Diagnosis Of Mitochondrial Diseases Research Articles

Prenatal diagnosis of severe mitochondrial diseases caused by nuclear gene defects: a study in Japan

Prenatal diagnoses of mitochondrial diseases caused by defects in nuclear DNA (nDNA) or mitochondrial DNA have been reported in several countries except for Japan. The present study aimed to clarify the status of prenatal genetic diagnosis of mitochondrial diseases caused by nDNA defects in Japan. A comprehensive genomic analysis was performed to diagnose more than 400 patients, of which, 13 families (16 cases) had requested prenatal diagnoses. Eight cases diagnosed with wild type homozygous or heterozygous variants same as either of the heterozygous parents continued the pregnancy and delivered healthy babies. Another eight cases were diagnosed with homozygous, compound heterozygous, or hemizygous variants same as the proband. Of these, seven families chose to terminate the pregnancy, while one decided to continue the pregnancy. Neonatal- or infantile-onset mitochondrial diseases show severe phenotypes and lead to lethality. Therefore, such diseases could be candidates for prenatal diagnosis with careful genetic counseling, and prenatal testing could be a viable option for families.

Advances in Diagnosis of Mitochondrial Diseases: Case Report of an Infant with Pearson Syndrome

Pearson syndrome (PS) is a mitochondrial disorder that presents in early infancy as a multisystemic disease affecting the bone marrow and pancreas. It may present with anemia, diarrhea, exocrine pancreatic dysfunction, and failure to thrive.[1] Delay in diagnosis can lead to severe morbidity and mortality in infancy. We report the case of a 9-month-old presenting with failure to thrive, severe macrocytic anemia and pancytopenia initially thought to have gastroesophageal reflux and feeding intolerance. Severe macrocytic anemia and pancytopenia prompted an early bone marrow evaluation. Abnormal bone marrow findings including vacuolated marrow precursors and ringed sideroblasts along with persistent mild lactic acidosis led to a rapid and extensive genetics workup. Whole exome sequencing including mitochondrial genome sequencing detected a 2.3 kb heteroplasmic deletion in m.12113_14421 encompassing the MT-ND5 gene consistent with the diagnosis of Pearson Syndrome. This case report highlights the advances in molecular genetic testing to diagnose patients with complex medical histories along the spectrum of mitochondrial diseases and the importance of early diagnosis to start treatment.

The Dimensions of Primary Mitochondrial Disorders.

The concept of a mitochondrial disorder was initially described in 1962, in a patient with altered energy metabolism. Over time, mitochondrial energy metabolism has been discovered to be influenced by a vast number of proteins with a multitude of functional roles. Amongst these, defective oxidative phosphorylation arose as the hallmark of mitochondrial disorders. In the premolecular era, the diagnosis of mitochondrial disease was dependent on biochemical criteria, with inherent limitations such as tissue availability and specificity, preanalytical and analytical artifacts, and secondary effects. With the identification of the first mitochondrial disease-causing mutations, the genetic complexity of mitochondrial disorders began to unravel. Mitochondrial dysfunctions can be caused by pathogenic variants in genes encoded by the mitochondrial DNA or the nuclear DNA, and can display heterogenous phenotypic manifestations. The application of next generation sequencing methodologies in diagnostics is proving to be pivotal in finding the molecular diagnosis and has been instrumental in the discovery of a growing list of novel mitochondrial disease genes. In the molecular era, the diagnosis of a mitochondrial disorder, suspected on clinical grounds, is increasingly based on variant detection and associated statistical support, while invasive biopsies and biochemical assays are conducted to an ever-decreasing extent. At present, there is no uniform biochemical or molecular definition for the designation of a disease as a “mitochondrial disorder”. Such designation is currently dependent on the criteria applied, which may encompass clinical, genetic, biochemical, functional, and/or mitochondrial protein localization criteria. Given this variation, numerous gene lists emerge, ranging from 270 to over 400 proposed mitochondrial disease genes. Herein we provide an overview of the mitochondrial disease associated genes and their accompanying challenges.

Multi-Omics Approaches to Improve Mitochondrial Disease Diagnosis: Challenges, Advances, and Perspectives.

Mitochondrial diseases (MD) are rare disorders caused by deficiency of the mitochondrial respiratory chain, which provides energy in each cell. They are characterized by a high clinical and genetic heterogeneity and in most patients, the responsible gene is unknown. Diagnosis is based on the identification of the causative gene that allows genetic counseling, prenatal diagnosis, understanding of pathological mechanisms, and personalized therapeutic approaches. Despite the emergence of Next Generation Sequencing (NGS), to date, more than one out of two patients has no diagnosis in the absence of identification of the responsible gene. Technologies currently used for detecting causal variants (genetic alterations) is far from complete, leading many variants of unknown significance (VUS) and mainly based on the use of whole exome sequencing thus neglecting the identification of non-coding variants. The complexity of human genome and its regulation at multiple levels has led biologists to develop several assays to interrogate the different aspects of biological processes. While one-dimension single omics investigation offers a peek of this complex system, the combination of different omics data allows the discovery of coherent signatures. The community of computational biologists and bioinformaticians, in order to integrate data from different omics, has developed several approaches and tools. However, it is difficult to understand which suits the best to predict diverse phenotypic outcome. First attempts to use multi-omics approaches showed an improvement of the diagnostic power. However, we are far from a complete understanding of MD and their diagnosis. After reviewing multi-omics algorithms developed in the latest years, we are proposing here a novel data-driven classification and we will discuss how multi-omics will change and improve the diagnosis of MD. Due to the growing use of multi-omics approaches in MD, we foresee that this work will contribute to set up good practices to perform multi-omics data integration to improve the prediction of phenotypic outcomes and the diagnostic power of MD.

Advanced pathologic study for definite diagnosis of mitochondrial cardiomyopathy

Abstract Mitochondrial cardiomyopathy (MCM) is usually recognized as one of the phenotypes of systemic mitochondrial disease. However if there are no cardiac symptoms, it is difficult to make a definite diagnosis because of various cardiovascular phenotypes and no diagnostic criteria in pathological examination. To add myocardial pathology to the diagnostic criteria for mitochondrial respiratory chain disorders, which is the gold standard in the diagnosis of mitochondrial diseases, we performed quantitative analysis of mitochondria using electron microscopy and immunohistopathologic analysis with respiratory chain enzyme antibodies. Ten patients with hypertrophic or restrictive cardiomyopathy who had undergone endomyocardial biopsy were studied. Respiratory chain enzymatic assay and genetic study were performed and four patients were diagnosed with MCM. Using electron microscopy with quantitative analysis, volume density of mitochondria within cardiac muscle cells was significantly increased in the MCM group compared to the non-MCM group (p=0.013). Immunohistopathologic results were compatible with the result of the respiratory chain enzymatic assay. These advanced pathological tests can distinguish MCM from other cardiomyopathies. Results of immunopathologic study Funding Acknowledgement Type of funding source: Public grant(s) – National budget only. Main funding source(s): The Practical Research Project for Rare/Intractable Diseases from the Japan Agency for Medical Research and Development, AMED.

Diagnostic value of serum biomarkers FGF21 and GDF15 compared to muscle sample in mitochondrial disease.

The aim of this study was to compare the value of serum biomarkers, fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15), with histological analysis of muscle in the diagnosis of mitochondrial disease. We collected 194 serum samples from patients with a suspected or known mitochondrial disease. Biomarkers were analyzed blinded using enzyme-labeled immunosorbent assay. Clinical data were collected using a structured questionnaire. Only 39% of patients with genetically verified mitochondrial disease had mitochondrial pathology in their muscle histology. In contrast, biomarkers were elevated in 62% of patients with genetically verified mitochondrial disease. Those with both biomarkers elevated had a muscle manifesting disorder and a defect affecting mitochondrial DNA expression. If at least one of the biomarkers was induced and the patient had a myopathic disease, a mitochondrial DNA expression disease was the cause with 94% probability. Among patients with biomarker analysis and muscle biopsy taken <12 months apart, a mitochondrial disorder would have been identified in 70% with analysis of FGF21 and GDF15 compared to 50% of patients whom could have been identified with muscle biopsy alone. Muscle findings were nondiagnostic in 72% (children) and 45% (adults). Induction of FGF21 and GDF15 suggest a mitochondrial etiology as an underlying cause of a muscle manifesting disease. Normal biomarker values do not, however, rule out a mitochondrial disorder, especially if the disease does not manifest in muscle. We suggest that FGF21 and GDF15 together should be first-line diagnostic investigations in mitochondrial disease complementing muscle biopsy.

Decoding mitochondrial heterogeneity in single muscle fibres by imaging mass cytometry

The study of skeletal muscle continues to support the accurate diagnosis of mitochondrial disease and remains important in delineating molecular disease mechanisms. The heterogeneous expression of oxidative phosphorylation proteins and resulting respiratory deficiency are both characteristic findings in mitochondrial disease, hence the rigorous assessment of these at a single cell level is incredibly powerful. Currently, the number of proteins that can be assessed in individual fibres from a single section by immunohistochemistry is limited but imaging mass cytometry (IMC) enables the quantification of further, discrete proteins in individual cells. We have developed a novel workflow and bespoke analysis for applying IMC in skeletal muscle biopsies from patients with genetically-characterised mitochondrial disease, investigating the distribution of nine mitochondrial proteins in thousands of single muscle fibres. Using a semi-automated analysis pipeline, we demonstrate the accurate quantification of protein levels using IMC, providing an accurate measure of oxidative phosphorylation deficiency for complexes I–V at the single cell level. We demonstrate signatures of oxidative phosphorylation deficiency for common mtDNA variants and nuclear-encoded complex I variants and a compensatory upregulation of unaffected oxidative phosphorylation components. This technique can now be universally applied to evaluate a wide range of skeletal muscle disorders and protein targets.

MITO-FIND: A study in 390 patients to determine a diagnostic strategy for mitochondrial disease

Mitochondrial diseases, due to nuclear or mitochondrial genome mutations causing mitochondrial dysfunction, have a wide range of clinical features involving neurologic, muscular, cardiac, hepatic, visual, and auditory symptoms. Making a diagnosis of a mitochondrial disease is often challenging since there is no gold standard and traditional testing methods have required tissue biopsy which presents technical challenges and most patients prefer a non-invasive approach. Since a diagnosis invariably involves finding a disease-causing DNA variant, new approaches such as next generation sequencing (NGS) have the potential to make it easier to make a diagnosis. We evaluated the ability of our traditional diagnostic pathway (metabolite analysis, tissue neuropathology and respiratory chain enzyme activity) in 390 patients. The traditional diagnostic pathway provided a diagnosis of mitochondrial disease in 115 patients (29.50%). Analysis of mtDNA, tissue neuropathology, skin electron microscopy, respiratory chain enzyme analysis using inhibitor assays, blue native polyacrylamide gel electrophoresis were all statistically significant in distinguishing patients between a mitochondrial and non-mitochondrial diagnosis. From these 390 patients who underwent traditional analysis, we recruited 116 patients for the NGS part of the study (36 patients who had a mitochondrial diagnosis (MITO) and 80 patients who had no diagnosis (No-Dx)). In the group of 36 MITO patients, nuclear whole exome sequencing (nWES) provided a second diagnosis in 2 cases who already had a pathogenic variant in mtDNA, and a revised diagnosis (GLUL) in one case that had abnormal pathology but no pathogenic mtDNA variant. In the 80 NO-Dx patients, nWES found non-mitochondrial diagnosis in 26 patients and a mitochondrial diagnosis in 1 patient. A genetic diagnosis was obtained in 53/116 (45.70%) cases that were recruited for NGS, but not in 11/116 (9.48%) of cases with abnormal mitochondrial neuropathology. Our results show that a non-invasive, bigenomic sequencing (BGS) approach (using both a nWES and optimized mtDNA analysis to include large deletions) should be the first step in investigating for mitochondrial diseases. There may still be a role for tissue biopsy in unsolved cases or when the diagnosis is still not clear after NGS studies.

A novel, pathogenic dinucleotide deletion in the mitochondrial MT-TY gene causing myasthenia-like features

Mitochondrial DNA (mtDNA)-related diseases often pose a diagnostic challenge and require rigorous clinical and laboratory investigation. Pathogenic variants in the mitochondrial tRNA gene MT-TY, which encodes the tRNATyr, are a rare cause of mitochondrial disease. Here we describe a novel m.5860delTA anticodon variant in the MT-TY gene in a patient who initially presented with features akin to a childhood onset myasthenic syndrome. Using histochemical, immunohistochemical and protein studies we demonstrate that this mutation leads to severe biochemical defects of mitochondrial translation, which is reflected in the early onset and progressive phenotype. This case highlights the clinical overlap between mtDNA-related diseases and other neuromuscular disorders, and demonstrates the potential pitfalls in analysis of next generation sequencing results, given whole exome sequencing of a blood DNA sample failed to make a genetics diagnosis. Muscle biopsy remains an important requirement in the diagnosis of mitochondrial disease and in establishing the pathogenicity of novel mtDNA variants.

Fibroblast growth factor 21 and grow differentiation factor 15 are sensitive biomarkers of mitochondrial diseases due to mitochondrial transfer-RNA mutations and mitochondrial DNA deletions.

Diagnosis of mitochondrial diseases (MDs) is challenging, since they are multisystemic disorders, characterized by a heterogeneous symptomatology. Recently, an increase in serum levels of fibroblast growth factor 21 (FGF21) and growth differentiation factor 15 (GDF15) has been found in the majority of patients with MDs compared with healthy controls. On the other hand, the finding of low FGF21 and GDF15 levels in some patients with MDs suggests that different types of respiratory chain defects may lead to different profiles of these two proteins. In this study, we aimed to validate the diagnostic reliability of FGF21 and GDF15 assays in MDs and to evaluate a possible correlation between serum levels of the two biomarkers with genotype of MD patients. Serum FGF21 and GDF15 levels were measured by a quantitative ELISA. Our results showed increased serum FGF21 and GDF15 levels in MD patients; however, GDF15 measurement seems to be more sensitive and specific for screening tests for MD than FGF21. Moreover, we showed a positive correlation with both FGF21 and GDF15 levels and the number of COX-negative fibers. Finally, we also demonstrated that the increase of FGF21 and GDF15 was related to MDs caused by mitochondrial translation defects, and multiple and single mtDNA deletions, but not to MDs due to mutations in the respiratory chain subunits.

Desminopathy: Novel Desmin Variants, a New Cardiac Phenotype, and Further Evidence for Secondary Mitochondrial Dysfunction.

Background: The pleomorphic clinical presentation makes the diagnosis of desminopathy difficult. We aimed to describe the prevalence, phenotypic expression, and mitochondrial function of individuals with putative disease-causing desmin (DES) variants identified in patients with an unexplained etiology of cardiomyopathy. Methods: A total of 327 Czech patients underwent whole exome sequencing and detailed phenotyping in probands harboring DES variants. Results: Rare, conserved, and possibly pathogenic DES variants were identified in six (1.8%) probands. Two DES variants previously classified as variants of uncertain significance (p.(K43E), p.(S57L)), one novel DES variant (p.(A210D)), and two known pathogenic DES variants (p.(R406W), p.(R454W)) were associated with characteristic desmin-immunoreactive aggregates in myocardial and/or skeletal biopsy samples. The individual with the novel DES variant p.(Q364H) had a decreased myocardial expression of desmin with absent desmin aggregates in myocardial/skeletal muscle biopsy and presented with familial left ventricular non-compaction cardiomyopathy (LVNC), a relatively novel phenotype associated with desminopathy. An assessment of the mitochondrial function in four probands heterozygous for a disease-causing DES variant confirmed a decreased metabolic capacity of mitochondrial respiratory chain complexes in myocardial/skeletal muscle specimens, which was in case of myocardial succinate respiration more profound than in other cardiomyopathies. Conclusions: The presence of desminopathy should also be considered in individuals with LVNC, and in the differential diagnosis of mitochondrial diseases.

Systematic analysis of a mitochondrial disease‐causing ND6 mutation in mitochondrial deficiency

BackgroundThe m.14487T>C mutation is recognized as a diagnostic mutation of mitochondrial disease during the past 16 years, emerging evidence suggests that mutant loads of m.14487T>C and disease phenotype are not closely correlated.MethodsImmortalized lymphocytes were generated by coculturing the Epstein–Barr virus and lymphocytes from m.14487T>C carrier Chinese patient with Leigh syndrome. Fifteen cytoplasmic hybrid (cybrid) cell lines were generated by fusing mtDNA lacking 143B cells with platelets donated by patients. Mitochondrial function was systematically analyzed at transcriptomic, metabolomic, and biochemical levels.ResultsUnlike previous reports, we found that the assembly of mitochondrial respiratory chain complexes, mitochondrial respiration, and mitochondrial OXPHOS function was barely affected in cybrid cells carrying homoplastic m.14487T>C mutation. Mitochondrial dysfunction associated transcriptomic and metabolomic reprogramming were not detected in cybrid carrying homoplastic m.14487T>C. However, we found that mitochondrial function was impaired in patient‐derived immortalized lymphocytes.ConclusionOur data revealed that m.14487T>C mutation is insufficient to cause mitochondrial deficiency; additional modifier genes may be involved in m.14487T>C‐associated mitochondrial disease. Our results further demonstrated that a caution should be taken by solely use of m.14487T>C mutation for molecular diagnosis of mitochondrial disease.

Advancement in the diagnosis of mitochondrial diseases

Advancement in the diagnosis of mitochondrial diseases

Dynamic derangement in amino acid profile during and after a stroke-like episode in adult-onset mitochondrial disease: a case report

BackgroundMaternally inherited diabetes and deafness, and mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes are examples of mitochondrial diseases that are relatively common in the adult population. Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes are assumed to be associated with decreases in arginine and citrulline. Biomarkers, such as growth differentiation factor-15, were developed to assist in the diagnosis of mitochondrial diseases.Case presentationA 55-year-old Japanese man, an insulin user, presented after a loss of consciousness. A laboratory test showed diabetic ketoacidosis. He and his mother had severe hearing difficulty. Bilateral lesions on magnetic resonance imaging, the presence of seizure, and an elevated ratio of lactate to pyruvate, altogether suggested a diagnosis of mitochondrial disease. Mitochondrial DNA in our patient’s peripheral blood was positive with a 3243A>G mutation, which is the most frequent cause of maternally inherited diabetes and deafness, and mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes. As a result, maternally inherited diabetes and deafness/mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes was diagnosed. We measured growth differentiation factor-15 and multiple amino acids in his blood, longitudinally during and after the stroke-like episode. Growth differentiation factor-15 was increased to an immeasurably high level on the day of the stroke-like episode. Although his diabetes improved with an increased dose of insulin, the growth differentiation factor-15 level gradually increased, suggesting that his mitochondrial insufficiency did not improve. Multiple amino acid species, including arginine, citrulline, and taurine, showed a decreased level on the day of the episode and a sharp increase the next day. In contrast, the level of aspartic acid increased to an extremely high level on the day of the episode, and decreased gradually thereafter.ConclusionsGrowth differentiation factor-15 can be used not only for the diagnosis of mitochondrial disease, but as an indicator of its acute exacerbation. A stroke-like episode of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes reflects a drastic derangement of multiple amino acids. The involvement of aspartic acid in the episodes should be explored in future studies.

5162Novel insights into desminopathy in the era of next generation sequencing

Abstract Aims The pleomorphic clinical presentation makes the diagnosis of desminopathy difficult. This disease of intermediate filaments causes not only a contractile dysfunction in cardiomyopathy and skeletal myopathy, but also a secondary mitochondrial dysfunction. We aimed to describe prevalence, phenotypic expression and mitochondrial function in desmin mutation carriers identified in a large cohort of patients with unexplained cardiomyopathy. Methods and results A representative cohort of 327 Czech patients with unexplained cardiomyopathy underwent whole exome sequencing. The cohort consisted of cases with familial and sporadic dilated cardiomyopathy (81%), left ventricular noncompaction cardiomyopathy (LVNC) (13%), and less frequently of restrictive (3%) or arrhythmogenic cardiomyopathy (3%). Clinical and laboratory data of desmin mutation carriers were collected. Morphology, desmin expression and mitochondrial function were studied in available myocardial and skeletal muscle specimens. Rare, conserved and possibly pathogenic desmin variants were identified in 6 (1.8%) probands. Two desmin mutations previously classified as variants of uncertain significance (p.K43E, p.S57L), one novel desmin variant (p.A210D) and two known pathogenic desmin mutations (p.R406W, p.R454W) were in affected individuals associated with characteristic pathological desmin aggregates in myocardial and/or skeletal biopsy samples. The individual with the novel desmin variant p.Q364H had decreased myocardial expression of desmin with absent desmin aggregates in myocardial/skeletal biopsy and presented with familial left ventricular non-compaction cardiomyopathy, a novel phenotype of desminopathy. Assessment of mitochondrial function in four carriers of desmin mutations with fresh-frozen skeletal and/or myocardial muscle specimens confirmed decreased metabolic capacity of mitochondrial respiratory chain complexes, which was in case of myocardial succinate respiration more profound than in end-stage heart failure of other etiologies. Genetic testing corrected an inappropriate clinical diagnosis in two probands previously diagnosed with mitochondrial disease and inflammatory cardiomyopathy. During a median follow-up of 56 months, 5 (83%) probands developed end-stage heart failure. Conclusions Desminopathy is a rare cause of cardiomyopathy and/or skeletal muscle myopathy with a pleomorphic clinical presentation and poor prognosis. The presence of desminopathy should be considered also in individuals with left ventricular non-compaction cardiomyopathy, and in the differential diagnosis of mitochondrial diseases and inflammatory cardiomyopathy. Acknowledgement/Funding Research grant of the Ministry of Health, Czech Republic [MZ 15-27682A], No. 00023001 (IKEM, Prague, CZ), the Czech Science Foundation [14-36804G].

Plasma FGF‐21 and GDF‐15 are elevated in different inherited metabolic diseases and are not diagnostic for mitochondrial disorders

Recently, the plasma cytokines FGF-21 and GDF-15 were described as cellular metabolic regulators. They share an endocrine function and are highly expressed in the liver under stress and during starvation. Several studies found that these markers have high sensitivity and specificity for the diagnosis of mitochondrial diseases, especially those with prominent muscular involvement. In our study, we aimed to determine whether these markers could help distinguish mitochondrial diseases from other groups of inherited diseases. We measured plasma FGF-21 and GDF-15 concentrations in 122 patients with genetically confirmed primary mitochondrial disease and 127 patients with non-mitochondrial inherited diseases. Although GDF-15 showed better analytical characteristics (sensitivity = 0.66, specificity = 0.64, area under the curve [AUC] = 0.88) compared to FGF-21 (sensitivity = 0.51, specificity = 0.76, AUC = 0.78) in the pediatric group of mitochondrial diseases, both markers were also elevated in a variety of non-mitochondrial diseases, especially those with liver involvement (Gaucher disease, galactosemia, glycogenosis types 1a, 1b, 9), organic acidurias and some leukodystrophies. Thus, the overall positive and negative predictive values were not acceptable for these measurements to be used as diagnostic tests for mitochondrial diseases (FGF-21 positive predictive value [PPV] = 34%, negative predictive value [NPV] = 73%; GDF-15 PPV = 47%, NPV = 28%). We suggest that FGF-21 and GDF-15 increase in patients with metabolic diseases with metabolic or oxidative stress and inflammation.

EKLIPse: a sensitive tool for the detection and quantification of mitochondrial DNA deletions from next-generation sequencing data

Accurate detection of mitochondrial DNA (mtDNA) alterations is essential for the diagnosis of mitochondrial diseases. The development of high-throughput sequencing technologies has enhanced the detection sensitivity of mtDNA pathogenic variants, but the detection of mtDNA rearrangements, especially multiple deletions, is still poorly processed. Here, we present eKLIPse, a sensitive and specific tool allowing the detection and quantification of large mtDNA rearrangements from single and paired-end sequencing data. The methodology was first validated using a set of simulated data to assess the detection sensitivity and specificity, and second with a series of sequencing data from mitochondrial disease patients carrying either single or multiple deletions, related to pathogenic variants in nuclear genes involved in mtDNA maintenance. eKLIPse provides the precise breakpoint positions and the cumulated percentage of mtDNA rearrangements at a given gene location with a detection sensitivity lower than 0.5% mutant. eKLIPse software is available either as a script to be integrated in a bioinformatics pipeline, or as user-friendly graphical interface to visualize the results through a Circos representation ( https://github.com/dooguypapua/eKLIPse ). Thus, eKLIPse represents a useful resource to study the causes and consequences of mtDNA rearrangements, for further genotype/phenotype correlations in mitochondrial disorders.

Rapid mitochondrial genome (MTDNA) sequencing: facilitating rapid diagnosis of mitochondrial diseases in paediatric acute care

Rapid mitochondrial genome (MTDNA) sequencing: facilitating rapid diagnosis of mitochondrial diseases in paediatric acute care

Muscle Involvement in a Large Cohort of Pediatric Patients with Genetic Diagnosis of Mitochondrial Disease.

Mitochondrial diseases (MD) are a group of genetic and acquired disorders which present significant diagnostic challenges. Here we report the disease characteristics of a large cohort of pediatric MD patients (n = 95) with a definitive genetic diagnosis, giving special emphasis on clinical muscle involvement, biochemical and histopathological features. Of the whole cohort, 51 patients harbored mutations in nuclear DNA (nDNA) genes and 44 patients had mutations in mitochondrial DNA (mtDNA) genes. The nDNA patients were more likely to have a reduction in muscle fiber succinate dehydrogenase (SDH) stains and in SDH-positive blood vessels, while a higher frequency of mtDNA patients had ragged red (RRF) and blue fibers. The presence of positive histopathological features was associated with ophthalmoplegia, myopathic facies, weakness and exercise intolerance. In 17 patients younger than two years of age, RRF and blue fibers were observed only in one case, six cases presented cytochrome c oxidase (COX) reduction/COX-fibers, SDH reduction was observed in five and all except one presented SDH-positive blood vessels. In conclusion, muscle involvement was a frequent finding in our series of MD patients, especially in those harboring mutations in mtDNA genes.

First-line Screening of OXPHOS Deficiencies Using Microscale Oxygraphy in Human Skin Fibroblasts: A Preliminary Study.

The diagnosis of mitochondrial diseases is a real challenge because of the vast clinical and genetic heterogeneity. Classically, the clinical examination and genetic analysis must be completed by several biochemical assays to confirm the diagnosis of mitochondrial disease. Here, we tested the validity of microscale XF technology in measuring oxygen consumption in human skin fibroblasts isolated from 5 pediatric patients with heterogeneous mitochondrial disorders. We first set up the protocol conditions to allow the determination of respiratory parameters including respiration associated with ATP production, proton leak, maximal respiration, and spare respiratory capacity with reproducibility and repeatability. Maximum respiration and spare capacity were the only parameters decreased in patients irrespective of the type of OXPHOS deficiency. These results were confirmed by high-resolution oxygraphy, the reference method to measure cellular respiration. Given the fact that microscale XF technology allows fast, automated and standardized measurements, we propose to use microscale oxygraphy among the first-line methods to screen OXPHOS deficiencies.