Bulk autophagy, but not mitophagy, is increased in cellular model of mitochondrial disease

Oxidative phosphorylation system (OXPHOS) deficiencies are rare diseases but constitute the most frequent inborn errors of metabolism. We analyzed the autophagy route in 11 skin fibroblast cultures derived from patients with well characterized and distinct OXPHOS defects. Mitochondrial membrane potential determination revealed a tendency to decrease in 5 patients' cells but reached statistical significance only in 2 of them. The remaining cells showed either no change or a slight increase in this parameter. Colocalization analysis of mitochondria and autophagosomes failed to show evidence of increased selective elimination of mitochondria but revealed more intense autophagosome staining in patients' fibroblasts compared with controls. Despite the absence of increased mitophagy, Parkin recruitment to mitochondria was detected in both controls' and patients' cells and was slightly higher in cells harboring complex I defects. Western blot analysis of the autophagosome marker LC3B, confirmed significantly higher levels of the protein bound to autophagosomes, LC3B-II, in patients' cells, suggesting an increased bulk autophagy in OXPHOS defective fibroblasts. Inhibition of lysosomal proteases caused significant accumulation of LC3B-II in control cells, whereas in patients' cells this phenomenon was less pronounced. Electron microscopy studies showed higher content of late autophagic vacuoles and lysosomes in OXPHOS defective cells, accompanied by higher levels of the lysosomal marker LAMP-1. Our findings suggest that in OXPHOS deficient fibroblasts autophagic flux could be partially hampered leading to an accumulation of autophagic vacuoles and lysosomesThis work was supported by grants PS09/01359, PI12/ 01683, PI11/00182 and CP11/00151 from Instituto de Salud Carlos III- Ministerio de Industria y Competitividad de España (ISCIIIMINECO, Spain), S2010/BMD-2361 and S2010/BMD-2402 from Comunidad de Madrid (CAM, Spain). AD is recipient of a research contract from ISCIII-MINECO (PI12/01683). MM is supported by a research contract ‘Miguel Servet’ ISCIII-MINECO (CP11/00151). AB is supported by a research contract (CIBERER-Spain

iTRAQ-Based Analysis of Progerin Expression Reveals Mitochondrial Dysfunction, Reactive Oxygen Species Accumulation and Altered Proteostasis

[Abstract] Introduction. Nuclear accumulation of a mutant form of the nuclear protein Lamin-A, called Progerin (PG) or Lamin AΔ50, occurs in Hutchinson-Gilford Progeria Syndrome (HGPS) or Progeria, an accelerated aging disease. One of the main symptoms of this genetic disorder is a loss of sub-cutaneous fat due to a dramatic lipodystrophy. Methods. We stably induced the expression of human PG and GFP -Green Fluorescent Protein- as control in 3T3L1 cells using a lentiviral system to study the effect of PG expression in the differentiation capacity of this cell line, one of the most used adipogenic models. Quantitative proteomics (iTRAQ) was done to study the effect of the PG accumulation. Several of the modulated proteins were validated by immunoblotting and real-time PCR. Mitochondrial function was analyzed by measurement of a) the mitochondrial basal activity, b) the superoxide anion production and c) the individual efficiency of the different complex of the respiratory chain. Results. We found that over-expression PG by lentiviral gene delivery leads to a decrease in the proliferation rate and to defects in adipogenic capacity when compared to the control. Quantitative proteomics analysis showed 181 proteins significantly (p < 0.05) modulated in PG-expressing preadipocytes. Mitochondrial function is impaired in PG-expressing cells. Specifically, we have detected an increase in the activity of the complex I and an overproduction of Superoxide anion. Incubation with Reactive Oxygen Species (ROS) scavenger agents drives to a decrease in autophagic proteolysis as revealed by LC3-II/LC3-I ratio. Conclusion. PG expression in 3T3L1 cells promotes changes in several Biological Processes, including structure of cytoskeleton, lipid metabolism, calcium regulation, translation, protein folding and energy generation by the mitochondria. Our data strengthen the contribution of ROS accumulation to the premature aging phenotype and establish a link between mitochondrial dysfunction and loss of proteostasis in HGPS.Xunta de Galicia; PS07/86Instituto de Salud Carlos III; PI11/0279

The homozygous R504C mutation in MTO1 gene is responsible for ONCE syndrome

We report clinical and biochemical finding from three unrelated patients presenting ONCE (Optic Neuropathy, Cardiomyopathy and Encephalopathy with lactic acidosis and combined oxidative phosphorylation deficiency) syndrome. Whole-exome sequencing (WES) of the three patients and the healthy sister of one of them was used to identify the carry gene. Clinical and biochemical findings were used to filter variants, and molecular, in silico and genetic studies were performed to characterize the candidate variants. Mitochondrial DNA (mtDNA) defects involving mutations, deletions or depletion were discarded, whereas WES uncovered a double homozygous mutation in the MTO1 gene (NM_001123226:c.1510C>T, p.R504C, and c.1669G>A, p.V557M) in two of the patients and the homozygous mutation p.R504C in the other. Therefore, our data confirm p.R504C as pathogenic mutation responsible of ONCE syndrome, and p.V557M as a rare polymorphic variant.post-print712 K

Multicentric Standardization of Protocols for the Diagnosis of Human Mitochondrial Respiratory Chain Defects

The study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Institutional Review Board of each institution: IBC U737 (C0000128), HCL U722 (HCB2017/0808), 12O U723 (CEI:18/487), VH U701 (PR(IR)63/2016) and UPO U729 (C.I. 2768-N-21)The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseasesThis work was supported by Instituto de Salud Carlos III (ISCIII), grants FIS PI17/00021, PI17/00359, PI18/00498, PI18/00451, PI18/01374, PI19/01772, PI20/00541, PI21/00229, PI21/00381 and PI21/00935 (ISCIII-FEDER “Cofinanciado por la Unión Europea”), Fundació Privada Cellex, Junta de Andalucía (UPO-1262247) and Ministerio de Ciencia e Innovación (MCINN) grant PID2019-110320RBI00. All participant centers are integrated in the Centro de Investigación Biomédica en Red (CIBER), Sección de Enfermedades Raras (CIBERER, an initiative of ISCIII), which is the founder of this present methodological stud

Multicentric Standardization of protocols for the diagnosis of human mitochondrial respiratory chain defects

The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases

Health Benefits of an Innovative Exercise Program for Mitochondrial Disorders

Purpose We determined the effects of an innovative 8-wk exercise intervention (aerobic, resistance, and inspiratory muscle training) for patients with mitochondrial disease. Methods Several end points were assessed in 12 patients (19–59 yr, 4 women) at pretraining, posttraining, and after 4-wk detraining: aerobic power, muscle strength/power and maximal inspiratory pressure (main end points), ability to perform activities of daily living, body composition, quality of life, and blood myokines (secondary end points). Results The program was safe, with patients’ adherence being 94% ± 5%. A significant time effect was found for virtually all main end points (P ≤ 0.004), indicating a training improvement. Similar findings (P ≤ 0.003) were found for activities of daily living tests, total/trunk/leg lean mass, total fat mass, femoral fracture risk, and general health perception. No differences were found for blood myokines, except for an acute exertional increase in interleukin 8 at posttraining/detraining (P = 0.002) and in fatty acid binding protein 3 at detraining (P = 0.002). Conclusions An intervention including novel exercises for mitochondrial disease patients (e.g., inspiratory muscle training) produced benefits in numerous indicators of physical capacity and induced a previously unreported shift toward a healthier body composition phenotype

A crowdsourcing database for the copy-number variation of the Spanish population

Background: Despite being a very common type of genetic variation, the distribution of copy-number variations (CNVs) in the population is still poorly understood. The knowledge of the genetic variability, especially at the level of the local population, is a critical factor for distinguishing pathogenic from non-pathogenic variation in the discovery of new disease variants. Results: Here, we present the SPAnish Copy Number Alterations Collaborative Server (SPACNACS), which currently contains copy number variation profiles obtained from more than 400 genomes and exomes of unrelated Spanish individuals. By means of a collaborative crowdsourcing effort whole genome and whole exome sequencing data, produced by local genomic projects and for other purposes, is continuously collected. Once checked both, the Spanish ancestry and the lack of kinship with other individuals in the SPACNACS, the CNVs are inferred for these sequences and they are used to populate the database. A web interface allows querying the database with different filters that include ICD10 upper categories. This allows discarding samples from the disease under study and obtaining pseudo-control CNV profiles from the local population. We also show here additional studies on the local impact of CNVs in some phenotypes and on pharmacogenomic variants. SPACNACS can be accessed at: http://csvs.clinbioinfosspa.es/spacnacs/. Conclusion: SPACNACS facilitates disease gene discovery by providing detailed information of the local variability of the population and exemplifies how to reuse genomic data produced for other purposes to build a local reference database

Identificación molecular de enfermedades OXPHOS: aproximaciones diagnósticas y bioinformáticas mediante secuenciación masiva

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Bioquímica. Fecha de lectura: 03-03-2020Las enfermedades del sistema de fosforilación oxidativa mitocondrial (EM‐OXPHOS) se pueden originar por mutaciones en genes codificados en dos genomas: el DNA mitocondrial (mtDNA) o el DNA nuclear (nDNA). Esto obliga a establecer estrategias de análisis que tengan en consideración las particularidades propias de cada genoma. Por lo general, estas enfermedades cursan con afectación multisistémica y presentan una amplia variabilidad clínica. Identificar la causa genética resulta fundamental para: a) evitar el peregrinaje de los pacientes entre distintos especialistas y los diferentes niveles del sistema sanitario, con el consiguiente retraso en el diagnóstico de certeza; b) clasificar adecuadamente la EM‐OXPHOS concreta, debido a que cada vez se conocen más cuadros clínicos con afectación de diferentes órganos y tejidos asociados a diferentes genes y mutaciones; y c) contribuir a identificar los posibles tratamientos aplicables específicamente a pacientes con una determinada causa genética. El objetivo de esta tesis doctoral es el desarrollo, mejora y aplicación de metódicas genético‐moleculares y bioinformáticas con el propósito de su traslación al ámbito del diagnóstico genético de las EM‐OXPHOS. Se han desarrollado y adaptado estrategias de análisis molecular para analizar el mtDNA en profundidad y detectar desde variaciones simples de un nucleótido hasta grandes deleciones, empleándose tanto métodos genéticos tradicionales, como aproximaciones de secuenciación masiva. En este último caso se ha desarrollado un pipeline completo de análisis de mtDNA a partir de datos de la plataforma PGM (Life Technologies). El estudio del genoma nuclear fue abordado inicialmente con paneles de genes, mediante la integración y optimización en scripts propios de un sistema de filtrado de variantes frecuentes, un informe de cobertura apropiado (que indica cobertura mínima en las regiones de interés) y el programa de anotación Annovar. Posteriormente, se realizó una aproximación mediante secuenciación del Exoma (WES) con la oportuna adaptación del citado sistema de anotación, la inclusión del análisis de mtDNA residual y, finalmente, la detección de variaciones en el número de copias (CNVs). El enfoque fundamentalmente traslacional de la investigación desarrollada tuvo en cuenta el balance coste/beneficio y su potencial viabilidad dentro del Sistema Sanitario. En total, y gracias a las estrategias de análisis empleadas, en el periodo de tiempo estudiado (5 años) se ha logrado el diagnóstico genético de 147 pacientes y se han encontrado 52 variantes distintas con significado incierto. Finalmente, se propone un diagrama de flujo que aborda el diagnóstico de estas patologías mediante la integración de las diferentes metodologías y tipos de muestras empleadas en la actualidad con el objetivo de orientar el diagnóstico hacia un uso racional de los recursos de acuerdo con la información fenotípica inicialMitochondrial disorders due to defects of oxidative phosphorylation system (OXPHOS disorders) are a group of rare diseases that can be produced by mutations in genes encoded by two genomes: mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). This fact makes necessary to establish analysis strategies which take into account the specific characteristics of each genome. Most of these diseases show multisystemic phenotypes with a wide clinical variability. Reaching an accurate genetic characterization is crucial: i) to avoid the diagnostic odyssey and consequently the delay in diagnosis, ii) to classify properly the mitochondrial disorder, as there are more and more clinical pictures with involvement of different organs and tissues that are associated with distinct genes and mutations, and iii) to contribute to identify possible treatments which can be particularly tailored for patients with a specific genetic cause. The purpose of this doctoral thesis is the development, improvement, and application of molecular genetic methods and bioinformatics approaches with the aim of being transferred to the area of clinical genetic diagnosis of OXPHOS disorders. In this work, strategies of molecular analysis were developed and adapted to analyze mtDNA in depth and to detect from single nucleotide variations to large deletions. During the process, traditional methods and massively parallel sequencing (MPS) technologies were applied. A complete pipeline for mtDNA analysis by MPS was developed using data from the PGM platform (Life Technologies). The study of the nuclear genome was approached initially by gene panels, through the integration and optimization of own scripts to establish a system for filtering frequent variants, to generate an appropriate coverage report (that indicates minimum coverage in the regions of interest), and the Annovar annotation system. Then, exome sequencing (WES) studies were carried out with the adaptation of the previously developed annotation system, including the analysis of residual mtDNA and a pipeline for the detection of copy number variants (CNVs). This work presents a translational focus, bearing in mind the cost‐benefit analysis and its viability within a Health System. Thus, the application of the developed tools for analysis, in the period of time studied (5 years), led to the genetic characterization of 147 patients, and 52 different variants of uncertain significance. In conclusion, a diagnostic flow chart for OXPHOS disorders is proposed based on initial phenotypic information and the integration of different methodologies and types of biological samples, with the aim of guiding the diagnosis under a rational use of resource

Noninvasive Prenatal Testing: Comparison of Two Mappers and Influence in the Diagnostic Yield

Objective. The aim of this study was to determine if the use of different mappers for NIPT may vary the results considerably. Methods. Peripheral blood was collected from 217 pregnant women, 58 pathological (34 pregnancies with trisomy 21, 18 with trisomy 18, and 6 with trisomy 13) and 159 euploid. MPS was performed following a manufacturer’s modified protocol of semiconductor sequencing. Obtained reads were mapped with two different software programs: TMAP and HPG-Aligner, comparing the results. Results. Using TMAP, 57 pathological samples were correctly detected (sensitivity 98.28%, specificity 93.08%): 33 samples as trisomy 21 (sensitivity 97.06%, specificity 99.45%), 16 as trisomy 18 (sensibility 88.89%, specificity 93.97%), and 6 as trisomy 13 (sensibility 100%, specificity 100%). 11 false positives, 1 false negative, and 2 samples incorrectly identified were obtained. Using HPG-Aligner, all the 58 pathological samples were correctly identified (sensibility 100%, specificity 96.86%): 34 as trisomy 21 (sensibility 100%, specificity 98.91%), 18 as trisomy 18 (sensibility 100%, specificity 98.99%), and 6 as trisomy 13 (sensibility 100%, specificity 99.53%). 5 false positives were obtained. Conclusion. Different mappers use slightly different algorithms, so the use of one mapper or another with the same batch file can provide different results