Renal Phenotype in Mitochondrial Diseases: A Multicenter Study.

Aims: This study aimed to investigate associations between renal and extrarenal manifestations of mitochondrial diseases and their natural history as well as predictors of renal disease severity and overall disease outcome. The secondary aim was to generate a protocol of presymptomatic assessment and monitoring of renal function in patients with a defined mitochondrial disease. Methods: A multicenter, retrospective cohort study was performed by the Mitochondrial Clinical and Research Network (MCRN). Patients of any age with renal manifestations associated with a genetically verified mitochondrial disease were included from 8 expert European centers specializing in mitochondrial diseases: Gothenburg, Oulu, Copenhagen, Bergen, Helsinki, Stockholm, Rotterdam, and Barcelona. Results: Of the 36 patients included, two-thirds had mitochondrial DNA-associated disease. Renal manifestations were the first sign of mitochondrial disease in 19%, and renal involvement was first identified by laboratory tests in 57% of patients. Acute kidney injury occurred in 19% of patients and was the first sign of renal disease in the majority of these. The most common renal manifestation was chronic kidney disease (75% with stage 2 or greater), followed by tubulopathy (44.4%), the latter seen mostly among patients with single large-scale mitochondrial DNA deletions. Acute kidney injury and tubulopathy correlated with worse survival outcome. The most common findings on renal imaging were increased echogenicity and renal dysplasia/hypoplasia. Renal histology revealed focal segmental glomerulosclerosis, nephrocalcinosis, and nephronophthisis. Conclusion: Acute kidney injury is a distinct renal phenotype in patients with mitochondrial disease. Our results highlight the importance to recognize renal disease as a sign of an underlying mitochondrial disease. Acute kidney injury and tubulopathy are 2 distinct indicators of poor survival in patients with mitochondrial diseases.

How do changes in the mtDNA and mitochondrial dysfunction influence cancer and cancer therapy? Challenges, opportunities and models.

Several mutations in nuclear genes encoding for mitochondrial components have been associated with an increased cancer risk or are even causative, e.g. succinate dehydrogenase (SDHB, SDHC and SDHD genes) and iso-citrate dehydrogenase (IDH1 and IDH2 genes). Recently, studies have suggested an eminent role for mitochondrial DNA (mtDNA) mutations in the development of a wide variety of cancers. Various studies associated mtDNA abnormalities, including mutations, deletions, inversions and copy number alterations, with mitochondrial dysfunction. This might, explain the hampered cellular bioenergetics in many cancer cell types. Germline (e.g. m.10398A>G; m.6253T>C) and somatic mtDNA mutations as well as differences in mtDNA copy number seem to be associated with cancer risk. It seems that mtDNA can contribute as driver or as complementary gene mutation according to the multiple-hit model. This can enhance the mutagenic/clonogenic potential of the cell as observed for m.8993T>G or influences the metastatic potential in later stages of cancer progression. Alternatively, other mtDNA variations will be innocent passenger mutations in a tumor and therefore do not contribute to the tumorigenic or metastatic potential. In this review, we discuss how reported mtDNA variations interfere with cancer treatment and what implications this has on current successful pharmaceutical interventions. Mutations in MT-ND4 and mtDNA depletion have been reported to be involved in cisplatin resistance. Pharmaceutical impairment of OXPHOS by metformin can increase the efficiency of radiotherapy. To study mitochondrial dysfunction in cancer, different cellular models (like ρ(0) cells or cybrids), in vivo murine models (xenografts and specific mtDNA mouse models in combination with a spontaneous cancer mouse model) and small animal models (e.g. Danio rerio) could be potentially interesting to use. For future research, we foresee that unraveling mtDNA variations can contribute to personalized therapy for specific cancer types and improve the outcome of the disease.

RYR1-related myopathies: a wide spectrum of phenotypes throughout life.

BACKGROUND AND PURPOSE: Although several recent studies have implicated RYR1 mutations as a common cause of various myopathies and the malignant hyperthermia susceptibility (MHS) trait, many of these studies have been limited to certain age groups, confined geographical regions or specific conditions. The aim of the present study was to investigate the full spectrum of RYR1-related disorders throughout life and to use this knowledge to increase vigilance concerning malignant hyperthermia. METHODS: A retrospective cohort study was performed on the clinical, genetic and histopathological features of all paediatric and adult patients in whom an RYR1 mutation was detected in a national referral centre for both malignant hyperthermia and inherited myopathies (2008-2012). RESULTS: The cohort of 77 non-related patients (detection rate 28%) included both congenital myopathies with permanent weakness and 'induced' myopathies such as MHS and non-anaesthesia-related episodes of rhabdomyolysis or hyperCKemia, manifested throughout life and triggered by various stimuli. Sixty-one different mutations were detected, of which 24 were novel. Some mutations are present in both dominant (MHS) and recessive modes (congenital myopathy) of inheritance, even within families. Histopathological features included an equally wide spectrum, ranging from only subtle abnormalities to prominent cores. CONCLUSIONS: This broad range of RYR1-related disorders often presents to the general paediatric and adult neurologist. Its recognition is essential for genetic counselling and improving patients' safety during anaesthesia. Future research should focus on in vitro testing by the in vitro contracture test and functional characterization of the large number of RYR1 variants whose precise effects currently remain uncertain.

Forty-Five Years of Duchenne Muscular Dystrophy in The Netherlands.

BACKGROUND: Duchenne muscular dystrophy (DMD) is a progressive muscle disease. No curative therapy is currently available, but in recent decades standards of care have improved. These improvements include the use of corticosteroids and mechanical ventilation. OBJECTIVE: To present a detailed population based report of the DMD disease course in The Netherlands (1980-2006) and evaluate the effect of changes in care by comparing it with an historical Dutch DMD cohort (1961-1974). METHODS: Information about DMD patients was gathered through the Dutch Dystrophinopathy Database using a standardized questionnaire and information from treating physicians. RESULTS: The study population involved 336 DMD patients (70% of the estimated prevalence), of whom 285 were still alive. Mean age at disease milestones was: diagnosis 4.3 years, wheelchair dependence 9.7 years, scoliosis surgery 14 years, cardiomyopathy (fractional shortening <27%) 15 years, mechanical ventilation 17 years and death 19 years. Within our cohort, corticosteroid use was associated with an increased age of wheelchair dependence from 9.8 to 11.6 years (p < 0.001). When comparing the recent cohort to the historical cohort, mean survival improved from 17 to 27 years (p < 0.001). CONCLUSION: The current study gives detailed information about the disease course of DMD patients, provides evidence for the positive effect of steroid treatment and mechanical ventilation and supports the use of patient registries as a valuable resource for evaluating improvements in care.

A multi-center comparison of diagnostic methods for the biochemical evaluation of suspected mitochondrial disorders.

A multicenter comparison of mitochondrial respiratory chain and complex V enzyme activity tests was performed. The average reproducibility of the enzyme assays is 16% in human muscle samples. In a blinded diagnostic accuracy test in patient fibroblasts and SURF1 knock-out mouse muscle, each lab made the correct diagnosis except for two complex I results. We recommend that enzyme activities be evaluated based on ratios, e.g. with complex IV or citrate synthase activity. In spite of large variations in observed enzyme activities, we show that inter-laboratory comparison of patient sample test results is possible by using normalization against a control sample.

Physical activity is the key determinant of skeletal muscle mitochondrial function in type 2 diabetes.

CONTEXT: Conflicting data exist on mitochondrial function and physical activity in type 2 diabetes mellitus (T2DM) development. OBJECTIVE: The aim was to assess mitochondrial function at different stages during T2DM development in combination with physical exercise in longstanding T2DM patients. DESIGN AND METHODS: We performed cross-sectional analysis of skeletal muscle from 12 prediabetic 11 longstanding T2DM male subjects and 12 male controls matched by age and body mass index. INTERVENTION: One-year intrasubject controlled supervised exercise training intervention was done in longstanding T2DM patients. MAIN OUTCOME MEASUREMENTS: Extensive ex vivo analyses of mitochondrial quality, quantity, and function were collected and combined with global gene expression analysis and in vivo ATP production capacity after 1 yr of training. RESULTS: Mitochondrial density, complex I activity, and the expression of Krebs cycle and oxidative phosphorylation system-related genes were lower in longstanding T2DM subjects but not in prediabetic subjects compared with controls. This indicated a reduced capacity to generate ATP in longstanding T2DM patients only. Gene expression analysis in prediabetic subjects suggested a switch from carbohydrate toward lipid as an energy source. One year of exercise training raised in vivo skeletal muscle ATP production capacity by 21 ± 2% with an increased trend in mitochondrial density and complex I activity. In addition, expression levels of ß-oxidation, Krebs cycle, and oxidative phosphorylation system-related genes were higher after exercise training. CONCLUSIONS: Mitochondrial dysfunction is apparent only in inactive longstanding T2DM patients, which suggests that mitochondrial function and insulin resistance do not depend on each other. Prolonged exercise training can, at least partly, reverse the mitochondrial impairments associated with the longstanding diabetic state.

Distinctive Phenotypic Abnormalities Associated with Submicroscopic 21q22 Deletion Including DYRK1A.

Partial monosomy 21 has been reported, but the phenotypes described are variable with location and size of the deletion. We present 2 patients with a partially overlapping microdeletion of 21q22 and a striking phenotypic resemblance. They both presented with severe psychomotor delay, behavioral problems, no speech, microcephaly, feeding problems with frequent regurgitation, idiopathic thrombocytopenia, obesity, deep set eyes, down turned corners of the mouth, dysplastic ears, and small chin. Brain MRI showed cerebral atrophy mostly evident in frontal and temporal lobes, widened ventricles and thin corpus callosum in both cases, and in one patient evidence of a migration disorder. The first patient also presented with epilepsy and a ventricular septum defect. The second patient had a unilateral Peters anomaly. Microarray analysis showed a partially overlapping microdeletion spanning about 2.5 Mb in the 21q22.1-q22.2 region including the DYRK1A gene and excluding RUNX1. These patients present with a recognizable phenotype specific for this 21q22.1-q22.2 locus. We searched the literature for patients with overlapping deletions including the DYRK1A gene, in order to define other genes responsible for this presentation.

Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome.

BACKGROUND: Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. METHODS AND RESULTS: A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers. CONCLUSIONS: A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.

Microcephaly and simplified gyral pattern of the brain associated with early onset insulin-dependent diabetes mellitus.

Two families are presented with a child suffering from microcephaly with a simplified gyral pattern of the brain (SGP) and early onset insulin dependent diabetes mellitus (IDDM). The first patient was diagnosed postmortally with Wolcott-Rallison syndrome, after her younger brother developed IDDM, and a homozygous mutation in the eukaryotic translation initiation factor 2-alpha kinase 3 was found. The younger brother did not undergo magnetic resonance imaging (MRI). The patient from the second family has no EIF2AK3 mutation. SGP is considered to arise from decreased neuronal proliferation or increased apoptosis at an early stage of embryonal development, but insight into the pathways involved is minimal. EIF2AK3 is involved in translation initiation. It has been proposed that loss of function mutations reduce the ability of the cell to respond to endoplasmic reticulum stress, resulting in apoptosis of pancreatic Langerhans cells. Our findings suggest that in some cases, early onset IDDM and SGP can arise from common mechanisms leading to increased apoptosis.

Novel mutations in three families confirm a major role of COL4A1 in hereditary porencephaly.

BACKGROUND: Porencephaly (cystic cavities of the brain) is caused by perinatal vascular accidents from various causes. Several familial cases have been described and autosomal dominant inheritance linked to chromosome 13q has been suggested. COL4A1 is an essential component in basal membrane stability. Mouse mutants bearing an in-frame deletion of exon 40 of Col4a1 either die from haemorrhage in the perinatal period or have porencephaly in survivors. A report of inherited mutations in COL4A1 in two families has shown that familial porencephaly may have the same cause in humans. OBJECTIVE: To describe three novel COL4A1 mutations. RESULTS: The three mutations occurred in three unrelated Dutch families. There were two missense mutations of glycine residues predicted to result in abnormal collagen IV assembly, and one mutation predicted to abolish the traditional COL4A1 start codon. The last mutation was also present in an asymptomatic obligate carrier with white matter abnormalities on brain magnetic resonance imaging. CONCLUSIONS: This observation confirms COL4A1 as a major locus for genetic predisposition to perinatal cerebral haemorrhage and porencephaly and suggests variable expression of COL4A1 mutations.

Increased risk for cardiorespiratory failure associated with the A3302G mutation in the mitochondrial DNA encoded tRNALeu(UUR) gene.

Screening the mitochondrial DNA of a 64-year-old woman with mitochondrial myopathy revealed 76% of the tRNA(Leu(UUR)) A3302G mutation in muscle. Muscle of her affected son carried 96% mutated mitochondrial DNA. Both patients were biopsied twice, showing isolated complex I deficiency in the son's first biopsy, additional increased (within normal range) complex II + III activities in his second biopsy, combined complex I, II + III deficiency in mothers first biopsy and additional complex IV deficiency in her second biopsy. After a stay in the mountains, the son died of cardiac arrhythmia. The A3302G mutation has been reported before and is associated with mitochondrial myopathy and cardiorespiratory failure. Pathogenesis is explained by abnormal mtRNA processing, which was also reported for the adjacent C3303T mutation associated with cardiomyopathy and/or skeletal myopathy. Our findings suggest that a high mutation load of the A3302G mutation can lead to fatal cardiorespiratory failure, likely triggered by low environmental oxygen pressure and exercise.

Hereditary porencephaly: clinical and MRI findings in two Dutch families.

Familial porencephaly is a rare disorder causing motor impairment, hemiplegia, mental retardation and epilepsy in variable degrees. Two families with porencephaly and apparently dominant inheritance are reported. Brain MRI findings are reviewed and described in seven affected individuals. Most patients also show white matter abnormalities in the cerebral hemisphere, also contralateral to the cystic lesion. In the first family an obligate carrier was identified who did not have a cystic lesion but clear abnormalities of the white matter. Although a predisposition for thrombophilia has previously been reported, we did not observe any genetic, environmental or epigenetic predisposition for the porencephaly. The lesions are most compatible with a deep venous thrombosis/ischemic event occurring in a late stage of pregnancy, not necessarily aggravated by perinatal asphyxia.

Pearson syndrome and the role of deletion dimers and duplications in the mtDNA.

Pearson syndrome is an often fatal multisystem disease associated with mitochondrial DNA rearrangements. Here we report a patient with a novel mtDNA deletion of 3.4 kb ranging from nucleotides 6097 to 9541 in combination with deletion dimers. The mutation percentage in different tissues (blood, muscle and liver) varied between 64% and 95%. After a remission period of about a year, the patient suddenly died at the age of 3 years owing to a severe lactic acidosis. A second patient with a previously reported deletion of 8 kb and a milder phenotype was found to have mitochondrial duplications and died at the age of 10 years. From these data and data from previous reports, we hypothesize that duplications might be beneficial in the clinical course of the disease and in life expectancy.

A PCR test for progressive external ophthalmoplegia and Kearns-Sayre syndrome on DNA from blood samples.

Progressive external ophthalmoplegia (PEO) and Kearns-Sayre syndrome (KSS) are caused by deletions in mitochondrial DNA. Identification of these deletions is important for diagnosis, prognosis and genetic counselling. As yet, the most frequently used test is Southern blot analysis of DNA isolated from a muscle biopsy. Here, we describe a sensitive PCR-based test for the identification of these deletions in DNA isolated from blood. The main advantage is that in the majority of cases a muscle biopsy is no longer necessary for the molecular diagnosis of PEO and KSS.

Clinical heterogeneity in respiratory chain complex III deficiency in childhood.

Six children are presented with an isolated complex III deficiency in muscle tissue. More specifically, oxidation rates and ATP+CrP production rates from both pyruvate and succinate as substrates and/or the activity of decylubiquinol:cytochrome c oxidoreductase were all markedly reduced. Complex III deficiency was also present in liver of two patients tested, but could not be demonstrated in cultured fibroblasts of four patients tested. Mitochondrial DNA, extracted from muscle, was analyzed; no deletions or common point mutations were found. Four patients presented with a multi-organ disorder. Among these patients three presented at neonatal age with neurological signs and lactate elevation in blood and CSF, of whom two had severe neonatal Fanconi syndrome. One child, aged seven years, had encephalomyopathy, ophthalmoplegia, retinopathy and Wolff-Parkinson-White syndrome. The remaining two patients exhibited myopathy only, within the first year of life. Thus, like in other respiratory chain disorders, patients with complex III deficiency may present at any age and show variable symptoms and outcome, ranging from neonatal death to failure to thrive only. Apparently there are no clinical findings which are specific for complex III deficiency.

Magnetic resonance imaging in classification of congenital muscular dystrophies with brain abnormalities.

A survey was performed of magnetic resonance imaging (MRI) findings in 21 patients with congenital muscular dystrophy (CMD) with cerebral abnormalities to evaluate the contribution of MRI to the classification of CMD patients. In 5 patients with Walker-Warburg syndrome (WWS), MRI showed hydrocephalus due to aqueduct stenosis, generalized cerebral cortical agyric or pachygyric polymicrogyria, diffuse cerebral hemispheric white matter abnormalities, and malformations of posterior fossa structures. In 4 patients with muscle-eye-brain disease, MRI showed cortical dysplasia, but less severe than in WWS. The cerebral white matter either was normal or contained multiple focal abnormalities. Malformations of posterior fossa structures were present. Eight patients, classified as having classic merosin-deficient CMD (MD-CMD), had diffuse cerebral hemispheric white matter abnormalities, no other abnormalities. One patient with MD-CMD had only a few, focal white matter abnormalities. Three CMD patients had occipital agyria, otherwise normal gyration, multifocal or more diffuse cerebral white matter changes, and variable hypoplasia of pons and vermis. Two of the 3 patients had negative muscle merosin staining. The conclusion of the study is that MRI is an important adjunct in the classification of CMD patients. CMD with occipital agyria can be regarded as a newly recognized, separate CMD subtype.

Canavan disease: neuromorphological and biochemical analysis of a brain biopsy specimen.

In this study we present a patient with Canavan disease or Van Bogaert and Bertrand type of spongiform leukodystrophy, proven by brain biopsy. We performed morphological studies and biochemical assays on fresh homogenates of the grey and white matter. Quantitative neuromorphological analysis of the cortex showed normal values except for poor dendritic arborization of the inner layers. No signs of neuronal damage were observed. The Na-K-ATPase activity was increased. Pyruvate and ketone bodies oxidation rates and the activity of cytochrome-c oxidase were normal. We conclude that there is neither a primary neuronal damage nor a primary mitochondrial dysfunction in the oxidative processes despite the abnormal morphology of mitochondria in this disease.