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  1. The definition of mitochondrial diseases used in this chapter defines these diseases as conditions in which a defect of the mitochondrial oxidative phosphorylation (OXPHOS) pathway is a key contributor to the pathophysiology of the disease. Classifying which genes fall under that definition remains debatable for some genes where the pathophysiology is not yet completely understood.

  2. The mitochondrial OXPHOS pathway consists of five heteropolymeric enzyme complexes in the mitochondrial inner membrane, containing more than 80 different proteins, plus the two electron carriers ubiquinone and cytochrome c. OXPHOS complexes can be further assembled into supercomplexes. A functional OXPHOS pathway relies on the interaction of two genomes. The mitochondrial DNA (mtDNA) encodes 13 of the OXPHOS proteins plus 2 rRNAs and 22 tRNAs required for translation of the mtDNA genome. The nuclear genome encodes hundreds of proteins essential for OXPHOS function. These include subunits and assembly factors for each complex plus all the machinery required for import, processing and quality control of mitochondrial proteins, replication and expression of mtDNA as well as synthesis of redox centers, electron carriers, cofactors, nucleotides and maintaining an appropriate membrane milieu for stability of the OXPHOS complexes. Given this genetic complexity, it is not surprising that mitochondrial diseases are now known to comprise at least 200 monogenic diseases.

  3. Mitochondrial diseases encompass a very large number of conditions with a huge clinical spectrum, with signs affecting any tissue in the body, with onset at any age, and with any type of inheritance. The functional, structural and genetic complexity of OXPHOS directly explains the wide diversity of mitochondrial diseases. In most cell types, in particular neuronal and muscle cells, OXPHOS is producing more than 90% of the cellular ATP. In parallel it has central roles in the maintenance of the cellular redox potential, inner membrane potential and reactive oxygen species metabolism.

  4. Many mitochondrial diseases are multisystemic, involving several tissues unrelated by functional or embryological link. However tissue-specificity is also frequent and the pathophysiological mechanisms remain largely hypothetical. In mitochondrial diseases affecting a single tissue, demonstration of the mitochondrial defect can be difficult because most histological and enzymatic analyses are made in muscle, which may be spared by the disease. In addition a mitochondrial defect, even severe but affecting a tissue with minor mass, will have little influence on the global metabolic status that is analysed in blood and urine samples.

  5. The initial steps in investigating possible mitochondrial disease involve assessment of family history and clinical features, with a focus on recognizing classical mitochondrial syndromes and multisystem disease, together with consideration of possible alternative diagnoses. In most cases, the next steps involve non-invasive studies on body fluids to detect any alteration in cellular redox ratio or accumulation of substrates upstream of the deficient metabolic step, such as lactate, pyruvate or intermediates of the Krebs cycle. Together with imaging, these investigations can guide whether specific single gene tests, gene panels or whole exome sequencing should be performed or if the patient should proceed to tissue biopsies.

  6. Traditionally, most patients with suspected mitochondrial disease have been investigated by performing a tissue biopsy, which would ideally be done on an affected tissue. Skeletal muscle biopsy is by far the most common tissue biopsied for investigations, of which there are two major categories. The first is histological analyses, in which tissue sections are studied by a series of stains to identify abnormal mitochondrial numbers, decreased activity of OXPHOS complexes II and IV, or in some cases abnormal mitochondrial morphology detected by electron microscopy. The second major category of testing is spectrophotometric enzyme assays to determine the activity of OXPHOS complexes. A number of other diagnostic tests are used less commonly, such as respirometry and determining the size and amount of each complex by immunochemical approaches. While histological and enzyme tests can provide definitive results, they lack high sensitivity and specificity. This primarily results from the issues of genetic complexity, tissue specificity and the expectation that most defects will be partial as complete OXPHOS defects would cause embryonic lethality. For example, most children with mitochondrial disease lack clear histological abnormalities and many OXPHOS enzyme defects have substantial residual activity, which can overlap with the normal range. The membrane localization as well as the structural and functional complexity of the OXPHOS enzymes also contribute technical difficulties to the assessment of OXPHOS function.

  7. Genetic diagnosis of mitochondrial diseases is complicated by the large number of candidate genes, located in the nuclear chromosomes or the mtDNA. An additional challenge is brought by the quantitative aspects of mtDNA alterations. Identification of the alteration alone is not sufficient for diagnosis but has to be complemented by its quantification because a low proportion of a heteroplasmic pathogenic point mutation or deletion can be observed in asymptomatic individuals. In addition the mtDNA copy number per cell can be altered, either abnormally decreased (mtDNA depletion syndromes) or increased (in parallel with mitochondrial proliferation). Traditionally, genetic testing for mitochondrial disease was guided by the clinical features, family history and biopsy results. Increasingly though, the use of gene panels or whole exome sequencing is becoming a front-line diagnostic test. If a clear genetic diagnosis is found, then the patient is spared the need for a tissue biopsy and if no diagnosis is found, biopsies can be performed to obtain additional information that may guide re-analysis of sequencing results. Prenatal diagnosis and preimplantation strategies for heteroplasmic mtDNA mutations can be offered to some families. However, they are faced with difficulty in predicting the outcome of intermediate mutant loads and some uncertainty with respect to mtDNA segregation during embryogenesis and postnatal life.

  8. In conclusion the diagnosis of mitochondrial diseases differs from that of most other metabolic diseases due to the highly diverse clinical presentations, the lack of specific/constant metabolic hallmarks, the potential for false positive and false negative results from histology and enzyme analyses, the large number of candidate genes, and the quantitative aspects of mitochondrial DNA genetics. As such it represents an extraordinary diagnostic challenge whose chances of success are significantly increased when it can rely on reciprocal and easy communication between the diagnostic laboratory, the physicians involved in the care and metabolic investigation of the patients and research groups essential for functional validation of novel genetic causes. In the absence of a gold standard diagnostic test, it is helpful to use diagnostic criteria that attempt to define the certainty of diagnosis based on objective assessment of evidence from multiple independent sources.

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