**4.5. Defects in mtDNA translation**

**4.3. Multiple deletions of mtDNA**

168 Mitochondrial DNA - New Insights

**4.4. Depletion of mtDNA**

mtDNA, highlighting two syndromes:

zyme deoxyguanosine kinase (dGK))

(p-53 inducible ribonucleotide reductase) [80, 82]

(which codes for a subunit of POLG) (**Figure 8**) [14, 80, 81].

From the clinical point of view, the multiple deletion syndromes of mtDNA are characterized by progressive external ophthalmoparesis (PEO), ptosis, and proximal muscle weakness associated with signs of involvement of other systems that include the peripheral nerves (sensory-motor neuropathy), the brain (ataxia, dementia, psychosis), the ear (sensorineural deafness), and the eye (cataracts). Genes involved in the homeostasis of the mitochondrial pool of nucleotides are associated with the presence of PEO and multiple deletions in mtDNA. They include ANT1 (encodes the adenosine nucleotide translocator), PEO1 (codes for a helicase known as Twinkle), ECGF1 (which codes for thymidine phosphorylase or TP), POLG (encodes for the catalytic subunit of mitochondrial gamma polymerase), and POLG2

Mitochondrial neurogastrointestinal encephalomyopathy or MNGIE is a multisystemic disease of autosomal recessive inheritance, of presentation in young adults secondary to mutations in TP (thymidine phosphorylase). It is characterized by PEO, neuropathy, leukoencephalopathy, and severe gastrointestinal dysmotility, leading to profound cachexia and early death. The decrease in thymidine phosphorylase activity leads to a defect in the synthesis of mtDNA, causing not only multiple deletions in the mtDNA but also depletion of same and point mutations that are reflected in the muscle, even though it expresses little TP. The damage, therefore, seems to be mediated by toxins. Thus, thymidine and deoxyuridine are toxic intermediaries that accumulate in the blood of these patients, and their elimination leads to clinical improvement. Different approaches have been carried out to favor the elimination of these toxic intermediaries from the blood, by means of hemodialysis,

platelet transfusion, and, finally, allogeneic bone marrow transplantation [35, 80].

Mutations in mitochondrial gamma polymerase (POLG) may occur in the form of PEO at the onset of adulthood and multiple deletions in mtDNA and be accompanied by ataxia, peripheral neuropathy, Parkinsonism, psychiatric symptoms, myoclonic epilepsy, and gastrointestinal symptoms. These disorders can have both dominant and recessive inheritances. An example of a clinical syndrome secondary to mutations in this gene is SANDO (sensory ataxic neuropathy, dysarthria, ophthalmoparesis). Mutations in POLG are also responsible for the Alpers syndrome in children, a recessively inherited disorder characterized by encephalopathy and severe hepatopathy and associated with mtDNA depletion [80].

Some mutations in POLG are responsible for a fatal hepatocerebral syndrome in children (Alpers syndrome) characterized by a profound depletion of mtDNA. Mutations in proteins that affect the control of the pool of nucleotides in mitochondria also produce depletion of

• Hepatocerebral syndrome caused by mutations in POLG or in DGUOK (encodes the en-

• Myopathic syndrome caused by mutations in TK2 (which codes for the mitochondrial form of thymidine kinase), in SUCLA2 (beta subunit of succinyl-CoA synthetase), or in RRM2B In the translation of the 13 subunits of the respiratory chain encoded in the mtDNA, the participation of many nuclear coding factors such as polymerases, ribosomal proteins, RNA-modifying enzymes and initiation, elongation, and termination factors, among others, is necessary. These defects result in deep combined deficits of all the respiratory chain complexes. Clinically, they manifest as necrotizing leukoencephalopathies, cardiomyopathies, and hepatocerebral syndromes among others. Genes involved include GFM1 (encodes a ribosomal elongation factor), MRPS16 (encodes the 16 subunit of the mitochondrial ribosomal protein), TSFM (encodes the mitochondrial elongation factor EFTs), and TUFM (encodes the elongation factor Tu) [80, 82, 83].

### **4.6. Mitochondrial secondary disease**

Even when a sophisticated biochemical analysis confirms mitochondrial dysfunction, it can be challenging to distinguish whether the cause of this dysfunction is a gene that directly affects the electron transport or is secondary to an unrelated genetic or environmental cause. Thus, the definitive diagnosis of mitochondrial disease cannot be based solely on biochemical findings, since the in vitro activity of the electron transport chain enzyme in a sample of patient tissue may be diminished as a consequence of other metabolic diseases or issues related to the handling of samples. Mitochondrial dysfunction that may or may not be clinically relevant is observed when the main defect lies in another metabolic pathway related to energy, such as the oxidation of fatty acids or the metabolism of amino acids. In addition, alteration of OXPHOS has been observed with decreased in vitro activity of the electron transport chain enzyme in up to 50% in tissue samples from patients with other metabolic diseases [84]. Of course, other diagnoses that have finally been confirmed in individuals with suspected mitochondrial disease and biochemical samples of mitochondrial dysfunction in vitro include disorders of copper metabolism (Menkes disease and Wilson's disease), lysosomal disorders (neuronal ceroid lipofuscinosis and Fabry disease), peroxisomal disorders, neurodegeneration associated with pantothenate kinase, holocarboxylase synthetase deficiency, molybdenum cofactor deficiency, and neonatal hemochromatosis [85]. It is increasingly accepted that the alteration of OXPHOS may contribute to the pathology in some genetic alterations that are not typically classified as mitochondrial or metabolic disorders, such as Rett syndrome, Aicardi-Goutières syndrome, various neuromuscular disorders, and Duchenne muscular dystrophy. In addition, the activities of the electron transport complexes in skeletal muscle can decrease in malnourished children, correcting to normal values after improvement of nutrition [41, 74, 86].

Medications and toxins can also significantly alter mitochondrial function. Sodium valproate can alter mitochondrial function by inducing carnitine deficiency, depression of intramitochondrial oxidation of fatty acids, and/or inhibition of OXPHOS, which should suggest the use of an alternative anticonvulsant in mitochondrial disease, especially in patients with POLG1 mutations. Other important examples of drugs that can induce mitochondrial dysfunction are retroviral nucleoside analogs in HIV infection, as well as salicylates that can alter the hepatic mitochondria in Reye syndrome. Since there are so many nonspecific clinical features that can raise the suspicion of mitochondrial diseases, the differential diagnosis can be very broad. The clinical presentation of mitochondrial disease in children can mimic other multisystem disorders, such as congenital disorders of glycosylation or Marinesco-Sjögren syndrome, or even be confused with a syndrome of vascular or immunological stroke. Although the clinical

analyzed earlier, is not sensitive or specific as an isolated biomarker in many mitochondrial disorders), tissue biopsy tests of the abnormal activity of the electron transport chain enzyme or an alteration of the respiratory capacity, and, if possible, the identification of a pathogenic mutation of mtDNA or nDNA. This process usually involves sophisticated tests that request invasive procedures, such as muscle or liver biopsy to obtain the tissue for assessment in specialized laboratories. These investigations may offer intermediate or ambiguous results, and the decrease in the activities of the enzymes of the electron transport chain may be secondary to non-respiratory chain disorders. To assist in the interpretation, two diagnostic schemes have been proposed for infants and children to classify the probability of a specific patient's mitochondrial disease as clear, probable, possible, or improbable. Recently, guidelines were proposed for the diagnosis and treatment of mitochondrial disorders in infants and children; however, these complex and sophisticated diagnostic algorithms are aimed for the metabolic specialist and have a limited clinical utility for the family physician who contemplates the start of the diagnostic evaluation of

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The diagnostic evaluation typically begins with the general clinical assessment and goes through the systematic, imaging, and metabolic screening tests up to the most specific biochemical and genetic determinations. This starts with the less invasive evaluations and moves on to the biopsy-based, more invasive analyses, as needed. Obviously, the complete diagnostic process can be complicated and including the early intervention of a local specialist in metabolism can be very useful. Recommendation to a metabolic specialist should occur whenever the symptoms and signs clearly suggest a mitochondrial disease, patients are potentially unstable with the classic features of metabolic disease, there is lactic acidosis in the blood or the cerebrospinal fluid (CSF), a pattern of maternal inheritance is observed, or anomalies are identified in the initial diagnostic assessment. Referral by a primary care physician is also prudent when a more elaborate study is necessary, such as a provocation test or a muscle biopsy with the study of the enzymes of the electron transport chain [79, 88]. If a biochemical diagnosis has been established but its molecular basis remains unknown, further study and genetic counseling should be coordinated by a specialist. Mitochondrial disease is clearly not a single entity but rather a heterogeneous disorder of energy dysfunction caused by hundreds of different mutations, deletions, duplications, and other defects of nuclear and mitochondrial genes. Thus, at present, there is no accepted and gene-based diagnostic algorithm that is useful for all patients or used by all metabolic specialists. The study of nDNA mutations can be performed on any tissue, including blood. However, most of the diagnostic study of nDNA genes should not be done a priori but guided by the clinical picture, the specific headlines, and the biochemical findings in a given patient. On the contrary, the most informative study of mtDNA mutations is performed in a muscle biopsy sample, although urinary sediment and buccal cells may also

It is important to recognize that dietary advice should always be offered in a specialized setting. In addition, although there are only a few viable therapeutic options for mitochondrial

disease, it is best to be offered by clinicians with experience in these disorders [89].

a specific patient [20, 21, 87].

be useful.

**Figure 9.** Medications and toxin in mitochondria: Alteration of mitochondrial function by exogenous metabolites; the main damage affects cell respiration, but also damage in the oxidation of fatty acids is observed. This image is a modification of QIAGEN's original [Torres-Sánchez ED].

and neuroimaging features of Leigh's syndrome often clearly suggest a mitochondrial disorder, other alterations may give rise to striatal necrosis, which should be taken into account. Similarly, clinical and neuroimaging findings may sometimes suggest other leukoencephalopathies or degenerative disorders (**Figure 9**) [85, 86].
