The alternative enzymes-bearing tunicates lack multiple widely distributed genes coding for peripheral OXPHOS subunits.

The respiratory chain alternative enzymes (AEs) NDX and AOX from the tunicate Ciona intestinalis (Ascidiacea) have been xenotopically expressed and characterized in human cells in culture and in the model organisms Drosophila melanogaster and mouse, with the purpose of developing bypass therapies to combat mitochondrial diseases in human patients with defective complexes I and III/IV, respectively. The fact that the genes coding for NDX and AOX have been lost from genomes of evolutionarily successful animal groups, such as vertebrates and insects, led us to investigate if the composition of the respiratory chain of Ciona and other tunicates differs significantly from that of humans and Drosophila, to accommodate the natural presence of AEs. We have failed to identify in tunicate genomes fifteen orthologous genes that code for subunits of the respiratory chain complexes; all of these putatively missing subunits are peripheral to complexes I, III and IV in mammals, and many are important for complex-complex interaction in supercomplexes (SCs), such as NDUFA11, UQCR11 and COX7A. Modeling of all respiratory chain subunit polypeptides of Ciona indicates significant structural divergence that is consistent with the lack of these fifteen clear orthologous subunits. We also provide evidence using Ciona AOX expressed in Drosophila that this AE cannot access the coenzyme Q pool reduced by complex I, but it is readily available to oxidize coenzyme Q molecules reduced by glycerophosphate oxidase, a mitochondrial inner membrane-bound dehydrogenase that is not involved in SCs. Altogether, our results suggest that Ciona AEs might have evolved in a mitochondrial inner membrane environment much different from that of mammals and insects, possibly without SCs; this correlates with the preferential functional interaction between these AEs and non-SC dehydrogenases in heterologous mammalian and insect systems. We discuss the implications of these findings for the applicability of Ciona AEs in human bypass therapies and for our understanding of the evolution of animal respiratory chain.

A case of mitochondrial DNA depletion syndrome type 11 - expanding the genotype and phenotype.

Mitochondrial DNA depletion syndrome type 11 (MTDPS11) is caused by pathogenic variants in MGME1 gene. We report a woman, 40-year-old, who presented slow progressive drop eyelid at 11-year-old with, learning difficulty and frequent falls. Phisical examination revealed: mild scoliosis, elbow hyperextensibility, flat feet, chronic progressive external ophthalmoplegia with upper eyelid ptosis, diffuse hypotonia, and weakness of arm abduction and neck flexion. Investigation evidenced mild serum creatine kinase increase and glucose intolerance; second-degree atrioventricular block; mild mixed-type respiratory disorder and atrophy and granular appearance of the retinal pigment epithelium. Brain magnetic resonance showed cerebellar atrophy. Muscle biopsy was compatible with mitochondrial myopathy. Genetic panel revealed a homozygous pathogenic variant in the MGME1 gene, consistent with MTDPS11 (c.862C>T; p.Gln288*). This case of MTDPS11 can contribute to the phenotypic characterization of this ultra-rare mitochondrial disorder, presenting milder respiratory and nutritional involvement than the previously reported cases, with possible additional features.

Leigh syndrome in an infant: autopsy and histopathology findings.

Leigh syndrome is an inherited neurodegenerative disorder of infancy that typically manifests between 3 and 12 months of age. The common neurological manifestations are developmental delay or regression, progressive cognitive decline, dystonia, ataxia, brainstem dysfunction, epileptic seizures, and respiratory dysfunction. Although the disorder is clinically and genetically heterogeneous, the histopathological and radiological features characteristically show focal and bilaterally symmetrical, necrotic lesions in the basal ganglia and brainstem. The syndrome has a characteristic histopathological signature that helps in clinching the diagnosis. We discuss these unique findings on autopsy and radiology in a young infant who succumbed to a subacute, progressive neurological illness suggestive of Leigh syndrome. Our case highlights that Leigh syndrome should be considered in the differential diagnosis of infantile-onset, subacute neuroregression with dystonia and seizures, a high anion gap metabolic acidosis, normal ketones, elevated lactates in blood, brain, and urine, and bilateral basal ganglia involvement.

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.

Clinical and molecular characterization of mitochondrial DNA disorders in a group of Argentinian pediatric patients.

OBJECTIVE: To describe the clinical and molecular features of a group of Argentinian pediatric patients with mitochondrial DNA (mtDNA) disorders, and to evaluate the results of the implementation of a classical approach for the molecular diagnosis of mitochondrial diseases. METHODS: Clinical data from 27 patients with confirmed mtDNA pathogenic variants were obtained from a database of 89 patients with suspected mitochondrial disease, registered from 2014 to 2020. Clinical data, biochemical analysis, neuroimaging findings, muscle biopsy and molecular studies were analyzed. RESULTS: Patients were 18 females and 9 males, with ages at onset ranging from 1 week to 14 years (median = 4 years). The clinical phenotypes were: mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome (n = 11), Leigh syndrome (n = 5), Kearns-Sayre syndrome (n = 3), Chronic Progressive External Ophthalmoplegia (n = 2), Leber hereditary optic neuropathy (n = 2), myoclonic epilepsy associated with ragged-red fibers (n = 1) and reversible infantile myopathy with cytochrome-C oxidase deficiency (n = 3). Most of the patients harbored pathogenic single nucleotide variants, mainly involving mt-tRNA genes, such as MT-TL1, MT-TE and MT-TK. Other point variants were found in complex I subunits, like MT-ND6, MT-ND4, MT-ND5; or in MT-ATP6. The m.13513G > A variant in MT-ND5 and the m.9185 T > C variant in MT-ATP6 were apparently de novo. The rest of the patients presented large scale-rearrangements, either the "common" deletion or a larger deletion. CONCLUSIONS: This study highlights the clinical and genetic heterogeneity of pediatric mtDNA disorders. All the cases presented with classical phenotypes, being MELAS the most frequent. Applying classical molecular methods, it was possible to achieve a genetic diagnosis in 30% of the cases, suggesting that this is an effective first approach, especially for those centers from low-middle income countries, leaving NGS studies for those patients with inconclusive results.

Miopatia mitocondrial e deficiência auditiva neurossensorial: estudo de caso / Mitochondrial myopathy and sensorineural hearing loss: case study

Resumo A miopatia mitocondrial é causada pela ausência e/ou insuficiência de uma enzina quaternária, L-carnitina, responsável por transportar ácidos graxos livres para a parte interna da mitocôndria. A função da mitocôndria é produzir energia, contribuindo para o bom funcionamento das células. A Lipidose Muscular é uma doença que provoca anomalias em enzimas que metabolizam gordura e por consequência causa acúmulo de toxinas de subprodutos com gordura nos tecidos. O objetivo deste trabalho é apresentar o estudo de caso da paciente B.D., 37 anos, diagnosticada com Lipidose Muscular aos seis anos, com deficiência de L-Carnitina e relatar o acompanhamento fonoaudiológico realizado em um serviço de saúde auditiva. A abertura de prontuário da paciente foi realizada em 05/03/1989. Foi prescrito pelo neurologista o uso contínuo de 2g/dia de L-carnitina. A mãe relatou que B.D. apresentava dificuldades em ouvir, pois era muito desatenta, o que foi mais evidente quando começou a frequentar a escola. Em 1988, a paciente foi diagnosticada com perda auditiva neurossensorial de grau moderado bilateral e começou a fazer uso de aparelhos de amplificação sonora individual retroauriculares em 1989. O desempenho escolar e comunicação melhoraram. Em 1998, passou a utilizar aparelhos tipo micro canal, o que a favoreceu esteticamente, promoveu melhora da localização sonora e maior ganho em altas frequências. Os limiares de audibilidade apresentaram uma leve piora e a paciente atualmente é pós-graduada e trabalha em uma grande instituição financeira. Conclui-se que o diagnostico neurológico e a intervenção fonoaudiológica precoces possibilitaram o adequado desenvolvimento de linguagem da paciente.

Abstract Mitochondrial myopathy is caused by the absence and/or insufficiency of L-carnitine, a quaternary enzyme responsible for transporting free fatty acids into the mitochondria. The primary function of the mitochondria is to produce energy, contributing to proper cell functioning. Muscular lipidosis causes abnormalities in enzymes that metabolize fat, resulting in the accumulation of harmful amounts of fats in tissues. The aim of this study was to present the case study of patient B.D., a 37-year-old woman diagnosed with muscular lipidosis with L-carnitine deficiency at 6 years old, and describe the speech-language follow-up performed at a hearing care clinic. The first entry in the patient's medical chart was on 03/05/1989, with continuous use of 2g/day of L-carnitine prescribed by a neurologist. The mother reported that B.D. had difficulty hearing and was inattentive, which became more evident when she started school. In 1988 the patient was diagnosed with moderate bilateral sensorineural hearing loss and began using behind-the-ear (BTE) hearing aids in 1989, after which her academic performance and communication improved. In 1998 she switched to Completely in Canal (CIC) hearing aids, which are more discreet, provided better sound localization and greater high frequency gain, although her hearing thresholds worsened slightly. She completed her graduate studies and currently works at a large financial institution. It was concluded that early neurological diagnosis and speech-language intervention enabled adequate language development in the patient.

Leigh syndrome in an infant: autopsy and histopathology findings

Leigh syndrome is an inherited neurodegenerative disorder of infancy that typically manifests between 3 and 12 months of age. The common neurological manifestations are developmental delay or regression, progressive cognitive decline, dystonia, ataxia, brainstem dysfunction, epileptic seizures, and respiratory dysfunction. Although the disorder is clinically and genetically heterogeneous, the histopathological and radiological features characteristically show focal and bilaterally symmetrical, necrotic lesions in the basal ganglia and brainstem. The syndrome has a characteristic histopathological signature that helps in clinching the diagnosis. We discuss these unique findings on autopsy and radiology in a young infant who succumbed to a subacute, progressive neurological illness suggestive of Leigh syndrome. Our case highlights that Leigh syndrome should be considered in the differential diagnosis of infantile-onset, subacute neuroregression with dystonia and seizures, a high anion gap metabolic acidosis, normal ketones, elevated lactates in blood, brain, and urine, and bilateral basal ganglia involvement.

Molecular Diagnosis in an Argentinean Mitochondrial Disorders Cohort

Abstract Mitochondrial diseases are multisystemic disorders characterized by an impairment of the mitochondrial respiratory chain. Diagnosis requires an approach that involves a high index of suspicion, molecular techniques and a careful selection of the tissue to be studied. Our goal was to develop and implement local strategies for diagnosing mitochondrial disorders, by standardizing procedures of molecular biology and nucleic acid sequencing. A prospective, analytical, observational study was conducted in a cohort of, a total of 82 patients with suspected mitochondrial disorder who were treated at our hospital between May 2008 and June 2019. We developed molecular diagnostic tools that included classical monogenic techniques and Next Generation Sequencing. We characterized the neurological and extra neurological manifestations noted in our cohort. Following the proposed algorithm, we obtained a molecular diagnostic performance of 54%, identifying mutations in 44 patients. mtDNA mutations were identified in 34 patients. Structural rearrangements in mitochondrial genome were found in 3 and 7 in nuclear genes, respectively. Our results confirm the utility of the proposed algorithm and the molecular tools used, as evidenced by a high diagnostic performance. This is of great value to a more efficient and comprehensive medical care of patients and families affected by mitochondrial disorders.

Anticuerpos antimitocondriales y anticuerpos contra antígenos extraíbles del núcleo en pacientes con cirrosis biliar primaria / Antimitochondrial Antibodies and antibodies against extractable nucleus antigens in patients with primary biliary cirrhosis

Resumen Objetivo: Determinar la frecuencia de anticuerpos antimitocondriales y de anticuerpos contra antígenos extraíbles del núcleo en pacientes con cirrosis biliar primaria. Material y métodos: Estudio de tipo cuantitativo, observacional y transversal, realizado en el Servicio de Inmunología del Hospital Nacional Arzobispo Loayza entre enero 2018 y marzo 2019. Se revisaron las historias clínicas de 30 pacientes con características presuntivas de cirrosis biliar primaria; para la detección de los anticuerpos antinucleares y anticuerpos antimitocondriales se empleó el kit inmunológico en sangre y observación con microscopio de inmunofluorescencia a 40X y para la detección de los anticuerpos contra antígenos extraíbles del núcleo se empleó el método Immunoblot. Resultados: Se estudiaron 30 pacientes con cirrosis biliar primaria, 20 fueron de sexo femenino (66,7%). El patrón de tinción más frecuente fue el citoplasmático moteado reticular en 17(56,7%), seguido del patrón citoplasmático moteado reticular y patrón moteado en 7(23,3%) pacientes, y en menor frecuencia el patrón citoplasmático moteado reticular y patrón centromérico. Nueve (42,9%) pacientes con cirrosis biliar primaria tenían anti-M2. Se demostró mayor frecuencia, 21(70%) de los pacientes con cirrosis biliar primaria tenían anticuerpos antimitocondriales. Conclusiones: Se encontró alta frecuencia de patrón citoplasmático moteado reticular en pacientes con cirrosis biliar primaria, se demostró asociación significativa con los anti-M2 y anticuerpos antimitocondriales.

Summary Objective: To determine the frequency of antimitochondrial antibodies and antibodies against extractable nucleus antigens in patients with primary biliary cirrhosis. Methods : A quantitative, observational and cross-sectional study was carried out at the Immunology Service of the Arzobispo Loayza National Hospital between January 2018 and March 2019. The medical records of 30 patients with presumptive characteristics of primary biliary cirrhosis were reviewed; for the detection of the antinuclear antibodies and antimitochondrial antibodies, the immunological kit was used in blood and observation with a 40X immunofluorescence microscope, and the Immunoblot method was used for the detection of the antibodies against extractable nucleus antigens. Results: Thirty patients with primary biliary cirrhosis disease were studied, 20 were female (66.7%). The most frequent staining pattern was the reticular mottled cytoplasmic in 17 (56.7%), followed by the reticular mottled cytoplasmic pattern and mottled pattern in 7 (23.3%) patients, and less frequently the reticular mottled cytoplasmic pattern and centromeric. Nine (42.9%) patients with primary biliary cirrhosis had anti-M2. In the present investigation, a higher frequency was demonstrated, 21 (70%) of the patients with primary biliary cirrhosis had antimitochondrial antibodies. Conclusions: A high frequency of reticular mottled cytoplasmic pattern was found in patients with primary biliary cirrhosis; a significant association with anti-M2 and antimitochondrial antibodies was demonstrated.

Neumopatía crónica secundaria al trastorno de la deglución en un paciente con miopatía mitocondrial / Chronic pneumopathy secondary to swallowing disorder in a patient with mitochondrial myopathy

RESUMEN Introducción: la enfermedad pulmonar crónica secundaria a la disfagia es una complicación frecuente en los pacientes con enfermedades neuromusculares. Las miopatías mitocondriales son un conjunto de enfermedades que pueden conducir a daño pulmonar progresivo, secundario al síndrome aspirativo crónico. Caso clínico: niño de 7 años con signos clínicos y radiológicos de enfermedad pulmonar crónica; además, con desnutrición crónica, debilidad muscular, disfonía y oculoparesia externa crónica multiplanar. Su padre tuvo síntomas similares desde la infancia y requirió alimentación con dieta espesa por trastorno de la deglución. Se confirma en el paciente la presencia de disfagia como la causa de la neumopatía crónica y se sospecha miopatía congénita hereditaria. En consecuencia, se realiza el diagnóstico de enfermedad mitocondrial con oculoparesia externa crónica, mediante la secuenciación del gen polimerasa gamma del ADN mitocondrial (POLG). Conclusiones: en los pacientes con neumopatía crónica se deben considerar las enfermedades neuromusculares en el diagnóstico diferencial. La miopatía mitocondrial con oculoparesia externa crónica progresiva, se asocia con trastorno de la deglución hasta en un 50 % de los casos. El diagnóstico temprano es importante para retardar el deterioro de la función pulmonar.

SUMMARY Introduction: Chronic lung disease secondary to dysphagia is a frequent complication in patients with neuromuscular diseases. Mitochondrial myopathies could lead to progressive lung damage due to chronic aspiration syndrome. Clinical case: Seven-year-old male with clinical and radiological signs of chronic lung disease, as well as low weight, weakness, dysphonia and multiplanar external oculoparesis. His father had similar symptoms during infancy and needed thickened liquid diet due to swallowing disorder. Dysphagia was confirmed as the cause of chronic lung disease and, therefore, hereditary congenital myopathy was suspected. Mitochondrial disease with chronic external oculoparesis was confirmed by molecular sequencing of the mitochondrial DNA gamma polymerase gene (POLG). Conclusion: Neuromuscular disorders may cause chronic lung disease. Mitochondrial myopathy with progressive chronic external oculoparesis is associated with swallowing disorder in 50 % of the cases. Early diagnosis is important to slow decline in lung function.

Infección asociada a catéter por Leclercia adecarboxylata y Raoultella ornithinolytica en un paciente con enfermedad mitocondrial / Leclercia adecarboxylata and Raoultella ornithinolytica catheter-related infection in a child with mitocondrial disease

Leclercia adecarboxylata y Raoultella ornithinolytica constituyen bacterias Gram-negativas emergentes. Los casos descritos son excepcionales. En los últimos años, las mejoras en las técnicas de diagnóstico microbiológico han permitido su detección y conocimiento. Se presenta el caso de un niño de 11 años con enfermedad mitocondrial, portador de catéter venoso central de larga duración, que desarrolló dos episodios de sepsis por L. adecarboxylata y R. ornithinolytica, respectivamente. En los casos de infección asociada al uso de catéter, es posible, en ocasiones, el tratamiento sin su retirada con evolución favorable. Es importante reconocer L. adecarboxylata y R. ornithinolytica como patógenos de diagnóstico cada vez más frecuentes, sobre todo, en pacientes inmunodeprimidos o con patologías crónicas asociadas.

Leclercia adecarboxylata and Raoultella ornithinolytica are emergent Gram-negative bacteria. Infections caused by these microorganisms are exceptional. Improvement of microbiologist techniques in the last years has enabled their detection and more accurate knowledge. We present the case of an 11-year-old boy with mitochondrial disease with a longterm central catheter who suffered from two sepsis caused by L. adecarboxylata and R. ornithinolytica, respectively. In catheter-related infections, sometimes it is possible to provide antimicrobial treatment without removal of catheter with good results, as in our patient. It is important to recognize L. adecarboxylata and R. ornithinolytica like increasingly frequent pathogenic bacteria, mostly in immunocompromised or chronic patients.

[Leclercia adecarboxylata and Raoultella ornithinolytica catheter-related infection in a child with mitocondrial disease]. / Infección asociada a catéter porLeclercia adecarboxylata yRaoultella ornithinolytica en un paciente con enfermedad mitocondrial.

Leclercia adecarboxylata and Raoultella ornithinolytica are emergent Gram-negative bacteria. Infections caused by these microorganisms are exceptional. Improvement of microbiologist techniques in the last years has enabled their detection and more accurate knowledge. We present the case of an 11-year-old boy with mitochondrial disease with a longterm central catheter who suffered from two sepsis caused by L. adecarboxylata and R. ornithinolytica, respectively. In catheter-related infections, sometimes it is possible to provide antimicrobial treatment without removal of catheter with good results, as in our patient. It is important to recognize L. adecarboxylata and R. ornithinolytica like increasingly frequent pathogenic bacteria, mostly in immunocompromised or chronic patients.

Leclercia adecarboxylata y Raoultella ornithinolytica constituyen bacterias Gram-negativas emergentes. Los casos descritos son excepcionales. En los últimos años, las mejoras en las técnicas de diagnóstico microbiológico han permitido su detección y conocimiento. Se presenta el caso de un niño de 11 años con enfermedad mitocondrial, portador de catéter venoso central de larga duración, que desarrolló dos episodios de sepsis por L. adecarboxylata y R. ornithinolytica, respectivamente. En los casos de infección asociada al uso de catéter, es posible, en ocasiones, el tratamiento sin su retirada con evolución favorable. Es importante reconocer L. adecarboxylata y R. ornithinolytica como patógenos de diagnóstico cada vez más frecuentes, sobre todo, en pacientes inmunodeprimidos o con patologías crónicas asociadas.

Mitochondrial stress triggers a pro-survival response through epigenetic modifications of nuclear DNA.

Mitochondrial dysfunction represents an important cellular stressor and when intense and persistent cells must unleash an adaptive response to prevent their extinction. Furthermore, mitochondria can induce nuclear transcriptional changes and DNA methylation can modulate cellular responses to stress. We hypothesized that mitochondrial dysfunction could trigger an epigenetically mediated adaptive response through a distinct DNA methylation patterning. We studied cellular stress responses (i.e., apoptosis and autophagy) in mitochondrial dysfunction models. In addition, we explored nuclear DNA methylation in response to this stressor and its relevance in cell survival. Experiments in cultured human myoblasts revealed that intense mitochondrial dysfunction triggered a methylation-dependent pro-survival response. Assays done on mitochondrial disease patient tissues showed increased autophagy and enhanced DNA methylation of tumor suppressor genes and pathways involved in cell survival regulation. In conclusion, mitochondrial dysfunction leads to a "pro-survival" adaptive state that seems to be triggered by the differential methylation of nuclear genes.

Quantitation of muscle pathology abnormalities in 18 patients with mitochondrial disorders / Quantificação das anormalidades patológicas musculares em 18 pacientes com desordens mitocondriais

ABSTRACT INTRODUCTION: Mitochondrial disorders can lead to the accumulation of mitochondria in muscle fibers, as indicated by ragged red (RRF) or ragged blue fibers when stained with modified Gomori trichrome or succinate dehydrogenase (SDH+), respectively, and, absence of activity of cytochrome c oxidase, COX negative fibers (COX-). The combined COX-SDH stain (COMBO+) can reveal even more COX-deficient fibers. OBJECTIVE: To quantify RRFs, SDH+, COX-, and COMBO+ fibers in muscle biopsies with mitochondrial findings. MATERIAL AND METHODS: We retrospectively selected 18 muscle biopsies with mitochondrial abnormalities based on the Walker criteria (percentage of RRFs/COX- fibers, and clinical picture), and/or the Sleigh criteria (percentage of RRFs, SDH+, and COX- fibers). RESULTS: Females represented 83.3%, with a mean age of 38.6 years (5 months-70 years). Patients were diagnosed with chronic progressive external ophthalmoplegia (CPEO, 66.7%), proximal myopathy (22.2%), idiopathic hyperCKemia (11.1%), Kearns-Sayre syndrome (5.6%), mitochondrial encephalomyopathy with ragged red fibers and stroke-like episodes (5.6%), and a dystrophic pattern (5.6%). Some cases of CPEO were combined with proximal myopathy. The quantitative pathologic findings were: RRFs, 3.95% ± 3.17%; SDH+, 7.55% ± 6.1%; COX-, 10.9% ± 7.2%; COMBO+, 14.22% ± 12.79%. We found a slight variation in the diameter of muscle fibers, no necrosis or proliferative connective tissue, few fibers with internal nuclei, and some cases with fiber type grouping. CONCLUSION: Pathologic events, grouped in ascending order of frequency, were RRFs, SDH+ fibers, COX- fibers, and COMBO+ fibers. These data emphasize the importance of the COMBO technique in revealing occult COX deficiency in muscle fibers.

RESUMO INTRODUÇÃO: Desordens mitocondriais são usualmente caracterizadas por: 1. acúmulo de mitocôndria nas fibras musculares que aparecem como fibras vermelhas rasgadas (FVR) ou azuis rasgadas quando coradas, respectivamente, pelo tricrômio modificado de Gomori ou pelo succinato desidrogenase (SDH+); 2. ausência de atividade da citocromo c oxidase (COX), originando fibras COX negativa (COX-). A combinação de colorações COX e SDH pode revelar ainda mais fibras COX deficiente (COMBO+). Objetivos: Quantificar FVR, SDH+, COX- e COMBO+ em biópsias musculares com anormalidades mitocondriais. MATERIAL E MÉTODOS: Foram analisadas retrospectivamente 18 biópsias com anormalidades mitocondriais com base no critério de Walker (percentagem de FVR/ COX- e quadro clínico) e/ou critério de Sleigh (percentagem de FVR, SDH+ e COX-). RESULTADOS: Sexo feminino representou 83, 3% e média de idade 38, 6 anos (5 meses a 70 anos). Oftalmoplegia externa progressiva crônica (OEPC) representou 66, 7%; miopatia proximal, 22, 2%; hiperCKemia idiopática, 11, 1%; síndrome de Kearns-Sayre, 5, 6%; encefalopatia mitocondrial com FVR e episódios semelhantes a acidente vascular cerebral, 5, 6%; e padrão distrófico, 5, 6%. Alguns casos de OEPC estavam associados à miopatia proximal. Achados patológicos quantitativos: FVR, 3, 95% ± 3, 17%; SDH+, 7, 55% ± 6, 1%; COX-, 10, 9% ± 7, 2%; COMBO+, 14, 22% ± 12, 79%. Encontramos leve variação de calibre das fibras musculares sem necrose ou proliferação de tecido conjuntivo, poucas fibras com núcleos internos e alguns casos com agrupamento de fibras. CONCLUSÃO: As anormalidades patológicas nas fibras musculares em ordem ascendente de frequência foram: FVR, SDH+, COX- e COMBO+. Nossos achados enfatizam a importância da técnica COMBO (COX + SDH) para aumento na frequência de fibras musculares COX deficiente ocultas.

Metabolic studies of a patient harbouring a novel S487L mutation in the catalytic subunit of AMPK.

AMP-activated protein kinase (AMPK) regulates many different metabolic pathways in eukaryote cells including mitochondria biogenesis and energy homeostasis. Here we identify a patient with hypotonia, weakness, delayed milestones and neurological impairment since birth harbouring a novel homozygous mutation in the AMPK catalytic α-subunit 1, encoded by the PRKAA1 gene. The homozygous mutation p.S487L in isoform 1 present in the patient is in a cryptic residue for AMPK activity. In the present study, we performed the characterization of mitochondrial respiratory properties of the patient, in comparison to healthy controls, through the culture of skin fibroblasts in order to understand some of the cellular consequences of the PRKAA1 mutation. In these assays, mitochondrial respiratory complex I showed lower activity, which was followed by a decrement in the mtDNA copy number, which is a probable consequence of the lower expression of PGC-1α and PRKAA1 itself as measured in our quantitative PCRs experiments. Confirming the effect of the patient mutation in respiration, transfection of patient fibroblasts with wild type PRKAA1 partially restore complex I level. The preliminary clinic evaluations of the patient suggested a metabolic defect related to the mitochondrial respiratory function, therefore treatment with CoQ10 supplementation dose started four years ago and a clear improvement in motor skills and strength has been achieved with this treatment.

Considerações médicas, éticas e jurídicas sobre decisões de fim de vida em pacientes pediátricos / Medical, ethical and legal considerations regarding end-of-life decisions in pediatric patients / Consideraciones médicas, éticas y jurídicas sobre decisiones de fin de la vida en pacientes pediátricos

Resumo A partir do caso do bebê Charlie Gard, discutem-se aspectos relativos à tomada de decisão médica em pediatria, sobretudo em relação a pacientes portadores de doenças incuráveis e terminais. Foram considerados princípios bioéticos e do cuidado paliativo, além de questões jurídicas relacionadas a autoridade parental e obstinação terapêutica, sob a perspectiva do ordenamento jurídico brasileiro. O processo de tomada de decisões referentes a cuidado de fim de vida em pediatria deve contemplar compartilhamento de responsabilidades entre equipe de saúde e pais, com a participação da criança sempre que possível, buscando o princípio do melhor interesse. Deve-se evitar a judicialização de questões médicas, situação associada a desgaste e sofrimento de todas as partes envolvidas. Conclui-se que a tomada de decisão de final de vida em pediatria deve se pautar na busca do direito a viver com dignidade, mas, sobretudo, de mantê-la até o fim.

Abstract To discuss, in the case of the baby Charlie Gard, aspects to be considered in medical decision making in pediatrics, especially in patients with incurable and terminal diseases. Bioethical principles and Palliative Care were considered, as well as legal issues related to parental authority and therapeutic obstinacy, from the perspective of the Brazilian legal system. Decisions related to end-of-life care in pediatrics should be a process of sharing responsibilities between the health team and parents, with the participation of the child whenever possible, seeking the principle of the best interest. Judicialization of medical issues must be avoided, as it is associated with attrition and suffering for all parties involved. End-of-life decision-making in pediatrics should be based on the search for the right to live with dignity, but, above all, to maintain it until the end of life.

Resumen A partir del caso del bebé Charlie Gard, se discuten aspectos relativos a la toma de decisiones médicas en pediatría, sobre todo en pacientes portadores de enfermedades incurables y terminales. Se consideraron los principios bioéticos y de los cuidados paliativos, además de las cuestiones jurídicas relacionadas con la autoridad parental y la obstinación terapéutica, desde la perspectiva del ordenamiento jurídico brasileño. El proceso de toma de decisiones referidas a los cuidados en el fin de la vida en pediatría debe contemplar responsabilidades compartidas entre el equipo de salud y los padres, con la participación del niño siempre que sea posible, buscando el principio del mejor interés. Se debe evitar la judicialización de cuestiones médicas, situación asociada a desgaste y sufrimiento para todas las partes involucradas. Se concluye que la toma de decisión de final de vida en pediatría debe guiarse por la búsqueda del derecho a vivir con dignidad, pero, sobre todo, de mantenerla hasta el final de la vida.

Evolução da disfagia em um caso de síndrome MELAS: o olhar dos cuidados paliativos / Dysphagia progression in a MELAS syndrome case: a palliative care perspective

RESUMO Este artigo teve por objetivo descrever a progressão da disfagia e a decisão pela via de alimentação em um caso de síndrome MELAS, sob o olhar dos cuidados paliativos. Trata-se de um caso do sexo feminino, que, por volta dos 26 anos de idade sofreu os primeiros sintomas da doença e teve sua função de deglutição progressivamente impactada. Foi realizado acompanhamento fonoaudiológico durante seis meses, com aplicação do protocolo de Avaliação da Segurança da Deglutição, da Functional Oral Intake Scale (FOIS) e gerenciamento da deglutição, com retornos ambulatoriais semanais e mensais. Em seis meses de seguimento, a paciente evoluiu de disfagia moderada a disfagia moderada a grave e variou entre os níveis 5 e 1 da FOIS. Manteve a alimentação por via oral, com restrição de consistências, manobra de deglutições múltiplas e controle de volume para ingestão de líquido, até que, ao final dos seis meses de seguimento, foi realizada gastrostomia. A alimentação por via oral em mais de uma consistência, porém com compensações, foi reduzida a uma alimentação exclusiva por via alternativa, ao longo do acompanhamento fonoaudiológico. Optou-se por manter a via oral de alimentação até a colocação da gastrostomia. A não sugestão de sonda nasoenteral se embasou no respeito à vontade da paciente e na possibilidade de alimentar-se, minimamente, de uma consistência por via oral.

ABSTRACT This article aims to describe a dysphagia progression and a choice of the feeding options in a case of MELAS syndrome, under the perspective of palliative care. It is a case in which a woman at the age of 26 years suffered the first symptoms of the disease and had the swallowing functionality progressively impacted. Speech-Language Therapy follow-up was performed at 6 months with the application of a swallowing safety assessment protocol, Functional Oral Intake Scale (FOIS) and swallowing management, with weekly and monthly outpatient returns. At six months of follow-up, the patient progressed from moderate dysphagia to moderate to severe dysphagia and ranged from levels 5 to 1 of FOIS. The patient maintained oral feeding with consistency restriction, dry swallowing maneuver, and control of volume for liquid intake until the end of the six months of follow-up, when gastrostomy was made. Oral feeding in more than one consistency but with compensations was reduced to exclusive non-oral feeding. We chose to maintain oral feeding until the gastrostomy was placed. Non-suggestion of nasoenteral tube was based on the patient's desire and the possibility of oral feeding in at least one food consistency.

Arginine and citrulline for the treatment of MELAS syndrome.

MELAS (mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes) syndrome is a maternally inherited mitochondrial disease with a broad spectrum of manifestations. In addition to impaired energy production, nitric oxide (NO) deficiency occurs in MELAS syndrome and leads to impaired blood perfusion in microvasculature that can contribute to several complications including stroke-like episodes, myopathy, and lactic acidosis. The supplementation of NO precursors, L-arginine and L-citrulline, increases NO production and hence can potentially have therapeutic utility in MELAS syndrome. L-citrulline raises NO production to a greater extent than L-arginine; therefore, L-citrulline may have a better therapeutic effect. The clinical effect of L-citrulline has not yet been studied and clinical studies on L-arginine, which are limited, only evaluated the stroke-like episodes aspect of the disease. Controlled studies are still needed to assess the clinical effects of L-arginine and L-citrulline on different aspects of MELAS syndrome.

Arginine and Citrulline for the Treatment of MELAS Syndrome

Abstract Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome is a maternally inherited mitochondrial disease with a broad spectrum of manifestations. In addition to impaired energy production, nitric oxide (NO) deficiency occurs in MELAS syndrome and leads to impaired blood perfusion in microvasculature that can contribute to several complications including stroke-like episodes, myopathy, and lactic acidosis. The supplementation of NO precursors, L-arginine and L-citrulline, increases NO production and hence can potentially have therapeutic utility in MELAS syndrome. L-citrulline raises NO production to a greater extent than L-arginine; therefore, L-citrulline may have a better therapeutic effect. The clinical effect of L-citrulline has not yet been studied and clinical studies on L-arginine, which are limited, only evaluated the stroke-like episodes' aspects of the disease. Controlled studies are still needed to assess the clinical effects of L-arginine and L-citrulline on different aspects of MELAS syndrome.