Impact of newborn screening for fatty acid oxidation disorders on neurological outcome: A Belgian retrospective and multicentric study.

Fatty acid oxidation (FAO) disorders are autosomal recessive genetic disorders affecting either the transport or the oxidation of fatty acids. Acute symptoms arise during prolonged fasting, intercurrent infections, or intense physical activity. Metabolic crises are characterized by alteration of consciousness, hypoglycemic coma, hepatomegaly, cardiomegaly, arrhythmias, rhabdomyolysis, and can lead to death. In this retrospective and multicentric study, the data of 54 patients with FAO disorders were collected. Overall, 35 patients (64.8%) were diagnosed after newborn screening (NBS), 17 patients on clinical presentation (31.5%), and two patients after family screening (3.7%). Deficiencies identified included medium-chain acyl-CoA dehydrogenase (MCAD) deficiency (75.9%), very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency (11.1%), long-chain hydroxyacyl-CoA dehydrogenase (LCHAD) deficiency (3.7%), mitochondrial trifunctional protein (MTP) deficiency (1.8%), and carnitine palmitoyltransferase 2 (CPT 2) deficiency (7.4%). The NBS results of 25 patients were reviewed and the neurological outcome of this population was compared with that of the patients who were diagnosed on clinical presentation. This article sought to provide a comprehensive overview of how NBS implementation in Southern Belgium has dramatically improved the neurological outcome of patients with FAO disorders by preventing metabolic crises and death. Further investigations are needed to better understand the physiopathology of long-term complications in order to improve the quality of life of patients and to ensure optimal management.

Tracer-based lipidomics enables the discovery of disease-specific candidate biomarkers in mitochondrial ß-oxidation disorders.

Carnitine derivatives of disease-specific acyl-CoAs are the diagnostic hallmark for long-chain fatty acid ß-oxidation disorders (lcFAOD), including carnitine shuttle deficiencies, very-long-chain acyl-CoA dehydrogenase deficiency (VLCADD), long-chain 3-hydroxyacyl-CoA dehydrogenase deficiency (LCHADD) and mitochondrial trifunctional protein deficiency (MPTD). The exact consequence of accumulating lcFAO-intermediates and their influence on cellular lipid homeostasis is, however, still unknown. To investigate the fate and cellular effects of the accumulating lcFAO-intermediates and to explore the presence of disease-specific markers, we used tracer-based lipidomics with deuterium-labeled oleic acid (D9-C18:1) in lcFAOD patient-derived fibroblasts. In line with previous studies, we observed a trend towards neutral lipid accumulation in lcFAOD. In addition, we detected a direct connection between the chain length and patterns of (un)saturation of accumulating acylcarnitines and the various enzyme deficiencies. Our results also identified two disease-specific candidate biomarkers. Lysophosphatidylcholine(14:1) (LPC(14:1)) was specifically increased in severe VLCADD compared to mild VLCADD and control samples. This was confirmed in plasma samples showing an inverse correlation with enzyme activity, which was better than the classic diagnostic marker C14:1-carnitine. The second candidate biomarker was an unknown lipid class, which we identified as S-(3-hydroxyacyl)cysteamines. We hypothesized that these were degradation products of the CoA moiety of accumulating 3-hydroxyacyl-CoAs. S-(3-hydroxyacyl)cysteamines were significantly increased in LCHADD compared to controls and other lcFAOD, including MTPD. Our findings suggest extensive alternative lipid metabolism in lcFAOD and confirm that lcFAOD accumulate neutral lipid species. In addition, we present two disease-specific candidate biomarkers for VLCADD and LCHADD, that may have significant relevance for disease diagnosis, prognosis, and monitoring.

Challenges and opportunities to bridge translational to clinical research for personalized mitochondrial medicine.

Mitochondrial disorders are a group of rare and heterogeneous genetic diseases characterized by dysfunctional mitochondria leading to deficient adenosine triphosphate synthesis and chronic energy deficit in patients. The majority of these patients exhibit a wide range of phenotypic manifestations targeting several organ systems, making their clinical diagnosis and management challenging. Bridging translational to clinical research is crucial for improving the early diagnosis and prognosis of these intractable mitochondrial disorders and for discovering novel therapeutic drug candidates and modalities. This review provides the current state of clinical testing in mitochondrial disorders, discusses the challenges and opportunities for converting basic discoveries into clinical settings, explores the most suited patient-centric approaches to harness the extraordinary heterogeneity among patients affected by the same primary mitochondrial disorder, and describes the current outlook of clinical trials.

Protein biomarkers GDF15 and FGF21 to differentiate mitochondrial hepatopathies from other pediatric liver diseases.

BACKGROUND: Mitochondrial hepatopathies (MHs) are primary mitochondrial genetic disorders that can present as childhood liver disease. No recognized biomarkers discriminate MH from other childhood liver diseases. The protein biomarkers growth differentiation factor 15 (GDF15) and fibroblast growth factor 21 (FGF21) differentiate mitochondrial myopathies from other myopathies. We evaluated these biomarkers to determine if they discriminate MH from other liver diseases in children. METHODS: Serum biomarkers were measured in 36 children with MH (17 had a genetic diagnosis); 38 each with biliary atresia, α1-antitrypsin deficiency, and Alagille syndrome; 20 with NASH; and 186 controls. RESULTS: GDF15 levels compared to controls were mildly elevated in patients with α1-antitrypsin deficiency, Alagille syndrome, and biliary atresia-young subgroup, but markedly elevated in MH (p<0.001). FGF21 levels were mildly elevated in NASH and markedly elevated in MH (p<0.001). Both biomarkers were higher in patients with MH with a known genetic cause but were similar in acute and chronic presentations. Both markers had a strong performance to identify MH with a molecular diagnosis with the AUC for GDF15 0.93±0.04 and for FGF21 0.90±0.06. Simultaneous elevation of both markers >98th percentile of controls identified genetically confirmed MH with a sensitivity of 88% and specificity of 96%. In MH, independent predictors of survival without requiring liver transplantation were international normalized ratio and either GDF15 or FGF21 levels, with levels <2000 ng/L predicting survival without liver transplantation (p<0.01). CONCLUSIONS: GDF15 and FGF21 are significantly higher in children with MH compared to other childhood liver diseases and controls and, when combined, were predictive of MH and had prognostic implications.

Atypical presentation of Pearson syndrome in an infant with suspected myelodysplastic syndrome.

BACKGROUND: Anemia exhibits complex causation mechanisms and genetic heterogeneity. Some cases result in poor outcomes with multisystemic dysfunction, including renal tubulopathy. Early diagnosis is crucial to improve management. CASE-DIAGNOSIS/TREATMENT: A 21-month-old female patient was admitted with severe anemia. Persistent neutropenia and dysplastic signs suggested myelodysplastic syndrome, but targeted gene panel results were negative. After multiple transfusions, spontaneous hematologic recovery was observed. At 4 years old, she presented failure to thrive, renal Fanconi syndrome, and severe metabolic acidosis. Differential diagnosis included Pearson syndrome (PS), a life-threatening condition associated with mitochondrial DNA (mtDNA), featuring anemia and pancreatic insufficiency. Further analysis revealed a ~ 7.5 kb mtDNA deletion. Until the age of 5, supportive care has been provided, without pancreatic insufficiency. CONCLUSIONS: This PS case highlights the importance of genetic testing, even in the absence of typical features. Understanding the nature of mitochondrial disorders enables treatment tailoring and counseling about the prognosis.

Clinical, Genetic, and Histological Characterization of Patients with Rare Neuromuscular and Mitochondrial Diseases Presenting with Different Cardiomyopathy Phenotypes.

Cardiomyopathies are mostly determined by genetic mutations affecting either cardiac muscle cell structure or function. Nevertheless, cardiomyopathies may also be part of complex clinical phenotypes in the spectrum of neuromuscular (NMD) or mitochondrial diseases (MD). The aim of this study is to describe the clinical, molecular, and histological characteristics of a consecutive cohort of patients with cardiomyopathy associated with NMDs or MDs referred to a tertiary cardiomyopathy clinic. Consecutive patients with a definitive diagnosis of NMDs and MDs presenting with a cardiomyopathy phenotype were described. Seven patients were identified: two patients with ACAD9 deficiency (Patient 1 carried the c.1240C>T (p.Arg414Cys) homozygous variant in ACAD9; Patient 2 carried the c.1240C>T (p.Arg414Cys) and the c.1646G>A (p.Ar549Gln) variants in ACAD9); two patients with MYH7-related myopathy (Patient 3 carried the c.1325G>A (p.Arg442His) variant in MYH7; Patient 4 carried the c.1357C>T (p.Arg453Cys) variant in MYH7); one patient with desminopathy (Patient 5 carried the c.46C>T (p.Arg16Cys) variant in DES); two patients with mitochondrial myopathy (Patient 6 carried the m.3243A>G variant in MT-TL1; Patient 7 carried the c.253G>A (p.Gly85Arg) and the c.1055C>T (p.Thr352Met) variants in MTO1). All patients underwent a comprehensive cardiovascular and neuromuscular evaluation, including muscle biopsy and genetic testing. This study described the clinical phenotype of rare NMDs and MDs presenting as cardiomyopathies. A multidisciplinary evaluation, combined with genetic testing, plays a main role in the diagnosis of these rare diseases, and provides information about clinical expectations, and guides management.

Diagnostic Challenges of Neuromuscular Disorders after Whole Exome Sequencing.

BACKGROUND: Whole-exome sequencing (WES) facilitates the diagnosis of hereditary neuromuscular disorders. To achieve an accurate diagnosis, physicians should interpret the genetic report carefully along with clinical information and examinations. We described our experience with (1) clinical validation in patients with variants found using WES and (2) a diagnostic approach for those with negative findings from WES. METHODS: WES was performed on patients with the clinical impression of hereditary neuromuscular disorders. Information on clinical manifestations, neurological examination, electrodiagnostic studies, histopathology of muscle and nerve, and laboratory tests were collected. RESULTS: Forty-one patients (Male/Female: 18/23, age of onset: 34.5±15.9) accepted WES and were categorized into four scenarios: (1) patients with a positive WES result, (2) patients with an inconclusive WES result but supporting clinical data, (3) negative findings from WES, but a final diagnosis after further work-up, and (4) undetermined etiology from WES and in further work-ups. The yield rate of the initial WES was 63.4% (26/41). Among these, seventeen patients had positive WES result, while the other nine patients had inconclusive WES result but supporting clinical data. Notably, in the fifteen patients with negative findings from WES, four patients (26.7%) achieved a diagnosis after further workup: tumor-induced osteomalacia, metabolic myopathy with pathogenic variants in mitochondrial DNA, microsatellite expansion disease, and vasculitis-related neuropathy. The etiologies remained undetermined in eleven patients (myopathy: 7, neuropathy: 4) after WES and further workup. CONCLUSIONS: It is essential to design genotype-guided molecular studies to correlate the identified variants with their clinical features. For patients who had negative findings from WES, acquired diseases, mitochondrial DNA disorders and microsatellite expansion diseases should be considered.

Methodologies in Mitochondrial Testing: Diagnosing a Primary Mitochondrial Respiratory Chain Disorder.

BACKGROUND: Mitochondria are cytosolic organelles within most eukaryotic cells. Mitochondria generate the majority of cellular energy in the form of adenosine triphosphate (ATP) through oxidative phosphorylation (OxPhos). Pathogenic variants in mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) lead to defects in OxPhos and physiological malfunctions (Nat Rev Dis Primer 2016;2:16080.). Patients with primary mitochondrial disorders (PMD) experience heterogeneous symptoms, typically in multiple organ systems, depending on the tissues affected by mitochondrial dysfunction. Because of this heterogeneity, clinical diagnosis is challenging (Annu Rev Genomics Hum Genet 2017;18:257-75.). Laboratory diagnosis of mitochondrial disease depends on a multipronged analysis that can include biochemical, histopathologic, and genetic testing. Each of these modalities has complementary strengths and limitations in diagnostic utility. CONTENT: The primary focus of this review is on diagnosis and testing strategies for primary mitochondrial diseases. We review tissue samples utilized for testing, metabolic signatures, histologic findings, and molecular testing approaches. We conclude with future perspectives on mitochondrial testing. SUMMARY: This review offers an overview of the current biochemical, histologic, and genetic approaches available for mitochondrial testing. For each we review their diagnostic utility including complementary strengths and weaknesses. We identify gaps in current testing and possible future avenues for test development.

LYRM7-associated mitochondrial complex III deficiency with non-cavitating leukoencephalopathy and stroke-like episodes.

Defects of respiratory chain complex III (CIII) result in characteristic but rare mitochondrial disorders associated with distinct neuroradiological findings. The underlying molecular defects affecting mitochondrial CIII assembly factors are few and yet to be identified. LYRM7 assembly factor is required for proper CIII assembly where it acts as a chaperone for the Rieske iron-sulfur (UQCRFS1) protein in the mitochondrial matrix and stabilizing it. We present here the seventeenth individual with LYRM7-associated mitochondrial leukoencephalopathy harboring a previously reported rare pathogenic homozygous LYRM 7 variant, c.2T>C, (p.Met1?). Like previously reported individuals, our 5-year-old male proband presented with recurrent metabolic and lactic acidosis, encephalopathy, and fatigue. Further, he has additional, previously unreported features, including an acute stroke like episode with bilateral central blindness and optic neuropathy, recurrent hyperglycemia and hypertension associated with metabolic crisis. However, he has no signs of psychomotor regression. He has been stable clinically with residual left-sided reduced visual acuity and amblyopia, and no more metabolic crises for 2-year-period while on the mitochondrial cocktail. Although the reported brain MRI findings in other affected individuals are homogenous, it is slightly different in our index, revealing evidence of bilateral almost symmetric multifocal periventricular T2 hyperintensities with hyperintensities of the optic nerves, optic chiasm, and corona radiata but with no cavitation or cystic changes. This report describes new clinical and radiological findings of LYRM7-associated disease. The report also summarizes the clinical and molecular data of previously reported individuals describing the full phenotypic spectrum.

A Patient with Mitochondrial Disease on Dialysis with Long-term Follow-up of Cardiomyopathy: An Autopsy Case Report.

A 59-year-old man developed diabetes at 24 years old and underwent hemodialysis at 42 years old. At 54 years old, cardiac dysfunction with left ventricular hypertrophy was detected, followed by complete atrioventricular block at 57 years old. The patient was diagnosed with mitochondrial disease based on a myocardial biopsy and the presence of a mitochondrial DNA mutation (3243A>G). He died of septic shock at 59 years old, and an autopsy confirmed mitochondrial cardiomyopathy. If progressive cardiac hypertrophy and conduction disturbances are observed in patients with diabetes mellitus on long-term hemodialysis, mitochondrial disease needs to be considered.

Clinical and genetic features of four patients with Pearson syndrome: An observational study.

ABSTRACT: Pearson syndrome (PS) is a multisystem mitochondrial cytopathy arising from deletions in mitochondrial DNA. Pearson syndrome is a sporadic disease that affects the hematopoietic system, pancreas, eyes, liver, and heart and the prognosis is poor. Causes of morbidity include metabolic crisis, bone marrow dysfunction, sepsis, and liver failure in early infancy or childhood. Early diagnosis may minimize complications, but suspicion of the disease is difficult and only mitochondrial DNA gene testing can identify mutations. There is no specific treatment for PS, which remains supportive care according to symptoms; however, hematopoietic stem cell transplantation may be considered in cases of bone marrow failure.We herein describe the clinical and genetic characteristics of four patients with PS. One patient presented with hypoglycemia, two developed pancytopenia, and the final patient had hypoglycemia and acute hepatitis as the primary manifestation. All patients had lactic acidosis. Additionally, all patients showed a variety of clinical features including coagulation disorder, pancreatic, adrenal, and renal tubular insufficiencies. Two patients with pancytopenia died in their early childhood. Our experience expands the phenotypic spectrum associated with PS and its clinical understanding.

Mitochondrial Retinopathy.

PURPOSE: To report the retinal phenotype and the associated genetic and systemic findings in patients with mitochondrial disease. DESIGN: Retrospective case series. PARTICIPANTS: Twenty-three patients with retinopathy and mitochondrial disease, including chronic progressive external ophthalmoplegia (CPEO), maternally inherited diabetes and deafness (MIDD), mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS), Kearns-Sayre syndrome, neuropathy, ataxia, and retinitis pigmentosa (NARP) syndrome, and other systemic manifestations. METHODS: Review of case notes, retinal imaging, electrophysiologic assessment, molecular genetic testing including protein modeling, and histologic analysis of muscle biopsy. MAIN OUTCOME MEASURES: Phenotypic characteristics of mitochondrial retinopathy. RESULTS: Genetic testing identified sporadic large-scale mitochondrial DNA deletions and variants in MT-TL1, MT-ATP6, MT-TK, MT-RNR1, or RRM2B. Based on retinal imaging, 3 phenotypes could be differentiated: type 1 with mild, focal pigmentary abnormalities; type 2 characterized by multifocal white-yellowish subretinal deposits and pigment changes limited to the posterior pole; and type 3 with widespread granular pigment alterations. Advanced type 2 and 3 retinopathy presented with chorioretinal atrophy that typically started in the peripapillary and paracentral areas with foveal sparing. Two patients exhibited a different phenotype: 1 revealed an occult retinopathy, and the patient with RRM2B-associated retinopathy showed no foveal sparing, no severe peripapillary involvement, and substantial photoreceptor atrophy before loss of the retinal pigment epithelium. Two patients with type 1 disease showed additional characteristics of mild macular telangiectasia type 2. Patients with type 1 and mild type 2 or 3 disease demonstrated good visual acuity and no symptoms associated with the retinopathy. In contrast, patients with advanced type 2 or 3 disease often reported vision problems in dim light conditions, reduced visual acuity, or both. Short-wavelength autofluorescence usually revealed a distinct pattern, and near-infrared autofluorescence may be severely reduced in type 3 disease. The retinal phenotype was key to suspecting mitochondrial disease in 11 patients, whereas 12 patients were diagnosed before retinal examination. CONCLUSIONS: Different types of mitochondrial retinopathy show characteristic features. Even in absence of visual symptoms, their recognition may facilitate the often challenging and delayed diagnosis of mitochondrial disease, in particular in patients with mild or nebulous multisystem disease.

Pediatric single large-scale mtDNA deletion syndromes: The power of patient reported outcomes.

There is a limited understanding of system-level clinical outcomes and interventions associated with single large-scale mitochondrial DNA deletion syndromes (SLSMDS). Additionally, no research exists that describes patient reported outcomes (PROs) of children with SLSMDS. A global and observational registry was established to understand the multi-systemic course of SLSMDS and track clinical outcomes. The development and design of the registry is described. Demographic characteristics, history and diagnoses, and system level prevalence of problems and interventions are reported for 42 children. System level problems and interventions include information on the following body systems: audiology, cardiac, endocrine, gastrointestinal (including pancreatic and hepatobiliary system), hematological, metabolic, neurological (including autonomic, mobility, & learning), ophthalmic, psychiatric, renal, and respiratory. Results emphasize the need of patient registries and suggest that the diagnostic odyssey and burden of disease for children with SLSMDS is significant. System-level findings may help families and clinical providers with diagnosis, prognostication, and treatment. A multidisciplinary team of clinical experts with a central coordinating specialist for children with SLSMDS is recommended.

Hypotonia-cystinuria 2p21 deletion syndrome: Intrafamilial variability of clinical expression.

Two siblings presented similarly with congenital hypotonia, lactic acidosis, and failure to thrive. Later in childhood, the brother developed cystinuria and nephrolithiasis whereas the older sister suffered from cystinuria and chronic neurobehavioral disturbances. Biopsied muscle studies demonstrated deficient cytochrome c oxidase activities consistent with a mitochondrial disease. Whole exome sequencing (WES), however, revealed a homozygous 2p21 deletion involving two contiquous genes, SLC3A1 (deletion of exons 2-10) and PREPL (deletion of exons 2-14). The molecular findings were consistent with the hypotonia-cystinuria 2p21 deletion syndrome, presenting similarly in infancy with mitochondrial dysfunction but diverging later in childhood and displaying intrafamilial phenotypic variability.

Diagnosis of primary mitochondrial disorders -Emphasis on myopathological aspects.

Mitochondrial disorders are one of the most common neurometabolic disorders affecting all age groups. The phenotype-genotype heterogeneity in these disorders can be attributed to the dual genetic control on mitochondrial functions, posing a challenge for diagnosis. Though the advancement in the high-throughput sequencing and other omics platforms resulted in a "genetics-first" approach, the muscle biopsy remains the benchmark in most of the mitochondrial disorders. This review focuses on the myopathological aspects of primary mitochondrial disorders. The utility of muscle biopsy is not limited to analyse the structural abnormalities; rather it also proves to be a potential tool to understand the deranged sub-cellular functions.

Protective Effects of Flavonoids Against Mitochondriopathies and Associated Pathologies: Focus on the Predictive Approach and Personalized Prevention.

Multi-factorial mitochondrial damage exhibits a "vicious circle" that leads to a progression of mitochondrial dysfunction and multi-organ adverse effects. Mitochondrial impairments (mitochondriopathies) are associated with severe pathologies including but not restricted to cancers, cardiovascular diseases, and neurodegeneration. However, the type and level of cascading pathologies are highly individual. Consequently, patient stratification, risk assessment, and mitigating measures are instrumental for cost-effective individualized protection. Therefore, the paradigm shift from reactive to predictive, preventive, and personalized medicine (3PM) is unavoidable in advanced healthcare. Flavonoids demonstrate evident antioxidant and scavenging activity are of great therapeutic utility against mitochondrial damage and cascading pathologies. In the context of 3PM, this review focuses on preclinical and clinical research data evaluating the efficacy of flavonoids as a potent protector against mitochondriopathies and associated pathologies.

Therapy Prospects for Mitochondrial DNA Maintenance Disorders.

Mitochondrial DNA depletion and multiple deletions syndromes (MDDS) constitute a group of mitochondrial diseases defined by dysfunctional mitochondrial DNA (mtDNA) replication and maintenance. As is the case for many other mitochondrial diseases, the options for the treatment of these disorders are rather limited today. Some aggressive treatments such as liver transplantation or allogeneic stem cell transplantation are among the few available options for patients with some forms of MDDS. However, in recent years, significant advances in our knowledge of the biochemical pathomechanisms accounting for dysfunctional mtDNA replication have been achieved, which has opened new prospects for the treatment of these often fatal diseases. Current strategies under investigation to treat MDDS range from small molecule substrate enhancement approaches to more complex treatments, such as lentiviral or adenoassociated vector-mediated gene therapy. Some of these experimental therapies have already reached the clinical phase with very promising results, however, they are hampered by the fact that these are all rare disorders and so the patient recruitment potential for clinical trials is very limited.

A novel MT-CO2 variant causing cerebellar ataxia and neuropathy: The role of muscle biopsy in diagnosis and defining pathogenicity.

Pathogenic variants in mitochondrial DNA (mtDNA) are associated with significant clinical heterogeneity with neuromuscular involvement commonly reported. Non-syndromic presentations of mtDNA disease continue to pose a diagnostic challenge and with genomic testing still necessitating a muscle biopsy in many cases. Here we describe an adult patient who presented with progressive ataxia, neuropathy and exercise intolerance in whom the application of numerous Mendelian gene panels had failed to make a genetic diagnosis. Muscle biopsy revealed characteristic mitochondrial pathology (cytochrome c oxidase deficient, ragged-red fibers) prompting a thorough investigation of the mitochondrial genome. Two heteroplasmic MT-CO2 gene variants (NC_012920.1: m.7887G>A and m.8250G>A) were identified, necessitating single fiber segregation and familial studies - including the biopsy of the patient's clinically-unaffected mother - to demonstrate pathogenicity of the novel m.7887G>A p.(Gly101Asp) variant and establishing this as the cause of the mitochondrial biochemical defects and clinical presentation. In the era of high throughput whole exome and genome sequencing, muscle biopsy remains a key investigation in the diagnosis of patients with non-syndromic presentations of adult-onset mitochondrial disease and fully defining the pathogenicity of novel mtDNA variants.

Acetyl-CoA-driven respiration in frozen muscle contributes to the diagnosis of mitochondrial disease.

BACKGROUND: Freezing human biopsies is common in clinical practice for storage. However, this technique disrupts mitochondrial membranes, hampering further analyses of respiratory function. To contribute to laboratorial diagnosis of mitochondrial diseases, this study sought to develop a respirometry approach using O2k (Oroboros Ins.) to measure the whole electron transport chain (ETC) activity in homogenates of frozen skeletal muscle biopsies. PATIENTS AND METHODS: We enrolled 16 patients submitted to muscle biopsy in the process of routine diagnostic investigation: four with mitochondrial disease and severe mitochondrial dysfunction; seven with exercise intolerance and multiple deletions of mitochondrial DNA, presenting mild to moderate mitochondrial dysfunction; five without mitochondrial disease, as controls. Whole homogenates of muscle fragments were prepared using grinder-type equipment. O2 consumption rates were normalized using citrate synthase activity. RESULTS: Transmission electron microscopy confirmed mitochondrial membrane discontinuation, indicating increased permeability of mitochondrial membranes in homogenates from frozen biopsies. O2 consumption rates in the presence of acetyl-CoA lead to maximum respiratory rates sensitive to rotenone, malonate and antimycin. This protocol of acetyl-CoA-driven respiration (ACoAR), applied in whole homogenates of frozen muscle, was sensitive enough to identify ETC abnormality, even in patients with mild to moderate mitochondrial dysfunction. We demonstrated adequate repeatability of ACoAR and found significant correlation between O2 consumption rates and enzyme activity assays of individual ETC complexes. CONCLUSIONS: We present preliminary data on a simple, low cost and reliable procedure to measure respiratory function in whole homogenates of frozen skeletal muscle biopsies, contributing to diagnosis of mitochondrial diseases in humans.

Diagnosing newborns with suspected mitochondrial disorders: an economic evaluation comparing early exome sequencing to current typical care.

PURPOSE: To determine the value of early exome sequencing (eES) relative to the current typical care (TC) in the diagnosis of newborns with suspected severe mitochondrial disorders (MitD). METHODS: We used a decision tree-Markov hybrid to model neonatal intensive care unit (NICU)-related outcomes and costs, lifetime costs and quality-adjusted life-years among patients with MitD. Probabilities, costs, and utilities were populated using published literature, expert opinion, and the Pediatric Health Information System database. Incremental cost-effectiveness ratios (ICER) and net monetary benefits (NMB) were calculated from lifetime costs and quality-adjusted life-years for singleton and trio eES, and TC. Robustness was assessed using univariate and probabilistic sensitivity analyses (PSA). Scenario analyses were also conducted. RESULTS: Findings indicate trio eES is a cost-minimizing and cost-effective alternative to current TC. Diagnostic probabilities and NICU length-of-stay were the most sensitive model parameters. Base case analysis demonstrates trio eES has the highest incremental NMB, and PSA demonstrates trio eES had the highest likelihood of being cost-effective at a willingness-to-pay (WTP) of $200,000 relative to TC, singleton eES, and no ES. CONCLUSION: Trio and singleton eES are cost-effective and cost-minimizing alternatives to current TC in diagnosing newborns suspected of having a severe MitD.