dldhcri3 zebrafish exhibit altered mitochondrial ultrastructure, morphology, and dysfunction partially rescued by probucol or thiamine.

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disease caused by variants in DLD, the E3 subunit of mitochondrial α-keto (or 2-oxo) acid dehydrogenase complexes. DLD disease symptoms are multisystemic, variably manifesting as Leigh syndrome, neurodevelopmental disability, seizures, cardiomyopathy, liver disease, fatigue, and lactic acidemia. While most DLD disease symptoms are attributed to dysfunction of the pyruvate dehydrogenase complex, the effects of other α-keto acid dehydrogenase deficiencies remain unclear. Current therapies for DLD deficiency are ineffective, with no vertebrate animal model available for preclinical study. We created a viable Danio rerio (zebrafish) KO model of DLD deficiency, dldhcri3. Detailed phenotypic characterization revealed shortened larval survival, uninflated swim bladder, hepatomegaly and fatty liver, and reduced swim activity. These animals displayed increased pyruvate and lactate levels, with severe disruption of branched-chain amino acid catabolism manifest as increased valine, leucine, isoleucine, α-ketoisovalerate, and α-ketoglutarate levels. Evaluation of mitochondrial ultrastructure revealed gross enlargement, severe cristae disruption, and reduction in matrix electron density in liver, intestines, and muscle. Therapeutic modeling of candidate therapies demonstrated that probucol or thiamine improved larval swim activity. Overall, this vertebrate model demonstrated characteristic phenotypic and metabolic alterations of DLD disease, offering a robust platform to screen and characterize candidate therapies.

N-Glycosylation of MRS2 balances aerobic and anaerobic energy production by reducing rapid mitochondrial Mg2+ influx in conditions of high glucose or impaired respiratory chain function.

N-linked glycoproteins function in numerous biological processes, modulating enzyme activities as well as protein folding, stability, oligomerization, and trafficking. While N-glycosylation of mitochondrial proteins has been detected by untargeted MS-analyses, the physiological existence and roles of mitochondrial protein N-linked glycosylation remain under debate. Here, we report that MRS2, a mitochondrial inner membrane protein that functions as the high flux magnesium transporter, is N-glycosylated to various extents depending on cellular bioenergetic status. Both N-glycosylated and unglycosylated isoforms were consistently detected in mitochondria isolated from mouse liver, rat and mouse liver fibroblast cells (BRL 3A and AFT024, respectively) as well as human skin fibroblast cells. Immunoblotting of MRS2 showed it was bound to, and required stringent elution conditions to remove from, lectin affinity columns with covalently bound concanavalin A or Lens culinaris agglutinin. Following peptide:N-glycosidase F (PNGase F) digestion of the stringently eluted proteins, the higher Mr MRS2 bands gel-shifted to lower Mr and loss of lectin affinity was seen. BRL 3A cells treated with two different N-linked glycosylation inhibitors, tunicamycin or 6-diazo-5-oxo-l-norleucine, resulted in decreased intensity or loss of the higher Mr MRS2 isoform. To investigate the possible functional role of MRS2 N- glycosylation, we measured rapid Mg2+ influx capacity in intact mitochondria isolated from BRL 3A cells in control media or following treatment with tunicamycin or 6-diazo-5-oxo-l-norleucine. Interestingly, rapid Mg2+ influx capacity increased in mitochondria isolated from BRL 3A cells treated with either N-glycosylation inhibitor. Forcing reliance on mitochondrial respiration by treatment with either galactose media or the glycolytic inhibitor 2-deoxyglucose or by minimizing glucose concentration similarly reduced the N-glycosylated isoform of MRS2, with a correlated concomitant increase in rapid Mg2+ influx capacity. Conversely, inhibiting mitochondrial energy production in BRL 3A cells with either rotenone or oligomycin resulted in an increased fraction of N-glycosylated MRS2, with decreased rapid Mg2+ influx capacity. Collectively, these data provide strong evidence that MRS2 N-glycosylation is directly involved in the regulation of mitochondrial matrix Mg2+, dynamically communicating relative cellular nutrient status and bioenergetic capacity by serving as a physiologic brake on the influx of mitochondrial matrix Mg2+ under conditions of glucose excess or mitochondrial bioenergetic impairment.

Long-Term POTS Outcomes Survey: Diagnosis, Therapy, and Clinical Outcomes.

BACKGROUND: Limited data exist on long-term outcomes in individuals with postural orthostatic tachycardia syndrome (POTS). We designed an electronic questionnaire assessing various aspects of outcomes among patients diagnosed and treated in a single-center pediatric POTS clinical program. METHODS AND RESULTS: The LT-POTS (Long Term POTS Outcomes Survey) included questions about quality of life, symptoms, therapies, education, employment, and social impact of disease. Patients age≤18 years at POTS diagnosis who were managed in the Children's Hospital of Philadelphia POTS Program were included. A total of 227 patients with POTS responded with sufficient data for interpretation. The mean age of respondents was 21.8±3.5 years. The median age of symptom onset was 13 (interquartile range 11-14) years, with mean 9.6±3.4 years symptom duration. Multiple cardiovascular, neurologic, and gastrointestinal symptoms were reported. Symptom prevalence and severity were worse for female patients, with 99% of patients reporting ongoing symptoms. Quality of life showed moderate function and limitation, with more severe limitations in energy/fatigue and general health. Nearly three quarters of patients had diagnostic delays, and over half were told that their symptoms were "in their head." Multiple medications were used and were felt to be effective, whereas fewer nonpharmacologic interventions demonstrated efficacy. Nearly 90% of patients required continued nonpharmacologic therapy to control symptoms. CONCLUSIONS: POTS is a chronic disorder leading to significant disability with a range of multisystem problems. Although symptoms can be modifiable, it rarely spontaneously resolves. Improved understanding of POTS presentation and therapeutic approaches may inform provider education, improve diagnostic success, and help patients self-advocate for appropriate medical management approaches.

Expanding the Spectrum of Congenital Myopathy Linked to Variants in the MYBPC1 Gene: A Clinical Report.

Objectives: Heterozygous missense variants in MYBPC1 have been recently identified in 13 patients from 6 families with congenital myopathy with tremor. All the patients had mild skeletal myopathy invariably associated with a distinctive myogenic tremor and hypotonia with gradual clinical improvement. However, no phenotypic description has been reported for the neonatal respiratory impairment that patients may suffer. Methods: We report 3 new patients from 2 independent families with congenital myopathy with tremor. Results: Tremors and respiratory distress associated with stridor should raise the diagnosis of congenital myopathy with tremors linked to MYBPC1-dominant variants in children with neonatal hypotonia. Discussion: Neonatal severe respiratory impairment requiring intensive noninvasive ventilation because of stridor is described in 2 patients. Stridor was previously reported in one other case and is part of the clinical features.

Bridging the clinical-research gap: Harnessing an electronic data capture, integration, and visualization platform to systematically assess prospective patient-reported outcomes in mitochondrial medicine.

PURPOSE: Optimizing individualized clinical care in heterogeneous rare disorders, such as primary mitochondrial disease (PMD), will require gaining more comprehensive and objective understanding of the patient experience by longitudinally tracking quantifiable patient-specific outcomes and integrating subjective data with clinical data to monitor disease progression and targeted therapeutic effects. METHODS: Electronic surveys of patient (and caregiver) reported outcome (PRO) measures were administered in REDCap within clinical domains commonly impaired in patients with PMD in the context of their ongoing routine care, including quality of life, fatigue, and functional performance. Descriptive statistics, group comparisons, and inter-measure correlations were used to evaluate system feasibility, utility of PRO results, and consistency across outcome measure domains. Real-time tracking and visualization of longitudinal individual-level and cohort-level data were facilitated by a customized data integration and visualization system, MMFP-Tableau. RESULTS: An efficient PRO electronic capture and analysis system was successfully implemented within a clinically and genetically heterogeneous rare disease clinical population spanning all ages. Preliminary data analyses demonstrated the flexibility of this approach for a range of PROs, as well as the value of selected PRO scales to objectively capture qualitative functional impairment in four key clinical domains. High inter-measure reliability and correlation were observed. Between-group analyses revealed that adults with PMD reported significantly worse quality of life and greater fatigue than did affected children, while PMD patients with nuclear gene disorders reported lower functioning relative to those with an mtDNA gene disorder in several clinical domains. CONCLUSION: Incorporation of routine electronic data collection, integration, visualization, and analysis of relevant PROs for rare disease patients seen in the clinical setting was demonstrated to be feasible, providing prospective and quantitative data on key clinical domains relevant to the patient experience. Further work is needed to validate specific PROs in diverse PMD patients and cohorts, and to formally evaluate the clinical impact and utility of harnessing integrated data systems to objectively track and integrate quantifiable PROs in the context of rare disease patient clinical care.

MSeqDR Quick-Mitome (QM): Combining Phenotype-Guided Variant Interpretation and Machine Learning Classifiers to Aid Primary Mitochondrial Disease Genetic Diagnosis.

The international Mitochondrial Disease Sequence Data Resource Consortium (MSeqDR) Quick-Mitome (QM) is a web-based platform enabling automated variant interpretation of whole-exome sequencing (WES) datasets for the genetic diagnosis of primary mitochondrial diseases (PMD). Designed specifically to address the unique dual genome nature of PMD etiologies, QM includes features for both nuclear and mitochondrial DNA (mtDNA) genome analysis. QM requires VCF variant lists, HPO ID clinical phenotypes, and pedigree files for multiple-sample VCF inputs. QM maps phenotypes to HPO terms before analysis. QM analysis requires 2 to 20 min for 100,000 variants on an 8-vCPU AWS server using Exomiser's "PASS_ONLY" mode for nuclear variants. QM ranks variants based on allele frequency, phenotype-gene association, functional impact, and inheritance mode. Variants are further annotated with multiple data sources such as OMIM, ClinVar, dbNSFP, gnoMAD, MITOMAP, and MSeqDR. In addition to standard Exomiser results, QM generates an Analysis Report and QM Integrated Report with add-on mtDNA-specific analyses, including haplogroup prediction with Phy-Mer, heteroplasmy calculation, and mvTool annotations. We developed the Mitochondrial Disease Variant (MDV) classifier using XGBoost to predict variant pathogenicity for PMD. The MDV classifier was trained on >120 features and performance benchmarking showed that it correctly classified >98% of nuclear gene variants as being pathogenic or benign, and predicted PMD-causing variants with 94% precision. The MSeqDR QM server is an open-access resource for phenotype-driven dual-genome analyses for PMD diagnosis by the global mitochondrial disease community. It is publicly available for non-commercial, non-clinical research use at https://mseqdr.org/quickmitome.php. © 2024 Wiley Periodicals LLC. Basic Protocol 1: Standardizing clinical phenotypes into human phenotype ontology (HPO) terms as the phenotype input for Quick-Mitome (QM) Basic Protocol 2: Prepare the pedigree input for multiple-sample VCF Basic Protocol 3: Quick-Mitome (QM) analysis Basic Protocol 4: Reviewing and understanding the QM Integrated Report and Analysis Report.

Kagami Ogata syndrome: a small deletion refines critical region for imprinting.

Kagami-Ogata syndrome is a rare imprinting disorder and its phenotypic overlap with multiple different etiologies hampers diagnosis. Genetic etiologies include paternal uniparental isodisomy (upd(14)pat), maternal allele deletions of differentially methylated regions (DMR) in 14q32.2 or pure primary epimutations. We report a patient with Kagami-Ogata syndrome and an atypical diagnostic odyssey with several negative standard-of-care genetic tests followed by epigenetic testing using methylation microarray and a targeted analysis of whole-genome sequencing to reveal a 203 bp deletion involving the MEG3 transcript and MEG3:TSS-DMR. Long-read sequencing enabled the simultaneous detection of the deletion, phasing, and biallelic hypermethylation of the MEG3:TSS-DMR region in a single assay. This case highlights the challenges in the sequential genetic testing paradigm, the utility of long-read sequencing as a single comprehensive diagnostic assay, and the smallest reported deletion causing Kagami-Ogata syndrome allowing important insights into the mechanism of imprinting effects at this locus.

Novel Development of Magnetic Resonance Imaging to Quantify the Structural Anatomic Growth of Diverse Organs in Adult and Mutant Zebrafish.

Zebrafish (Danio rerio) is a widely used vertebrate animal for modeling genetic diseases by targeted editing strategies followed by gross phenotypic and biomarker characterization. While larval transparency permits microscopic detection of anatomical defects, histological adult screening for organ-level defects remains invasive, tedious, inefficient, and subject to technical artifact. Here, we describe a noninvasive magnetic resonance imaging (MRI) approach to systematically screen adult zebrafish for anatomical growth defects. An anatomical atlas of wild-type (WT) zebrafish at 5-31 months post-fertilization was created by ex vivo MRI with a 9.4 T magnet. Volumetric growth over time was measured of animals and major organs, including the brain, spinal cord, heart, eyes, optic nerve, ear, liver, kidneys, and swim bladder. Subsequently, surf1-/-, fbxl4-/-, and opa1+/- mitochondrial disease mutant adult zebrafish were quantitatively studied to compare organ volumes with age-matched WT zebrafish. Results demonstrated that MRI enabled noninvasive, high-resolution, rapid screening of mutant adult zebrafish for overall and organ-specific growth abnormalities. Detailed volumetric analyses of three mitochondrial disease mutants delineated specific organ differences, including significantly increased brain growth in surf1-/- and opa1+/-, and marginally significant decreased heart and spinal cord volumes in surf1-/- mutants. This is interesting as we know neurological involvement can be seen in SURF1-/- patients with ataxia, dystonia, and lesions in basal ganglia, as well as in OPA1+/- patients with spasticity, ataxia, and hyperreflexia indicative of neuropathology. Similarly, cardiomyopathy is a known sequelae of cardiac pathology in patients with SURF1-/--related disease. Future studies will define MRI signaling patterns of organ dysfunction to further delineate specific pathology.

Optimized Nutrition in Mitochondrial Disease Correlates to Improved Muscle Fatigue, Strength, and Quality of Life.

We sought to prospectively characterize the nutritional status of adults ≥ 19 years (n = 22, 27% males) and children (n = 38, 61% male) with genetically-confirmed primary mitochondrial disease (PMD) to guide development of precision nutritional support strategies to be tested in future clinical trials. We excluded subjects who were exclusively tube-fed. Daily caloric requirements were estimated using World Health Organization (WHO) equations to predict resting energy expenditure (REE) multiplied by an activity factor (AF) based on individual activity levels. We developed a Mitochondrial Disease Activity Factors (MOTIVATOR) score to encompass the impact of muscle fatigue typical of PMD on physical activity levels. PMD cohort daily diet intake was estimated to be 1,143 ± 104.1 kcal in adults (mean ± SEM, 76.2% of WHO-MOTIVATOR predicted requirement), and 1,114 ± 62.3 kcal in children (86.4% predicted). A total of 11/22 (50%) adults and 18/38 (47.4%) children with PMD consumed ≤ 75% predicted daily Kcal needs. Malnutrition was identified in 16/60 (26.7%) PMD subjects. Increased protein and fat intake correlated with improved muscle strength in those with insufficient daily Kcal intake (≤ 75% predicted); higher protein and fat intake correlated with decreased muscle fatigue; and higher protein, fat, and carbohydrate intake correlated with improved quality of life (QoL). These data demonstrate the frequent occurrence of malnutrition in PMD and emphasize the critical need to devise nutritional interventions to optimize clinical outcomes.

Multiple Independent Gene Disorders Causing Bardet-Biedl Syndrome, Congenital Hypothyroidism, and Hearing Loss in a Single Indian Patient.

We report a 20-year-old, female, adopted Indian patient with over 662 Mb regions of homozy-gosity who presented with intellectual disability, ataxia, schizophrenia, retinal dystrophy, moder-ate-to-severe progressive sensorineural hearing loss (SNHL), congenital hypothyroidism, cleft mi-tral valve with mild mitral valve regurgitation, and dysmorphic features. Exome analysis first on a clinical basis and subsequently on research reanalysis uncovered pathogenic variants in three nu-clear genes following two modes of inheritance that were causal to her complex phenotype. These included (1) compound heterozygous variants in BBS6 potentially causative for Bardet-Biedl syn-drome 6; (2) a homozygous, known pathogenic variant in the stereocilin (STRC) gene associated with nonsyndromic deafness; and (3) a homozygous variant in dual oxidase 2 (DUOX2) gene asso-ciated with congenital hypothyroidism. A variant of uncertain significance was identified in a fourth gene, troponin T2 (TNNT2), associated with cardiomyopathy but not the cleft mitral valve, with mild mitral regurgitation seen in this case. This patient was the product of an apparent first-degree relationship, explaining the multiple independent inherited findings. This case high-lights the need to carefully evaluate multiple independent genetic etiologies for complex pheno-types, particularly in the case of consanguinity, rather than presuming unexplained features are expansions of known gene disorders.

Efficacy and Safety of Elamipretide in Individuals With Primary Mitochondrial Myopathy: The MMPOWER-3 Randomized Clinical Trial.

BACKGROUND AND OBJECTIVES: Primary mitochondrial myopathies (PMMs) encompass a group of genetic disorders that impair mitochondrial oxidative phosphorylation, adversely affecting physical function, exercise capacity, and quality of life (QoL). Current PMM standards of care address symptoms, with limited clinical impact, constituting a significant therapeutic unmet need. We present data from MMPOWER-3, a pivotal, phase-3, randomized, double-blind, placebo-controlled clinical trial that evaluated the efficacy and safety of elamipretide in participants with genetically confirmed PMM. METHODS: After screening, eligible participants were randomized 1:1 to receive either 24 weeks of elamipretide at a dose of 40 mg/d or placebo subcutaneously. Primary efficacy endpoints included change from baseline to week 24 on the distance walked on the 6-minute walk test (6MWT) and total fatigue on the Primary Mitochondrial Myopathy Symptom Assessment (PMMSA). Secondary endpoints included most bothersome symptom score on the PMMSA, NeuroQoL Fatigue Short-Form scores, and the patient global impression and clinician global impression of PMM symptoms. RESULTS: Participants (N = 218) were randomized (n = 109 elamipretide; n = 109 placebo). The m0ean age was 45.6 years (64% women; 94% White). Most of the participants (n = 162 [74%]) had mitochondrial DNA (mtDNA) alteration, with the remainder having nuclear DNA (nDNA) defects. At screening, the most frequent bothersome PMM symptom on the PMMSA was tiredness during activities (28.9%). At baseline, the mean distance walked on the 6MWT was 336.7 ± 81.2 meters, the mean score for total fatigue on the PMMSA was 10.6 ± 2.5, and the mean T score for the Neuro-QoL Fatigue Short-Form was 54.7 ± 7.5. The study did not meet its primary endpoints assessing changes in the 6MWT and PMMSA total fatigue score (TFS). Between the participants receiving elamipretide and those receiving placebo, the difference in the least squares mean (SE) from baseline to week 24 on distance walked on the 6MWT was -3.2 (95% CI -18.7 to 12.3; p = 0.69) meters, and on the PMMSA, the total fatigue score was -0.07 (95% CI -0.10 to 0.26; p = 0.37). Elamipretide treatment was well-tolerated with most adverse events being mild to moderate in severity. DISCUSSION: Subcutaneous elamipretide treatment did not improve outcomes in the 6MWT and PMMSA TFS in patients with PMM. However, this phase-3 study demonstrated that subcutaneous elamipretide is well-tolerated. TRIAL REGISTRATION INFORMATION: Trial registered with clinicaltrials.gov, Clinical Trials Identifier: NCT03323749; submitted on October 12, 2017; first patient enrolled October 9, 2017. CLINICALTRIALS: gov/ct2/show/NCT03323749?term = elamipretide&draw = 2&rank = 9. CLASSIFICATION OF EVIDENCE: This study provides Class I evidence that elamipretide does not improve the 6MWT or fatigue at 24 weeks compared with placebo in patients with primary mitochondrial myopathy.

Expert Panel Curation of 113 Primary Mitochondrial Disease Genes for the Leigh Syndrome Spectrum.

OBJECTIVE: Primary mitochondrial diseases (PMDs) are heterogeneous disorders caused by inherited mitochondrial dysfunction. Classically defined neuropathologically as subacute necrotizing encephalomyelopathy, Leigh syndrome spectrum (LSS) is the most frequent manifestation of PMD in children, but may also present in adults. A major challenge for accurate diagnosis of LSS in the genomic medicine era is establishing gene-disease relationships (GDRs) for this syndrome with >100 monogenic causes across both nuclear and mitochondrial genomes. METHODS: The Clinical Genome Resource (ClinGen) Mitochondrial Disease Gene Curation Expert Panel (GCEP), comprising 40 international PMD experts, met monthly for 4 years to review GDRs for LSS. The GCEP standardized gene curation for LSS by refining the phenotypic definition, modifying the ClinGen Gene-Disease Clinical Validity Curation Framework to improve interpretation for LSS, and establishing a scoring rubric for LSS. RESULTS: The GDR with LSS across the nuclear and mitochondrial genomes was classified as definitive for 31 of 114 GDRs curated (27%), moderate for 38 (33%), limited for 43 (38%), and disputed for 2 (2%). Ninety genes were associated with autosomal recessive inheritance, 16 were maternally inherited, 5 were autosomal dominant, and 3 were X-linked. INTERPRETATION: GDRs for LSS were established for genes across both nuclear and mitochondrial genomes. Establishing these GDRs will allow accurate variant interpretation, expedite genetic diagnosis of LSS, and facilitate precision medicine, multisystem organ surveillance, recurrence risk counseling, reproductive choice, natural history studies, and determination of eligibility for interventional clinical trials. ANN NEUROL 2023;94:696-712.

N-acetylcysteine and cysteamine bitartrate prevent azide-induced neuromuscular decompensation by restoring glutathione balance in two novel surf1-/- zebrafish deletion models of Leigh syndrome.

SURF1 deficiency (OMIM # 220110) causes Leigh syndrome (LS, OMIM # 256000), a mitochondrial disorder typified by stress-induced metabolic strokes, neurodevelopmental regression and progressive multisystem dysfunction. Here, we describe two novel surf1-/- zebrafish knockout models generated by CRISPR/Cas9 technology. While gross larval morphology, fertility, and survival into adulthood appeared unaffected, surf1-/- mutants manifested adult-onset ocular anomalies and decreased swimming activity, as well as classical biochemical hallmarks of human SURF1 disease, including reduced complex IV expression and enzymatic activity and increased tissue lactate. surf1-/- larvae also demonstrated oxidative stress and stressor hypersensitivity to the complex IV inhibitor, azide, which exacerbated their complex IV deficiency, reduced supercomplex formation, and induced acute neurodegeneration typical of LS including brain death, impaired neuromuscular responses, reduced swimming activity, and absent heartrate. Remarkably, prophylactic treatment of surf1-/- larvae with either cysteamine bitartrate or N-acetylcysteine, but not other antioxidants, significantly improved animal resiliency to stressor-induced brain death, swimming and neuromuscular dysfunction, and loss of heartbeat. Mechanistic analyses demonstrated cysteamine bitartrate pretreatment did not improve complex IV deficiency, ATP deficiency, or increased tissue lactate but did reduce oxidative stress and restore glutathione balance in surf1-/- animals. Overall, two novel surf1-/- zebrafish models recapitulate the gross neurodegenerative and biochemical hallmarks of LS, including azide stressor hypersensitivity that was associated with glutathione deficiency and ameliorated by cysteamine bitartrate or N-acetylcysteine therapy.

Dichloroacetate and thiamine improve survival and mitochondrial stress in a C. elegans model of dihydrolipoamide dehydrogenase deficiency.

Dihydrolipoamide dehydrogenase (DLD) deficiency is a recessive mitochondrial disorder caused by depletion of DLD from α-ketoacid dehydrogenase complexes. Caenorhabditis elegans animal models of DLD deficiency generated by graded feeding of dld-1(RNAi) revealed that full or partial reduction of DLD-1 expression recapitulated increased pyruvate levels typical of pyruvate dehydrogenase complex deficiency and significantly altered animal survival and health, with reductions in brood size, adult length, and neuromuscular function. DLD-1 deficiency dramatically increased mitochondrial unfolded protein stress response induction and adaptive mitochondrial proliferation. While ATP levels were reduced, respiratory chain enzyme activities and in vivo mitochondrial membrane potential were not significantly altered. DLD-1 depletion directly correlated with the induction of mitochondrial stress and impairment of worm growth and neuromuscular function. The safety and efficacy of dichloroacetate, thiamine, riboflavin, 5-aminoimidazole-4-carboxamide-1-ß-d-ribofuranoside (AICAR), l-carnitine, and lipoic acid supplemental therapies empirically used for human DLD disease were objectively evaluated by life span and mitochondrial stress response studies. Only dichloroacetate and thiamine showed individual and synergistic therapeutic benefits. Collectively, these C. elegans dld-1(RNAi) animal model studies demonstrate the translational relevance of preclinical modeling of disease mechanisms and therapeutic candidates. Results suggest that clinical trials are warranted to evaluate the safety and efficacy of dichloroacetate and thiamine in human DLD disease.

Pathogenic mtDNA variants, in particular single large-scale mtDNA deletions, are strongly associated with post-lingual onset sensorineural hearing loss in primary mitochondrial disease.

In this retrospective cohort study of 193 consecutive subjects with primary mitochondrial disease (PMD) seen at the Children's Hospital of Philadelphia Mitochondrial Medicine Frontier Program, we assessed prevalence, severity, and time of onset of sensorineural hearing loss (SNHL) for PMD cases with different genetic etiologies. Subjects were grouped by genetic diagnosis: mitochondrial DNA (mtDNA) pathogenic variants, single large-scale mtDNA deletions (SLSMD), or nuclear DNA (nDNA) pathogenic variants. SNHL was audiometrically confirmed in 27% of PMD subjects (20% in mtDNA pathogenic variants, 58% in SLSMD and 25% in nDNA pathogenic variants). SLSMD had the highest odds ratio for SNHL. SNHL onset was post-lingual in 79% of PMD cases, interestingly including all cases with mtDNA pathogenic variants and SLSMD, which was significantly different from PMD cases caused by nDNA pathogenic variants. SNHL onset during school age was predominant in this patient population. Regular audiologic assessment is important for PMD patients, and PMD of mtDNA etiology should be considered as a differential diagnosis in pediatric patients and young adults with post-lingual SNHL onset, particularly in the setting of multi-system clinical involvement. Pathogenic mtDNA variants and SLSMD are less likely etiologies in subjects with congenital, pre-lingual onset SNHL.

Qualitative exploration of the lived experience of adults diagnosed with primary mitochondrial disease.

Primary mitochondrial disease (PMD) encompasses a heterogeneous group of energy deficiency disorders that are typically progressive, with affected individuals experiencing an average of 16 multisystem symptoms. Clinical trials are emerging, but current treatment options remain limited. In PMD, the effect of specific disease factors and their relationship to meaning-based coping has not been studied. Given the connection between prognostic uncertainty and psychological distress in other patient populations, we explored the lived experience of adults with PMD. Adults with PMD caused by pathogenic variant(s) in nuclear or mitochondrial genes impairing mitochondrial function were interviewed. Interview questions addressed the lived experience with PMD, diagnostic journey, practical learnings at the time of diagnosis, suggestions for supportive information to provide at diagnosis, diagnosis impact on daily living and self-care, and sources of support and hope. Focus group transcripts were analyzed using thematic analysis. Four themes (diagnostic challenges, adaptations to daily living, social implications, and meaning-based coping) and several subthemes (the importance of being hopeful and benefit finding) emerged. Most participants reported strong family support (9/14) and identified a benefit (9/14) derived from their PMD diagnosis, while (5/14) did not identify any benefits. Benefit finding, reframing, and maintaining a positive attitude emerged as common coping in adults living with PMD. Understanding how adults with PMD cope is essential to provide anticipatory guidance and ongoing support for those struggling with their disease diagnosis, progression, and broader life impact. Our findings suggest that adult PMD patients prefer healthcare providers to inquire about their emotional well-being and meaning based coping with PMD.

Dichloroacetate improves mitochondrial function, physiology, and morphology in FBXL4 disease models.

Pathogenic variants in the human F-box and leucine-rich repeat protein 4 (FBXL4) gene result in an autosomal recessive, multisystemic, mitochondrial disorder involving variable mitochondrial depletion and respiratory chain complex deficiencies with lactic acidemia. As no FDA-approved effective therapies for this disease exist, we sought to characterize translational C. elegans and zebrafish animal models, as well as human fibroblasts, to study FBXL4-/- disease mechanisms and identify preclinical therapeutic leads. Developmental delay, impaired fecundity and neurologic and/or muscular activity, mitochondrial dysfunction, and altered lactate metabolism were identified in fbxl-1(ok3741) C. elegans. Detailed studies of a PDHc activator, dichloroacetate (DCA), in fbxl-1(ok3741) C. elegans demonstrated its beneficial effects on fecundity, neuromotor activity, and mitochondrial function. Validation studies were performed in fbxl4sa12470 zebrafish larvae and in FBXL4-/- human fibroblasts; they showed DCA efficacy in preventing brain death, impairment of neurologic and/or muscular function, mitochondrial biochemical dysfunction, and stress-induced morphologic and ultrastructural mitochondrial defects. These data demonstrate that fbxl-1(ok3741) C. elegans and fbxl4sa12470 zebrafish provide robust translational models to study mechanisms and identify preclinical therapeutic candidates for FBXL4-/- disease. Furthermore, DCA is a lead therapeutic candidate with therapeutic benefit on diverse aspects of survival, neurologic and/or muscular function, and mitochondrial physiology that warrants rigorous clinical trial study in humans with FBXL4-/- disease.

Community Consensus Guidelines to Support FAIR Data Standards in Clinical Research Studies in Primary Mitochondrial Disease.

Primary mitochondrial diseases (PMD) are genetic disorders with extensive clinical and molecular heterogeneity where therapeutic development efforts have faced multiple challenges. Clinical trial design, outcome measure selection, lack of reliable biomarkers, and deficiencies in long-term natural history data sets remain substantial challenges in the increasingly active PMD therapeutic development space. Developing "FAIR" (findable, accessible, interoperable, reusable) data standards to make data sharable and building a more transparent community data sharing paradigm to access clinical research metadata are the first steps to address these challenges. This collaborative community effort describes the current landscape of PMD clinical research data resources available for sharing, obstacles, and opportunities, including ways to incentivize and encourage data sharing among diverse stakeholders. This work highlights the importance of, and challenges to, developing a unified system that enables clinical research structured data sharing and supports harmonized data deposition standards across clinical consortia and research groups. The goal of these efforts is to improve the efficiency and effectiveness of drug development and improve understanding of the natural history of PMD. This initiative aims to maximize the benefit for PMD patients, research, industry, and other stakeholders while acknowledging challenges related to differing needs and international policies on data privacy, security, management, and oversight.

Early developmental delay in Leigh syndrome spectrum disorders is associated with poor clinical prognosis.

BACKGROUND: Leigh spectrum syndrome (LSS) is a primary mitochondrial disorder characterized by neurodevelopmental regression and metabolic stroke typically in early life. Developmental delay (DD) is known to follow episodes of neurologic regression in LSS, although primary developmental delay (pDD) has been rarely reported. We hypothesized that pDD precedes regression in a broader subset of LSS individuals and may associate with worse long-term educational outcomes. METHODS: From a retrospective cohort, subjects with pathogenic variant(s) in a nuclear or mitochondrial gene associated with LSS and consistent clinical manifestations and neuroradiological findings. Detailed developmental histories and neurologic outcomes were extracted. RESULTS: Of 69 LSS subjects, 47 (68.1%) had a history of pDD and 53 (76.8%) had neurodevelopmental regression. We identified 3 distinct developmental phenotypes: [1] pDD followed by regression (N = 31/69, 44.9%), [2] pDD without subsequent regression (16/69, 23.2%), [3] regression without pDD (N = 22/69, 31.9%). A history of pDD was associated with earlier disease onset (p = 0.0003) and worse educational outcomes (OR 22.14). CONCLUSION: LSS is associated with multiple developmental phenotypes and pDD is associated with negative educational outcomes. pDD occurring prior to neurologic regression suggests that mitochondrial energetics impact developmental trajectories prior to acute metabolic failure and regression, providing an opportunity for earlier diagnosis and/or therapeutic intervention.

Variants in ADD1 cause intellectual disability, corpus callosum dysgenesis, and ventriculomegaly in humans.

PURPOSE: Adducins interconnect spectrin and actin filaments to form polygonal scaffolds beneath the cell membranes and form ring-like structures in neuronal axons. Adducins regulate mouse neural development, but their function in the human brain is unknown. METHODS: We used exome sequencing to uncover ADD1 variants associated with intellectual disability (ID) and brain malformations. We studied ADD1 splice isoforms in mouse and human neocortex development with RNA sequencing, super resolution imaging, and immunoblotting. We investigated 4 variant ADD1 proteins and heterozygous ADD1 cells for protein expression and ADD1-ADD2 dimerization. We studied Add1 functions in vivo using Add1 knockout mice. RESULTS: We uncovered loss-of-function ADD1 variants in 4 unrelated individuals affected by ID and/or structural brain defects. Three additional de novo copy number variations covering the ADD1 locus were associated with ID and brain malformations. ADD1 is highly expressed in the neocortex and the corpus callosum, whereas ADD1 splice isoforms are dynamically expressed between cortical progenitors and postmitotic neurons. Human variants impair ADD1 protein expression and/or dimerization with ADD2. Add1 knockout mice recapitulate corpus callosum dysgenesis and ventriculomegaly phenotypes. CONCLUSION: Our human and mouse genetics results indicate that pathogenic ADD1 variants cause corpus callosum dysgenesis, ventriculomegaly, and/or ID.