New variants expand the neurological phenotype of COQ7 deficiency.

The protein encoded by COQ7 is required for CoQ10 synthesis in humans, hydroxylating 3-demethoxyubiquinol (DMQ10) in the second to last steps of the pathway. COQ7 mutations lead to a primary CoQ10 deficiency syndrome associated with a pleiotropic neurological disorder. This study shows the clinical, physiological, and molecular characterization of four new cases of CoQ10 primary deficiency caused by five mutations in COQ7, three of which have not yet been described, inducing mitochondrial dysfunction in all patients. However, the specific combination of the identified variants in each patient generated precise pathophysiological and molecular alterations in fibroblasts, which would explain the differential in vitro response to supplementation therapy. Our results suggest that COQ7 dysfunction could be caused by specific structural changes that affect the interaction with COQ9 required for the DMQ10 presentation to COQ7, the substrate access to the active site, and the maintenance of the active site structure. Remarkably, patients' fibroblasts share transcriptional remodeling, supporting a modification of energy metabolism towards glycolysis, which could be an adaptive mechanism against CoQ10 deficiency. However, transcriptional analysis of mitochondria-associated pathways showed distinct and dramatic differences between patient fibroblasts, which correlated with the extent of pathophysiological and neurological alterations observed in the probands. Overall, this study suggests that the combination of precise genetic diagnostics and the availability of new structural models of human proteins could help explain the origin of phenotypic pleiotropy observed in some genetic diseases and the different responses to available therapies.

Imbalanced mitochondrial dynamics contributes to the pathogenesis of X-linked adrenoleukodystrophy.

The peroxisomal disease adrenoleukodystrophy (X-ALD) is caused by loss of the transporter of very-long-chain fatty acids (VLCFAs), ABCD1. An excess of VLCFAs disrupts essential homeostatic functions crucial for axonal maintenance, including redox metabolism, glycolysis and mitochondrial respiration. As mitochondrial function and morphology are intertwined, we set out to investigate the role of mitochondrial dynamics in X-ALD models. Using quantitative 3D transmission electron microscopy, we revealed mitochondrial fragmentation in corticospinal axons in Abcd1- mice. In patient fibroblasts, an excess of VLCFAs triggers mitochondrial fragmentation through the redox-dependent phosphorylation of DRP1 (DRP1S616). The blockade of DRP1-driven fission by the peptide P110 effectively preserved mitochondrial morphology. Furthermore, mRNA inhibition of DRP1 not only prevented mitochondrial fragmentation but also protected axonal health in a Caenorhabditis elegans model of X-ALD, underscoring DRP1 as a potential therapeutic target. Elevated levels of circulating cell-free mtDNA in patients' CSF align this leukodystrophy with primary mitochondrial disorders. Our findings underscore the intricate interplay between peroxisomal dysfunction, mitochondrial dynamics and axonal integrity in X-ALD, shedding light on potential avenues for therapeutic intervention.

Mutations of GEMIN5 are associated with coenzyme Q10 deficiency: long-term follow-up after treatment.

GEMIN5 exerts key biological functions regulating pre-mRNAs intron removal to generate mature mRNAs. A series of patients were reported harboring mutations in GEMIN5. No treatments are currently available for this disease. We treated two of these patients with oral Coenzyme Q10 (CoQ10), which resulted in neurological improvements, although MRI abnormalities remained. Whole Exome Sequencing demonstrated compound heterozygosity at the GEMIN5 gene in both cases: Case one: p.Lys742* and p.Arg1016Cys; Case two: p.Arg1016Cys and p.Ser411Hisfs*6. Functional studies in fibroblasts revealed a decrease in CoQ10 biosynthesis compared to controls. Supplementation with exogenous CoQ10 restored it to control intracellular CoQ10 levels. Mitochondrial function was compromised, as indicated by the decrease in oxygen consumption, restored by CoQ10 supplementation. Transcriptomic analysis of GEMIN5 patients compared with controls showed general repression of genes involved in CoQ10 biosynthesis. In the rigor mortis defective flies, CoQ10 levels were decreased, and CoQ10 supplementation led to an improvement in the adult climbing assay performance, a reduction in the number of motionless flies, and partial restoration of survival. Overall, we report the association between GEMIN5 dysfunction and CoQ10 deficiency for the first time. This association opens the possibility of oral CoQ10 therapy, which is safe and has no observed side effects after long-term therapy.

Foetal Haemoglobin as a Marker of Bone Marrow Suppression Secondary to Anti-Kell Alloimmunisation.

Anti-Kell alloimmunisation is a potentially severe minor blood group type incompatibility, not only as a cause of haemolytic disease of the foetus and newborn, but also due to the destruction of red blood cells (RBC) and mature form in the bone marrow with the subsequent hyporegenerative anaemia. In severe cases and when the foetus shows signs of anaemia, an intrauterine transfusion (IUT) may be necessary. When repeated, this treatment can suppress erythropoiesis and worsen the anaemia. We report the case of a newborn who required four IUTs plus an additional RBC transfusion at one month of life due to late onset anaemia. The identification of an adult haemoglobin profile with a complete absence of foetal haemoglobin in the patient's newborn screening samples at 2 and 10 days of life warned us of a possible late anaemia. The newborn was successfully treated with transfusion, oral supplements and subcutaneous erythropoietin. A blood sample taken at 4 months of life showed the expected haemoglobin profile for that age with a foetal haemoglobin of 17.7%. This case illustrates the importance of a close follow-up of these patients, as well as the usefulness of the haemoglobin profile screening as a tool for anaemia assessment.

Laboratory testing for mitochondrial diseases: biomarkers for diagnosis and follow-up.

The currently available biomarkers generally lack the specificity and sensitivity needed for the diagnosis and follow-up of patients with mitochondrial diseases (MDs). In this group of rare genetic disorders (mutations in approximately 350 genes associated with MDs), all clinical presentations, ages of disease onset and inheritance types are possible. Blood, urine, and cerebrospinal fluid surrogates are well-established biomarkers that are used in clinical practice to assess MD. One of the main challenges is validating specific and sensitive biomarkers for the diagnosis of disease and prediction of disease progression. Profiling of lactate, amino acids, organic acids, and acylcarnitine species is routinely conducted to assess MD patients. New biomarkers, including some proteins and circulating cell-free mitochondrial DNA, with increased diagnostic specificity have been identified in the last decade and have been proposed as potentially useful in the assessment of clinical outcomes. Despite these advances, even these new biomarkers are not sufficiently specific and sensitive to assess MD progression, and new biomarkers that indicate MD progression are urgently needed to monitor the success of novel therapeutic strategies. In this report, we review the mitochondrial biomarkers that are currently analyzed in clinical laboratories, new biomarkers, an overview of the most common laboratory diagnostic techniques, and future directions regarding targeted versus untargeted metabolomic and genomic approaches in the clinical laboratory setting. Brief descriptions of the current methodologies are also provided.

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.

Advancing in Schaaf-Yang syndrome pathophysiology: from bedside to subcellular analyses of truncated MAGEL2.

BACKGROUND: Schaaf-Yang syndrome (SYS) is caused by truncating mutations in MAGEL2, mapping to the Prader-Willi region (15q11-q13), with an observed phenotype partially overlapping that of Prader-Willi syndrome. MAGEL2 plays a role in retrograde transport and protein recycling regulation. Our aim is to contribute to the characterisation of SYS pathophysiology at clinical, genetic and molecular levels. METHODS: We performed an extensive phenotypic and mutational revision of previously reported patients with SYS. We analysed the secretion levels of amyloid-ß 1-40 peptide (Aß1-40) and performed targeted metabolomic and transcriptomic profiles in fibroblasts of patients with SYS (n=7) compared with controls (n=11). We also transfected cell lines with vectors encoding wild-type (WT) or mutated MAGEL2 to assess stability and subcellular localisation of the truncated protein. RESULTS: Functional studies show significantly decreased levels of secreted Aß1-40 and intracellular glutamine in SYS fibroblasts compared with WT. We also identified 132 differentially expressed genes, including non-coding RNAs (ncRNAs) such as HOTAIR, and many of them related to developmental processes and mitotic mechanisms. The truncated form of MAGEL2 displayed a stability similar to the WT but it was significantly switched to the nucleus, compared with a mainly cytoplasmic distribution of the WT MAGEL2. Based on the updated knowledge, we offer guidelines for the clinical management of patients with SYS. CONCLUSION: A truncated MAGEL2 protein is stable and localises mainly in the nucleus, where it might exert a pathogenic neomorphic effect. Aß1-40 secretion levels and HOTAIR mRNA levels might be promising biomarkers for SYS. Our findings may improve SYS understanding and clinical management.

Technical Aspects of Coenzyme Q10 Analysis: Validation of a New HPLC-ED Method.

The biochemical measurement of the CoQ status in different tissues can be performed using HPLC with electrochemical detection (ED). Because the production of the electrochemical cells used with the Coulochem series detectors was discontinued, we aimed to standardize a new HPLC-ED method with new equipment. We report all technical aspects, troubleshooting and its performance in different biological samples, including plasma, skeletal muscle homogenates, urine and cultured skin fibroblasts. Analytical variables (intra- and inter-assay precision, linearity, analytical measurement range, limit of quantification, limit of detection and accuracy) were validated in calibrators and plasma samples and displayed adequate results. The comparison of the results of a new ERNDIM external quality control (EQC) scheme for the plasma CoQ determination between HPLC-ED (Lab 1) and LC-MS/MS (Lab 2) methods shows that the results of the latter were slightly higher in most cases, although a good consistency was generally observed. In conclusion, the new method reported here showed a good analytical performance. The global quality of the EQC scheme results among different participants can be improved with the contribution of more laboratories.

The Genetic Landscape of Mitochondrial Diseases in Spain: A Nationwide Call.

The frequency of mitochondrial diseases (MD) has been scarcely documented, and only a few studies have reported data in certain specific geographical areas. In this study, we arranged a nationwide call in Spain to obtain a global estimate of the number of cases. A total of 3274 cases from 49 Spanish provinces were reported by 39 centres. Excluding duplicated and unsolved cases, 2761 patients harbouring pathogenic mutations in 140 genes were recruited between 1990 and 2020. A total of 508 patients exhibited mutations in nuclear DNA genes (75% paediatric patients) and 1105 in mitochondrial DNA genes (33% paediatric patients). A further 1148 cases harboured mutations in the MT-RNR1 gene (56% paediatric patients). The number of reported cases secondary to nuclear DNA mutations increased in 2014, owing to the implementation of next-generation sequencing technologies. Between 2014 and 2020, excepting MT-RNR1 cases, the incidence was 6.34 (95% CI: 5.71-6.97) cases per million inhabitants at the paediatric age and 1.36 (95% CI: 1.22-1.50) for adults. In conclusion, this is the first study to report nationwide epidemiological data for MD in Spain. The lack of identification of a remarkable number of mitochondrial genes necessitates the systematic application of high-throughput technologies in the routine diagnosis of MD.

Plasma idebenone monitoring in Friedreich's ataxia patients during a long-term follow-up.

INTRODUCTION AND OBJECTIVES: Despite the growing interest and the potential benefits of idebenone as a repurposed drug for different orphan conditions, data regarding its monitoring are scarce. Our main goal was to report plasma idebenone values in a cohort of Friedreich's ataxia (FRDA) patients during a long-term follow-up. Taking advantage of this, we also assessed cardiological and neurological status together with idebenone values and genetic background. METHODS: Long-term follow-up retrospective study in 27 FRDA patients with a disease onset at the paediatric age treated with idebenone by compassionate use. Plasma idebenone was measured by HPLC with electrochemical detection. RESULTS: Median plasma idebenone values increased when doses were increased, but apparently linearity was lost in the highest dose group. Marked intraindividual and interindividual differences were observed among patients. We did not find a consistent positive effect after analysis of paired data at the beginning and the end of the study. We only found a correlation between some cardiological measures and the duration of idebenone therapy at high doses, but with uncertain significance. CONCLUSIONS: The large variations observed among the different individuals involved in this study should be considered for optimization of individual dosage regimens.

Circulating Cell-Free Mitochondrial DNA in Cerebrospinal Fluid as a Biomarker for Mitochondrial Diseases.

BACKGROUND: Mitochondrial diseases (MD) are genetic metabolic disorders that impair normal mitochondrial structure or function. The aim of this study was to investigate the status of circulating cell-free mitochondrial DNA (ccfmtDNA) in cerebrospinal fluid (CSF), together with other biomarkers (growth differentiation factor-15 [GDF-15], alanine, and lactate), in a cohort of 25 patients with a molecular diagnosis of MD. METHODS: Measurement of ccfmtDNA was performed by using droplet digital PCR. RESULTS: The mean copy number of ccfmtDNA was approximately 6 times higher in the MD cohort compared to the control group; patients with mitochondrial deletion and depletion syndromes (MDD) had the higher levels. We also detected the presence of both wild-type mtDNA and mtDNA deletions in CSF samples of patients with single deletions. Patients with MDD with single deletions had significantly higher concentrations of GDF-15 in CSF than controls, whereas patients with point mutations in mitochondrial DNA presented no statistically significant differences. Additionally, we found a significant positive correlation between ccfmtDNA levels and GDF-15 concentrations (r = 0.59, P = 0.016). CONCLUSION: CSF ccfmtDNA levels are significantly higher in patients with MD in comparison to controls and, thus, they can be used as a novel biomarker for MD research. Our results could also be valuable to support the clinical outcome assessment of MD patients.

Coenzyme Q10 Treatment Monitoring in Different Human Biological Samples.

Coenzyme Q10 (CoQ) treatment monitoring is a matter of debate since CoQ distribution from plasma to blood cells and tissues is not fully understood. We aimed to analyze the CoQ levels in a wide set of human biological samples (plasma, blood mononuclear cells (BMCs), platelets, urinary cells, and skeletal muscle) from a group of 11 healthy male runners before and after CoQ supplementation. The CoQ content in the different samples was analyzed by HPLC coupled to electrochemical detection. No significant differences were observed in the CoQ levels measured in the BMCs, platelets, and urine after the one-month treatment period. Plasma CoQ (expressed in absolute values and values relative to total cholesterol) significantly increased after CoQ supplementation (p = 0.003 in both cases), and the increase in CoQ in muscle approached significance (p = 0.074). CoQ levels were increased in the plasma of all supplemented subjects, and muscle CoQ levels were increased in 8 out of 10 supplemented subjects. In conclusion, the analysis of CoQ in plasma samples seems to be the best surrogate biomarker for CoQ treatment monitoring. Moreover, oral CoQ administration was effective for increasing muscle CoQ concentrations in most subjects.