Mitochondrial Disease Research Articles

MELAS Presenting as Bilateral Symmetric Occipital and Temporal Cortices Lesions: A Case Report and Literature Review.

Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episode (MELAS) is one of the most common maternally inherited mitochondrial diseases. The stroke-like episode affecting the cortical cortex is the hallmark of MELAS; however, it rarely presents as simultaneously bilateral symmetric cortices lesions. We reported a case of MELAS in a 46-year-old female patient with bilateral symmetric occipital and internal temporal cortices involvements on brain magnetic resonance imaging (MRI). A literature review of MELAS patients and a retrospective analysis were performed. She had a family history of diabetes. Although she denied a history of diabetes, elevated blood glucose was noted after admission, and diabetes was diagnosed. Laboratory examination revealed elevated lactate acid and creatine kinase levels in blood. Cranial computed tomography (CT) image demonstrated basal ganglia calcification, as well as subtle decreased attenuation in bilateral symmetric occipital and internal temporal cortices. Brain magnetic resonance imaging (MRI) demonstrated symmetric gyriform hyperintensity in bilateral occipital lobes and internal temporal lobes in both grey and white matter on fluid-attenuated inversion recovery (FLAIR) images with restricted diffusion on diffusion weighted images (DWI). A genetic test revealed a point mutation in the mtDNA(3243A > G) by blood examination. Literature review showed that there were 231 eligible patients with MELAS identified from 212 published papers. Symmetric cortical involvements were seen in 15 (6.5%) patients on brain MRI. MELAS should be considered as a potential diagnosis in the patients with bilateral symmetric stroke-like cortices lesions.

Targeted nanopore sequencing using the Flongle device to identify mitochondrial DNA variants

Variants in mitochondrial genomes (mtDNA) can cause various neurological and mitochondrial diseases such as mitochondrial myopathy, encephalopathy, lactic acidosis, stroke-like episodes (MELAS). Given the 16 kb length of mtDNA, continuous sequencing is feasible using long-read sequencing (LRS). Herein, we aimed to show a simple and accessible method for comprehensive mtDNA sequencing with potential diagnostic applications for mitochondrial diseases using the compact and affordable LRS flow cell “Flongle.” Whole mtDNA amplification (WMA) was performed using genomic DNA samples derived from four patients with mitochondrial diseases, followed by LRS using Flongle. We compared these results to those obtained using Cas9 enrichment. Additionally, the accuracy of heteroplasmy rates was assessed by incorporating mtDNA variants at equimolar levels. Finally, mtDNA from 19 patients with Parkinson’s disease (PD) was sequenced using Flongle to identify disease risk-associated variants. mtDNA variants were detected in all four patients with mitochondrial diseases, with results comparable to those obtained from Cas9 enrichment. Heteroplasmy levels were accurately detected (r2 > 0.99) via WMA using Flongle. A reported variant was identified in three patients with PD. In conclusion, Flongle can simplify the traditionally cumbersome and expensive mtDNA sequencing process, offering a streamlined and accessible approach to diagnosing mitochondrial diseases.

Beyond the cochlea: exploring the multifaceted nature of hearing loss in primary mitochondrial diseases

Abstract Primary mitochondrial diseases, with diverse systemic manifestations, often present with auditory impairments due to mitochondrial dysfunction. This study provides an in-depth exploration of auditory deficits in primary mitochondrial diseases, highlighting the impact of various pathogenic variants on both cochlea and neural/central auditory functions. An observational study involving 72 adults with primary mitochondrial diseases was conducted. Participants underwent extensive audiological evaluations including Pure-Tone Audiometry, Tympanometry, Acoustic Reflex Thresholds, Quick Speech-in-Noise Test, Listening in Spatialized Noise-Sentences test, Auditory-evoked Brainstem Responses, and Distortion Product Otoacoustic Emissions. Multivariate Analysis of Covariance and logistic regression analyses assessed the influence of various pathogenic DNA variants, accounting for age, cognitive status via the Montreal Cognitive Assessment, and disease severity through the Newcastle Mitochondrial Disease Adult Scale. Participants with the pathogenic m.3243A>G/T variants (m.3243A>G n=40; m.3243A>T n=1) exhibited significant elevations in Pure-Tone Audiometry thresholds, especially at high frequencies, suggesting cochlea involvement. Notably, the Listening in Spatialized Noise-Sentences test showed significant spatial processing deficits in the m.3243A>G/T group, possibly indicating a unique mutation-specific impact on central auditory processing. Auditory-evoked Brainstem Responses results highlighted a higher likelihood of auditory brainstem response abnormalities in this group, further substantiating neural/central auditory pathway involvement. This study emphasises the heterogeneous nature of hearing impairment in primary mitochondrial diseases, with a genotype-phenotype correlation, particularly in the m.3243A>G/T group. These insights advocate for personalised, genotype-specific auditory assessments and targeted management strategies. Conventional hearing aids and cochlear implants are ineffective for those with central auditory dysfunctions related to mitochondrial mutations. There is an urgent need for innovative rehabilitation strategies catering for both cochlear and neural/central auditory pathways.

Mitochondrial DNA Alterations in Glioblastoma and Current Therapeutic Targets.

Metabolic reprogramming within tumor cells involves a shift towards either glycolysis or mitochondrial respiration, depending on the stage of tumor progression. Consequently, irreversible dysfunction of the mitochondria is considered a crucial mechanism driving the progression mechanism. While numerous mutations in mitochondrial DNA (mtDNA) have been identified across various tumor types, including glioblastoma, many studies have been limited in the scope, focusing on small segments of mtDNA or utilizing sequencing methods with restricted sensitivity. As a result, several potentially significant mtDNA mutations may have been underestimated, along with their heteroplasmic states, which play a crucial role in determining the phenotypic impact of mtDNA mutation. Although both somatic and germline mtDNA mutations have been observed in different tumor types, research on the mtDNA mutations linked to glioblastoma remains scarce. The mitochondrial genome encodes thirteen protein-coding genes that are essential for the proper functioning of respiratory complex chains. Alterations in mitochondrial function manifest at various levels, including structural and functional changes, impacting mitogenic, hemodynamic, bioenergetic, and apoptotic signaling pathways. These alterations often signify a reduced efficiency of the oxidative phosphorylation system and energy production in tumor cells. As the crucial role of mitochondrial dysfunction in glioma development grows, mitochondria have emerged as promising targets for therapy aimed at overcoming chemoresistance and eliminating cancer cells. This brief review outlines the association between mtDNA alteration and glioblastoma, as well as the current advancements in therapeutic strategies targeting mtDNA alterations.

TRANscranial direct current stimulation for FOcal Refractory epilepsy in mitochondrial disease (TRANSFORM): delayed-start, randomised, double-blinded, placebo-controlled study

BackgroundFocal epilepsy is common in children and adults with mitochondrial disease. Seizures are often refractory to pharmacological treatment and, in this patient group, frequently evolve to refractory focal status epilepticus (also known as epilepsia partialis continua). Where this occurs, the long-term prognosis is poor. Transcranial DC stimulation (tDCS) is a promising, non-invasive, adjunctive treatment alternative to common surgical procedures. Limited recruitment of study participants with this rare disease and the ethical challenges of administering a treatment to one group and not another, while maintaining strict methodological rigour can pose challenges to the design of a clinical study.MethodWe designed the first delayed start, double-blinded, sham-controlled study to evaluate the efficacy of tDCS as an adjunctive treatment for focal epilepsy. We will include participants with a genetically confirmed diagnosis of mitochondrial disease with drug-resistant focal epilepsy aged ≥ 2 years, aiming to collect 30 episodes of focal status epilepticus, each treated for a maximum period of 14 days. The early start intervention arm will receive tDCS from day 1. The delayed start intervention arm will receive sham stimulation until crossover on day 3. Our primary endpoint is a greater than 50% reduction from baseline (on day 0) in seizure frequency assessed by 3x daily reporting, accelerometery, and video monitoring. Changes in the underlying epileptogenic focus within the brain related to the tDCS intervention will be assessed by magnetic resonance imaging (MRI) and/or electroencephalography (EEG).DiscussionStudy results in favour of treatment efficacy would support development of tDCS into a mainstream treatment option for focal epileptic seizures related to mitochondrial disease.Trials registrationISRCTN: 18,241,112; registered on 16/11/2021.

Digestive system and mitochondrial diseases in children

The article discusses the features of lesions of the digestive system in children with mitochondrial diseases. Mitochondria play an important role in cellular metabolism as they are responsible for producing the majority of cellular energy in the form of adenosine triphosphate. Mutations in mitochondrial genes responsible for the functioning of mitochondria can lead to various forms of mitochondrial diseases. These diseases may present with the following clinical symptoms: muscle weakness, movement disorders, neurological symptoms, impaired motility and absorption from the gastrointestinal tract. Diagnosis of mitochondrial diseases can be challenging due to genetic and clinical heterogeneity. Treatment currently remains a pressing problem, as research in this area is being actively conducted and new methods are emerging aimed at the therapy and treatment of these rare diseases.

Clinical characteristics of children with MT-TK gene m.8344A>G variation

Objective: To summarize the clinical characteristics of children carrying the m.8344A>G variant of MT-TK gene. Methods: A case series study was conducted to retrospectively collect data of 22 children with mitochondrial disease caused by MT-TK gene m.8344A>G variation who were treated at the Department of Neurology of Beijing Children's Hospital of Capital Medical University from January 2012 to January 2024. Their clinical data, laboratory tests, muscle pathology, genetic testing, and the follow-up results were analyzed. Pearson correlation analysis was used for correlation analysis. Results: Among the 22 children, there were 13 boys and 9 girls. The age of onset was 5.00 (2.75, 9.00) years. Fifteen children had myoclonic epilepsy with ragged-red fibers (MERRF), 3 had Leigh syndrome (LS), and 4 had LS-MERRF overlap syndrome (LS-MERRF). Myoclonus presented and worsened progressively in all 15 MERRF children, with 10 as the initial symptom and 5 developing progressively during the disease course. Myoclonus was predominantly focal, worsening with fine motor tasks or stress. Electroencephalogram monitoring in the 15 MERRF children revealed myoclonic seizures in 10 children, with 6 classified as myoclonic epilepsy, and 4 as subcortical myoclonus. Two children had generalized myoclonic seizures, and 1 each had absence seizures and generalized seizures. Twelve children had cerebellar ataxia, 10 children exhibited exercise intolerance, and 8 children had muscle weakness. Magnetic resonance imaging (MRI) revealed periventricular white matter involvement in 1 child and bilateral hippocampal involvement in 1 child, likely due to frequent seizures. All 3 children with LS exhibited developmental regressions, accompanied with 2 symptoms include cerebellar ataxia, muscle weakness, and dysphagia. The clinical manifestations of 4 LS-MERRF overlap children presented with combined features of MERRF and LS. Cranial MRI in the 7 LS and LS-MERRF children showed brainstem involvement (all affecting the midbrain) in 6 children and basal ganglia involvement in 4 children. Among the 22 children, 12 had m.8344A>G variant levels >90%, 3 had >80%-90%, 4 had >70%-80%, and 3 had >60%-70%. Higher variant level correlated with the LS phenotype and earlier onset age (r=0.47, -0.50; P=0.018 and 0.029, respectively). Sanger sequencing in 19 mothers revealed m.8344A>G variations in 18, with 4 showing exercise intolerance. Follow-up of 13 children on antimyoclonic treatment showed>75% reduction in seizures with levetiracetam monotherapy in 2 children, with combination therapy required in others. Most achieved >50% seizures reduction within 2 years, but the effectiveness declined with disease progression. Conclusions: The m.8344A>G variant is rare, with MERRF being the most common phenotype, while LS and LS-MERRF are less common. Children with higher ratio of the m.8344A>G variant are more likely to present LS phenotype. Myoclonus, primarily focal, is a key feature, with levetiracetam as the first-line treatment and benzodiazepines recommended for refractory cases.

Potential role of heteroplasmic mitochondrial DNA mutations in modulating the subtype-specific adaptation of oral squamous cell carcinoma to cisplatin therapy

Cancer cells are constantly evolving to adapt to environmental changes, particularly during exposure to drug treatment. In this work, we aimed to characterize genetic and epigenetic changes in mitochondrial DNA (mtDNA) that may increase the resistance of oral squamous cell carcinoma (OSCC) to cisplatin. We first derived drug-resistant cells from two human OSCC cell lines, namely SAS and H103, by continual cisplatin treatments for about 4 months. To determine mtDNA changes induced by cisplatin, we performed nanopore sequencing and quantitative polymerase chain reaction analysis of mtDNA extracted from the cells pre- and post-treatment. We also assessed the mitochondrial functions of the cells and their capacity to generate intracellular reactive oxygen species (ROS). We found that in the cisplatin-resistant cells derived from SAS, there was a reduction in mtDNA content and significant enrichment of a m.3910G > C mutation in the MT-ND1 gene. However, such changes were not detected in cisplatin-resistant H103 cells. The cisplatin treatment also altered methylation patterns in both SAS and H103 cells and decreased their sensitivity to ROS-induced cytotoxicity. We suggest that the sequence alterations and epigenetic changes in mtDNA and the reduction in mtDNA content could be key drivers of cisplatin resistance in OSCC. These mtDNA alterations may participate in cellular adaptation that serves as a response to adverse changes in the environment, particularly exposure to cytotoxic agents. Importantly, the observed mtDNA changes may be influenced by the distinct genetic landscapes of various cancer subtypes. Overall, this study reveals significant insights into cisplatin resistance driven by complex mtDNA dynamics, particularly in OSCC. This underscores the need for targeted therapies tailored to the genetic profiles of individual OSCC patients to improve disease prognosis.

Genetics of Exertional Heat Illness: Revealing New Associations and Expanding Heterogeneity

Environmental heat stress represents a pervasive threat to warfighters, athletes, and occupational workers, impacting performance and increasing the risk of injury. Exertional heat illness (EHI) is a spectrum of clinical disorders of increasing severity. While frequently predictable, EHI can occur unexpectedly and may be followed by long-term comorbidities, including cardiovascular dysfunction and exercise intolerance. The objective of this study was to assess genetic factors contributing to EHI. Whole-exome sequencing was performed in a cohort of 53 cases diagnosed with EHI. Rare variants in prioritized gene sets were analyzed and classified per published guidelines. Clinically significant pathogenic and potentially pathogenic variants were identified in 30.2% of the study cohort. Variants were found in 14 genes, including the previously known RYR1 and ACADVL genes and 12 other genes (CAPN3, MYH7, PFKM, RYR2, TRPM4, and genes for mitochondrial disorders) reported here for the first time in EHI. Supporting structural and functional studies of the TRPM4 p.Arg905Trp variant show that it impairs the thermal sensitivity of the TRPM4 channel, revealing a potentially new molecular mechanism contributing to EHI susceptibility. Our study demonstrates associations between EHI and genes implicated in muscle disorders, cardiomyopathies, thermoregulation, and oxidative phosphorylation deficiencies. These results expand the genetic heterogeneity of EHI and shed light on its molecular pathogenesis.

Neonatal Hemochromatosis Secondary to an Inborn Error of Metabolism (Mitochondrial Disorder): Post- mortem Diagnosis in the Fetus of a Young Primipara

Abstract Introduction/Objective Neonatal hemochromatosis (NH), an extremely rare congenital phenotype of acute hepatic failure with hepatic and extrahepatic siderosis, is primarily (98%) caused by gestational alloimmune liver disease (GALD) and, rarely (2%), by infection, metabolic disorders, bile acid defects, and mitochondrial hepatopathy. This report presents an extremely rare case of NH caused by an inborn error of metabolism (suspected mitochondrial dysfunction). This low-birth-weight female was born with acute liver failure, metabolic acidosis, multisystem failure, and progressive lactate acidosis, and expired on day 7. On autopsy, liver was less than 50% of normal size and without nodularity. Microscopic architecture was preserved with erythropoiesis, canalicular bile plugs, microvesicular steatosis (oil red O stain) and abundant coarsely granular siderosis (iron stain). GALD was unlikely because of the absence of the typical hepatocytic injury, fibrosis or biliary destruction, and the negative immunohistochemistry for MAC (C5b-C9). Extrahepatic siderosis was demonstrated in thyroid follicles, thymic Hassall corpuscles, myocardium, and adenohypophysis. Sanger sequencing and deletion testing of mitochondrial genome on a maternal buccal biopsy identified a homoplasmic variant (m.15482 T>C p.(S246P) in maternal MT-CYB gene but without molecular diagnosis of a mitochondrial DNA disorder. Infant plasma had increased C6DC, C5, C12-OH acylcarnitine, alanine, and proline. Urine contained elevated organic acids (lactate/pyruvate, 2-ketoacids, 3-methylglutarate, 3-methylglutaconate), concerning acute mitochondrial respiratory chain dysfunction. Methods/Case Report: Case Study Results (if a Case Study enter NA) NA Conclusion Diagnosis of NH warrants exclusion of the most common etiology, GALD. Mitochondrial genetic mutations are the most difficult to diagnosis because of heterogeneity and typically undiagnosed maternal defects. This is the first report of NH with a homoplasmic maternal variant of uncertain significance. Neonatal lactic acidosis is the strongest clinical clue for mitochondrial dysfunction and should trigger phenotyping, maternal mitochondrial genetic variants (mtDNA, nuDNA), functional, biochemical, and genetic studies.

Clinical and Genetic Spectrum of Patients With Mitochondrial Disease in a Pediatric Egyptian Cohort: Novel Variants and Phenotypic Expansion.

Mitochondrial disorders exhibit clinical and genetic diversity. Nearly 400 distinct genes, located in both the mitochondrial and nuclear genomes, harbor pathogenic variants that can produce a broad spectrum of mitochondrial diseases. This work aims to explore the genetic etiology of a cohort of Egyptian pediatric patients who were clinically suspected of having a mitochondrial disorder. A total of 49 patients from 44 unrelated families were studied. Selection criteria included age below 18 years and meeting Morava criteria (a score ≥ 3). The mitochondrial disease criteria (MDC) have been developed to quantify the clinical picture and evaluate the probability of an underlying mitochondrial disorder Exome sequencing, including mitochondrial genome sequencing, was carried out for each participant. Causative variants likely responsible for the phenotypes were identified in 68% of the study population. The mitochondrial subgroup constituted 41% of the studied population with a median age of 4 years. No primary pathogenic variants in mitochondrial DNA were detected. Pathogenic or likely pathogenic variants in eight mitochondrial genes were identified in 78% of the mitochondrial cohort. Additionally, seven novel variants were identified. Nonmitochondrial diagnoses accounted for 27% of the study population. In 32% of cases, disease-causing variants were not identified. The current study underscores the diverse phenotypic and genetic landscape of mitochondrial disorders among Egyptian patients.

Auditory and vestibular function in mitochondrial patients harbouring the m.3243A>G variant.

Hearing impairment is a frequent clinical feature in patients with mitochondrial disease harbouring the pathogenic variant, m.3243A>G. However, auditory neural dysfunction, its perceptual consequences and implications for patient management are not established. Similarly, the association with vestibular impairment has not yet been explored. This case-control study investigated in 12 adults with genetically confirmed m.3243A>G adults [9 females; 45.5 ± 16.3 years (range 18-66); 47.1 ± 21.5 hearing level, dB] compared with 12 age, sex and hearing level-matched controls with sensory (cochlear level) hearing loss [9 females; 46.6 ± 11.8 years (range 23-59); 47.7 ± 25.4 hearing level, dB]. Participants underwent a battery of electroacoustic, electrophysiologic and perceptual tests, which included pure tone audiometry, otoacoustic emissions, auditory brainstem responses, auditory temporal processing measures, monaural/binaural speech perception, balance and vestibular testing and self-reported questionnaires (dizziness and hearing disability). Our findings showed evidence of auditory neural abnormality and perceptual deficits greater than expected for cochlear pathology. Compared with matched controls with sensory hearing loss, adults with mitochondrial disease harbouring m.3243A>G had abnormal electrophysiologic responses from the VIII nerve and auditory brainstem (P = 0.005), an impaired capacity to encode rapidly occurring acoustic signal changes (P = 0.005), a reduced ability to localize sound sources (P = 0.028) and impaired speech perception in background noise (P = 0.008). Additionally, vestibular dysfunction (P = 0.011), greater perceived dizziness (P = 0.001) and reduced stance time (balance, P = 0.009) were also seen in participants with m.3243A>G mitochondrial disease when compared with matched counterparts. This pilot study revealed that auditory evaluation including evoked potential responses from the auditory nerve/brainstem and speech perception in noise tests should form an important part of the management for individuals with m.3243A>G-related mitochondrial disease. Those presenting with hearing impairment and symptoms concerning balance and dizziness should undergo vestibular testing and appropriate management.

TARS2 c.470 C > G is a chinese-specific founder mutation in three unrelated families with mitochondrial encephalomyopathy

Biallelic pathogenic variants in TARS2 lead to combined oxidative phosphorylation deficiency, subtype 21 (COXPD21, MIM #615918), which is a rare mitochondrial encephalomyopathy (ME) characterized by early-onset severe axial hypotonia, limb hypertonia, psychomotor developmental delay, epilepsy and brain anomalies. To date, approximately 28 individuals with COXPD21 and 28 TARS2 variants have been identified. In this study, we reported additional four individuals from three unrelated Chinese families with mitochondrial encephalomyopathy caused by pathogenic variants in TARS2, and described the novel clinical phenotypes and genotypic information. In addition to two novel variants (c.512G > A, p.Arg171Lys; c.988dup, p.Arg330Lysfs*4), one previously reported variant (c.470 C > G, p.Thr157Arg) recurred in six Chinese individuals with COXPD21 but was not present in populations of other races. Our findings expanded the mutation spectrum of TARS2 and confirmed that c.470 C > G is a Chinese-specific founder mutation. The novel phenotypes, including reduced fetal movement, eye anomalies and sleep irregularities, observed in our patients enriched the clinical characteristics of COXPD21.

Clinico-Radiological and Genotypic Spectrum of Nuclear Mitochondriopathies.

Mitochondrial disorders are a diverse group of diseases caused by mutations in genes encoded by either nuclear or mitochondrial DNA. In a group of patients with nuclear mitochondriopathies, the authors analysed the clinico-radiological and genotypic spectrum. The study included 25 patients with a genetic diagnosis of nuclear mitochondrial cytopathy who were seen over a 5 y period. There were 25 patients in the study cohort (Mean age of presentation- 14 mo). Biallelic mutations involving nuclear mitochondrial genes were identified in all 25 of them. In 13 and 9 patients, respectively, respiratory chain defects (complex I and complex IV) and mitochondrial DNA depletion syndromes were identified. Twelve novel variants were identified. Interestingly, NDUFV1 with a south Indian founder variant c.1156C > T (p.Arg386Cys) was the commonest variant. Accurate phenotyping combined with next generation sequencing aids in the precise diagnosis of mitochondrial nuclear gene defects and provides the opportunity for appropriate counseling.

Taurine hypomodification underlies mitochondrial tRNATrp-related genetic diseases.

Escherichia coli MnmE and MnmG form a complex (EcMnmEG), generating transfer RNA (tRNA) 5-carboxymethylaminomethyluridine (cmnm5U) modification. Both cmnm5U and equivalent 5-taurinomethyluridine (τm5U, catalyzed by homologous GTPBP3 and MTO1) are found at U34 in several human mitochondrial tRNAs (hmtRNAs). Certain mitochondrial DNA (mtDNA) mutations, including m.3243A>G in tRNALeu(UUR) and m.8344A>G in tRNALys, cause genetic diseases, partially due to τm5U hypomodification. However, whether other mtDNA variants in different tRNAs cause a defect in τm5U biogenesis remains unknown. Here, we purified naturally assembled EcMnmEG from E. coli. Notably, EcMnmEG was able to incorporate both cmnm5U and τm5U into hmtRNATrp (encoded by MT-TW), providing a valuable basis for directly monitoring the effects of mtDNA mutations on U34 modification. In vitro, several clinical hmtRNATrp pathogenic mutations caused U34 hypomodification. A patient harboring an m.5541C>T mutation exhibited hmtRNATrp τm5U hypomodification. Moreover, using mtDNA base editing, we constructed two cell lines carrying m.5532G>A or m.5545C>T mutations, both of which exhibited hmtRNATrp τm5U hypomodification. Taurine supplementation improved mitochondrial translation in patient cells. Our findings describe the third hmtRNA species with mutation-related τm5U-hypomodification and provide new insights into the pathogenesis and intervention strategy for hmtRNATrp-related genetic diseases.

Disease models of Leigh syndrome: From yeast to organoids.

Leigh syndrome (LS) is a severe mitochondrial disease that results from mutations in the nuclear or mitochondrial DNA that impairs cellular respiration and ATP production. Mutations in more than 100 genes have been demonstrated to cause LS. The disease most commonly affects brain development and function, resulting in cognitive and motor impairment. The underlying pathogenesis is challenging to ascertain due to the diverse range of symptoms exhibited by affected individuals and the variability in prognosis. To understand the disease mechanisms of different LS-causing mutations and to find a suitable treatment, several different model systems have been developed over the last 30 years. This review summarizes the established disease models of LS and their key findings. Smaller organisms such as yeast have been used to study the biochemical properties of causative mutations. Drosophila melanogaster, Danio rerio, and Caenorhabditis elegans have been used to dissect the pathophysiology of the neurological and motor symptoms of LS. Mammalian models, including the widely used Ndufs4 knockout mouse model of complex I deficiency, have been used to study the developmental, cognitive, and motor functions associated with the disease. Finally, cellular models of LS range from immortalized cell lines and trans-mitochondrial cybrids to more recent model systems such as patient-derived induced pluripotent stem cells (iPSCs). In particular, iPSCs now allow studying the effects of LS mutations in specialized human cells, including neurons, cardiomyocytes, and even three-dimensional organoids. These latter models open the possibility of developing high-throughput drug screens and personalized treatments based on defined disease characteristics captured in the context of a defined cell type. By analyzing all these different model systems, this review aims to provide an overview of past and present means to elucidate the complex pathology of LS. We conclude that each approach is valid for answering specific research questions regarding LS, and that their complementary use could be instrumental in finding treatment solutions for this severe and currently untreatable disease.

In response to: "Involvement of the endocrine system is common in mitochondrial disorders and requires long-term comprehensive investigations".

In response to: "Involvement of the endocrine system is common in mitochondrial disorders and requires long-term comprehensive investigations".

Can endogenous ethylene glycol production occur in humans? A detailed investigation of adult monozygotic twin sisters

Introduction To the best of our knowledge, clinically significant endogenous ethylene glycol production has never been reported in humans, very seldom reported in other animals or microorganisms, and then only under rare and specific conditions. We describe the detailed investigations we undertook in two adult monozygotic twin sisters to ascertain whether they were producing endogenous ethylene glycol. Methods Two previously healthy monozygotic adult twin sisters presented with recurrent episodes of apparent ethylene glycol poisoning beginning at age 35, requiring chronic hemodialysis to remove ethylene glycol and its metabolites as well as to restore metabolic homeostasis. The sisters denied ingestion or exposure to ethylene glycol. At their request, they were admitted to hospital under strict supervision to exclude surreptitious ingestion of ethylene glycol and to evaluate the need for treatment. Hemodialysis was withheld during this prospective study. Twin A was admitted for 14 days and twin B for 11 days. Serial biochemical analyses were performed in blood and urine. Clinical exome sequencing and mitochondrial deoxyribonucleic acid sequencing were also completed. Results In both twins, ethylene glycol was detected in urine, along with intermittent increases in concentrations of lactate, glycolate, and glycine in blood and/or urine. Blood ethylene glycol concentrations, however, remained <62 mg/L (<1 mmol/L) but became positive soon after discharge. The oxalate concentration remained normal in blood and urine. Plasma and urine amino acid profiles showed intermittent small increases in glycine, serine, taurine, proline, and/or alanine concentrations. Exome sequencing and mitochondrial deoxyribonucleic acid sequencing were non-diagnostic. Neither twin has been admitted with metabolic acidosis nor ethylene glycol poisoning since chronic hemodialysis was started. Twin A developed a calcium oxalate dihydrate lithiasis. Discussion Mitochondrial disease, methylmalonic/propionic/isovaleric aciduria, primary hyperoxaluria, and analyte error were all excluded in these twins, as were obvious common environmental exposures. Conclusion Detailed investigations were performed in adult monozygotic twin sisters to ascertain whether they were producing endogenous ethylene glycol. Alternative explanations were excluded to the very best of our efforts and knowledge. Global metabolomics, gut microbiome analyses, and whole genome sequencing are pending.

Compensatory activity of the PC-ME1 metabolic axis underlies differential sensitivity to mitochondrial complex I inhibition.

Deficiencies in the electron transport chain (ETC) lead to mitochondrial diseases. While mutations are distributed across the organism, cell and tissue sensitivity to ETC disruption varies, and the molecular mechanisms underlying this variability remain poorly understood. Here we show that, upon ETC inhibition, a non-canonical tricarboxylic acid (TCA) cycle upregulates to maintain malate levels and concomitant production of NADPH. Our findings indicate that the adverse effects observed upon CI inhibition primarily stem from reduced NADPH levels, rather than ATP depletion. Furthermore, we find that Pyruvate carboxylase (PC) and ME1, the key mediators orchestrating this metabolic reprogramming, are selectively expressed in astrocytes compared to neurons and underlie their differential sensitivity to ETC inhibition. Augmenting ME1 levels in the brain alleviates neuroinflammation and corrects motor function and coordination in a preclinical mouse model of CI deficiency. These studies may explain why different brain cells vary in their sensitivity to ETC inhibition, which could impact mitochondrial disease management.

Resolving Phenotypic Variability in Mitochondrial Diseases: Preliminary Findings of a Proteomic Approach

The introduction of new sequencing approaches into clinical practice has radically changed the diagnostic approach to mitochondrial diseases, significantly improving the molecular definition rate in this group of neurogenetic disorders. At the same time, there have been no equal successes in the area of in-depth understanding of disease mechanisms and few innovative therapeutic approaches have been proposed recently. In this regard, the identification of the molecular basis of phenotypic variability in primary mitochondrial disorders represents a key aspect for deciphering disease mechanisms with important therapeutic implications. In this study, we present data from proteomic investigations in two subjects affected by mitochondrial disease characterized by a different clinical severity and associated with the same variant in the TWNK gene, encoding the mitochondrial DNA and RNA helicase with a specific role in the mtDNA replisome. Heterozygous pathogenic variants in this gene are associated with progressive external ophthalmoplegia and ptosis, usually with adult onset. The overall results suggest an imbalance in glucose metabolism and ROS production/regulation, with possible consequences on the phenotypic manifestations of the enrolled subjects. Although the data will need to be validated in a large cohort, proteomic investigations have proven to be a valid approach for a deep understanding of these neurometabolic disorders.