Diagnosis Of Mitochondrial Disorders Research Articles

Critically unwell infants and children with mitochondrial disorders diagnosed by ultrarapid genomic sequencing

PurposeTo characterize the diagnostic and clinical outcomes of a cohort of critically ill infants and children with suspected mitochondrial disorders (MD) undergoing ultrarapid genomic testing as part of a national program. MethodsUltrarapid genomic sequencing was performed in 454 families (genome sequencing: n = 290, exome sequencing +/− mitochondrial DNA sequencing: n = 164). In 91 individuals, MD was considered, prompting analysis using an MD virtual gene panel. These individuals were reviewed retrospectively and scored according to modified Nijmegen Mitochondrial Disease Criteria. ResultsA diagnosis was achieved in 47% (43/91) of individuals, 40% (17/43) of whom had an MD. Seven additional individuals in whom an MD was not suspected were diagnosed with an MD after broader analysis. Gene-agnostic analysis led to the discovery of 2 novel disease genes, with pathogenicity validated through targeted functional studies (CRLS1 and MRPL39). Functional studies enabled diagnosis in another 4 individuals. Of the 24 individuals ultimately diagnosed with an MD, 79% had a change in management, which included 53% whose care was redirected to palliation. ConclusionUltrarapid genetic diagnosis of MD in acutely unwell infants and children is critical for guiding decisions about the need for additional investigations and clinical management.

Serum fibroblast growth factor 21 and growth differentiation factor 15: Two sensitive biomarkers in the diagnosis of mitochondrial disorders

Mitochondrial disorders are often difficult to diagnose because of diverse clinical phenotypes. FGF-21 and GDF-15 are metabolic hormones and promising biomarkers for the diagnosis of these disorders. This study has systematically evaluated serum FGF-21 and GDF-15 levels by ELISA in a well-defined cohort of patients with definite mitochondrial disorders (n = 30), neuromuscular disease controls (n = 36) and healthy controls (n = 36) and aimed to ascertain their utility in the diagnosis of mitochondrial disorders. Both serum FGF-21 and GDF-15 were significantly elevated in patients with mitochondrial disorders, especially in those with muscle involvement. The levels were higher in patients with mitochondrial deletions (both single and multiple) and translation disorders compared to respiratory chain subunit or assembly factor defects.

The utility of next-generation sequencing technologies in diagnosis of Mendelian mitochondrial diseases and reflections on clinical spectrum.

Diagnostic process of mitochondrial disorders (MD) is challenging because of the clinical variability and genetic heterogeneity of these conditions. Next-Generation Sequencing (NGS) technology offers a high-throughput platform for nuclear MD. We included 59 of 72 patients that undergone WES and targeted exome sequencing panel suspected to have potential PMDs. Patients who were included in the analysis considering the possible PMD were reviewed retrospectively and scored according to the Mitochondrial Disease Criteria Scale. Sixty-one percent of the patients were diagnosed with whole-exome sequencing (WES) (36/59) and 15% with targeted exome sequencing (TES) (9/59). Patients with MD-related gene defects were included in the mito group, patients without MD-related gene defects were included in the nonmito group, and patients in whom no etiological cause could be identified were included in the unknown etiology group. In 11 out of 36 patients diagnosed with WES, a TES panel was applied prior to WES. In 47 probands in 39 genes (SURF1, SDHAF1, MTO1, FBXL4, SLC25A12, GLRX5, C19oRF12, NDUFAF6, DARS2, BOLA3, SLC19A3, SCO1, HIBCH, PDHA1, PDHAX, PC, ETFA, TRMU, TUFM, NDUFS6, WWOX, UBCD TREX1, ATL1, VAC14, GFAP, PLA2G6, TPRKB, ATP8A2, PEX13, IGHMBP2, LAMB2, LPIN1, GFPT1, CLN5, DOLK) (20 mito group, 19 nonmito group) 59 variants (31 mito group, 18 nonmito group) were detected. Seven novel variants in the mito group (SLC25A12, GLRX5, DARS2, SCO1, PC, ETFA, NDUFS6), nine novel variants in the nonmito group (IVD, GCDH, COG4, VAC14, GFAP, PLA2G6, ATP8A2, PEX13, LPIN1) were detected. We explored the feasibility of identifying pathogenic alleles using WES and TES in MD. Our results show that WES is the primary method of choice in the diagnosis of MD until at least all genes responsible for PMD are found and are highly effective in facilitating the diagnosis process.

Identification of Disease-Causing Mutations by Functional Complementation of Patient-Derived Fibroblast Cell Lines.

Diagnosis of mitochondrial disorders is still hampered by their phenotypic and genotypic heterogeneity. In many cases, exome sequencing, the state-of-the-art method for genetically diagnosing mitochondrial disease patients, does not allow direct identification of the disease-associated gene but rather results in a list of variants in candidate genes. Here, we present a method to validate the disease-causing variant based on functional complementation assays. First, cell lines expressing a wild-type cDNA of the candidate genes are generated by lentiviral infection of patient-derived fibroblasts. Next, oxidative phosphorylation is measured by the Seahorse XF analyzer to assess rescue efficiency.

When to Suspect and How to Diagnose Mitochondrial Disorders?

Disorders of the mitochondrial respiratory chain are an exceedingly diverse group. The clinical features can affect any tissue or organ and occur at any age, with any mode of inheritance. The diagnosis of mitochondrial disorders requires knowledge of the clinical phenotypes and access to a wide range of laboratory techniques. A few syndromes are associated with a specific genetic defect and in these cases it is appropriate to proceed directly to an appropriate test of blood or urine. In most cases, however, the best strategy starts with biochemical and histochemical studies on a muscle biopsy. Appropriate molecular genetic studies can then be chosen, based on these results and the clinical picture. Unfortunately, there is currently limited availability of respiratory chain studies in India. Exome sequencing is undertaken increasingly often; without preceding mitochondrial studies, this can lead to misleading results.

Growth differentiation factor 15 as a useful biomarker for mitochondrial disorders

ObjectiveThe diagnosis of mitochondrial disorders (MDs) is occasionally difficult because patients often present with solitary, or a combination of, symptoms caused by each organ insufficiency, which may be the result of respiratory chain enzyme deficiency. Growth differentiation factor 15 (GDF‐15) has been reported to be elevated in serum of patients with MDs. In this study, we investigated whether GDF‐15 is a more useful biomarker for MDs than several conventional biomarkers.MethodsWe measured the serum levels of GDF‐15 and fibroblast growth factor 21 (FGF‐21), as well as other biomarkers, in 48 MD patients and in 146 healthy controls in Japan. GDF‐15 and FGF‐21 concentrations were measured by enzyme‐linked immunosorbant assay and compared with lactate, pyruvate, creatine kinase, and the lactate‐to‐pyruvate ratio. We calculated sensitivity and specificity and also evaluated the correlation based on two rating scales, including the Newcastle Mitochondrial Disease Rating Scale (NMDAS).ResultsMean GDF‐15 concentration was 6‐fold higher in MD patients compared to healthy controls (2,711 ± 2,459 pg/ml vs 462.5 ± 141.0 pg/mL; p < 0.001). Using a receiver operating characteristic curve, the area under the curve was significantly higher for GDF‐15 than FGF‐21 and other conventional biomarkers. Our date suggest that GDF‐15 is the most useful biomarker for MDs of the biomarkers examined, and it is associated with MD severity.InterpretationOur results suggest that measurement of GDF‐15 is the most useful first‐line test to indicate the patients who have the mitochondrial respiratory chain deficiency. Ann Neurol 2015;78:Ann Neurol 2015;78:679–696

Diagnosis of mitochondrial disorders in children with next generation sequencing

To explore the application value of next generation sequencing (NGS) in the diagnosis of mitochondrial disorders. According to mitochondrial disease criteria, genomic DNA was extracted using standard procedure from peripheral venous blood of patients with suspected mitochondrial disease collected from neurological department of Beijing Children's Hospital Affiliated to Capital Medical University between October 2012 and February 2014. Targeted NGS to capture and sequence the entire mtDNA and exons of the 1 000 nuclear genes related to mitochondrial structure and function. Clinical data were collected from patients diagnosed at a molecular level, then clinical features and the relationship between genotype and phenotype were analyzed. Mutation was detected in 21 of 70 patients with suspected mitochondrial disease, in whom 10 harbored mtDNA mutation, while 11 nuclear DNA (nDNA) mutation. In 21 patients, 1 was diagnosed congenital myasthenic syndrome with episodic apnea due to CHAT gene p.I187T homozygous mutation, and 20 were diagnosed mitochondrial disease, in which 10 were Leigh syndrome, 4 were mitochondrial encephalomyopathy with lactic acidosis and stroke like episodes syndrome, 3 were Leber hereditary optic neuropathy (LHON) and LHON plus, 2 were mitochondrial DNA depletion syndrome and 1 was unknown. All the mtDNA mutations were point mutations, which contained A3243G, G3460A, G11778A, T14484C, T14502C and T14487C. Ten mitochondrial disease patients harbored homozygous or compound heterozygous mutations in 5 genes previously shown to cause disease: SURF1, PDHA1, NDUFV1, SUCLA2 and SUCLG1, which had 14 mutations, and 7 of the 14 mutations have not been reported. NGS has a certain application value in the diagnosis of mitochondrial diseases, especially in Leigh syndrome atypical mitochondrial syndrome and rare mitochondrial disorders.

Whole exome sequencing and whole mitochondrial genome sequencing for molecular diagnosis of mitochondrial disorders: Lessons from 865 Cases

Whole exome sequencing and whole mitochondrial genome sequencing for molecular diagnosis of mitochondrial disorders: Lessons from 865 Cases

Mitochondrial genome sequencing: A valuable addition to whole exome sequencing for the molecular diagnosis of mitochondrial disorders

Mitochondrial genome sequencing: A valuable addition to whole exome sequencing for the molecular diagnosis of mitochondrial disorders

OP4 – 2557: Retrospective study of clinical presentation and diagnostic yield of cohort of children with suspected mitochondrial disease

Objective The diagnosis of mitochondrial disorders (MD) is complex and protracted, due to clinical and genetic heterogeneity, requiring collation of radiological, biochemical, histological and genetic investigations. Collaboration with mitochondrial expert neurologists, biochemists and geneticists in the UK helps to achieve the best diagnostic rates of MD. The aim of the study was to determine the clinical characteristics of children with suspected and confirmed MD, review the yield of investigations and recommend best practice. Methods All children with suspected MD seen August 2008 to July 2014, within Cambridge paediatric neuroscience, had expert review of their presentation and investigations at an annual multidisciplinary mitochondrial group meeting by paediatric neurology, biochemistry, genetics, neuropathology, mitochondrial genetics, academic mitochondrial specialists within our tertiary centre, London, Oxford and Newcastle national mitochondrial centres. Results 28 children were identified, age range: 14 days–13 years (7 had died at 14 days–4 years), who were predominantly of Caucasian origin. The most common clinical presentation was developmental delay (71%) and refractory seizures (64%), MRI was suggestive of MD in 17%. MD was genetically confirmed in 20% and on respiratory chain abnormality (RCE) in 7%. RCE analysis proved beneficial in detecting abnormalities when initial histology was normal or indicative of alternative pathology. 25% of children have been recruited for next generation exome sequencing. Conclusion Diagnostic delay in MD is common due to the complex nature of investigations. The low rate of genetic diagnosis in our cohort is similar to other reported case series. Next generation sequencing and biomarkers such as FGF21 are promising diagnostic tools that may improve diagnostic accuracy. Expert review of the cases by the designated mitochondrial disease centres in the UK improves diagnostic accuracy.

The spectrum of pyruvate oxidation defects in the diagnosis of mitochondrial disorders.

Pyruvate oxidation defects (PODs) are among the most frequent causes of deficiencies in the mitochondrial energy metabolism and represent a substantial subset of classical mitochondrial diseases. PODs are not only caused by deficiency of subunits of the pyruvate dehydrogenase complex (PDHC) but also by various disorders recently described in the whole pyruvate oxidation route including cofactors, regulation of PDHC and the mitochondrial pyruvate carrier. Our own patients from 2000 to July 2014 and patients identified by a systematic survey of the literature from 1970 to July 2014 with a pyruvate oxidation disorder and a genetically proven defect were included in the study (n=628). Of these defects 74.2% (n=466) belong to PDHC subunits, 24.5% (n=154) to cofactors, 0.5% (n=3) to PDHC regulation and 0.8% (n=5) to mitochondrial pyruvate import. PODs are underestimated in the field of mitochondrial diseases because not all diagnostic centres include biochemical investigations of PDHC in their routine analysis. Cofactor and transport defects can be missed, if pyruvate oxidation is not measured in intact mitochondria routinely. Furthermore deficiency of the X-chromosomal PDHA1 can be biochemically missed depending on the X-inactivation pattern. This is reflected by an increasing number of patients diagnosed recently by genetic high throughput screening approaches. PDHC deficiency including regulation and import affect mainly the glucose dependent central and peripheral nervous system and skeletal muscle. PODs with combined enzyme defects affect also other organs like heart, lung and liver. The spectrum of clinical presentation of PODs is still expanding. PODs are a therapeutically interesting group of mitochondrial diseases since some can be bypassed by ketogenic diet or treated by cofactor supplementation. PDHC kinase inhibition, chaperone therapy and PGC1α stimulation is still a matter of further investigations.

Neurofibromatosis Type 1: A Novel NF1 Mutation Associated with Mitochondrial Complex I Deficiency

Background. Neurofibromatosis type 1 is a multisystemic, progressive disease, with an estimated incidence of 1/3500-2500. Mitochondrial diseases are generally multisystemic and may be present at any age, and the global prevalence is 1/8500. The diagnosis of these disorders is complex because of its clinical and genetic heterogeneity. Case Report. We present a rare case of the association of these two different genetic diseases, in which a heterozygous missense mutation in the NF1 gene was identified which had not yet been described (p.M1149 V). Additionally, the patient is suspected of carrying an unspecified mutation causing respiratory chain complex I deficiency. Clinical presentation included hypotonia, global development delay, reduced growth rate, progressive microcephaly, and numerous café-au-lait spots. Discussion. To the best of our knowledge this is the first report of complex I deficiency in a patient with neurofibromatosis type 1. It is very important to maintain a high index of suspicion for the diagnosis of mitochondrial disorders. In this patient, both the laboratory screening and muscle histology were normal and only the biochemical study of muscle allowed us to confirm the diagnosis.

Clinical re-sequencing for the diagnosis of mitochondrial disorders reveals high genetic heterogeneity

Clinical re-sequencing for the diagnosis of mitochondrial disorders reveals high genetic heterogeneity

Whole mitochondrial genome amplification and next generation sequencing for the diagnosis of mitochondrial disorders: Yield of 613 cases

Whole mitochondrial genome amplification and next generation sequencing for the diagnosis of mitochondrial disorders: Yield of 613 cases

Utilization of clinical diagnostic exome sequencing for the molecular diagnosis of mitochondrial disorders and therapeutic implications

 More than half of the patients with a suspicion of mitochondrial disease remain undiagnosed, accentuating the great potential and continuing challenges in the molecular diagnosis of mitochondrial diseases. Mutations in non-coding regions, epigenetic changes, oligenic inheritance, and yet-to-be-discovered novel genes may contribute to the phenotypes in patients with a negative results for Mendelian disorder based on whole exome sequencing.

Improved diagnosis of mitochondrial disorders by next generation sequencing approach

Improved diagnosis of mitochondrial disorders by next generation sequencing approach

Fibroblast growth factor 21: a novel biomarker for human muscle-manifesting mitochondrial disorders

Diagnosis of mitochondrial disorders is challenging, because of their highly variable clinical manifestations and age-of-onset and the shortage of specific diagnostic tools. Recent molecular studies have found that serum fibroblast growth factor 21 (FGF21) has potential to be a biomarker for muscle-manifesting mitochondrial disease, as well as for follow-up of disease progression and effect of intervention. Serum FGF21 as a biomarker is compared to conventional serum diagnostic tools for mitochondrial disorders. Mitochondrial disorders are a large group of different progressive disorders, with the age-of-onset from neonatal life to late adulthood, and symptoms originating from any organ system but sharing an underlying cause of mitochondrial dysfunction. The prevalence of these disorders is about 1:2000, varying somewhat between different countries. Serum diagnostic tools include lactate, pyruvate, their ratio, creatine kinase and amino acids. However, none of these markers are both sensitive and specific. Increased levels of FGF21 cytokine were recently found in the serum of patients, who have a muscle-manifesting mitochondrial disease, thus providing a promising, novel, sensitive and specific biomarker for these disorders.

Transition to Next Generation Analysis of the Whole Mitochondrial Genome: A Summary of Molecular Defects

The diagnosis of mitochondrial disorders is challenging because of the clinical variability and genetic heterogeneity. Conventional analysis of the mitochondrial genome often starts with a screening panel for common mitochondrial DNA (mtDNA) point mutations and large deletions (mtScreen). If negative, it has been traditionally followed by Sanger sequencing of the entire mitochondrial genome (mtWGS). The recently developed "Next-Generation Sequencing" (NGS) technology offers a robust high-throughput platform for comprehensive mtDNA analysis. Here, we summarize the results of the past 6 years of clinical practice using the mtScreen and mtWGS tests on 9,261 and 2,851 unrelated patients, respectively. A total of 344 patients (3.7%) had mutations identified by mtScreen and 99 (3.5%) had mtDNA mutations identified by mtWGS. The combinatorial analyses of mtDNA and POLG revealed a diagnostic yield of 6.7% in patients with suspected mitochondrial disorders but no recognizable syndromes. From the initial mtWGS-NGS cohort of 391 patients, 21 mutation-positive cases (5.4%) have been identified. The mtWGS-NGS provides a one-step approach to detect common and uncommon point mutations, as well as deletions. Additionally, NGS provides accurate, sensitive heteroplasmy measurement, and the ability to map deletion breakpoints. A new era of more efficient molecular diagnosis of mtDNA mutations has arrived.

First tier molecular diagnosis of mitochondrial disorders — The experience of a mitochondrial diagnostic laboratory pre‐NextGen era

First tier molecular diagnosis of mitochondrial disorders — The experience of a mitochondrial diagnostic laboratory pre‐NextGen era

Comprehensive analysis of entire mitochondrial genome by long-range PCR and next generation sequencing for the diagnosis of mitochondrial disorders: Yield of 216 cases

Comprehensive analysis of entire mitochondrial genome by long-range PCR and next generation sequencing for the diagnosis of mitochondrial disorders: Yield of 216 cases