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Resumen: Objetivo: El objetivo de este estudio fue analizar el proceso diagnóstico de los pacientes con fibrosis pulmonar idiopática en España, desde el inicio de los síntomas hasta el diagnóstico y tratamiento antifibrótico, en relación con la publicación de las sucesivas guías de práctica clínica. Material y métodos: Estudio multicéntrico, observacional, ambispectivo, en el que se analizaron los pacientes incluidos en el registro de la fibrosis pulmonar idiopática de la Sociedad Española de Neumología y Cirugía Torácica. Para ello se habilitó un cuaderno electrónico de recogida de datos en la web de la sociedad. Se recogieron variables sociodemográficas y clínicas al diagnóstico y seguimiento de los pacientes. Resultados: Desde enero de 2012 hasta diciembre de 2019 se incluyeron 1.064 pacientes, siendo finalmente analizados 929. El proceso diagnóstico varió en función del año en el que se realizó el diagnóstico y el patrón radiológico observado en la tomografía computarizada de alta resolución. En 244 (26,3%) pacientes, el diagnóstico se realizó con tomografía computarizada de alta resolución de tórax y evaluación clínica. La biopsia quirúrgica se utilizó hasta en el 50,2% de los casos diagnosticados antes del 2011, y en un 14,2% a partir de 2018. La mediana de tiempo que transcurre desde el inicio de los síntomas hasta el diagnóstico es de 360 días (RIC 120-720), siendo mayor de 2 años en el 21,0% de los pacientes. Recibieron tratamiento antifibrótico al 79,4% de los pacientes. El tiempo desde el diagnóstico hasta el inicio del tratamiento fue de 309 ± 596,5 días, con una mediana de 49 (RIC 0-307). Conclusiones: El proceso diagnóstico, incluyendo el tiempo hasta el diagnóstico y el tipo de pruebas utilizadas, ha ido cambiando desde 2011 hasta 2019, probablemente debido el avance en la investigación clínica y la publicación de guías consenso diagnóstico-terapéuticas. Abstract: Objective: The objective of the study was to analyze the diagnostic process and the time until the start of treatment of patients with idiopathic pulmonary fibrosis in relation to the publication of successive clinical practice guide. Material and methods: Multicenter, observational, ambispective study, in which patients includes in the idiopathic pulmonary fibrosis registry of the Spanish Society of Pulmonologist and Thoracic Surgery were analyzed. An electronic data collection notebook was enabled on the society's website. Sociodemographic and clinical variables were collected at diagnosis and follow-up of the patients. Results: From January 2012 to december 2019, 1064 patients were included in the registry, with 929 finally analyzed. The diagnosis process varied depending on the year in which it was performed, and the radiological pattern observed in the high-resolution computed tomography. Up to 26.3% of the cases (244) were diagnosed with chest high-resolution computed tomography and clinical evaluation. Surgical biopsy was used up to 50.2% of cases diagnosed before 2011, while it has been used in 14.2% since 2018. The median time from the onset of symptoms to diagnosis was 360 days (IQR 120-720), taking more than 2 years in the 21.0% of patients. A percentage of 79.4 of patients received antifibrotic treatment. The average time from diagnosis to the antifibrotic treatment has been 309 ± 596.5 days, with a median of 49 (IQR 0-307). Conclusions: The diagnostic process, including the time until diagnosis and the type of test used, has changed from 2011 to 2019, probably due to advances in clinical research and the publication of diagnostic-therapeutic consensus guidelines.

Achromatopsia is an autosomal recessive disorder, in which cone photoreceptors undergo progressive degeneration, causing color blindness and poor visual acuity, among other significant eye affectations. It belongs to a group of inherited retinal dystrophies that currently have no treatment. Although functional improvements have been reported in several ongoing gene therapy studies, more efforts and research should be carried out to enhance their clinical application. In recent years, genome editing has arisen as one of the most promising tools for personalized medicine. In this study, we aimed to correct a homozygous PDE6C pathogenic variant in hiPSCs derived from a patient affected by achromatopsia through CRISPR/Cas9 and TALENs technologies. Here, we demonstrate high efficiency in gene editing by CRISPR/Cas9 but not with TALENs approximation. Despite a few of the edited clones displaying heterozygous on-target defects, the proportion of corrected clones with a potentially restored wild-type PDE6C protein was more than half of the total clones analyzed. In addition, none of them presented off-target aberrations. These results significantly contribute to advances in single-nucleotide gene editing and the development of future strategies for the treatment of achromatopsia.

Encephalomyopathic mitochondrial DNA (mtDNA) depletion syndrome 13 (MTDPS13) is a rare genetic disorder caused by defects in F-box leucine-rich repeat protein 4 (FBXL4). Although FBXL4 is essential for the bioenergetic homeostasis of the cell, the precise role of the protein remains unknown. In this study, we report two cases of unrelated patients presenting in the neonatal period with hyperlactacidemia and generalized hypotonia. Severe mtDNA depletion was detected in muscle biopsy in both patients. Genetic analysis showed one patient as having in compound heterozygosis a splice site variant c.858+5G>C and a missense variant c.1510T>C (p.Cys504Arg) in FBXL4. The second patient harbored a frameshift novel variant c.851delC (p.Pro284LeufsTer7) in homozygosis. To validate the pathogenicity of these variants, molecular and biochemical analyses were performed using skin-derived fibroblasts. We observed that the mtDNA depletion was less severe in fibroblasts than in muscle. Interestingly, the cells harboring a nonsense variant in homozygosis showed normal mtDNA copy number. Both patient fibroblasts, however, demonstrated reduced mitochondrial transcript quantity leading to diminished steady state levels of respiratory complex subunits, decreased respiratory complex IV (CIV) activity, and finally, low mitochondrial ATP levels. Both patients also revealed citrate synthase deficiency. Genetic complementation assays established that the deficient phenotype was rescued by the canonical version of FBXL4, confirming the pathological nature of the variants. Further analysis of fibroblasts allowed to establish that increased mitochondrial mass, mitochondrial fragmentation, and augmented autophagy are associated with FBXL4 deficiency in cells, but are probably secondary to a primary metabolic defect affecting oxidative phosphorylation.

Neuronal differentiation appears to be dependent on oxidative phosphorylation capacity. Several drugs inhibit oxidative phosphorylation and might be detrimental for neuronal differentiation. Some pregnant women take these medications during their first weeks of gestation when fetal nervous system is being developed. These treatments might have later negative consequences on the offspring’s health. To analyze a potential negative effect of three widely used medications, we studied in vitro dopaminergic neuronal differentiation of cells exposed to pharmacologic concentrations of azidothymidine for acquired immune deficiency syndrome; linezolid for multidrug-resistant tuberculosis; and atovaquone for malaria. We also analyzed the dopaminergic neuronal differentiation in brains of fetuses from pregnant mice exposed to linezolid. The drugs reduced the in vitro oxidative phosphorylation capacity and dopaminergic neuronal differentiation. This differentiation process does not appear to be affected in the prenatally exposed fetus brain. Nevertheless, the global DNA methylation in fetal brain was significantly altered, perhaps linking an early exposure to a negative effect in older life. Uridine was able to prevent the negative effects on in vitro dopaminergic neuronal differentiation and on in vivo global DNA methylation. Uridine could be used as a protective agent against oxidative phosphorylation-inhibiting pharmaceuticals provided during pregnancy when dopaminergic neuronal differentiation is taking place.

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Mitochondrial ATAD3A is an ATPase Associated with diverse cellular Activities (AAA) domain containing enzyme, involved in the structural organization of the inner mitochondrial membrane and of increasing importance in childhood disease. In humans, two ATAD3A paralogs arose by gene duplication during evolution: ATAD3B and ATAD3C. Here we investigate the cellular activities of the ATAD3C paralog that has been considered a pseudogene. We detected unique ATAD3C peptides in HEK 293T cells, with expression similar to that in human tissues, and showed that it is an integral membrane protein that exposes its carboxy-terminus to the intermembrane space. Overexpression of ATAD3C, but not of ATAD3A, in fibroblasts caused a decrease in cell proliferation and oxygen consumption rate, and an increase of cellular ROS. This was due to the incorporation of ATAD3C monomers in ATAD3A complex in the mitochondrial membrane reducing its size. Consistent with a negative regulation of ATAD3A function in mitochondrial membrane organization, ATAD3C expression led to increased accumulation of respiratory chain dimeric CIII in the inner membrane, to the detriment to that assembled in respiratory supercomplexes. Our results demonstrate a negative dominant role of the ATAD3C paralog with implications for mitochondrial OXPHOS function and suggest that its expression regulates ATAD3A in the cell., Competing Interests: Declaration of competing interest The authors declare no competing interests., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

Achromatopsia is an autosomal recessive disorder, in which cone photoreceptors undergo progressive degeneration, causing color blindness and poor visual acuity, among other significant eye affectations. It belongs to a group of inherited retinal dystrophies that currently have no treatment. Although functional improvements have been reported in several ongoing gene therapy studies, more efforts and research should be carried out to enhance their clinical application. In recent years, genome editing has arisen as one of the most promising tools for personalized medicine. In this study, we aimed to correct a homozygous PDE6C pathogenic variant in hiPSCs derived from a patient affected by achromatopsia through CRISPR/Cas9 and TALENs technologies. Here, we demonstrate high efficiency in gene editing by CRISPR/Cas9 but not with TALENs approximation. Despite a few of the edited clones displaying heterozygous on-target defects, the proportion of corrected clones with a potentially restored wild-type PDE6C protein was more than half of the total clones analyzed. In addition, none of them presented off-target aberrations. These results significantly contribute to advances in single-nucleotide gene editing and the development of future strategies for the treatment of achromatopsia.

Neuronal differentiation appears to be dependent on oxidative phosphorylation capacity. Several drugs inhibit oxidative phosphorylation and might be detrimental for neuronal differentiation. Some pregnant women take these medications during their first weeks of gestation when fetal nervous system is being developed. These treatments might have later negative consequences on the offspring's health. To analyze a potential negative effect of three widely used medications, we studied in vitro dopaminergic neuronal differentiation of cells exposed to pharmacologic concentrations of azidothymidine for acquired immune deficiency syndrome; linezolid for multidrug-resistant tuberculosis; and atovaquone for malaria. We also analyzed the dopaminergic neuronal differentiation in brains of fetuses from pregnant mice exposed to linezolid. The drugs reduced the in vitro oxidative phosphorylation capacity and dopaminergic neuronal differentiation. This differentiation process does not appear to be affected in the prenatally exposed fetus brain. Nevertheless, the global DNA methylation in fetal brain was significantly altered, perhaps linking an early exposure to a negative effect in older life. Uridine was able to prevent the negative effects on in vitro dopaminergic neuronal differentiation and on in vivo global DNA methylation. Uridine could be used as a protective agent against oxidative phosphorylation-inhibiting pharmaceuticals provided during pregnancy when dopaminergic neuronal differentiation is taking place.

Many patients suffering late-onset Alzheimer disease show a deficit in respiratory complex IV activity. The de novo pyrimidine biosynthesis pathway connects with the mitochondrial respiratory chain upstream from respiratory complex IV. We hypothesized that these patients would have decreased pyrimidine nucleotide levels. Then, different cell processes for which these compounds are essential, such as neuronal membrane generation and maintenance and synapses production, would be compromised. Using a cell model, we show that inhibiting oxidative phosphorylation function reduces neuronal differentiation. Linking these processes to pyrimidine nucleotides, uridine treatment recovers neuronal differentiation. To unmask the importance of these pathways in Alzheimer disease, we firstly confirm the existence of the de novo pyrimidine biosynthesis pathway in adult human brain. Then, we report altered mRNA levels for genes from both de novo pyrimidine biosynthesis and pyrimidine salvage pathways in brain from patients with Alzheimer disease. Thus, uridine supplementation might be used as a therapy for those Alzheimer disease patients with low respiratory complex IV activity.

Aim: To perform a deep phenotype characterisation in a pedigree of 3 siblings with Leigh syndrome and compound heterozygous NDUFAF6 mutations., Method: A multi-gene panel of childhood-onset basal ganglia neurodegeneration inherited conditions was analysed followed by functional studies in fibroblasts., Results: Three siblings developed gait dystonia in infancy followed by rapid progression to generalised dystonia and psychomotor regression. Brain magnetic resonance showed symmetric and bilateral cytotoxic lesions in the putamen and proliferation of the lenticular-striate arteries, latter spreading to the caudate and progressing to cavitation and volume loss. We identified a frameshift novel change (c.554_558delTTCTT; p.Tyr187AsnfsTer65) and a pathogenic missense change (c.371T>C; p.Ile124Thr) in the NDUFAF6 gene, which segregated with an autosomal recessive inheritance within the family. Patient mutations were associated with the absence of the NDUFAF6 protein and reduced activity and assembly of mature complex I in fibroblasts. By functional complementation assay, the mutant phenotype was rescued by the canonical version of the NDUFAF6. A literature review of 14 NDUFAF6 patients showed a consistent phenotype of an early childhood insidious onset neurological regression with prominent dystonia associated with basal ganglia degeneration and long survival., Interpretation: NDUFAF6-related Leigh syndrome is a relevant cause of childhood onset dystonia and isolated bilateral striatal necrosis. By genetic complementation, we could demonstrate the pathogenicity of novel genetic variants in NDUFAF6., (Copyright © 2019 Elsevier Inc. All rights reserved.)