UNIVERSITA’ DI PISA
FACOLTA’ DI MEDICINA E CHIRURGIA
Scuola di Specializzazione in Neurologia
Mitochondrial DNA mutations and
related phenotypes. The experience of
the Italian Network.
Candidato: Dott. Daniele ORSUCCI
INDICE
1. RIASSUNTO DELLA TESI IN LINGUA ITALIANA ... 4
2. ENGLISH ABSTRACT ... 6
3. INTRODUCTION ... 8
3.1 Mitochondria and mitochondrial diseases ... 8
3.2 The diagnostic approach ... 15
3.3 Muscle biopsy and biochemical studies ... 20
3.4 Molecular studies ... 24
3.5 Therapeutic approaches ... 27
4. BACKGROUND AND AIMS ... 28
4.1 General aims ... 28
4.2 The A8344G mutation of the mitochondrial DNA ... 29
4.3 The A3243G mutation of the mitochondrial DNA ... 30
4.4 Mitochondrial DNA single deletion ... 31
5. METHODS ... 33
6. RESULTS ... 36
6.1 Patients enrolled in the Italian Database ... 36
6.2 Mitochondrial DNA mutations in our cohort ... 37
6.3 Mitochondrial DNA point mutations: A8344G ... 38
6.4 Mitochondrial DNA point mutations: A3243G ... 47
7. DISCUSSION ... 64
7.1 The A8344G mutation of the mitochondrial genome ... 64
7.2 The A3243G mutation of the mitochondrial genome ... 68
7.3 Mitochondrial DNA single deletion ... 73
8. CONCLUSION ... 75
9. ACKNOWLEDGEMENTS ... 78
10. ABREVIATIONS ... 79
11. REFERENCES ... 80
1. RIASSUNTO DELLA TESI IN LINGUA ITALIANA
Background. Le malattie mitocondriali sono tra le malattie genetiche più
frequenti. A dispetto degli straordinari progressi nella comprensione delle loro basi molecolari, le possibilità di trattamento sono ad oggi estremamente limitate. La rarità di queste malattie rappresenta una delle principali problematiche. Lo sviluppo di un registro informatico dei pazienti con malattia mitocondriale può consentire una miglior comprensione dei fenotipi e della storia naturale di queste patologie.
Obiettivi e metodi. Lo studio delle correlazioni genotipo-fenotipo nelle malattie
mitocondriali è un compito complesso. Le difficoltà sono evidenti: i sintomi possibili sono molteplici, la variabilità clinica è ampia anche all’interno di un singolo genotipo e la potenza statistica è bassa negli studi monocentrici a causa della rarità di queste condizioni. Obiettivo di questo lavoro è una migliore definizione dei correlati genotipo-fenotipo in un ampio gruppo di adulti e bambini con malattia mitocondriale da mutazione del DNA mitocondriale (mtDNA). Questo lavoro sarà condotto separatamente per i differenti genotipi (mutazioni puntiformi A8344G e A3243G e delezioni singole del mtDNA), al fine di definire meglio i fenotipi di tre delle più comuni mutazioni del mtDNA.
Risultati. Con questo approccio, siamo stati in grado di ridefinire i fenotipi dei
pazienti con una di queste mutazioni patogenetiche del mtDNA, rivisitando le definizioni di condizioni come la MELAS (encefalomiopatia mitocondriale associata ad acidosi lattica ed episodi stroke-like: pazienti con evidenza istologica, biochimica o molecolare di malattia mitocondriale che presentano episodi
stroke-like), MERRF (encefalomiopatia mioclonica con ragged red fibers: sindrome mitocondriale in cui il mioclono è la caratteristica clinica prominente, che non presenta i criteri per rientrare tra le altre sindromi encefalopatiche ben definite), sindrome di Kearns-Sayre (KSS: ptosi e/o oftalmoparesi da delezione singola del mtDNA in associazione ad almeno una delle seguenti caratteristiche: retinopatia, atassia, difetti di conduzione cardiaci, ipoacusia, deficit di crescita/bassa statura, coinvolgimento cognitivo, cardiomiopatia) ed oftalmoplegia esterna progressiva da delezione singola (ptosi e/o oftalmoparesi da delezione singola del mtDNA che non rientra tra i nuovi criteri della KSS né tra i criteri della sindrome di Pearson).
Conclusioni. Grandi studi multicentrici sono necessari per caratterizzare meglio il
quadro clinico e la storia naturale di queste malattie, al fine di identificare misure di cui possano beneficiare i pazienti affetti da queste malattie croniche, tuttora incurabili.
2. ENGLISH ABSTRACT
Background. Mitochondrial disorders are among the most common genetic
disorders. In contrast to the extraordinary progress in our understanding of the molecular bases, we are still extremely limited in our ability to treat these conditions. Small patient populations represent the major impediment to progress. The development of a web-based register of patients with mitochondrial disease can allow us to better understand the phenotypes and the natural history of these diseases.
Aims and methods. Studying genotype-phenotype relationship in mitochondrial
disorders is a complex task. The difficulties are obvious: there are a mass of possible clinical symptoms, the clinical variability is large even in individuals with the same genotype and the statistical power is low in single-center studies given the rarity of these conditions. The purpose of this work is to better define the genotype-phenotype correlations in a large group of adults and children with molecularly-confirmed mitochondrial DNA (mtDNA)-related mitochondrial disease. This work will be separately conducted for the different genotypes (i.e., A8344G, A3243G, mtDNA single deletion), in order to better define the clinical phenotypes of three of the most common mtDNA mutations.
Results. With this approach, we could more accurately define the phenotypes of
patients harbouring one of these pathogenic mtDNA mutations, revisiting the
classic definitions of conditions such as MELAS (mitochondrial
encephalomyopathy, lactic acidosis and stroke-like episodes syndrome: patients with histological, biochemical and/or molecular evidence of mitochondrial disease
who experience stroke-like episodes), MERRF (myoclonic encephalomyopathy with ragged red fibers: mitochondrial syndrome where myoclonus is the prominent clinical feature, and which does not meet the criteria of other well-defined mitochondrial encephalophatic syndromes), Kearns-Sayre syndrome (KSS: ptosis and/or ophthalmoparesis due to a mtDNA single large-scale deletion and at least one of the following features: retinopathy, ataxia, cardiac conduction defects, hearing loss, failure to thrive/short stature, cognitive involvement, cardiomyopathy) and single-deletion PEO (ptosis and/or ophthalmoparesis due to a mtDNA single large-scale deletion not fulfilling the “new” KSS criteria nor criteria for Pearson syndrome).
Conclusion. Large, multicenter studies are needed to better characterize the
clinical picture and natural history of these diseases, in order to identify some countermeasures capable of benefit the patients suffering with these chronic, still incurable disorders.
3. INTRODUCTION
3.1 Mitochondria and mitochondrial diseases
Mitochondria evolved from the aerobic bacteria which about 1.5 billion years ago populated primordial eukaryotic cells, thus endowing the host cells with oxidative phosphorilation (much more efficient than anaerobic glycolysis) (DiMauro and Schon 2003). The most crucial task of the mitochondrion is the generation of energy as adenosine triphosphate (ATP), by means of the electron transport chain (ETC). The ETC consists of five multimeric protein complexes located in the inner mitochondrial membrane (DiMauro and Schon 2003), and also requires two electron carriers, coenzyme Q10 (CoQ10, or ubiquinone) and cytochrome c (cyt c). Electrons are transported along the complexes to molecular oxygen (O2),
finally producing water (Figure 1). At the same time, protons are pumped across the mitochondrial inner membrane, from the matrix to the intermembrane space, by the complexes I, III, and IV. This process creates an electrochemical proton gradient. ATP is produced by the influx of these protons back through the complex V, or ATP synthase (the “rotary motor”) (Noji and Yoshida 2001). This metabolic pathway is under control of both nuclear (nDNA) and mitochondrial (mtDNA) genomes (DiMauro and Schon 2003). The mtDNA encodes information for mitochondrial transfer RNAs (tRNAs), for ribosomal RNAs (rRNAs), and for a few of subunits of the ETC. The rest of the mitochondrial proteins are encoded by genes in the nuclear chromosomes, and finally imported into the mitochondrion (DiMauro and Schon 2003).
Mitochondrial diseases (MD) are a group of disorders caused by impairment of the mitochondrial ETC. The effects of mutations which affect the ETC may be multisystemic, with involvement of visual and auditory pathways, heart, central nervous system (CNS), and skeletal muscle. The estimated prevalence of MD is 1-2 in 10000 (Schaefer et al. 1-2008). MD are, therefore, one of the most common inherited neuromuscular disorders.
The genetic classification of MD distinguishes disorders due to defects in mtDNA from those due to defects in nDNA (Filosto and Mancuso 2007) (Table 1). The first ones are inherited according to the rules of mitochondrial genetics (maternal inheritance, heteroplasmy and the threshold effect, mitotic segregation). Each cell contains multiple copies of mtDNA (polyplasmy), which in normal individuals are identical to one another (homoplasmy). Heteroplasmy refers to the coexistence of two populations of mtDNA, normal and mutated. Mutated mtDNA in a given tissue has to reach a minimum critical number before oxidative metabolism is impaired severely enough to cause dysfunction (threshold effect) (DiMauro and Schon 2003). The pathogenic threshold varies from tissue to tissue according to the relative dependence of each tissue on oxidative metabolism (DiMauro and Schon 2003). CNS, skeletal muscle, heart, endocrine glands, the retina, the renal tubule and the auditory sensory cells, frequent targets of MD, have a high energy demand and, therefore, a lower threshold for a critical load of mtDNA mutation. Most mtDNA-related disorders are multisystemic (DiMauro et al. 2004).
Other factors, including nuclear genetic background, mtDNA polymorphisms and additional mtDNA (or nuclear) mutations may influence the expression of mtDNA mutations (Swalwell et al. 2008). For instance, mtDNA 12S rRNA A1555G mutation is one of the most important causes of aminoglycoside-induced
and non-syndromic deafness. Chinese “A1555G” families carrying mitochondrial haplogroup B exhibited higher penetrance and expressivity of hearing loss (Lu et al. 2009). These observations suggested that mtDNA polymorphisms may modulate the variable penetrance and expressivity of deafness among families carrying this mutation. Gene-environment interactions could have a role in mtDNA-related disorders as well. A study identified a strong and consistent association between visual loss and smoking in Leber hereditary optic neuropathy (LHON), independent of gender and alcohol intake, leading to a clinical penetrance of 93% in men who smoked (Kirkman et al. 2009). There was a trend towards increased visual failure with alcohol, but only with a heavy intake (Kirkman et al. 2009). Therefore, carriers of a “LHON” mtDNA mutation should be strongly advised not to smoke and to moderate their alcohol intake.
The sporadic occurrence of a MD, such as progressive external ophthalmoplegia (PEO), Kearns-Sayre syndrome (KSS, ophthalmoplegia associated to pigmentary retinopathy and cardiac conduction block) and Pearson syndrome (refractory sideroblastic anemia associated with pancreatic insufficiency) is suggestive for a single, sporadic, mtDNA deletion (Filosto and Mancuso 2007). The percentage and location of the single deletion could differ between these syndromes, and the moment when the deletion is produced probably determines the affected tissues and the phenotype, as well as the percentage and location of the deletion (Lopez-Gallardo et al. 2009).
MD related to nDNA are caused by mutations in structural components or ancillary proteins of the ETC, by defects of the membrane lipid milieu, of CoQ10 biosynthetic genes and by defects in intergenomic signaling (associated to mtDNA depletion or multiple deletions) (DiMauro and Schon 2003). Nuclear
mutations affecting the ETC are often autosomal recessive. Tissue involvement is less variable than in mtDNA-related disorders, and the onset is earlier (often soon after birth). Most causes of Leigh syndrome involve mutations in genes encoding subunits or assembly proteins of the ETC (DiMauro et al. 2004).
A plausible pathogenic mechanism of MD is oxidative stress (see Figure 1), but to date all of the evidence underlying the relationship between mitochondrial dysfunction and oxidative stress has been obtained from in vitro studies (Vives-Bauza et al. 2006) or from studies on patient biopsies (Hargreaves et al. 2005). Surprisingly, very few reports have examined this relationship directly in blood samples from patients with MD. Hargreaves and co-workers found a deficiency of reduced glutathione (GSH) in skeletal muscle from patients with MD, either as a consequence of diminished ATP availability or of increased oxidative stress, or both (Hargreaves et al. 2005). More recently, Atkuri and co-workers (2009) found intracellular GSH deficiency using high-dimensional flow cytometry and biochemical analysis of freshly obtained blood samples from patients with MD and organic acidemias. GSH levels were low in T-lymphocyte subsets, monocytes and neutrophils from patients who were not on antioxidant supplements but were normal in blood cells from similar subjects on antioxidant supplements (Atkuri et al. 2009).
Our group reported that protein oxidative damage was increased in patients with MD, especially in the more severely affected ones, supporting the hypothesis of the vicious circle involving mitochondrial dysfunction and oxidative stress in MD (Mancuso et al. 2010). In detail, we examined oxidative stress biomarkers (advanced oxidation protein products [AOPP] and ferric reducing antioxidant power [FRAP]) in blood samples from 27 patients with MD and 42 controls.
AOPP levels were greater in patients than in controls (P < 0.00001), whereas FRAP levels did not differ between the two groups (Mancuso et al. 2010). Subsequently, we performed a double-blind cross-over study to evaluate if 30-day supplementation with a whey-based cysteine donor could modify these markers. As already seen, GSH deficiency has been reported in MD, and the biosynthesis of GSH depends on cysteine availability. Clinical scales and lactate at rest and after aerobic incremental exercise were not modified by the 30-day supplementation, but the cysteine donor modified with a high significant P-value the considered oxidative stress markers, as well as total GSH levels (Mancuso et al. 2010). Oxidative stress biomarkers may be useful to detect redox imbalance in mitochondrial diseases and to provide non-invasive tools to monitor disease status.
The field of mitochondrial genetics has greatly benefited from the creation of cell and animal models of mtDNA mutation (Khan et al. 2007). In the cytoplasmic hybrid (“cybrid”) technique, culturable cells depleted of endogenous mtDNA are repopulated with mitochondria (with their own mtDNA) from patients (King and Attardi 1989). Phenotypic differences among cybrid lines are caused by the donor mtDNA, and not by nDNA or environmental factors. Also animal models of mtDNA function and mutation are of great importance in elucidating mitochondrial biology (Khan et al. 2007). Because of the technical difficulties of introducing mtDNA into cells, however, only a few such models exist (Li et al. 2006). Intra-mitochondrial delivery of restriction enzymes has been used to effect changes in the heteroplasmic frequency of mtDNA variants in cell culture and in an animal model (Bayona-Bafaluy et al. 2005). A technique analogous to cloning has been used to generate mtDNA mice, a clone stably expressing a large deletion (Inoue et al. 2000). Mice created by the introduction of a proofreading-deficient
form of mtDNA-specific polymerase gamma (POLG) present an accelerated accumulation of randomly dispersed point mutations and large deletions (Longley et al. 2005). Available animal models of mtDNA mutation argue the importance of mtDNA to aging and disease, and improved knowledge of MD (Khan et al. 2007). Better techniques of mtDNA transfer will be necessary for further progress.
Figure 1. Mitochondrial metabolism. Schematic representation of mitochondrial
biochemical pathways, representing relationship between energy production (TCA and electron transport chain), reactive oxygen species production (superoxide O2˙¯ , hydroxyl radical OH˙), apoptosis regulation. Electron transport chain complexes: I (NADH:ubiquinone reductase), II (succinate:ubiquinone reductase), III (ubiquinone-cyt c oxidoreductase), IV (cytochrome c oxidase), F0V (ATP
synthase). ADP, adenosine 5'-diphosphate; ATP, adenosine 5'-triphosphate; AIF, apoptosis inducing factor; ANT, adenine nucleotide transporter; CAD, caspase-activated DNase; CytC, cytochrome c; GPX, glutathione peroxidase; LDH, lactic dehydrogenase; MnSOD, manganese superoxide dismutase; NAD+/NADH, oxidized/reduced nicotinamide adenine dinucleotide; OAA, oxalacetic acid; PDH, pyruvate dehydrogenase; Pi, inorganic phosphorus; TCA, tricarboxylic acid cycle; VDAC, voltage-dependent anion channel. From MITOMAP: A Human
Mitochondrial Genome Database (http://www.mitomap.org), allowed
Table 1. Genetic classification of well characterized mitochondrial diseases (MD). MELAS, mitochondrial encephalomyopathy with lactic acidosis and
stroke-like episodes; MERRF, myoclonic epilepsy with ragged red fibers; MNGIE, Mitochondrial neurogastrointestinal encephalomyopathy; MLASA, mitochondrial myopathy and sideroblastic anemia; NARP, neuropathy, ataxia, retinitis pigmentosa; PEO, progressive external ophthalmoplegia (Modified from Filosto and Mancuso, 2007).
Disorders of mitochondrial genome
MD caused by nuclear gene defects Sporadic rearrangements
Kearns-Sayre syndrome (KSS)
Pearson syndrome Sporadic PEO
Diabetes and deafness
Sporadic point mutations
PEO MELAS Exercise intolerance Isolated myopathy Maternal-inherited mtDNA point mutations
Genes encoding structural proteins
Leber hereditary optic neuropathy (LHON) NARP
Maternal-inherited Leigh syndrome
Genes encoding tRNAs
MELAS MERRF Cardiomyopathy and myopathy (MIMyCa) PEO Isolated myopathy Diabetes and deafness Sensorineural hearing loss Hypertrophic
cardiomyopathy Tubulopathy
Genes encoding rRNAs
Aminoglycosides-induced deafness
Hypertrophic cardiomyopathy
Defects of genes encoding for structural proteins of the complexes of respiratory chain
Leigh Syndrome Cardiomyopathy
Paraganglioma, pheochromocytomas Multisystemic syndromes
Defects of genes encoding factors involved in the assembling complexes of respiratory chain (“Assembly genes”)
Leigh Syndrome
Multisystemic Syndromes
Defects of genes altering mtDNA stability and integrity (intergenomic communication)
Autosomal PEO
Early-onset parkinsonism Multisystemic syndromes MNGIE
mtDNA depletion syndromes
Coenzyme Q10 deficiency
Cerebellar form Myopathic form Encephalomyopathy
Infantile multisystemic disease Leigh syndrome
Defects of the lipid milieu
Barth syndrome
Syndromes due to defects of mitochondrial ribonucleic acid
MLASA
Defects of mitochondrial fission or fusion
Autosomal dominant optic atrophy (ADOA) Charcot-Marie-Tooth (CMT) 2A
3.2 The diagnostic approach
The diagnostic process of MD does not differ from that employed for other neuromuscular diseases, and should start from patient and family history and physical and neurologic examination (DiMauro et al. 2004). “Red flags” for MD are short stature, sensorineural hearing loss, ptosis, ophthalmoplegia, axonal neuropathy, diabetes mellitus, hypertrophic cardiomyopathy, renotubular acidosis, migraine (DiMauro et al. 2004). Diagnosis of MD requires a complex approach: measurements of serum lactate, electromyography, magnetic resonance spectroscopy (MRS), muscle histology and enzymology, and genetic analysis.
Serum creatine kinase (CK) levels are usually normal or only moderately elevated in MD, but exceptions are possible (Nishigaki et al. 2002).
In MD there is an impairment of ETC and oxidative phosphorylation, leading up to defective aerobic system. Thus, muscle is forced to apply an anaerobic metabolism, with enhanced lactate production. Lactic acidosis is a common finding in MD. In MELAS, resting plasma lactate levels correlated with muscle mtDNA mutation A3243G load (Jeppesen et al. 2006). Lactic acidemia is also present in pyruvate dehydrogenase (PDH) deficiency. The blood lactate-to-pyruvate (L:P) molar ratio is useful for differentiating MD (L:P > 25) from PDH deficiency in newborns with lactic acidosis (Debray et al. 2007).
In patients with MD exercise testing should stress the aerobic metabolism. In our experience, we adopt an incremental aerobic exercise cycle ergometer protocol (Siciliano et al. 2007). The sensitivity of lactate increase after exercise seems to be lower if compared to resting lactate (Jeppesen et al. 2003). A simple, non-invasive screening tool for MD is the aerobic forearm exercise test (Meulemans et al.
2007). A recent study on 41 normal controls, fifteen MD patients and twenty subjects with other myopathies showed a sensitivity of only 20% and a specificity of 95% versus normal controls, but only of 75% versus diseased controls (Hanisch et al. 2006). Forearm ischemic test is more sensitive for MD (Tarnopolsky et al. 2003), but less specific.
The serum alanine level has been proposed in differential diagnosis of hypotonic newborns with lactic acidemia. No significant serum alanine level elevation was found in transient lactic acidemia after mild hypoxia, whilst increased serum alanine was a sensitive marker of mitochondrial dysfunction (Morava et al. 2006). For unclear reasons, in MELAS and NARP (neuropathy, ataxia, and retinitis pigmentosa) citrulline levels are reduced, with an inverse correlation between arginine and citrulline (Naini et al. 2005).
Measurement of plasmatic thymidine and deoxyuridine levels by a gradient-elution high performance liquid chromatography (HPLC) can be useful in selected MD cases (Valentino et al. 2007; Lara et al. 2007). Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disease caused by mutation in the nuclear gene ECGF1 (or TP, thymidine phosphorylase), and is characterized by ptosis and ophthalmoparesis, gastrointestinal dysmotility, peripheral neuropathy and leukoencephalopathy (Valentino et al. 2007; Lara et al. 2007). It is associated to both mtDNA depletion and multiple deletions.
Muscular imaging studies, of additional value in the diagnosis of patients with suspected MD, include CT, magnetic resonance imaging (MRI), and ultrasonography. MRI studies showed that changes of muscle architecture, similar to those found in muscular dystrophy patients, occurred consistently in patients
with a high mutation load for single large-scale deletions of mtDNA, but were absent in patients with the A3243AG “MELAS” mtDNA point mutation (Olsen et al. 2003). Pillen and co-workers (2006) investigated the diagnostic value of quantitative skeletal muscle ultrasonography in children suspected of having MD, and observed a sensitivity of 25-46%, with a specificity of 85-100% (Pillen et al. 2006).
Patients with different MD have characteristic brain MRI patterns (Bianchi et al. 2007). Rapid progression of atrophy involving all structures of the brain with variable involvement of deep gray and white matter were the most frequent MRI findings in forty children with ETC defects (Kim et al. 2008). The type of biochemical defect in the ETC did not correlate with MRI findings in these patients (Kim et al. 2008).
MR spectroscopy (MRS) is a frequency analysis of the MR signal that allows an evaluation of brain and muscle metabolism in vivo. Proton (1H) and phosphorous (31P) are the two main nuclear species explored with MRS.
1H MRS plays an important role in demonstrating an oxidative metabolism impairment in the brain, since it can show lactate accumulation in CNS (a doublet peak at 1.33 ppm). Lactate may be considered a metabolic marker of MD only in a proper clinical framework and in brain areas which appear normal in MRI (Bianchi et al. 2007). Ventricular cerebrospinal fluid may be a specific and sensitive site for screening by spectroscopy (Bianchi et al. 2007). In mitochondrial encephalopathies there is a reduction in N-acetylaspartate and choline, index of neuronal loss or dysfunction. These specific metabolic profiles are highly specific for MD. An additional abnormal signal at 0.9 ppm has been also observed
(Bianchi et al. 2003); it may be related to the accumulation of branched aminoacids.
31P MRS measures PCr and inorganic phosphate (Pi) fluctuations during muscular exercise, and can monitor some aspects of oxidative metabolism. The most useful indicator of MD are the findings of combined low PCr/Pi ratio at rest and low post-exercise recovery, and the delay in post exercise adenosine diphosphate (ADP) recovery. Nevertheless, 31P MRS of skeletal muscle does not seem to be a sensitive diagnostic test for mitochondrial myopathy. In a study of seven patients with single large-scale deletion, nine with point mutations of mtDNA and fourteen healthy subjects, PCr/Pi and ATP production after exercise were similar in patients and healthy subjects (Jeppesen et al. 2007). For the described reasons, muscular 31P MRS is still experimental in clinical practice, but could represent an useful tool for monitoring the response to therapies.
Also nuclear medicine can be useful in some cases of MD. The imaging of striatal dopamine transporters by a single-photon-emission tomography (SPECT) study (DATSCAN) can be useful in MD patients with extrapyramidal signs, such as tremor (Mancuso et al. 2008a). High-resolution regional cerebral blood flow obtained through Tc-99m ethylcysteinate dimer SPECT can better localize and assess the extent of brain damage in patients with suspected MD and only subtle changes on MRI (Narla et al. 2008). Reduced regional glucose metabolism has been observed in the fronto-temporal region of two siblings with mtDNA multiple deletions and a MNGIE-like disorder, by means of positron emission tomography with fluorine 18-labeled deoxyglucose (FDG-PET) (Lehnhardt et al. 2008).
14 patients with the mtDNA A3243G “MELAS” mutation, not experiencing acute stroke-like episodes, and 14 age-matched controls underwent FDG-PET, as well
as PET with [(15)O]H2O and [(15)O]O2 (Lindroos et al. 2009). Cerebral metabolic
rate of oxygen was decreased by 26% in the grey as well as the white matter of patients with the A3243G mutation, and decreased metabolic rate of glucose was found with predilection to the posterior regions (Lindroos et al. 2009).
A study with [(62)Cu]-diacetyl-bis(N4-methylthiosemicarbazone) FDG-PET has been performed to visualize the regional oxidative stress, glucose metabolism and blood flow in brain lesions of stroke-like episodes in MELAS, and could demonstrate oxidative stress following hyperemia along with increased glucose metabolism during MELAS stroke-like episodes (Ikawa et al. 2009).
3.3 Muscle biopsy and biochemical studies
The main pathological features of mitochondrial myopathies are ragged red fibers (RRF), accumulation of structurally altered mitochondria (Filosto et al. 2007) (Figures 2 and 3). RRF are usually present in case of alteration of functional genes (i.e. tRNAs), but rarely when structural genes are involved (such as in LHON) (Filosto et al. 2007). Further alterations are evident by the reaction for mitochondrial enzymes SDH (succinate dehydrogenase, or complex II) and COX (cytochrome c oxidase, or complex IV). SDH staining can show the presence of subsarcolemmal or diffuse accumulation of mitochondria (“ragged blue fibers”) and alterations of the intermyofibrillar reticulum (Figure 4). COX staining can show the presence of COX negative fibers (Figure 5). Combining the assessment of SDH with COX on the same section can be useful to identify fibers devoid of COX. Interestingly, in both MELAS and MERRF (myoclonus epilepsy with RRF) SDH staining usually reveals strongly SDH-reactive blood vessels (SSVs). However, these are typically positive in MELAS whereas they are COX-negative in MERRF. Anti-COX subunit antibodies allow the comparison of nuclear protein synthesis (COX IV) and mitochondrial protein synthesis (COX I, II and III) in single fibers, leading to the detection of the site of the dysfunction (nuclear or mitochondrial) (Filosto et al. 2007). Immunohistochemistry using antimitochondrial antibody can be performed in order to observe the ultrastructure of the skeletal muscle and other tissues (Yuri et al. 2008). Among the morphological methods, electron microscopy was historically instrumental in the diagnosis of MD confirming the presence of affected fibers with abnormal mitochondria. The changes may affect the number, the size and the shape of mitochondria, with bizarre and giant forms, and also the pattern of the cristae and
the presence of crystalline or osmiophilic inclusions. However, with the development of molecular and genetic characterization of MD, the contribution of electron microscopy in the diagnosis of MD has been questioned. Nevertheless, consistent patterns can be recognized (Kyriacou and Kyriakides 2006).
Some MELAS (and rarely MERRF) patients have unremarkably biopsy findings, but a biochemical defect of respiratory complexes; thus, normal muscle histology do not rule out MD, especially in patients with tRNA mutations (Mancuso et al. 2008b; McFarland et al. 2002).
Apart from histopathological findings, muscle biopsy can add important information about the molecular and biochemical defect responsible of the disease. A mutation in a nDNA or mtDNA structural gene commonly results in deficient activity of the affected enzyme, whereas the impairment of mitochondrial protein synthesis (mutations in tRNA, single or multiple deletions, and mitochondrial depletion) reduce the activity of respiratory complexes I, III, IV and V (DiMauro et al. 2004). Biochemical spectrophotometric investigations include analysis of respiratory complexes activity, and can be performed in tissue homogenates. Polarographic studies include measurement of ETC activity, and can also determine the efficiency of substrate transport and the integrity of mitochondrial membranes.
Correct function of the ETC requires proper assembly of the approximately eighty proteins in the complexes. There are numerous assembly factors. Blue native electrophoresis has become a crucial tool to investigate ETC-related defects in MD patients (Calvaruso et al. 2008). In addition, ETC-assembly profiles can be obtained by two dimensional blue native/SDS gel electrophoresis, which provides
additional information for identifying disease-causing mutations and insight in the role of specific proteins in the biogenesis of the ETC (Calvaruso et al. 2008).
Figure 2. Subsarcolemmal accumulation of structurally altered mitochondria. Two pathological fibers are shown (Hematoxylin-Eosin).
Figure 3. Ragged red fibers (RRF). Two RRF are present in the figure
Figure 4. Subsarcolemmal accumulation of structurally altered mitochondria. A pathological, “pre-ragged blue” fiber is shown (SDH [succinate
dehydrogenase] staining).
Figure 5. Cytochrome c oxidase (COX)-negative fibers. Many COX-negative
3.4 Molecular studies
Genetic studies on blood cells are more useful in nDNA than in mtDNA-associated disorders. MtDNA mutations are more easily found in muscular cells. Whilst the most common MERRF-associated mutation (A8344G) is frequently detectable in blood, other tRNA mutations are sometimes present in very low amounts in circulating cells (DiMauro et al. 2004). Other easily accessible tissues are urinary sediment, oral mucosa, hair follicles and cultured skin fibroblasts (DiMauro et al. 2004).
In leukocytes, alterations in the copy number of mtDNA are related to the proportion of mtDNA with a point mutation or large-scale deletion. This may serve as biomarker of disease progression of MELAS and MERRF (Liu et al. 2006). In relation to controls, the copy number of mtDNA in leukocytes of MELAS or MERRF patients resulted higher at a young age but lower at an advanced age (Liu et al. 2006). Patients with multi-system disorders had lower mtDNA copy numbers in leukocytes (Liu et al. 2006). Also higher proportions of mtDNA with 4.9 kb deletion were found in leukocytes of MERRF patients with multi-system involvement (Liu et al. 2006).
Being able to predict which patients are likely to develop complications of MD could be essential for optimal clinical management. An important issue is the detection of the tissues suitable for molecular analysis that better reflect the level of mutation in clinically affected tissue. Five tissues (leukocytes, skin fibroblasts, hair roots, urinary sediment, and cheek mucosa) have been studied in 61 individuals from 22 families harbouring the A3243G “MELAS” mutation (Shanske et al. 2004). Although mutational loads varied widely among these tissues, as a rule DNA from urinary sediment had the highest and blood the lowest
proportion of mutant genomes. Furthermore, fibroblasts and cheek mucosa tended to have higher mutation loads than blood but lower than urinary sediment. Therefore, urinary sediment and cheek mucosa could be tissues of choice for the diagnosis of mtDNA mutations (Shanske et al. 2004). Other groups obtained similar findings (Ma et al. 2009). Whittaker and co-workers (2009) studied 24 adult patients harbouring the A3243G mutation in order to investigate whether urine was a useful tissue for assessing disease severity. Despite very weak correlation between both blood and muscle mutation load and disease severity, the highest correlation with clinical score was with A3243G mutation load in urinary epithelium (P < 0.012), which could replace muscle biopsy as the most suitable tissue for the initial diagnosis in patients suspected of harbouring the A3243G mutation (Whittaker et al. 2009).
Southern blot analysis (Schon et al. 2002) and real-time quantitative polymerase chain reaction (PCR) are effective methods which simultaneously detect mtDNA deletions and quantify total mtDNA content in skeletal muscle, using specific probes. Multiple deletions are caused by nuclear gene defects and subsequent intergenomic signaling impairment. The most frequent clinical presentations of multiple deletions is autosomal PEO, caused by mutations in nuclear genes ANT-1 (adenine nucleotide traslocator 1), Twinkle (a mtDNA helicase), POLG-1 or -2 (Spinazzola and Zeviani 2007), OPA1 (Hudson et al. 2008; Stewart et al. 2008),
RRM2B (Tyynismaa et al. 2009) and rarely other genes. MtDNA depletion
syndrome is transmitted as an autosomal recessive trait. Possible phenotypes are hepato-cerebral syndromes (including Alpers syndrome); encephalomyopathy and anemia; myopathy (Filosto and Mancuso 2007). Mutations in different nuclear genes are involved. MNGIE is associated to both mtDNA depletion and multiple deletions. Single deletions are usually sporadic. The most frequent clinical
syndrome due to single deletion is sporadic PEO. About one-third of these patients harbors a “common deletion” of 4.9-kb (Filosto and Mancuso 2007).
In muscle samples, gene analysis for mtDNA mutations commonly associated to MD can also be performed, i.e. PCR/restriction fragment length polymorphism (RFLP) analysis. Because of the segregation phenomenon, these analysis in circulating cells have low use. The following steps are denaturing HPLC or direct sequencing (DiMauro et al. 2004).
3.5 Therapeutic approaches
The treatment of MD is still inadequate, despite great progress in the molecular understanding of these disorders. Apart from symptomatic treatments, therapies that have been attempted include ETC cofactors, other metabolites secondarily decreased in MD, antioxidants and agents acting on lactic acidosis. However, their role in the treatment of the majority of MD remains unclear (Chinnery et al. 2006). Administration of metabolites and cofactors is the mainstay of real-life therapy and is especially important in disorders due to primary deficiencies of specific compounds, such as CoQ10.
There is currently no clear evidence supporting the use of any intervention in MD (Chinnery et al. 2006), and further research is needed. There have been very few randomized controlled clinical trials for the treatment of MD. Those that have been performed were short, and involved fewer than twenty study participants with heterogeneous phenotypes (Chinnery et al. 2006).
ETC cofactors include nicotinamide (vitamin B3), thiamine (vitamin B1), riboflavin (vitamin B2), succinate, and CoQ10. Despite isolated reports of improvement, there is no clear evidence that these dietary supplements improve the symptoms or alter the course of disease for the majority of patients with MD (Chinnery et al. 2006).
One of the “cocktails” of choice may be a combination of L-carnitine (1,000 mg three times a day) and CoQ10 (at least 300 mg a day), with the rationale of restoring free carnitine levels and exploiting the oxygen radical scavenger properties of CoQ10.
4. BACKGROUND AND AIMS
4.1 General aims
Studying genotype-phenotype relationship in mitochondrial disorders is a complex task. The difficulties are obvious: there are a mass of possible clinical symptoms, the clinical variability is large even in individuals with the same genotype and the statistical power is low in single-center studies given the rarity of these conditions. The study of the genotype-phenotype correlation can be best conducted using a comprehensive approach by a multicenter consortium that can establish a large database of patients to be characterized by uniform clinical standards and by state-of-the art molecular methods.
The purpose of this work is to better define the genotype-phenotype correlations in a large group of adults and children with molecularly-confirmed mtDNA-related mitochondrial disease. This work will be separately conducted for the different genotypes (i.e., A8344G, A3243G, mtDNA single deletion), in order to better define the clinical phenotypes of three of the most common mtDNA mutations.
4.2 The A8344G mutation of the mitochondrial DNA
Myoclonic epilepsy with ragged-red fibers (MERRF) is a mitochondrial syndrome, originally described in 1980 (Fukuhara et al. 1980) and mostly characterized by generalized seizures, myoclonus and ataxia, with variable onset and associated with various mitochondrial DNA (mtDNA) point mutations, the most frequent of which is the A8344G change in the gene encoding tRNA lysine (Noer et al. 1991). Early reports showed that the A8344G transition was highly correlated to the MERRF phenotype (Shoffner et al. 1990), although it can also be associated with other phenotypes, for instance Leigh disease, or myopathy with multiple lipomatosis (Silvestri et al. 1993). The absence of the mutation in some typical MERRF patients suggested that other mutations may produce the same phenotype (Silvestri et al. 1993), including the T8356C which also affects the tRNA lysine gene (Silvestri et al. 1992).
MERRF is very rare, as is the A8344G mutation (<0.5/100,000 - 4% of all pathogenic mtDNA mutations) (Remes et al. 2003; Schaefer et al. 2008). Therefore, most of the previous studies describing the clinical features of MERRF patients and/or subjects carrying the A8344G mutation have been based on single case/family reports (Wiedemann et al. 2008) or case series with very few patients (Lorenzoni et al. 2011). Some early reviews of literature are also available (Chinnery et al. 1997), but recent data are lacking, and clinical heterogeneity is probably higher than previously suspected (Graf et al. 1993).
The primary aim of this study is the clinical characterization of a cohort of mitochondrial patients with the A8344G mutation. The secondary aim is the thorough revision of previously reported clinical data about this mutation.
4.3 The A3243G mutation of the mitochondrial DNA
The A3243G mutation is one of the most common point mutations of the mitochondrial genome (mtDNA). The clinical syndromes that have been
historically ascribed to this substitution include mitochondrial
encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), maternally inherited deafness and diabetes (MIDD) and progressive external ophthalmoplegia (PEO) (Nesbitt et al. 2013), but heterogeneity is high (Kaufmann et al. 2009). Other reported features include isolated myopathy, cardiomyopathy, seizures, migraine, ataxia, cognitive impairment, gastro-intestinal dysmotility and short stature (Nesbitt et al. 2013). This phenotypic variability is incompletely understood; at least in part it may be due to heteroplasmy, with varying proportions of mutant and wild-type mtDNA molecules in different tissues (Kaufmann et al. 2009). However, other factors, environmental and/or genetic, must be involved.
Here, we revise the phenotypic spectrum associated with the A3243G mutation based on a cohort of carriers of the mutation from the database of the “Nation−wide Italian Collaborative Network of Mitochondrial Diseases”, in order to answer the following questions: I) Is the distinction in three phenotypes (MELAS; PEO; MIDD) fully justified or somehow arbitrary? II) Are some of the most frequent clinical features mutually associated? III) Which factors can explain the marked clinical heterogeneity of these patients? IV) Which clinical features may direct towards this molecular diagnosis?
4.4 Mitochondrial DNA single deletion
Mitochondrial DNA single deletion is one of the major causes of mitochondrial disease. It is commonly associated with PEO, Kearns-Sayre syndrome (KSS: PEO with multisystem involvement and specific diagnostic criteria) (Pitceathly et al. 2012), and Pearson syndrome (pediatric refractory sideroblastic anemia associated with pancreatic insufficiency), but its variability is incompletely understood.
The above mentioned conditions are the three sporadic clinical syndromes classically associated with large-scale deletions of mtDNA. However, it is increasingly recognised that there is a continuum of clinical phenotypes associated with single mtDNA deletions, and a large number of variants may exist (Pitceathly et al. 2012).
Single large-scale deletions of mtDNA were the first mtDNA genetic defects to be described and associated with human mitochondrial disorders. In 1988 large-scale mtDNA single deletions were identified in skeletal muscle from nine of 25 patients with mitochondrial myopathy (Holt et al. 1988) and in seven of seven patients with KSS (Zeviani et al. 1988). Some years later single deletions were also reported in Pearson syndrome (Morikawa et al. 1993).
Most of the previous studies describing the clinical features of single-deletion patients have been based on single case reports (Campos et al. 1996; Mancuso et al. 2008a; Santorelli et al. 1996) or case series with very few patients (Magalhaes et al. 1996; Kiyomoto et al. 1997; Vilarinho et al. 1998; Schroder et al. 2000). Some reviews of literature (Lopez-Gallardo et al. 2009) and larger surveys (Grady
et al. 2014; Yamashita et al. 2008) are also available, and clinical heterogeneity is probably higher than previously suspected.
Here, we revise the phenotypic spectrum associated with single deletions based on a cohort of carriers of the mutation from the database of the “Nation−wide Italian Collaborative Network of Mitochondrial Diseases”, in order to answer the following questions: I) Is the distinction in three phenotypes justified and clinically useful? II) How the classic diagnostic criteria could be updated?
5. METHODS
We reviewed the clinical, morphological and laboratory features of the carriers of mitochondrial DNA mutations (with special regard to the A8344G and A3243G point mutations and single deletions) enrolled in the database of the “Nation−wide Italian Collaborative Network of Mitochondrial Diseases”. In those patients, the diagnosis of mitochondrial disease was supported by clinical, histological, biochemical and/or molecular findings (Bernier et al. 2002).
The purposes of this work was to better define the genotype-phenotype correlations in a large group of adults and children with molecularly-confirmed mtDNA-related mitochondrial disease. This work was separately conducted for the different genotypes (i.e., A8344G, A3243G, mtDNA single deletion), in order to better define the clinical phenotypes of three of the most common mtDNA mutations.
A similar approach, but phenotype-based rather than genotype-based, was previously used for MERRF and myoclonus (Mancuso et al. 2014b). “Myoclonic epilepsy” is supposed to be the cardinal clinical feature of MERRF (myoclonic epilepsy with ragger red fibers). MERRF has been associated with various mtDNA mutations, most commonly the A8344G. Myoclonus can also be present in other mitochondrial conditions. Based on the database of the “Nation−wide Italian Collaborative Network of Mitochondrial Diseases”, financed by Telethon GUP09004, we reviewed the clinical and molecular data of mitochondrial patients with myoclonus among their clinical features. Myoclonus was a rather uncommon clinical feature of mitochondrial diseases (3.6%). Only one out of three patients
with MERRF has the A8344G mutation. Myoclonus is not inextricably linked to epilepsy in MERRF patients, and therefore the term “myoclonic epilepsy” could be inadequate and potentially misleading. We concluded that the practical definition of “MERRF” as the mitochondrial syndrome where myoclonus is the prominent clinical feature, and which doesn’t meet the criteria of other well-defined encephalophatic syndromes, seems more useful in the clinical practice as well as in the scientific literature. The acronym “MERRF” is probably too entrenched to be eliminated, but we suggested that MERRF should mean “myoclonic encephalomyopathy with ragged red fibers” (Mancuso et al. 2014b).
Given the lack of a standardized and uniform nomenclature in the previous literature, this work has redefined in a similar way the clinical features needed for the above-mentioned syndromic diagnoses (in patients with molecularly confirmed mtDNA-related disease due to the A8344G or to the A3243G point mutation or to a single mtDNA deletion) with the aim of making more uniform and comparable future studies.
Data were expressed as means ± standard deviation. The numeric data were compared by unpaired two-tailed t-testing (or one-way ANOVA when the groups were >2) or correlated by Spearman's coefficient of rank correlation. Proportions were compared by Fisher’s exact test. Categorical data were associated by means of the Fisher’s exact test. Bonferroni’s correction for multiple tests has been utilized when appropriate. A P value <0.05 was considered as significant. Data analysis was carried out using MedCalc® ans SPSS®.
The establishment of the database, and its use for scientific purposes, was approved by the local Ethical Committees of all the involved Centers, which
obtained written consent from each patient, in accordance with the ethical standards of the 1964 Declaration of Helsinki.
The database creation has been supported by a Telethon grant (GUP09004).
For the A8344G mutation, a systematic revision of previously reported data has also been included. Individuals harboring the A8344G mutation were identified through a Pubmed search of the scientific literature. Additional articles were identified in other databases such as Omim (http://www.ncbi.nlm.nih.gov/omim) and Mitomap (http://www.mitomap.org). Studies with an incomplete clinical description, as well as those reporting Italian patients, that could be included in our cohort, were excluded. Through this approach, we identified 75 articles (referenced in the Appendix).
6. RESULTS
6.1 Patients enrolled in the Italian Database
1197 patients were present in the database of the Italian network of Mitochondrial Diseases on January 31, 2013. Males were 574 (48.0%), females 623 (52%). The clinical picture was fully available for 1100 patients (mean age at onset 24.4 ± 20.5 years; age at last evaluation 39.8 ± 22.3 years; childhood onset [before age 16] 43.8%), the clinical features of whom are represented in Table 2.
Table 2. Clinical features of mitochondrial patients at last evaluation (39.8 ± 22.3 years). The table shows the features present in at least 5% of cases. Cataract,
stroke-like episodes, myoclonus, tremor, parkinsonism, dystonia, diskinesias, psychiatric involvement, vomiting, hypothyroidism, anaemia were rarer (2-5%).
Patients % (N = 1100) Ptosis/ophthalmoparesis 471 42.8 Muscle weakness 410 37.3 Hearing loss 261 23.7 Exercise intolerance 222 20.2 Optic neuropathy 210 19.1 Muscle wasting 195 17.7 Cerebellar ataxia 175 15.9 Hypotonia 174 15.8 Cognitive involvement 160 14.5 Neuropathy 130 11.8 Swallowing impairment 129 11.7 Epileptic seizures 123 11.2 Muscle pain 117 10.6 Pyramidal involvement 108 9.8 Diabetes 93 8.5 Cardiomyopathy 81 7.4 Migraine 79 7.2 Retinopathy 73 6.6 Respiratory impairment 68 6.2 Gastroint. dysmotility 58 5.3
6.2. Mitochondrial DNA mutations in our cohort
Among our 1197 patients, 731 (61.1%) harboured a mtDNA pathogenic point mutation (500 subjects, 41.8%) or single deletion (231 patients, 19.3%). Apart from the mutations associated to Leber hereditary optic neuropathy, A3243G and A8344G mutations were the two most frequent point mutations, as expected. The data are summarized in Table 3.
Table 3. Mitochondrial DNA mutations in our database
Subjects % (N = 1197) Single deletion 231 19.3 A3243G 133 11.1 “Leber” mutations 121 10.1 A8344G 42 3.5 T8993G 20 1.7 Others 184 15.4
6.3 Mitochondrial DNA point mutations: A8344G
6.3.1 The A8344G mutation in our database
A total of 42 patients harboring the A8344G mutation (25 patients from ten families and 17 other subjects) were identified among the 1197 mitochondrial patients (3.5%) present in our database. They represented the 8.4% (42/500) of all individuals with mtDNA point mutations. Male subjects were 22/42 (52.4%). 15/42 patients (35.7%) had childhood onset (<16 years). Of note, among the 17 subjects without molecularly-confirmed relatives, only five had a family history potentially indicative of a mtDNA-related disease.
The clinical picture was not available for three patients, who have not been considered in the following analysis. 4/39 (10.3%) subjects were asymptomatic mutation carriers (age at last evaluation 37.0±9.9 years; range 26-50). Heteroplasmy levels of asymptomatic carriers were 56.7±11.5% in blood and 55.8±25.1% in urine, not different from the symptomatic patients.
At the last evaluation, 38/39 subjects were adult (>16 year-old). The cardinal clinical feature of MERRF syndrome is deemed to be “myoclonic epilepsy” (DiMauro et al. 2002). However, Fisher’s exact test revealed no association between myoclonus and generalized seizures in the 38 adult 8344A>G mutation carriers with known clinical picture; contrariwise, myoclonus and cerebellar ataxia were associated (Table 4).
The mean age at onset of the 35 symptomatic patients was 30.1±18.4 years (range 0-66; 11/35 with clinical onset before age 16). Table 5 summarizes the clinical features at onset.
6.3.2 Disease progression
At the last evaluation, mean age and disease duration were 45.6±15.3 (range 5-72), and 21.6±15.3 (range 2-59) years, respectively. A 5-year-old boy had a complex syndrome characterized by consciousness disturbance, cognitive impairment, pyramidal signs, tremor, swallowing impairment, lactic acidosis, facial dysmorphism and cataract. The clinical features of the 34 adult patients are summarized in Figure 6 and described below.
Multiple lipomatosis was present in 11/34 patients (32.4%), only two of whom had isolated lipomatosis with no myopathic complaints (six cases) and/or CNS involvement (five cases). Of the four patients with isolated lipomatosis at onset, two developed myopathy but none CNS symptoms (mean follow-up 20.0 years, range 3-48).
19/34 (55.9%) showed clinical CNS involvement at the last evaluation (generalized seizures 12/34 [35.3%], cerebellar ataxia 8/34 [23.5%], myoclonus 8/34 [23.5%], cognitive involvement 2/34 [5.9%], stroke-like episodes 1/34 [2.9%]). 14 of these 19 patients also had myopathic complaints. Neither age at onset, nor age at last evaluation, nor disease duration could predict the development of CNS symptoms
Signs and symptoms of neuromuscular dysfunction were present in 26/34 patients (76.5%): muscle weakness 20/34 (58.8%), exercise intolerance 15/34 (44.1%), increased CK levels 15/34 (44.1%), eyelid ptosis 10/34 (29.4%) with ophthalmoparesis in two cases (5.9%), muscle wasting 7/34 (20.6%), muscle pain 6/34 (17.6%), cardiomyopathy with arrhythmia 4/34 (11.8%), arrhythmia without
cardiomyopathy 2/34 (5.9%), peripheral neuropathy 5/34 (14.7%), swallowing impairment 1/34 (2.9%), respiratory (ventilatory) impairment 1/34 (2.9%). Neither age at onset, nor age at last evaluation, nor disease duration significantly differ between patients with or without clinically evident myopathic involvement.
Moreover, at the last clinical evaluation, 12/34 patients had hearing loss (35.3%), 4/34 diabetes mellitus (11.8%), 3/34 migraine (8.8%), 2/34 hypothyroidism (5.9%), 1/34 cataract, 1/34 isolated hypogonadism, 1/34 psychiatric involvement, 1/34 gastrointestinal dysmotility (2.9% each).
Some patients with mild phenotypes were reported including a 50-year-old patient with eyelid ptosis, two 63 and 28-year-old subjects with isolated multiple lipomatosis and a 27-year-old patient with isolated hypogonadism.
Four patients, all with CNS involvement (including generalized seizures in all cases and myoclonus in two cases), were deceased (mean age 37.5±18.5 years, range 20-54; disease duration 9.8±4.6 years, range 5-15) because of heart failure (two cases), respiratory failure, or intractable lactic acidosis (one case each).
Basal lactate levels was increased in 65.0% of the cases, but neither CNS involvement, nor other clinical features, including age at onset, were related with this laboratory marker.
6.3.3 The A8344G mutation: neuroradiological features
Recent (<5 years before the last examination) MRI data were available for 17 patients with the 8344A>G mutation, being normal in ten (58.8%). White matter abnormalities were present in 4/17 (23.5%) patients, one of whom with no
clinically evident CNS involvement; two cases had also cortical/subcortical atrophy. Other MRI findings included periaqueductal lesions, cysts or vacuolated lesions, brainstem atrophy, and stroke-like lesions (1/17 [5.9%] each).
6.3.4 The A8344G mutation: histological findings
Muscle biopsy findings were available for 27 patients. RRF were present in 26 cases (96.3%), including four patients with no clinically evident myopathic involvement. COX-negative fibers were present in 20 cases (74.1%), but failed to predict neuromuscular involvement. Lipid storage was noted in five biopsies (18.5%), and was neither associated with multiple lipomatosis nor with other clinical features.
6.3.5 Heteroplasmy levels
Heteroplasmy levels of the A8344G mutation were available in muscle for 22 patients (mutation load 67.0±16.1%), in blood for 16 patients (mutation load 57.6±16.1%) and in urinary sediment for 11 patients (mutation load 59.4±16.3%). No correlation was observed between these levels and age at onset. Heteroplasmy levels in muscle, blood and urinary sediment were not mutually related, but a positive trend was obtained by comparing the levels in blood and urinary sediment (rho = 0.734, P = 0.052 [significance level after Bonferroni’s correction 0.017]).
Only a non-significant trend was obtained by associating clinically evident myopathic involvement and muscle mutation load (69.6±15.0% in 17 patients
with clinical myopathy versus 58.0±17.9% in five patients without clinically evident myopathy [P = 0.16]).
6.3.6 Systematic revision of the literature
Table 6 shows the clinical data of our 39 mutation carriers with known clinical pictures, as well as those of 282 previously reported A8344G-positive individuals (male/female ratio 0.85; mean age at the last evaluation 34.2±19.1 years; supplementary references in the Appendix). The asymptomatic subjects were included for both groups.
Considering all the 321 patients so far available including our dataset and previously published cases, at the mean age of ≈35 years, the clinical picture was characterized by the following signs/symptoms, in descending order: myoclonus, muscle weakness, ataxia (35-45% of patients); generalized seizures, hearing loss (25-34.9%); cognitive impairment, multiple lipomatosis, neuropathy, exercise intolerance (15-24.9%); increased CK levels, ptosis/ophthalmoparesis, optic atrophy, cardiomyopathy, muscle wasting, respiratory impairment, diabetes, muscle pain, tremor, migraine (5-14.9%).
Increased CK levels and exercise intolerance were more frequently reported in our patients than in the published cases. However, in the great majority of the previous reports, CK was only mentioned when increased, but it is unclear in how many cases this test was performed and found normal or was not performed at all. Moreover, exercise intolerance is a subtle symptom which could be missed if not specifically assessed (Mancuso et al. 2012a). Again, it is unclear whether this sign
was systematically searched for in the previous studies. Ataxia and myoclonus were less frequent in our own series.
Figure 6. Clinical features of the symptomatic A8344G patients aged >16 when evaluated. White: onset; black: last evaluation. For details, see text. Ex.
Table 4. Is myoclonus associated with epileptic seizures and/or ataxia in A8344G adult carriers? (A) No association between myoclonus and epilepsy in
adult subjects with the A8344G mutation (including asymptomatic subjects). Total n = 38, P = 0.176 (Fisher’s exact test), which is not significant (significance level 0.025 after Bonferroni’s correction). (B) Relationship between myoclonus and ataxia in adult subjects with the A8344G mutation. Total n = 38, P = 0.0245 (Fisher’s exact test), which is significant after the correction (significance level 0.025).
(A) Myoclonus: no Myoclonus: yes
Seizures: no 23 3 26
Seizures: yes 8 4 12
31 7
(B) Myoclonus: no Myoclonus: yes
Ataxia: no 27 3 30
Ataxia: yes 4 4 8
Table 5. Clinical features at the onset in A8344G patients. The mean age at
onset of the 35 symptomatic patients was 30.1±18.4 years (range 0-66; 11/35 with clinical onset before age 16). The table summarizes the clinical features at onset, and distinguishes the subjects with adult vs childhood onset. Of note, none of the six patients with multiple lipomatosis had CNS or multisystemic involvement at onset, which in all six occurred in adulthood (36.3±11.3 years; range 20-66; not significantly different from the other patients). Two of them had also myopathic symptoms. 13/35 patients (37.1%) had clear CNS involvement at onset, which was 25.7±18.9 years (range 0-54), not statistically different from the other patients. Four of them also had myopathic symptoms at onset. Neuromuscular signs and symptoms were present at onset in 20/35 patients (57.1%).
Overall (n = 35) Onset >16 years (n = 24) Onset ≤ 16 years (n = 11) Muscle weakness 12 (34.3%) 9 (37.5%) 3 (27.3%) Increased CK 9 (25.7%) 8 (33.3%) 1 (9.1%) Exercise intolerance 6 (17.1%) 4 (16.7%) 2 (18.2%) Multiple lipomatosis 6 (17.1%) 6 (25.0%) - Eyelid ptosis 5 (14.3%) 3 (12.5%) 2 (18.2%) Seizures 5 (14.3%) 2 (8.3%) 3 (27.3%) Ataxia 5 (14.3%) 4 (16.7%) 1 (9.1%) Muscle pain 4 (11.4%) 4 (16.7%) - Hearing loss 4 (11.4%) 4 (16.7%) - Myoclonus 3 (8.6%) 2 (8.3%) 1 (9.1%) Cardiomyopathy 3 (8.6%) 2 (8.3%) 1 (9.1%) Ophthalmoparesis 2 (5.7%) 2 (8.3%) - Cognitive inv. 2 (5.7%) 1 (4.2%) 1 (9.1%) Migraine 2 (5.7%) 1 (4.2%) 1 (9.1%) Myoglobinuria 1 (2.9%) 1 (4.2%) - Neuropathy 1 (2.9%) 1 (4.2%) - Respiratory imp. 1 (2.9%) 1 (4.2%) - Swallowing imp. 1 (2.9%) 1 (4.2%) - Hypogonadism 1 (2.9%) 1 (4.2%) - Diabetes 1 (2.9%) 1 (4.2%) - Psychiatric inv. 1 (2.9%) 1 (4.2%) - Cataract 1 (2.9%) - 1 (9.1%)
Table 6. Clinical features of the A8344G carriers. For further details, see text. Our cohort (n = 39) Literature revision (n = 282) Total (n = 321) Muscle weakness 20 (51.3%) 118 (41.8%) 138 (43.0%) Increased CK 15 (38.5%) 30 (10.6%) 45 (14.0%) Exercise intolerance 15 (38.5%) 40 (14.2%) 55 (17.1%) Seizures 12 (30.8%) 94 (33.3%) 106 (33.0%) Hearing loss 12 (30.8%) 87 (30.9%) 99 (30.8%) Multiple lipomatosis 11 (28.2%) 50 (17.7%) 61 (18.9%) Ptosis/ophthalmoparesis 10 (25.6%) 19 (6.7%) 29 (9.0%) Ataxia 8 (20.5%) 129 (45.7%) 137 (42.7%) Myoclonus 8 (20.5%) 134 (47.5%) 142 (44.2%) Muscle wasting 7 (17.9%) 17 (6.0%) 24 (7.5%) Muscle pain 6 (15.4%) 13 (4.6%) 19 (5.9%) Arrhytmia 6 (15.4%) 7 (2.5%) 13 (4.0%) Neuropathy 5 (12.8%) 52 (18.4%) 57 (17.8%) Cardiomyopathy 4 (10.3%) 20 (7.1%) 24 (7.5%) Diabetes 4 (10.3%) 16 (5.7%) 20 (6.2%) Cognitive involvement 3 (7.7%) 64 (22.7%) 67 (20.9%) Migraine 3 (7.7%) 14 (5.0%) 17 (5.3%) Swallowing impairment 2 (5.1%) 8 (2.8%) 10 (3.1%) Hypothyroidism 2 (5.1%) 2 (0.7%) 4 (1.2%) Cataract 2 (5.1%) 1 (0.4%) 3 (0.9%) Respiratory impairment 1 (2.6%) 20 (7.1%) 21 (6.5%) Tremor 1 (2.6%) 17 (6.0%) 18 (5.6%) Pyramidal signs 1 (2.6%) 9 (3.2%) 10 (3.1%) Psychiatric involvement 1 (2.6%) 8 (2.8%) 9 (2.8%) Stroke-like episodes 1 (2.6%) 7 (2.5%) 8 (2.5%) Gastrointestinal dismotil. 1 (2.6%) 2 (0.7%) 3 (0.9%) Optic atrophy - 25 (8.8%) 25 (7.8%) Retinopathy - 6 (2.1%) 6 (1.9%)
6.4 Mitochondrial DNA point mutations: A3243G
6.4.1 Relative frequency of the A3243G mutation in our database
A total of 133 patients harboring the A3243G mutation was identified among the 1197 mitochondrial patients (11.1%) enlisted in our database. They represented the 26.6% (133/500) of all individuals with mtDNA point mutations. The clinical features were not available for seven patients, who therefore have not been considered for the following analysis, performed on the remaining 126 individuals.
Patients’ mean age at the last evaluation was 36.0 ± 20.7 years. Men were 65/126 (51.6%). Eight of 126 (6.3%) subjects were asymptomatic carriers (age at last evaluation was 28.4 ± 27.9 years; range 1-77).
6.4.2 Clinical features
Figure 7 and Table 7 and summarize the clinical features at onset and at the last evaluation. The mean age at onset of the 118 symptomatic patients was 23.4 ± 15.6 years with 44.1% of the cases having a childhood (<16 years) onset. Hearing loss was the most common complaint at the onset, followed by generalized seizures and diabetes (see Table 7).
At the last evaluation, mean age and disease duration were 36.0 ± 20.7 (range 1-77) and 18.4 ± 16.6 years, respectively. Considering our 126 patients, at the mean age of ≈36 years, the clinical picture was characterized by the following signs/symptoms, starting from the most frequents: hearing loss (>50% of
patients); diabetes, stroke-like episodes (40-49.9%); muscle weakness,
generalized seizures, exercise intolerance, heart disease (30-39.9%);
ptosis/ophthalmoparesis, migraine, cognitive impairment, muscle wasting (20-29.9%); ataxia, increased CK levels, failure to thrive/short stature (15-19.9%); gastrointestinal dysmotility, hypotonia, pyramidal signs, peripheral neuropathy, muscle pain, retinopathy (10-14.9%).
Seventeen patients, all with central nervous system (CNS) involvement (stroke-like episodes reported in 12 cases) died at a mean age of 37.2±22.3 years (range 6-76) because of cardiocirculatory failure (four cases), stroke (in three), status epilepticus or intractable lactic acidosis (each in one case). The cause of death remained unknown for eight patients.
6.4.3 Stroke-like episodes
Stroke-like episodes are crucial for the syndromic diagnosis of MELAS. Here, we define as “MELAS” the group of patients with clinical and/or radiological evidence of stroke-like episodes. “Non-MELAS” patients are defined as individuals carrying the A3243G who have not experienced stroke-like episodes.
Fifty-one out of 126 A3243G carriers (40.5%), at the mean age of 36 years, had suffered stroke-like episodes (see Table 7). Age at onset, age at last control and disease duration were not different between “MELAS” and “non-MELAS” groups. Furthermore, heteroplasmy levels could not predict stroke-like episodes, and no difference in the proportion of patients with ragged red fibers and COX-negative fibers was observed between the two groups. Lactate increase was
