Physiopathological bases of the disease caused by HACE1 mutations: alterations in autophagy, mitophagy and oxidative stress response

Recessive HACE1 mutations are associated with a severe neurodevelopmental disorder (OMIM: 616756). However, the physiopathologycal bases of the disease are yet to be completely clarified. Whole-exome sequencing identified homozygous HACE1 mutations (c.240C>A, p.Cys80Ter) in a patient with brain atrophy, psychomotor retardation and 3-methylglutaconic aciduria, a biomarker of mitochondrial dysfunction. To elucidate the pathomechanisms underlying HACE1 deficiency, a comprehensive molecular analysis was performed in patient fibroblasts. Western Blot demonstrated the deleterious effect of the mutation, as the complete absence of HACE1 protein was observed. Immunofluorescence studies showed an increased number of LC3 puncta together with the normal initiation of the autophagic cascade, indicating a reduction in the autophagic flux. Oxidative stress response was also impaired in HACE1 fibroblasts, as shown by the reduced NQO1 and Hmox1 mRNA levels observed in H2O2-treated cells. High levels of lipid peroxidation, consistent with accumulated oxidative damage, were also detected. Although the patient phenotype could resemble a mitochondrial defect, the analysis of the mitochondrial function showed no major abnormalities. However, an important increase in mitochondrial oxidative stress markers and a strong reduction in the mitophagic flux were observed, suggesting that the recycling of damaged mitochondria might be targeted in HACE1 cells. In summary, we demonstrate for the first time that the impairment of autophagy, mitophagy and oxidative damage response might be involved in the pathogenesis of HACE1 deficiency

Leigh syndrome is the main clinical characteristic of PTCD3 deficiency

Mitochondrial translation defects are a continuously growing group of disorders showing a large variety of clinical symptoms including a wide range of neurological abnormalities. To date, mutations in PTCD3, encoding a component of the mitochondrial ribosome, have only been reported in a single individual with clinical evidence of Leigh syndrome. Here, we describe three additional PTCD3 individuals from two unrelated families, broadening the genetic and phenotypic spectrum of this disorder, and provide definitive evidence that PTCD3 deficiency is associated with Leigh syndrome. The patients presented in the first months of life with psychomotor delay, respiratory insufficiency and feeding difficulties. The neurologic phenotype included dystonia, optic atrophy, nystagmus and tonic-clonic seizures. Brain MRI showed optic nerve atrophy and thalamic changes, consistent with Leigh syndrome. WES and RNA-seq identified compound heterozygous variants in PTCD3 in both families: c.[1453-1G>C];[1918C>G] and c.[710del];[902C>T]. The functional consequences of the identified variants were determined by a comprehensive characterization of the mitochondrial function. PTCD3 protein levels were significantly reduced in patient fibroblasts and, consistent with a mitochondrial translation defect, a severe reduction in the steady state levels of complexes I and IV subunits was detected. Accordingly, the activity of these complexes was also low, and high-resolution respirometry showed a significant decrease in the mitochondrial respiratory capacity. Functional complementation studies demonstrated the pathogenic effect of the identified variants since the expression of wild-type PTCD3 in immortalized fibroblasts restored the steady-state levels of complexes I and IV subunits as well as the mitochondrial respiratory capacity. Additionally, minigene assays demonstrated that three of the identified variants were pathogenic by altering PTCD3 mRNA processing. The fourth variant was a frameshift leading to a truncated protein. In summary, we provide evidence of PTCD3 involvement in human disease confirming that PTCD3 deficiency is definitively associated with Leigh syndrome.© 2022 The Authors. Brain Pathology published by John Wiley & Sons Ltd on behalf of International Society of Neuropathology

Cardiac and placental mitochondrial characterization in a rabbit model of intrauterine growth restriction

BACKGROUND: Intrauterine growth restriction (IUGR) is associated with cardiovascular remodeling persisting into adulthood. Mitochondrial bioenergetics, essential for embryonic development and cardiovascular function, are regulated by nuclear effectors as sirtuins. A rabbit model of IUGR and cardiovascular remodeling was generated, in which heart mitochondrial alterations were observed by microscopic and transcriptomic analysis. We aimed to evaluate if such alterations are translated at a functional mitochondrial level to establish the etiopathology and potential therapeutic targets for this obstetric complication. METHODS: Hearts and placentas from 16 IUGR-offspring and 14 controls were included to characterize mitochondrial function. RESULTS: Enzymatic activities of complexes II, IV and II + III in IUGR-hearts (-11.96 ± 3.16%; -15.58 ± 5.32%; -14.73 ± 4.37%; p < 0.05) and II and II + III in IUGR-placentas (-17.22 ± 3.46%; p < 0.005 and -29.64 ± 4.43%; p < 0.001) significantly decreased. This was accompanied by a not significant reduction in CI-stimulated oxygen consumption and significantly decreased complex II SDHB subunit expression in placenta (-44.12 ± 5.88%; p < 0.001). Levels of mitochondrial content, Coenzyme Q and cellular ATP were conserved. Lipid peroxidation significantly decreased in IUGR-hearts (-39.02 ± 4.35%; p < 0.001), but not significantly increased in IUGR-placentas. Sirtuin3 protein expression significantly increased in IUGR-hearts (84.21 ± 31.58%; p < 0.05) despite conserved anti-oxidant SOD2 protein expression and activity in both tissues. CONCLUSIONS: IUGR is associated with cardiac and placental mitochondrial CII dysfunction. Up-regulated expression of Sirtuin3 may explain attenuation of cardiac oxidative damage and preserved ATP levels under CII deficiency. GENERAL SIGNIFICANCE: These findings may allow the design of dietary interventions to modulate Sirtuin3 expression and consequent regulation of mitochondrial imbalance associated with IUGR and derived cardiovascular remodeling

Systematic Collaborative Reanalysis of Genomic Data Improves Diagnostic Yield in Neurologic Rare Diseases

Altres ajuts: Generalitat de Catalunya, Departament de Salut; Generalitat de Catalunya, Departament d'Empresa i Coneixement i CERCA Program; Ministerio de Ciencia e Innovación; Instituto Nacional de Bioinformática; ELIXIR Implementation Studies (CNAG-CRG); Centro de Investigaciones Biomédicas en Red de Enfermedades Raras; Centro de Excelencia Severo Ochoa; European Regional Development Fund (FEDER).Many patients experiencing a rare disease remain undiagnosed even after genomic testing. Reanalysis of existing genomic data has shown to increase diagnostic yield, although there are few systematic and comprehensive reanalysis efforts that enable collaborative interpretation and future reinterpretation. The Undiagnosed Rare Disease Program of Catalonia project collated previously inconclusive good quality genomic data (panels, exomes, and genomes) and standardized phenotypic profiles from 323 families (543 individuals) with a neurologic rare disease. The data were reanalyzed systematically to identify relatedness, runs of homozygosity, consanguinity, single-nucleotide variants, insertions and deletions, and copy number variants. Data were shared and collaboratively interpreted within the consortium through a customized Genome-Phenome Analysis Platform, which also enables future data reinterpretation. Reanalysis of existing genomic data provided a diagnosis for 20.7% of the patients, including 1.8% diagnosed after the generation of additional genomic data to identify a second pathogenic heterozygous variant. Diagnostic rate was significantly higher for family-based exome/genome reanalysis compared with singleton panels. Most new diagnoses were attributable to recent gene-disease associations (50.8%), additional or improved bioinformatic analysis (19.7%), and standardized phenotyping data integrated within the Undiagnosed Rare Disease Program of Catalonia Genome-Phenome Analysis Platform functionalities (18%)

Identificación y caracterización de nuevos defectos del metabolismo energético mitocondrial asociados a aciduria 3-metilglutacónica y a deficiencia del complejo I

[spa] Las enfermedades del metabolismo energético mitocondrial son un grupo de patologías con una gran heterogeneidad clínica, bioquímica y genética. Aunque en la última década la implementación de las técnicas de “Next generation sequencing” (NGS) ha permitido la identificación de un importante número de genes asociados a estas enfermedades, el diagnóstico de estos pacientes sigue siendo muy complejo. Por ello, la identificación y el estudio de biomarcadores para estas enfermedades es fundamental, ya que permite dirigir la interpretación de los datos genéticos y el diagnóstico hacia vías metabólicas o funciones celulares concretas. Un biomarcador ampliamente aceptado como indicador de alteración mitocondrial es la elevación de ácido 3-metilglutacónico (3-MGA) en orina, que es el hilo conductor de esta tesis doctoral. Los niveles elevados y persistentes de este metabolito se asocian a defectos en proteínas asociadas a la membrana mitocondrial entre las que se encuentran ciertas deficiencias del complejo V de la cadena respiratoria mitocondrial (CRM). Por otro lado, la alteración más frecuente entre las patologías mitocondriales es la deficiencia del complejo I. En base a los conocimientos previos sobre la afectación mitocondrial en estos trastornos, la hipótesis de esta tesis es que la implementación de herramientas de caracterización mitocondrial facilitará la priorización e identificación de las variantes genéticas causantes de la enfermedad. Nuestros estudios se han centrado en la caracterización de la morfología mitocondrial, el ensamblaje de los complejos y supercomplejos de la CRM y la respiración celular. Además, en cada uno de los casos incluidos en este trabajo, se han realizado estudios más específicos relacionados con el gen alterado, dando en su conjunto una amplia visión de las bases moleculares subyacentes en cada patología. En esta tesis se han estudiado un total de 36 pacientes con sospecha de enfermedad mitocondrial en los que se han realizado análisis genéticos y estudios funcionales en células de los pacientes o en modelos celulares. En 14 pacientes se observó aciduria 3-metilglutacónica (3-MG), habiéndose conseguido un diagnóstico genético definitivo en 11 de ellos. En 17 de los 22 pacientes restantes, los estudios funcionales realizados han permitido confirmar la causalidad de las variantes identificadas. Asimismo, estos estudios han descartado la patogenicidad de las variantes candidatas en 5 casos. El estudio de las bases moleculares de las enfermedades causadas por mutaciones en TIMM50, HACE1, NDUFAF4 y NDUFA8 ha dado lugar a las 4 publicaciones que conforman el grueso de esta tesis doctoral. En el primer artículo, presentamos un paciente con síndrome de Leigh, atrofia óptica, neutropenia, cardiomiopatía y aciduria 3-MG en el que se identificaron dos mutaciones en heterocigosis compuesta en el gen TIMM50 (c.341G>A; p.Arg114Gln y c.805G>A; p.Gly269Ser). Este gen codifica para una proteína implicada en el transporte de proteínas desde el citosol hacia el interior de la mitocondria. Los estudios realizados en fibroblastos del paciente y en un modelo celular generado mediante CRISPR/Cas9 han demostrado que la deficiencia de TIMM50 da lugar a una disfunción mitocondrial severa que afecta al mantenimiento de la morfología mitocondrial, al ensamblaje del sistema OXPHOS y a la capacidad respiratoria mitocondrial. En el segundo trabajo se ha estudiado un paciente con sospecha de enfermedad mitocondrial que presentaba atrofia cerebral, retraso psicomotor y aciduria 3-MG. Sin embargo, mediante NGS se identificó una mutación en homocigosis en el gen HACE1 (c.240C>A; p.Cys80Ter), que codifica una proteína citoplasmática con actividad ubiquitin-ligasa. Los estudios funcionales realizados han demostrado que la deficiencia de HACE1 da lugar a alteraciones en las vías de respuesta a estrés oxidativo mediadas por NRF2 y defectos en los procesos de autofagia y mitofagia. Además, nuestros resultados han asociado por primera vez, la deficiencia de HACE1 a alteraciones de la función mitocondrial y a aciduria 3-MG. En los dos últimos artículos de la tesis se caracterizan 2 familias con deficiencia del complejo I de la CRM. En la primera, los dos hermanos afectos presentaron encefalopatía, dismorfia, acidosis láctica y aciduria 3-MG desde el nacimiento. Fueron exitus a los pocos meses de vida. Mediante los estudios genéticos, se identificaron mutaciones en homocigosis en el gen NDUFAF4 (c.478G>T; p.Glu160Ter), que codifica un factor de ensamblaje del complejo I de la CRM. Los estudios funcionales demostraron una reducción drástica del ensamblaje del complejo I y de los supercomplejos de alto peso molecular, alteraciones en la morfología mitocondrial y deficiencia de la respiración mitocondrial. En la segunda familia, se identificaron mutaciones en homocigosis en el gen NDUFA8 (c.293G>T; p.Arg98Leu), que codifica una subunidad del complejo I. La particularidad de estos pacientes reside en la progresión sorprendentemente favorable de la enfermedad, siendo prácticamente asintomáticos a la edad de 6 y 9 años. Este fenotipo es atípico tratándose de una deficiencia del complejo I de la CRM. Sin embargo, los estudios funcionales mostraron una importante deficiencia en el ensamblaje del complejo I y de los supercomplejos. La patogenicidad de la mutación fue demostrada mediante estudios de complementación funcional en fibroblastos de los pacientes. En resumen, los estudios funcionales realizados han permitido confirmar que las variantes identificadas son la causa de la enfermedad y profundizar en el conocimiento de las bases moleculares de estas enfermedades

Physiopathological Bases of the Disease Caused by HACE1 Mutations: Alterations in Autophagy, Mitophagy and Oxidative Stress Response

Recessive HACE1 mutations are associated with a severe neurodevelopmental disorder (OMIM: 616756). However, the physiopathologycal bases of the disease are yet to be completely clarified. Whole-exome sequencing identified homozygous HACE1 mutations (c.240C&gt;A, p.Cys80Ter) in a patient with brain atrophy, psychomotor retardation and 3-methylglutaconic aciduria, a biomarker of mitochondrial dysfunction. To elucidate the pathomechanisms underlying HACE1 deficiency, a comprehensive molecular analysis was performed in patient fibroblasts. Western Blot demonstrated the deleterious effect of the mutation, as the complete absence of HACE1 protein was observed. Immunofluorescence studies showed an increased number of LC3 puncta together with the normal initiation of the autophagic cascade, indicating a reduction in the autophagic flux. Oxidative stress response was also impaired in HACE1 fibroblasts, as shown by the reduced NQO1 and Hmox1 mRNA levels observed in H2O2-treated cells. High levels of lipid peroxidation, consistent with accumulated oxidative damage, were also detected. Although the patient phenotype could resemble a mitochondrial defect, the analysis of the mitochondrial function showed no major abnormalities. However, an important increase in mitochondrial oxidative stress markers and a strong reduction in the mitophagic flux were observed, suggesting that the recycling of damaged mitochondria might be targeted in HACE1 cells. In summary, we demonstrate for the first time that the impairment of autophagy, mitophagy and oxidative damage response might be involved in the pathogenesis of HACE1 deficiency

Over-Mutated Mitochondrial, Lysosomal and TFEB-Regulated Genes in Parkinson&rsquo;s Disease

The association between Parkinson&rsquo;s disease (PD) and mutations in genes involved in lysosomal and mitochondrial function has been previously reported. However, little is known about the involvement of other genes or cellular mechanisms. We aim to identify novel genetic associations to better understand the pathogenesis of PD. We performed WES in a cohort of 32 PD patients and 30 age-matched controls. We searched for rare variants in 1667 genes: PD-associated, related to lysosomal function and mitochondrial function and TFEB-regulated. When comparing the PD patient cohort with that of age matched controls, a statistically significant burden of rare variants in the previous group of genes were identified. In addition, the Z-score calculation, using the European population database (GnomAD), showed an over-representation of particular variants in 36 genes. Interestingly, 11 of these genes are implicated in mitochondrial function and 18 are TFEB-regulated genes. Our results suggest, for the first time, an involvement of TFEB-regulated genes in the genetic susceptibility to PD. This is remarkable as TFEB factor has been reported to be sequestered inside Lewy bodies, pointing to a role of TFEB in the pathogenesis of PD. Our data also reinforce the involvement of lysosomal and mitochondrial mechanisms in PD

Biallelic mutations in NDUFA8 cause complex I deficiency in two siblings with favorable clinical evolution

Isolated complex I (CI) deficiency is the most common cause of oxidative phosphorylation (OXPHOS) dysfunction. Whole-exome sequencing identified biallelic mutations in NDUFA8 (c.[293G>T]; [293G>T], encoding for an accessory subunit of CI, in two siblings with a favorable clinical evolution. The individuals reported here are practically asymptomatic, with the exception of slight failure to thrive and some language difficulties at the age of 6 and 9years, respectively. These observations are remarkable since the vast majority of patients with CI deficiency, including the only NDUFA8 patient reported so far, showed an extremely poor clinical outcome. Western blot studies demonstrated that NDUFA8 protein was strongly reduced in the patients' fibroblasts and muscle extracts. In addition, there was a marked and specific decrease in the steady-state levels of CI subunits. BN-PAGE demonstrated an isolated defect in the assembly and the activity of CI with impaired supercomplexes formation and abnormal accumulation of CI subassemblies. Confocal microscopy analysis in fibroblasts showed rounder mitochondria and diminished branching degree of the mitochondrial network. Functional complementation studies demonstrated disease-causality for the identified mutation as lentiviral transduction with wild-type NDUFA8 cDNA restored the steady-state levels of CI subunits and completely recovered the deficient enzymatic activity in immortalized mutant fibroblasts. In summary, we provide additional evidence of the involvement of NDUFA8 as a mitochondrial disease-causing gene associated with altered mitochondrial morphology, CI deficiency, impaired supercomplexes formation, and very mild progression of the disease

Mutations in TRAPPC11 are Associated with a Congenital Disorder of Glycosylation

Congenital disorders of glycosylation (CDG) are a heterogeneous and rapidly growing group of diseases caused by abnormal glycosylation of proteins and/or lipids. Mutations in genes involved in the homeostasis of the endoplasmic reticulum (ER), the Golgi apparatus and the vesicular trafficking from the ER to the ER-Golgi intermediate compartment (ERGIC) have been found to be associated with CDG. Here, we report a patient with defects in both N- and O-glycosylation combined with a delayed vesicular transport in the Golgi apparatus due to mutations in TRAPPC11, a subunit of the TRAPPIII complex. TRAPPIII is implicated in the anterograde transport from the ER to the ERGIC as well as in the vesicle export from the Golgi apparatus. This report expands the spectrum of genetic alterations associated with CDG, providing new insights for the diagnosis and the understanding of the physiopathological mechanisms underlying glycosylation disorders. This article is protected by copyright. All rights reserved.status: publishe