Diagnosis Of Mitochondrial Diseases Research Articles

20.: A new diagnostic paradigm for mitochondrial disease

Mitochondrial disease is usually diagnosed with a combined approach of clinical investigation and histological or enzymatic analysis of muscle biopsy or genetic testing. With the discovery of new serological biomarkers and the development of accurate next-generation sequencing (NGS) methods, the opportunity to change the diagnostic paradigm for this group of disorders presents. In this study, we aim to test the accuracy of a new method to diagnose mitochondrial disease, using a combined approach of serum fibroblast growth factor-21 (FGF-21) level testing and NGS methods. We measured serum FGF-21 levels in 70 patients with mitochondrial disease using an enzyme-linked immunosorbent assay based assay. We also performed long-range polymerase chain reaction (PCR) on urinary epithelial cells from the same patients. PCR products were subjected to massively parallel sequencing (NGS) to provide at least thousand-fold coverage of the mtDNA. Serum FGF-21 levels were elevated in patients with mitochondrial disease. This was a more sensitive biomarker of mitochondrial disease than serum lactate, pyruvate creatine kinase or serum lactate/pyruvate ratios. Long-range PCR was able to detect single and multiple deletions. NGS sequencing was able to determine the breakpoints for single deletions, as well as identify point mutations in the mtDNA. Our findings demonstrate that the diagnosis of mitochondrial disease can be performed by using a combined approach of clinical evaluation, serum FGF-21 levels and next generation sequencing. Adoption of this approach would avoid the need for muscle biopsy in most patients and thus represent a shift in the diagnostic paradigm for mitochondrial disease.

Role of Plasma Amino Acids and Urinary Organic Acids in Diagnosis of Mitochondrial Diseases in Children

BackgroundDiagnostic difficulty in mitochondrial diseases (MD) results not only from the wide spectrum of symptoms and signs but also from the absence of a reliable screening or diagnostic biomarker. AimTo investigate the likelihood of MD in patients with symptoms and signs impressive of MD through quantitative measurement of plasma amino acids, and urinary organic acids. MethodsTwenty patients with symptoms and signs suggestive of MD were further evaluated by quantitative plasma amino acids and urinary organic acids assay and neuroimaging. ResultsPlasma amino acid results revealed elevation of alanine in 11, glycine in five, and proline in two patients. Abnormal urinary organic acid analysis was present in six patients; increased urinary lactate (20%), dicarboxylicaciduria (15%), and urinary ketone bodies (10%). Upon enrollment our patients scored as possible MD according to the MD scoring system. At the end of the study, five patients still scored as possible MD, eight patients as probable MD, and seven patients as definite MD. All patients with definite MD had elevated serum lactate. In three patients, elevated urinary lactate was the only abnormality. Alanine was elevated in all patients with definite MD, whereas proline was elevated in only one. Magnetic resonance imaging of the brain showed atrophic changes in one patient and bilateral basal ganglia hyperintensity in another. ConclusionUrinary organic acids and quantitative plasma amino acids can help in the diagnosis of MD, especially when the economic burden and absence of specialized centers limits the diagnosis.

Role of brain magnetic resonance spectroscopy in the evaluation of suspected mitochondrial diseases in children: Experience in 30 pediatric cases

IntroductionMitochondrial diseases are a group of inherited disorders caused by derangement of mitochondrial respiration. MR spectroscopy (MRS) has been shown to detect abnormal accumulation of lactate in brain parenchyma and CSF in patients with mitochondrial disorders, but the frequency of detection is largely unknown. Aim of the workTo evaluate the role of brain MR spectroscopy in the assessment of suspected mitochondrial diseases in the pediatric age group. Patients and methodsThirty children with suspected mitochondrial diseases were examined by MRS. Examination was done using multisection technique and multiple echo times mainly short (35ms) and intermediate (144ms). Mitochondrial disease criteria scoring system was used to confirm the suspected diagnosis. ResultsAll patients showed elevated lactate peak with the CSF being the most sensitive (100%). Among the 30 patients, 26 (86.7%) had elevated levels of blood lactate/pyruvate ratio. Conventional MRI showed highly suggestive features in 15 patients while non specific findings were detected in 11 patients and 4 showed normal appearing brain. ConclusionsMRS provides a noninvasive tool for the diagnosis of mitochondrial diseases, especially in children with non specific findings on MRI, normal appearing MRI or a normal blood lactate/pyruvate ratio.

Points to Consider in the Clinical Use of NGS Panels for Mitochondrial Disease: An Analysis of Gene Inclusion and Consent Forms

Mitochondrial next generation sequencing (NGS) panels offer single-step analysis of the numerous nuclear genes involved in the structure, function, and maintenance of mitochondria. However, the complexities of mitochondrial biology and genetics raise points for consideration in clinical use of these tests. To understand the current status of mitochondrial genetic testing, we assessed the gene offerings and consent forms of mitochondrial NGS panels available from seven US-based clinical laboratories. The NGS panels varied markedly in number of genes (101-1204 genes), and the proportion of genes causing "classic" mitochondrial diseases and their phenocopies ranged widely between labs (18 %-94 % of panel contents). All panels included genes not associated with classic mitochondrial diseases (6 %-28 % of panel contents), including genes causing adult-onset neurodegenerative disorders, cancer predisposition, and other genetic syndromes or inborn errors of metabolism. Five of the panels included genes that are not listed in OMIM to be associated with a disease phenotype (5 %-49 % of panel contents). None of the consent documents reviewed had options for patient preference regarding receipt of incidental findings. These findings raise points of discussion applicable to mitochondrial diagnostics, but also to the larger arenas of exome and genome sequencing, including the need to consider the boundaries between clinical and research testing, the necessity of appropriate informed consent, and the responsibilities of clinical laboratories and clinicians. Based on these findings, we recommend careful evaluation by laboratories of the genes offered on NGS panels, clear communication of the predicted phenotypes, and revised consent forms to allow patients to make choices about receiving incidental findings. We hope that our analysis and recommendations will help to maximize the considerable clinical utility of NGS panels for the diagnosis of mitochondrial disease.

Index of Suspicion

The reader is encouraged to write possible diagnoses for each case before turning to the discussion. We invite readers to contribute case presentations and discussions. Please inquire first by contacting Dr. Deepak Kamat at DKamat@med.wayne.edu. A 2-year-old boy with a history of eczema and food allergies to nuts and soy presents to the emergency department with a 10-day history of generalized edema. The swelling began in the periorbital area but soon progressed to involve his entire body. There have been no fevers or any recent illnesses other than 1 day of cough and a few watery stools. He has had no new exposures to foods or other substances. His family history is remarkable for multiple allergies and eczema. Physical examination reveals a temperature of 98.8°F (37.1oC), heart rate of 98 beats per minute, respiratory rate of 36 breaths per minute, blood pressure of 103/65 mm Hg, and an oxygen saturation of 97% on room air. His weight is 12.6 kg (34th percentile). He is noted to have a moderate amount of periorbital edema, decreased breath sounds at the lung bases bilaterally, a protuberant abdomen, and 2+ pitting edema on his lower extremities. The rest of his physical examination findings are within normal limits. Laboratory evaluation reveals a white blood cell count of 23,800/μL (23.8 × 109/L), with 37% neutrophils, 49% lymphocytes, 4% bands, 7% monocytes, and 3% eosinophils; hemoglobin level of 13.3 g/dL (133 g/L); and platelet count of 427 × 103/μL (427 × 109/L). Serum electrolyte, blood urea nitrogen, and creatinine levels are normal. Sodium level is 132 mEq/L (132 mmol/L). Serum albumin level is 1.7 g/dL (17 g/L) (reference range, 3.4-5.4 g/dL [34-54 g/L]). Total protein level is 3.8 g/dL (38 g/L). Aspartate aminotransferase, alanine aminotransferase, and coagulation profiles …

DIAGNOSTIC CRITERIA FOR PEDIATRIC MITOCHONDRIAL DISORDERS

Our body’s nucleated cells contain 500-2000 mitochondria. In the liver mitochondria are specialized to detoxify ammonia in urea cycle, mitochondria are also required, for neurotransmitter metabolism. Mitochondrial encephalomyopathies are genetically, biochemically and clinically heterogeneous group of disorders associated with abnormalities of oxidative phosphorylation. The final diagnosis relied on clinical and molecular criteria. Diagnostic criteria for pediatric mitochondrial disorders have been modified from an adult classification system. This criterion is the modified walker criteria. Because mitochondrial disease have a natural history of heterogenous groups of disorders, the modified walker criteria is useful to evaluate the major clinical findings of infants and children with diagnosis of mitochondrial diseases. By using the modified walker criteria, we are able to gather a large group of pediatric patients with a definite diagnosis of a mitochondrial disorder and study their clinical histories.

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.

THE EFFECTS OF HIGH INTENSITY INTERVAL TRAINING ON CLINICAL SYMPTOMS AND FUNCTIONAL CAPACITY IN ADULTS WITH NEUROMUSCULAR DISEASE

BackgroundNeuromuscular disease includes a spectrum of inflammatory and mitochondrial myopathies where dysfunction in the energy–producing mitochondria can cause muscle weakness, exercise intolerance and fatigue. Continuous endurance training has been shown...

A novel mutation in STXBP1 causing epileptic encephalopathy (late onset infantile spasms) with partial respiratory chain complex IV deficiency

STXBP1 (MUNC18.1), encoding syntaxin binding protein 1, has been reported in Ohtahara syndrome, a rare epileptic encephalopathy with suppression burst pattern on EEG, in patients with infantile spasms and in a few patients with nonsyndromic mental retardation without epilepsy.We report a patient who presented late onset infantile spasms. Epilepsy was controlled but the patient developed severe mental delay. A first diagnosis of mitochondrial disease was based on clinical presentation and on a partial deficit of respiratory chain complex IV, but molecular screening for mitochondrial genes was negative. The sequencing of STXBP1 gene found a de novo nonsense mutation (c.585C>G/p.Tyr195X).This observation widens the clinical spectrum linked to STXBP1 mutations with the description of a patient with late onset infantile spasms. It raises the question of the value of epilepsy genes screening in patients with uncertain, partial or unconfirmed mitochondrial dysfunction.

P.17.16 Enophthalmos and MELAS

A female patient with genetically confirmed MELAS was seen when 32 old for headache of two types: daily diffuse headache and occasional right-sight migrainous.She also complained of memory loss, asthenia, episodes of dizziness resulting sometimes in falls. She never presented any seizure. Her examination revealed a right-sight sensory-motor weakness and an intellectual impairment on formal psychological testing. A CT scan showed a left temporo-sylvian arachnoid cyst with ipsilateral frontal sulcal effacement and ventricular mass effect. Cystoperitoneal shunting resulted in disappearance of right-sight headache episodes, but did not change the other symptoms, which progressively worsened. In addition, a conspicuous bilateral orbital deepening appeared within 2 months following surgery. Post-operative CT scan and brain magnetic resonance imaging revealed only temporal cyst and “ enophthalmos ”. The diagnosis of mitochondrial disease was suspected because of the familial history. A large spectrum of causes generates enophthalmos. Either the orbital volume increases or the volume of soft tissues in the orbit decreases. Few articles report enophtalmos secondary to ventriculoperitoneal shunt. The mechanism of enophthalmos in these cases has never been elucidated, but theories have included fat atrophy and expansion of the bony orbit. In our patient, neither valve pressure nor valve position were abnormal, RMI did not find any usual sign of intracranial hypotension: no thickening of the meninges; lateral ventricles and basal cisterns were present and brain did not collapse. A reduction of orbital content was discovered, due to a bilateral ocular fat atrophy and extra ocular muscles atrophy. The relation between enophthalmia and mitochondriopathies has not been reported in the literature. Fat tissue is not used to be a target-tissue of mitochondriopathies except their implication in diffuse fat atrophy in HIV patient treated with specific treatment.

Exome sequencing of a patient with suspected mitochondrial disease reveals a likely multigenic etiology

BackgroundThe clinical features of mitochondrial disease are complex and highly variable, leading to challenges in establishing a specific diagnosis. Despite being one of the most commonly occurring inherited genetic diseases with an incidence of 1/5000, ~90% of these complex patients remain without a DNA-based diagnosis. We report our efforts to identify the pathogenetic cause for a patient with typical features of mitochondrial disease including infantile cataracts, CPEO, ptosis, progressive distal muscle weakness, and ataxia who carried a diagnosis of mitochondrial disease for over a decade.MethodsWhole exome sequencing and bioinformatic analysis of these data were conducted on the proband.ResultsExome sequencing studies showed a homozygous splice site mutation in SETX, which is known to cause Spinocerebellar Ataxia, Autosomal Recessive 1 (SCAR1). Additionally a missense mutation was identified in a highly conserved position of the OCRL gene, which causes Lowe Syndrome and Dent Disease 2.ConclusionsThis patient’s complex phenotype reflects a complex genetic etiology in which no single gene explained the complete clinical presentation. These genetic studies reveal that this patient does not have mitochondrial disease but rather a genocopy caused by more than one mutant locus. This study demonstrates the benefit of exome sequencing in providing molecular diagnosis to individuals with complex clinical presentations.

Mitochondrial encephalopathy: a case report and review of the literature

Objective To investigate the clinical, imaging and pathological characteristics of one case of mitochondrial encephalopathy. Methods The data of physical examination, electromyopraphy (EMG), cranial CT and MRI, electroencephalography (EEG), serologic examination, and light microscopy and electron microscopy of muscle biopsy specimens of one case of mitochondrial encephalopathy were collected. The related literatures were reviewed. Results A 52-year-old female patient was admitted with an 8-month of seizures, numbness of the limbs for three times. The clinical manifestations were mainly focal seizures with intelligence disturbance and positive bilateral pyramidal tract sign. The imaging examination was considered to be ischemic stroke and cerebral atrophy. EEG revealed moderate and severe abmormal brain wave. In cerebrospinal fluid (CSF) examination, the intracranial pressure (ICP) was initially 2.01 kPa and finally 1.03 kPa, red blood cell count 1.04 × 10 6/L, white blood cell count 5 × 10 6/L (with an increase in neutrophilic granulocyte proportion), protein 0.70 g/L, chlorides 103.30 mmol/L and IgG 51.90 mg/L. Serum high-sensitivity C-reactive protein (hsCRP) was 15.40 mg/L, and erythrocyte sedimentation rate (ESR) 23 mm/h. Brain biopsy revealed cortical neuron swelling, loss of natural conical shape. The number of capillary increased and astrocytes proliferated reactively. Immunohistochemical examination showed that there were positive Mito granular materials inside the swelling and normal cytoplasm of neurons. The proliferative astrocytes were positive for glial fibrillany acid protein (GFAP). The electronic microscopic examination revealed that a large number of abnormal mitochondria various in shape and size were accumulated between subsarcolemma and myofibrilla. The mitochondrial cristae were flattened or prolonged and twining in concentricity. Osmiophilic bodies and crystalloid inclusion bodies were found in mitochondria. Conclusion Mitochondrial encephalopathy is a rare disease which may be misdiagnosed as ischemic stroke. The diagnosis of mitochondrial disease must be based upon clinical manifestations and laboratory examinations including serum enzymology, biochemistry, molecular biochemistry, muscular biopsy, etc.

Transient ischemic attack-like episodes without stroke-like lesions in MELAS

A stroke-like episode is a core symptom in mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes (MELAS). Proton magnetic resonance spectroscopy (1H-MRS) is useful in the diagnosis of mitochondrial diseases. We report an 8-year-old girl with MELAS, presenting with a seizure and blindness. 1H-MRS showed a strikingly elevated lactate peak in the right occipital region, where no abnormal signals appeared on either T2-W or diffusion-weighted MRI. She recovered completely within a day. We describe this mild clinical condition with abnormal lactate peak in normal-appearing gray matter as a transient ischemic attack-like episode in MELAS.

Mitochondrial Cardioencephalomyopathy Due to a Novel SCO2 Mutation in a Brazilian Patient

To review all patients with SCO2 mutations and to describe a Brazilian patient with cardioencephalomyopathy carrying compound heterozygous mutations in SCO2, one being the known pathogenic p.E140K mutation and the other a novel 12-base pair (bp) deletion at nucleotides 1519 through 1530 (c.1519_1530del). Case report and literature review. University hospital. Infant girl presenting with an encephalomyopathy, inspiratory stridor, ventilator failure, progressive hypotonia, and weakness, leading to death. Clinical features, neuroimaging findings, muscle biopsy with histochemical analysis, and genetic studies. This infant girl was the first child of healthy, nonconsanguineous parents. She developed progressive muscular hypotonia and ventilatory failure. At the end of the first month of life, she developed cardiomegaly and signs of cardiac failure. Routine blood tests showed lactic acidosis and mild elevation of the creatine kinase level. Brain magnetic resonance imaging showed increased T2 and fluid-attenuated inversion recovery signals in the putamen bilaterally. Nerve conduction studies showed severe axonal sensorimotor neuropathy. Muscle biopsy revealed a neurogenic pattern with mitochondrial proliferation and total absence of cytochrome- c oxidase histochemical stain. Sequencing of SCO2 showed that the patient had compound heterozygote SCO2 mutations: the previously described c.1541G>A (p.E140K) mutation and a novel 12-bp deletion at nucleotides 1519 through 1530 (c.1519_1530del). The patient died at age 45 days. Our findings and the literature review indicate that it is important to consider the diagnosis of mitochondrial disease in newborns with hypotonia and cardiomyopathy. In our case, the accurate diagnosis of SCO2 mutations is particularly important for genetic counseling.

Role of creatine as biomarker of mitochondrial diseases

Recent investigations have suggested creatine (Cr) as an additional biomarker of mitochondrial diseases. With the aim of corroborating previous findings, we have studied plasma Cr in a cohort of 33 patients with different mitochondrial diseases. Cr was clearly increased in 9 out of 33 patients. Therefore, positive patients represent only 28% of the total number, suggesting that Cr is not a sensitive biomarker of mitochondrial diseases although it does present an acceptable specificity (83%). High plasma Cr, together with other biomarkers, might be useful to reinforce the diagnosis of mitochondrial diseases.

Early cardiac evaluation in children with non‐specific mitochondrial disease with isolated mitochondrial respiratory chain complex I defect

We evaluated echocardiography and electrocardiography (ECG) results in children with non-specific mitochondrial disease (MD) in order to study early cardiac involvement, a well-known complication of the disease. Among non-specific MD children whose isolated mitochondrial respiratory chain complex I defect was confirmed by muscle biopsy and satisfied the criteria of MD, 27 who had no cardiac symptoms were evaluated by echocardiography and ECG. Three (11.1%) out of the 27 non-specific MD patients had left ventricular ejection fraction of less than 55% and two of them (7.4%) had fractional shortening of less than 26%. ECG abnormalities were observed in 16 of the non-specific MD patients (59.3%). Prolongation of heart rate-corrected QT interval was seen in 11 (40.7%) and widening of the QRS interval in eight (29.6%). Left ventricular ejection fraction and fractional shortening of the patients were significantly decreased compared with those in the control group while heart rate-corrected QT interval was prolonged in the former group. QRS interval was more widened in non-specific MD patients, but without statistical significance. The potentially severe cardiac involvement observed in our subjects suggests that early cardiac evaluation after confirming the diagnosis of MD and regular follow-up tests should be strongly recommended in children even in cases without typical cardiac manifestations.

Treatment for mitochondrial disorders

Treatment for mitochondrial disorders

Diagnosis of mitochondrial disorders applying massive pyrosequencing

Mitochondrial disorders are a frequent cause of neurological disability affecting children and adults. Traditionally, molecular diagnosis of mitochondrial diseases was mostly accomplished by the use of Sanger sequencing and PCR-RFLP. However, there are particular drawbacks associated with the use of these methods. Recent multidisciplinary advances have led to new sequencing methods that may overcome these limitations. Our goal was to explore the use of a next generation sequencing platform in the molecular diagnosis of mitochondrial diseases reporting our findings in adult patients that present with a clinical-pathological diagnosis of a mitochondrial encephalomyopathy. Complete genomic sequences of mitochondrial DNA were obtained by 454 massive pyrosequencing from blood samples. The analysis of these sequences allowed us to identify two diagnostic pathogenic mutations and 74 homoplasmic polymorphisms, useful for obtaining high-resolution mitochondrial haplogroups. In summary, molecular diagnosis of mitochondrial disorders could be efficiently done from readily accessible samples, such as blood, with the use of a new sequencing platform.

Plasma thiol status is altered in children with mitochondrial diseases

Objective. This study was undertaken to investigate thiol metabolism as a marker of oxidative stress and antioxidative defence capacity in a cohort of children with biochemically and/or genetically confirmed mitochondrial disease. Previous studies suggest that lower glutathione levels, which have been shown to further compromise mitochondrial function, may occur in these diseases. Better understanding of the pathogenesis of mitochondrial diseases is important in order to improve their treatment. Methods. We studied plasma and erythrocyte glutathione and cysteine levels, the activities of erythrocyte glutathione peroxidase (GPx), glutathione reductase (GR), glucose 6-phosphate dehydrogenase G6PDH) and glutathione S-transferase (GST), as well as the levels of erythrocyte thiobarbituric acid-reactive species (TBA-RS) and protein carbonyls in 10 children with a biochemical and/or genetic diagnosis of mitochondrial disease and six controls. Results. Levels of reduced cysteine (CYSH) as well as reduced to oxidised cysteine ratio were lower in plasma of patients with mitochondrial diseases (p = 0.008 and p = 0.02, respectively). Plasma levels of reduced glutathione (GSH) were low in patients with mitochondrial diseases, mostly below the detection limit. We did not detect significant differences in erythrocyte thiols or glutathione-related enzyme activities. Conclusion. Plasma thiols and their redox state are altered in patients with mitochondrial diseases, suggesting an increase in oxidative stress and depletion of antioxidant supplies. If confirmed in further studies, this relative thiol deficiency could be an important factor in the pathophysiology of mitochondrial diseases.

Non-invasive evaluation of buccal respiratory chain enzyme dysfunction in mitochondrial disease: Comparison with studies in muscle biopsy

Making a diagnosis of mitochondrial disease (MD) is extremely challenging and often employs the analysis of respiratory complex (RC) activities in biopsied skeletal muscle. Given both the invasive nature and expense of biopsied-muscle based testing for mitochondrial defects, buccal swab enzyme analysis has been explored as an alternative approach to the more invasive muscle biopsy. Case studies have recently suggested that buccal swabs from patients can be used to accurately assess mitochondrial enzyme activities including RC I and RC IV using a dipstick methodology combined with spectrophotometric analysis. In this study, forty patients with suspected MD who have previously been found to have significant defects in either RC I or RC IV in skeletal muscle were assessed by buccal swab analysis and compared to enzyme values obtained with unaffected controls (n=106) in the same age range. Buccal citrate synthase was used as an indicator of overall mitochondrial content, correlating well with overall buccal mitochondrial frataxin levels and was found to be elevated above control levels in 28% of the patients in this cohort. Of 26 cases with significant muscle RC I deficiency, 20 displayed significantly reduced levels of buccal RC I activity. All 7 of the patients with muscle RC IV deficiency showed significant buccal RC IV defect and 6 of the 7 patients with combined defects in muscle RC I and IV activity levels also exhibited analogous deficiencies in both buccal RC I and RC IV activities. In conclusion, the relatively high correlation (over 82%) of buccal and muscle RC deficiencies further supports the validity of this non-invasive approach as a potentially useful tool in the diagnosis of MD.