Review articleTreatment of mitochondrial disorders
Introduction
Mitochondrial disorders (MIDs) are due to mutations in the mitochondrial or nuclear DNA (mtDNA, nDNA, mitochondrial MIDs, nuclear MIDs), resulting in impaired respiratory chain (RC) or oxidative phosphorylation (OXPHOS) function. Phenotypically, MIDs present as single- or multi-system diseases, with onset between birth and senescence.1, 2 Single organ affection usually turns into multi-system involvement during the disease course. MIDs predominantly manifest in tissues/organs with high-energy requirement3 and are aggravated by fever, infection, stress, toxic agents, or certain drugs.4 Systems and organs most frequently clinically or subclinically affected in MIDs are the peripheral nervous system (PNS), the central nervous system (CNS), the endocrine glands, and the heart.5 Various combinations of organ affections constitute mitochondrial syndromes (syndromic MIDs) for which well known acronyms have been adopted.6 Treatment of MIDs is a challenge since the available options are scarce, since MID patients frequently develop adverse reactions to certain mitochondrion-toxic agents, and since only few randomized and controlled studies have been carried out, which demonstrate an effect of any of the symptomatic or supportive measures. After a short introduction to phenotype, genetics, and diagnosis of MIDs, the following review aims to give an overview on recent advances and current knowledge about the treatment of MIDs.
Clinically, MIDs manifest as single organ disorder or as multi-system disease, affecting the peripheral nervous system, the central nervous system, the eyes, ears, endocrine organs, heart, intestines, kidneys, bone marrow, or the dermis.6 Various typical combinations of clinical manifestations resulted in the definition of various mitochondrial syndromes, for which well known acronyms have been coined (MELAS, MERRF, LHON, NARP, MILS, KSS, mtCPEO, PS, LS, AD-CPEO, AR-CPEO, GRACILE, MNGIE, SANDO, SCAE, MLASA, XLASA, DDS (MTS), AHS, IOSCA, MEMSA, MIRAS, DIDMOAD, ADOAD, LBSL) (Table 1). In the majority of MIDs, however, the phenotype does not fit into one of these syndromes (non-syndromic MIDs (nsMIDs)).
MIDs may have a genetic etiology or may be acquired. The actual review mainly deals only with genetic MIDs, which may be either due to mtDNA or nDNA mutations.
Human mtDNA is a 16.5 kb circular minichromosome built up of the complementary H and L strands. mtDNA contains 13 genes encoding for subunits of RC complexes (RCC) I (ND1-4, ND4L, ND5-6), III (cytochrome b), IV (COXI-III), and V (ATPase6, ATPase8), and 24 genes encoding for 22 tRNAs and two rRNAs.7 Only the D-loop is a non-coding stretch, containing the promoters for L- and H-strand transcription. All tRNAs required for mitochondrial protein synthesis are encoded on mtDNA.8
Mitochondrial genetics differs from nuclear genetics in the following points: (1) mtDNA is maternally inherited. (2) Mitochondria are polyploid, containing 2–10 mtDNA copies per organelle, and each cell contains hundreds of mitochondria. (3) In the normal cell all mtDNA copies are identical (homoplasmy). The propensity of mtDNA to mutate randomly, however, results in the coexistence of wild-type mtDNA and mutant mtDNA in a single cell and organ (heteroplasmy). (4) During oogenesis mitochondria carrying mutant mtDNA are stochastically distributed to daughter cells, resulting in varying mutation loads between different oocytes, generations and tissues and increasing the phenotype variability of MIDs (bottleneck effect). (5) Because of mitotic segregation (the proportion of mutant mtDNA in daughter cells following cell division may shift due to a random drift and the phenotype may change accordingly) and polyploidy phenotypic expression is dependent on a threshold effect (usually 60–90%),8 such that the load of mutant mtDNA copies needs to exceed a certain amount that the effect of a mutation can no longer be compensated by wild-type mtDNA. (6) All coding sequences are contiguous with each other without introns.9 (7) The mtDNA genetic code slightly differs from the universal genetic code. (8) Expression of mtDNA genes relies not only on the mitochondrial transcription machinery but also on the interplay between nuclear encoded transcription and translation factors with mitochondrial tRNAs and rRNAs. (9) Phenotypic variability is additionally dependent on the pathogenicity of a mutation, the affected gene, and the reliance of an organ on mitochondrial energy supply. So far, ∼200 mtDNA point mutations have been reported.10 (10) mtDNA is normally not methylated.8
mtDNA mutations can be classified as single large-scale rearrangements (partial deletions or duplications) or point mutations. Large-scale rearrangements usually are sporadic, while point mutations usually are maternally inherited. Large-scale rearrangements affect several genes and are invariably heteroplasmic, whereas point mutations affect mit and sin genes and can be heteroplasmic or homoplasmic, like in LHON or certain tRNA(Ile) mutations.2, 7, 9, 11, 12 Phenotype expression of mtDNA mutations often requires the influence of nuclear modifier genes, environmental factors, or the presence of mtDNA haplotypes (polymorphisms). Clusters of mtDNA variants may act as predisposing haplotypes, increasing the risk of disease. Most frequently, mtDNA mutations are heteroplasmic and only rarely homoplasmic. Pathogenic nDNA mutations are likely to be more numerous than pathogenic mtDNA mutations.10
nDNA mutations are classified as follows: (1) Mutations in nuclearly encoded RC subunits (LS). (2) Mutations in ancillary proteins, such as RC subunit assembly factors (LS, GRACILE). (3) Mutations in genes affecting the maintenance or expression of mtDNA leading to faulty intergenomic communication and thus breakage syndromes (AD-CPEO, AR-CPEO, MNGIE, SANDO, SCAE, AHS), depletion syndromes (MDS, nsMID, myopathy, encephalomyopathy, multi-system disease), or translation defects (MLASA). (4) Mutations in biosynthetic enzymes for lipids or cofactors (Barth syndrome). (5) Mutations in genes involved in the coenzyme-Q (CoQ) metabolism (LS). (6) Mutations in genes resulting in defective mitochondrial trafficking or transport machinery (DDS/MTS, XLASA). (7) Mutations in genes encoding proteins involved in the mitochondrial biogenesis, such as fusion or fission of mitochondria (OPA, CMT2A) (Table 2).13
The genotype–phenotype-correlation in MIDs is generally poor.7 Whether mutated RC proteins represent new targets for the immune system remains speculative. However, there are indications that some mtDNA mutations create new antigens due to altered hydrophobicity.14
The main function of mitochondria is the production of energy in form of heat or ATP. To accomplish this goal ingested carbohydrates are metabolized via aerobic glycolysis, with pyruvate as the end-product and fat is hydrolyzed. Pyruvate enters the mitochondrion through a symport system in the wake of hydrogen ions, which flow into the matrix along their electrochemical gradient. There pyruvate is oxidized via the PDH complex into acetyl-CoA, which enters the Krebs cycle. Free fatty acids (FFA) enter the mitochondrion via a complex carrier system provided by carnitine-palmitoyl-transferase I and II. Inside the mitochondrion FFA undergo beta-oxidation, with acetyl-CoA as the end-product. Hydrogen ions from the Krebs cycle or beta-oxidation are transferred to either NAD+, generating NADH or to flavin adenine dinucleotide (FAD) from succinate in the Krebs cycle, generating FADH2. NADH transfers electrons to RCCI. FADH2 transfers electrons from succinate to RCCII or from the reduced electron transfer protein to CoQ.
The golden standard of diagnosing MIDs is genetic testing, why all effort should be taken to find the genetic defect. Due to the huge amount of undetected nDNA genes involved in mitochondrial metabolism, however, search for the genetic cause of MID often remains unsuccessful. In such cases the diagnosis relies on the documentation of a biochemical defect in the RC or another mitochondrial metabolic pathway. Diagnostic work-up starts with a comprehensive individual and family history, followed by a clinical neurologic, ophthalmologic, otologic, endocrinologic, cardiologic, gastroenterologic, nephrologic, hematologic, or dermatologic investigation. Instrumental investigations should be additionally applied to detect subclinical phenotypic manifestations of MIDs. Emergency laboratory should include glucose, lactate, ammonia, arterial blood gases, acyl-carnitine, amino acids in the serum, and organic acids in the urine.15
Based upon this information the clinician then decides whether the individual phenotype conforms to any of the syndromic MIDs or represents a nsMID. If a syndromic MID, such as CPEO, KSS, or PS is suspected a Southern blot or RFLP should be carried out to look for single or multiple mtDNA deletions. If multiple mtDNA deletions are detected, a search for mutations in the POLG1, POLG2, PEO1, ANT1, TYMP, or OPA1 genes should follow. If a syndromic MID, such as MELAS, MERRF, LHON, NARP, or MILS is suspected, DNA-micro-arrays, real-time PCR, single-gene sequencing of an affected tissue should be carried out. If no mutation is detected, mtDNA sequencing is the next step. If the phenotype suggests a syndromic MID due to a nDNA gene mutation (GRACILE, AD-CPEO, AR-CPEO, SANDO, SCAE, AHS, MNGIE, LS, MLASA, Barth syndrome, DDS, XLASA, or CMT2A), the corresponding genes should be sequenced.
In the presence of a non-syndromic phenotype, biochemical investigations of the most affected tissues should clarify if a single or multiple biochemical defect(s) is (are) present. In case of a single autosomally inherited biochemical defect, sequencing of genes encoding for structural subunits or assembly factors of RCCI, RCCIII, RCCIV, and RCCV, or for enzymes of the coenzyme-Q biosynthesis should be undertaken. If the single biochemical defect is maternally inherited, one should proceed with mtDNA sequencing. If multiple autosomally inherited biochemical defects are present, a Southern blot should clarify if there is depletion of mtDNA. If Southern blotting detects mtDNA depletion and the primary affected organ is the skeletal muscle, sequencing of genes such as TK2, SUCLG1, SUCLA2, or RRM2B is recommended. If Southern blotting detects mtDNA depletion and the primary affected organ is the liver, sequencing of genes such as POLG, PEO1, DGUOK, or MPV17 is recommended. If Southern blotting detects no mtDNA depletion sequencing of mutated genes involved in the mitochondrial protein synthesis machinery is necessary.
Section snippets
Treatment
There is no causal treatment of MIDs in humans, only symptomatic therapy of various manifestations can be offered so far. Because of the involvement of multiple organs, variable expression, and chronic progressive course of MIDs, an individualized, integrated, multi-disciplinary approach needs to be adopted. This includes specialist nurses, speech, occupational, or physiotherapists, as well as medical professionals for neurology, psychiatry, ophthalmology, oto-rhino-laryngology, endocrinology,
Conclusions
Though there is no causal therapy of MIDs yet available, there are a number of promising therapeutic concepts under development and investigation, which might reach clinical applicability. These include up-regulation of endogenous ROS-scavengers, such as superoxide dismutase or glutathione, stem cell therapy, or gene therapy. Among the strategies of gene therapy reduction of the heteroplasmy rate appears, at the moment, the most promising approach. A further important approach is the detection
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