutations identified in the FBXL4 gene were verified by direct automatic sequencing on an ABI3500 genetic analyzer (Thermo Fisher Scientific) using BigDye Terminator v.1.1 (Thermo Fisher Scientific). Specific oligonucleotide primers were used for polymerase chain reaction (PCR) (sequence available on request). Alignment and comparison of data was carried out in accordance with the NM_012160 transcript.

Clinical observation

The child was born at term to a somatically healthy woman with a burdened obstetric-gynecological and infectious history. The marriage is unrelated. The family has one healthy child. Pregnancy proceeded with an exacerbation of salpingo-oophoritis in the first trimester, pulpitis with an increase in temperature up to 38°C, and ended with spontaneous childbirth.

The child was born with a body weight of 2555 g, a length of 49 cm, an Apgar score of 8/9 points. Antenatally, hydronephrosis on the right side, subependymal cysts of the brain, and partial intestinal obstruction against the background of polyhydramnios were diagnosed. The first hours of life had the character of a "period of relative well-being", however, given the antenatal pathology, the child was transferred to the department of surgery, resuscitation and neonatal intensive care for examination.

By the end of the first day of life, the condition deteriorated sharply, there was a pronounced depression syndrome, muscle hypotension, hemodynamic deterioration, respiratory disorders that required artificial lung ventilation. According to the acid-base state and gas composition of the blood, decompensated metabolic lactic acidosis was noted (pH 7.12; pCO2 12.6 mm Hg; pO2 71.9 mm Hg; BE -24.2 mmol/l; lactate 19.0 mmol/l). Based on the history data, it was impossible to exclude the presence of an infectious process, and the child was prescribed antibacterial and immunomodulating therapy. In the clinical analysis of blood, leukocytosis was noted with a shift of the formula to the left, a decrease in the hemoglobin content, the level of platelets was within the normative values (Table 1).

At the same time, markers of the systemic inflammatory response (C-reactive protein and procalcitonin) were negative (0.24 mg/l and 10 ng/ml, respectively), and no foci of infection were detected during the examination. In order to exclude congenital pneumonia, an X-ray examination was performed, the results of which did not reveal specific changes. Based on the results of lumbar puncture, meningitis was ruled out. Clinical analysis of urine also did not reveal

Table 1. The parameters of the clinical blood test of the patient

inflammatory changes. In addition, negative results of microbiological cultures of blood and urine, scrapings from the pharynx and serological testing for TORCH infection were obtained.

In the neurological status, there was a syndrome of severe depression, there were no meningeal symptoms, there was an intermittent divergent strabismus, severe diffuse muscular hypotension. The therapy included the metabolic drug sodium megluminate succinate (Reamberin) and a stimulator of the synthesis of acetylcholine and phosphatidylcholine - choline alfosscerate (Cholitilin). Against the background of ongoing post-syndromic treatment, there was a positive trend; by the 8th day of life, the child was removed from respiratory therapy. According to the results of a clinical blood test, inflammatory changes stopped, inflammation markers C-reactive protein and procalcitonin remained within the normal range. However, the child retained signs of severe muscle hypotension, CNS depression syndrome, and lactic acidosis (9.5 mmol/l). It is important to note that the lactate level never decreased to normal values and was undulating throughout the entire period of hospital stay (Fig. 1).

The discrepancy between clinical signs of sepsis with severe decompensated lactic acidosis, negative markers of systemic inflammatory response and response to treatment was a reason to suspect a metabolic disorder. The range of differential diagnostics included diseases occurring in the neonatal period according to the type of "sepsis-like" symptom complex with lactic acidosis: a group of metabolic disorders of amino acids, organic acids, defects in p-oxidation of fatty acids, respiratory diseases

Parameters 2nd day of life Reference values (1-7th day of life) 8th day of life Reference values (> 7 days of life)

Erythrocytes, -1012/l 4.03 5.5-7.0 4.42 4.5-5.5

Hemoglobin, g/l 137 160-190 136 180-240

Hematocrit 40.9 0.41-0.56 38.1 0.41-0.56

Platelets, -199/l 236 218-419 213 218-419

Leukocytes, -109/l 49.11 5.0-30.0 11.72 8.5-14.0

Neutrophils, -109/l 27.514 6-20 4.342 1.5 - 7.0

Neutrophil index 0.44< 0,25 0,16 < 0,25

Stab, % 16 5-12 6 1-5

Segmented, % 56 50-70 47 35-55

Eosinophils, % 0 1-4 3 1-4

Monocytes, % 9 4-10 18 6-14

Lymphocytes, % 10 16-32 32 30-50

mitochondrial chain and glycogen type I disease (Girke's disease). The child was tested with feeding, based on the determination of the concentration of glucose and lactate in the blood after a hungry pause and 20-30 minutes after feeding. According to the results of this study, the level of fasting blood glucose was reduced, and the level of lactate was increased; after feeding, an increase in glucose levels and a pronounced increase in lactatemia were noted (Table 2).

The first-line examination group included tests that determined the spectrum of amino acids and acylcarnitines in the blood and organic acids in the urine, as well as plasma mitochondrial biomarkers FGF-21 and GDF-15. An increased content of alanine, leucine and ornithine was found in the blood (Table 3). The spectrum of acylcarnitines in the blood was within the normal range, which made it possible to exclude diseases from the group of defects in ß-oxidation of fatty acids. Urinalysis revealed an increase in the levels of lactate, fumaric acid, 3-hydroxybutyrate, pyruvate, succinate, and 4-hydroxyphenylpyruvate (see Table 3). These changes may indicate mitochondrial

Table 2. Results of a sample with feeding

Rice. 1. Dynamics of blood lactate concentration (in mmol/l). Fig. 1. Dynamics of blood lactate concentration.

nom violation and fumaric aciduria.

A molecular genetic study of the nucleotide sequence of the FH gene was carried out, the mutations of which cause the development of fumaric aciduria. No deviations from the norm were found.

The concentration of mitochondrial markers FGF-21 and GDF-15 in blood plasma was increased and amounted to 720 pg/ml (normal 0-330 pg/ml) and 15715 pg/ml (normal 0-2000 pg/ml), respectively.

At the age of 8 days of life, the child underwent an MRI of the brain, the results of which revealed a symmetrical lesion of the subcortical nuclei in the form of cystic changes, which is highly

Parameter Before meals 20-30 minutes after meals

BE, mmol/l - 6.2 - 7.7

Glucose, mmol/l 2.1 2.7

Lactate, mmol/l 5.8 9.2

p CO2, mmHg 33.4 29.2

Table 3. The level of patient's amino acids in the blood and organic acids in the urine

Parameter Lower limit of normal Upper limit of normal Value in the patient

Amino acids in blood, nmol/l

Alanine 85 750 1139.327

Leucine 35 300 405.533

Ornithine 29 400 409.205

Organic acids in urine, mol per mol of creatinine

Lactate 0.00 25.00 82.9

Fumaric acid 0.00 2.00 274.2

3-hydroxybutyrate 0.00 3.00 18.2

Pyruvate 0.00 12.00 13.7

Succinate 0.50 16.00 103.4

4-hydroxyphenylpyruvate 0.00 2.00 39.5

pathognomonic sign of mitochondrial diseases. The consequences of hemorrhage in the lateral ventricles of the brain were also revealed (Fig. 2).

Given the clinical and laboratory symptom complex, a mitochondrial disease from the group of infantile encephalomyopathies was suspected. Using targeted sequencing, the child was analyzed the coding sequence of 62 nuclear genes, mutations in which lead to the development of mitochondrial pathology. Two compound heterozygous mutations c.A1694G:p were identified in the FBXL4 gene. D565G (in exon 8) and c.627_633del:p.V209fs (in exon 4). Mutation c.A1694G:p.D565G

Rice. 2. MRI of the brain of a child aged 8 days of life. A - T2 weighted image in the axial plane. White arrows show cysts along the contours of the lateral ventricles, which are a characteristic sign of mitochondrial diseases. Red arrows located in a highly conserved region show the products of hemoglobin biodegradation in the lumen of the ventricular LRR (Leucine-Rich Repeat) domain and system (consequences of intraventricular hemorrhage).

B - the tomogram was made in the Flair mode in the axial plane. White arrows show cysts in the paraventricular areas and in the projection of the subcortical nuclei, which is typical for mitochondrial diseases. Fig. 2. MRI of the child's brain at the age of 8 days of life. A - T2 weighted image in the axial plane. White arrows show cysts along the contours of the lateral ventricles, which are a characteristic feature of mitochondrial diseases. Red arrows show the products of biodegradation of hemoglobin in the lumen of the ventricular system (consequences of intraventricular hemorrhage). B - the tomogram is performed in the Flair in the axial plane. White arrows show cysts in paraventricular regions and in the projection of the subcortical nuclei, which is characteristic of mitochondrial diseases.

previously described in the literature. The second mutation was found for the first time in our patient, and its pathogenicity is not in doubt, since it leads to a shift in the reading frame and the formation of a premature stop codon.

At the age of 42 days, the child was discharged home in a moderate condition. In the follow-up, there were signs of CNS depression, severe muscle hypotension, a tendency to ptosis, decompensated metabolic lactic acidosis, psychomotor retardation, dysphagia, a monotonous flat weight curve, frequent recurrent respiratory infections, which later led to the development of multiple organ failure and death. at the age of 11 months of life.

Discussion

In our observation, hydronephrosis on the right side, subependymal cysts of the brain, and partial intestinal obstruction against the background of polyhydramnios were diagnosed prenatally. This pattern during prenatal ultrasound diagnostics is described for mutations in the FBXL4 gene. In approximately 10% of cases, polyhydramnios occurs against the background of congenital diseases, including hereditary metabolic diseases. In the observation of M. Van Rij et al. the patient was also diagnosed with severe polyhydramnios at 30 weeks of fetal development and an organic lesion of the brain structure was found in the form of cerebellar hypoplasia, subependymal cysts, and expansion of the cerebral cisterna magna. Prenatal detection of subependymal cysts of the brain was also reported in the observation of T. Baroy et al. . In mitochondrial diseases, cases of prenatal diagnosis of hydronephrosis have also been described.

The child's condition deteriorated sharply by the end of the 1st day of life after the period of the "light period", there was a pronounced syndrome of depression, muscle hypotension, respiratory disorders (requiring artificial lung ventilation), deterioration of hemodynamics, decompensated metabolic lactic acidosis. The neonatal manifestation of the mtDNA depletion syndrome in more than 80% of cases is described as a pronounced syndrome of depression, muscle hypotension, encephalopathy, dysphagia with episodes of regurgitation in combination with an increased level of lactate and metabolic acidosis that occurs after a period of "light period". Pathogenetically, an increase in the level of lactate is due to the fact that with a functional violation of the respiratory chain, the redox balance in the cytoplasm changes, which leads to a disruption in the functioning of the Krebs cycle due to an excess of NADH relative to NAD+. This process leads to an increase in the concentration of lactate, an increase in the molar ratio of lactate / pyruvate and the concentration of ketone bodies in the blood. According to the literature, the level of lactate in children with type 13 mtDNA wasting syndrome ranges from 6.3 to 21 mmol/l. There is an increase in the level of lactate in the cerebrospinal fluid. The normal molar ratio of lactate / pyruvate composition is

lays<20, тогда как, по данным M. Van Rij и соавт., у детей с мутациями в гене FBXL4 этот показатель составил 71 . У нашей пациентки уровень пирува-та не исследовался.

In our observation, hyperlactatemia was the leading laboratory characteristic of the disease, but this symptom is not highly specific. The reasons for the increase in the concentration of lactate in the blood can also be perinatal asphyxia, congenital heart defects, sepsis, liver and kidney diseases, defects in fatty acid p-oxidation, organic aciduria, impaired biotin metabolism, carbohydrate metabolism, etc., which presents great difficulties for early diagnosis of pathology.

When examining a patient, there was a discrepancy between the clinical signs of an infectious process with decompensated lactic acidosis and negative markers of a systemic inflammatory response, combined with the absence of foci of infection and bacteremia. Against the background of posidromic therapy, there was some improvement in the child's condition, but neurological disorders and severe lactic acidosis persisted, which made it possible to suspect a metabolic disease. In the study of the spectrum of amino acids, an increased concentration of alanine, leucine and ornithine was revealed, which is often found in lactic acidosis. The level of lactate in the urine, as well as the metabolites of the Krebs cycle (fumaric acid, pyruvate, succinate) was significantly increased, which is also characteristic of a number of mitochondrial diseases. Similar changes in organic acids in urine were described by M.C. Van Rij et al. in clinical observation

a child with mtDNA wasting syndrome type 13.

In our patient, the plasma concentration of FGF-21 exceeded the upper limit of normal by 2 times, and the concentration of GDF-15 was more than 7 times. These data are consistent with recent publications that GDF-15 is a more sensitive marker of mitochondrial pathology. In hepato-cerebral forms of the mtDNA depletion syndrome, the level of both markers increases on average 15 times above the normal limit. At the age of 8 days of life, the child underwent an MRI of the brain, which revealed highly specific signs of the encephalomyopathic form of mitochondrial disease: symmetrical lesions of the subcortical nuclei in the form of cystic changes.

Thus, this paper presents a case of a patient with a neonatal manifestation of a mitochondrial disease, type 13 mtDNA depletion syndrome, caused by mutations in the FBXL4 gene. The first signs of the disease were nonspecific and had the character of a sepsis-like symptom complex that appeared after a period of a light gap in the child's condition. There was a pronounced syndrome of depression, muscle hypotension, as well as persistent lactic acidosis, an increase in the level of mitochondrial biomarkers FGF-21 and GDF-15 in blood plasma, and symmetrical lesions in subcortical structures on MRI of the brain. Currently, there is no pathogenetic treatment for mtDNA depletion syndrome, but the identification of the patient's genotype provides a basis for prenatal diagnosis, which will help prevent the rebirth of a sick child in the family.

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