28th Annual Conference of the United Mitochondrial Disease Foundation Abstracts - Mitochondrial Medicine 2025 St. Louis, MO – June 18-21, 2025

Grafstein J 1* , Howe C 1 , Van Hove J 2 , Malicdan MCV 3 , Gahl WA 1,4 , Toro C 1 1 Undiagnosed Diseases Program, NHGRI, NIH, Bethesda, MD, USA, 2 Department of Pathology and Laboratory Medicine, Children’s Hospital Colorado, Aurora, CO, USA, 3 Human Biochemical Genetics Section, Medical Genetics Branch, NHGRI, NIH, Bethesda, MD, USA, 4 Section of Biochemical Genetics, NHGRI, NIH, Bethesda, MD, USA *(Corresponding author’s email [email protected] ) Abstract: Mitochondrial dysfunction, including defects in mtDNA replication or mitochondrial nucleotide metabolism, impairs cellular aerobic energy generation. Mitochondrial dysfunction can take a particularly high toll on tissues that require a great deal of energy, such as the central nervous system and skeletal muscle. Because of the clinical heterogeneity of mitochondrial disorders, the path to diagnosis can be long and arduous, as the mean time to diagnosis is nearly a decade long. We present a 56-year-old woman with a progressive neurological syndrome associated with severe cerebellar ataxia, disabling bilateral tinnitus, S/P bowel resection due to chronic colonic inertia (hypomotility) necessitating chronic ostomy, dysphagia, dysarthria, gait issues, cognitive decline, visual impairment, refractory depression, and debilitating fatigue. Her symptoms first manifested in 2002, when she was just 34 years old, as colonic hypomotility. Over time, she has suffered from progressive vision loss and profound neurosensory hearing loss bilaterally status post (S/P) cochlear implants. Initially thought to be a case of autoimmune-mediated cerebellar ataxia, she was not responsive to immune modulators. mtDNA content on a muscle biopsy revealed a significant mitochondrial depletion syndrome. The multisystemic and pleiotropic nature of her symptoms highlights frequent challenges to diagnosing mitochondrial disorders and the need for comprehensive, multidisciplinary expertise. Abstract #: 2025PA-0000000161 Presenter: Pham Thi Hong Anh

Cortes DRE 1,2 , Hartwick S 1 , Becker-Szurszewski T 1 , West D 1 , Coulson NW 1 , Goetzman ES 1 , Christodoulou, AG 3 , Wu, YL 1,2 1 Departments of Pediatrics, University of Pittsburgh, USA, 2 Department of Bioengineering, University of Pittsburgh, USA, 3 Department of Radiological Sciences, University of California, Los Angeles, USA *(Corresponding author’s email [email protected] ) Abstract: Mitochondrial dysfunction is a critical element for wide ranges of inborn and acquired brain pathological conditions, such as mitochondrial respiratory chain disorders (MRCD), traumatic brain injury (TBI), epilepsy, stroke, neurodegenerative diseases, childhood-onset epileptic encephalopathy and psychiatric disorders. Neurons are highly sensitive to changes in oxygen and mitochondrial metabolism because oxidative phosphorylation, not glycolysis, powers presynaptic and postsynaptic activity, and broadly neurodevelopment, neurogenesis and synaptic plasticity. However, no non-invasive method is available that can probe in vivo mitochondrial functions in the intact brain in a spatially specific manner. Mitochondrial Complex I Deficiency is a devastating progressive neuro-degenerative disease, caused by genetic defects in the Complex I of the mitochondrial electron transport chain (ETC). It is unknown why genetic defects of the mitochondrial Complex I can result in very heterogeneous clinical presentations with very different life expectancy in Leigh Syndrome, LHON, MERRF, and MELAS. Differential neurological symptoms suggest different brain regions are affected differently. However, genetic testing cannot diagnose the ETC functions in the brain. There is a major knowledge gap in identifying brain-region specific mechanisms underlying heterogeneous neurological syndromes due to a lack of in vivo tools for brain-region specific functional assessment of ETC in live brains for differential diagnosis and therapeutic efficacy. Brain lesions on anatomical T 2 -weighted FLAIR MRI are found in Complex I disorders. However, many non-specific factors can contribute to T 2 hyperintensity, such as brain tissue loss, edema, neuroinflammation, or demyelination. Thus, it cannot be a specific biomarker for Complex I disorders. More importantly, by the time brain lesions are detectable by anatomical MRI, irreversible damage to that brain region has already occurred; it is too late for intervention, thus cannot be used to evaluate therapeutic efficacy. There is a lack of sensitive and robust tools to evaluate therapeutic efficacy in live brains before irreversible brain tissue damage occurs. We have successfully developed a novel 4D Oxy-wavelet MRI (US patents 11,828,825 and 18,636,419) capable of probing in vivo mitochondrial dysfunction in intact live brains in a spatially specific manner with both high spatial [0.000474 mm 3 ] and temporal (~ 14 msec) resolutions. We will present the preclinical utility of 4D Oxy-wavelet MRI in probing mitochondrial dysfunction in rodent models of genetic MRCD, early-onset epileptic encephalopathy, as well as fetal radiation injury, fetal alcohol spectrum disorder, traumatic brain injury, neurodegenerative disease, and acquired epilepsy. Abstract #: 2025PA-0000000043 Presenter: Lía Mayorga

Yadav T 1 , Baker S 1 , Rutter J 1,2 1 University of Utah, Salt Lake City, UT, USA, 2 Howard Hughes Medical Institute [email protected] Abstract: The inner mitochondrial membrane (IMM) harbors transmembrane (TM) proteins involved in critical cellular functions. The unique structure, composition, and prokaryotic origins of the IMM impose unique biochemical constraints on these proteins. Thus, IMM-targeted TM proteins need to balance stability in the IMM with the ability to traverse the aqueous environment of the cytosol and intermembrane space. We hypothesized that these constraints might be broadly observed across eukaryotic evolution as mitochondrial DNA (mtDNA) genes relocated to the nucleus. Since mitochondrial defects cause severe disorders, understanding these evolutionary changes in IMM proteins is imperative. This project aims to identify sequence adaptations in IMM TM proteins that balance targeting efficiency with membrane stability over eukaryotic evolution, and test if these adaptations can be leveraged for mitochondrial gene therapy. We performed bioinformatic and AlphaFold analyses to map changes across mitochondrial evolution, combining sequence information with 3D structural information. This hybrid approach allowed us to perform markedly improved alignments than what traditional sequence alignment would yield. We carried out three independent analyses of the compositional landscape of IMM TMDs employing various IMM protein datasets. First, we evaluated if human IMM TM proteins possess unique biochemical features that stabilize them in the IMM. Next, we aligned orthologous proteobacterial and eukaryotic IMM TMDs to assess evolutionary compositional changes. Then, we analyzed shifts during mitochondrial evolution in eukaryotes and examined mtDNA-encoded proteins that were relocated to the nucleus. Finally, we tested the feasibility of our findings for mitochondrial gene therapy by allotopically (i.e., from the nucleus) expressing mtDNA-encoded proteins and assessing their targeting in HEK293Ts using confocal microscopy. We found that over evolution, IMM-targeted TM proteins reduce hydrophobicity by substituting their aliphatic residues with Thr- which we term Aliphatic to Threonine Substitutions (ATS). We observed the same preference for Thr in all three analyses of mitochondrial TM proteins. This strategy allows mitochondrial TM protein targeting to balance targeting, which requires lower hydrophobicity with IMM stability, which necessitates high hydrophobicity. The amphipathic nature of Thr in ATS successfully lowers hydrophobicity without compromising membrane stability. Thr is a unique amino acid since it is neither too hydrophilic nor too hydrophobic, and this unique advantage is why ATS has been employed so extensively across eukaryotic evolution to achieve IMM protein targeting. Interestingly, independent nuclear transfers of the same protein ATP6, were accompanied by the same ATS-mediated reduction in hydrophobicity, indicating its efficiency. Hence, we tested whether the ATS strategy could be used for expressing mtDNA-encoded proteins since efficient targeting is a major hurdle for mitochondrial gene therapy. As proof-of-principle, our experiments showed that when an ATS-substituted ATP6 protein is expressed in HEK293Ts, it successfully targets to the mitochondria without cytoplasmic aggregation or ER mislocalization. In summary, our findings can inform gene therapies for mitochondrial disorders and reveal conserved principles of mitochondrial proteome evolution. Abstract #: 2025PA-0000000157 Presenter: Madeleine Hesselgesser

McGinn DE 1* , Flickinger J 1 , Sultan L 2 , Morgan T 2 , Gendler L 2 , Rahaman I 1 , Nguyen S 1 , Ogiso E 1 , Morisue J 1 , Serai S 3,4 , Nguyen J 5 , Senthil K 6 , George-Sankoh I 1 , Chinwalla A 7 , Xiao R 8,9 , Anderson VE 1 , Ischiropoulos H 10 , Nadkarni VM 6,12 , Falk MJ 1,12 , Adams JA 11 , Zolkipli-Cunningham Z 1,12 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA, 2 Center for Pediatric Contrast Ultrasound, Department of Radiology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, 3 Department of Radiology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, 4 Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA: 5 Section of Musculoskeletal Imaging, Department of Radiology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, 6 Department of Anesthesiology, Critical Care and Pediatrics, The Children's Hospital of Philadelphia, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA, 7 Department of Biomedical and Health Information, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, 8 Division of Biostatistics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, 9 Epidemiology and Informatics, Department of Biostatistics, University of Pennsylvania, Philadelphia, PA, USA, 10 Children's Hospital of Philadelphia Research Institute and Departments of Pediatrics and Pharmacology, Raymond and Ruth Perelman School of Medicine at the University of Pennsylvania, Philadelphia, PA, USA, 11 Division of Neonatology, Mount Sinai Medical Center, Miami Beach, FL, United States, 12 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA(* [email protected] ) Abstract: Primary mitochondrial disease (PMD) affects high-energy-demanding organs, leading to exercise intolerance, fatigue, and muscle weakness 1 , often resulting in a sedentary lifestyle. Periodic acceleration (pGz) passive exercises, involving head-to-footward motion of a supine body using a specialized bed (pGz-BED) and passive jogging using a specialized rhythmic-device (Gentle Jogger [GJ]), have demonstrated cardiovascular benefits in other patient populations by inducing pulsatile shear stress, which upregulates endothelial nitric oxide synthase and nitric oxide 2,3 . This study evaluates the feasibility and physiological impact of pGz-BED and GJ passive exercise strategies compared to conventional active cardiopulmonary exercise testing (CPET) in PMD. Older children and adults (10-60 years) with PMD and healthy controls completed three separate exercise interventions: (i) conventional CPET, (ii) pGz-BED passive exercise, and (iii) GJ passive exercise. Participants avoided strenuous activity 48 hours prior to each study visit. The primary outcome was maximal oxygen consumption (VO₂ max ), a key measure of mitochondrial aerobic capacity, assessed before and after intervention. Secondary outcomes included the pulse waveform a/b ratio (a biomarker of nitric oxide-mediated vasodilation 4 ), contrast-enhanced ultrasound (CEUS) imaging for muscular flow assessment, and serum lactate/pyruvate, glutathione, ketone bodies panel, and nitric oxide metabolites. Outcome measures were compared between the intervention types and between PMD and healthy participants. 18 subjects have enrolled to date, including 8 PMD and 10 control subjects. Following conventional exercise, VO₂ max increased as expected in both PMD (change (δ),17.9 ± 6.55, n=2) and control (26.2 ± 4.81, n=3) subjects. However, following pGz-exercise (pGz-BED and GJ), VO₂ levels showed only a modest increase in PMD (0.17 ± 0.11, n=5) subjects and did not increase for controls (0.06 ± 0.05, n=4). An increase in the a/b ratio was observed in PMD subjects after both active (2.4 ± 1.4) and pGz-exercise (0.39 ± 0.23). Notably, following pGz-exercise, a/b ratio was higher in PMD subjects compared to controls (0.14 ± 0.10) trending towards significance (p=0.08). CEUS imaging demonstrated a significant increase in perfusion index (PI) in PMD subjects (7.35 ± 1.1, n=3) compared to controls (0.02 ± 0.37, n=2) following GJ intervention (p=0.03). PMD and control subjects had similar increase in PI following active exercise. Metabolic markers revealed no change in lactate levels following passive exercise, whereas plasma lactate markedly increased [~5 fold] following CPET for all subjects. However, the Beta-Hydroxybutyrate(BHB)/Acetoacetate(AcAc) ratio, a measure of NADH/NAD⁺ redox balance, was elevated (2.94, normal 0.48-2.56) in PMD subjects following pGz-exercise compared to controls (2.49), suggesting altered mitochondrial redox status. PMD and healthy controls had similarly elevated BHB/AcAc ratios following active exercise. Preliminary findings suggest that pGz-exercise induces a vasodilatory response in PMD patients, as evidenced by increased a/b ratio and PI on CEUS imaging, which may be linked to underlying systemic endothelial dysfunction not previously recognized in PMD. Furthermore, results demonstrate PMD patients had similar, though less pronounced, physiologic responses to pGz-exercise compared to active exercise. These findings highlight passive exercise as a feasible intervention to quantify dysfunction in PMD. It may also merit further investigation into its long-term cardiovascular and metabolic benefits as a treatment intervention in PMD. References We will be conducting Gentle Jogger assessments in the Clinical Research Pavilion at the 2025 annual conference. 1. Zolkipli-Cunningham Z, Xiao R, Stoddart A, McCormick EM, Holberts A, Burrill N, McCormack S, Williams L, Wang X, Thompson JLP, Falk MJ. Mitochondrial disease patient motivations and barriers to participate in clinical trials. PLoS One. 2018 May 17;13(5):e0197513. doi: 10.1371/journal.pone.0197513. PMID: 29771953; PMCID: PMC5957366. 2. J. A. Adams, M. J. Mangino, J. Bassuk, P. Kurlansky, and M. A. Sackner, “Regional blood flow during periodic acceleration,” Crit. Care Med., vol. 29, no. 10, pp. 1983–1988, Oct. 2001, doi: 10.1097/00003246-200110000-00022. 3. M. Fujita et al., “Periodic acceleration enhances release of nitric oxide in healthy adults,” Int. J. Angiol., vol. 14, no. 1, pp. 11–14, Feb. 2005, doi: 10.1007/s00547-005-2013-2. 4. Adams, J.A., Lopez, J.R., Banderas, V. et al. A single arm trial using passive simulated jogging for blunting acute hyperglycemia. Sci Rep 11, 6437 (2021). https://doi.org/10.1038/s41598-021-85579-7 Abstract #: 2025PA-0000000106 Presenters: Arnold Z. Olali

Melissa A. Walker MD, PhD 1,2,3,4* , Henry A. Shull AB 1 , Maria Miranda PhD 1,2,3 , Vamsi K. Mootha MD 1,2,3 1 Department of Molecular Biology, Massachusetts General Hospital, Boston, Massachusetts, USA, 2 Department of Systems Biology, Harvard Medical School, Boston, Massachusetts, USA, 3 Broad Institute, Cambridge, Massachusetts, USA, 4 Department of Neurology, Massachusetts General Hospital, Boston, Massachusetts, USA* [email protected] Abstract: In contrast to the nuclear genome, mitochondrial DNA (mtDNA) copy number (CN) measurements are highly variable by tissue type, physiologic status, and disease state. Past observational studies have reported variable associations of peripheral blood mtDNA CN and several physiologic states and diseases, provoking hypotheses relating mitochondrial function or dysfunction to these pathologies. However, peripheral blood is composed of multiple distinct cell types, the proportions of which vary across individuals, clinical status, and time. In this context, our ongoing work seeks to elucidate absolute mtDNA CN distribution by blood cell type. Here we present results of per cell absolute quantification of mtDNA CN by quantitative polymerase chain reaction (qPCR) and fluorescence confocal microscopy of 10 flow sorted blood cell types, hematopoietic stem cells, and hematologic malignancy-derived cell lines. Qualitative assessment of mtDNA in these cell types was additionally performed via fluorescence confocal microscopy. Finally, we use concurrent clinical grade complete blood counts for each blood sample to estimate the relative contribution of each cell type to total peripheral blood mtDNA CN. Based on these data, we conclude that anucleate blood cells contain meaningful mtDNA content, and in bulk account for a significant proportion of whole blood mtDNA. Consequently, variation in blood composition must be considered in any comparison of whole blood mtDNA copy number across individuals or groups. Abstract #: 2025PA-0000000099 Presenter: Margaret Means, MD

Swankita Godara 1 , Annaliese Andsager 1 , Morgan Schroeder 1 , Divine Idahosa 1 , Fatoumata Dabo 1 , Brian Kennedy 2 , Matt Kaeberlein 3 , Temugin Berta 4 , Katherine E. Vest 4 , Anthony S. Grillo 1 1 Department of Chemistry, University of Cincinnati, USA, 2 Department of Biochemistry, National University of Singapore, Singapore, 3 Department of Laboratory Medicine and Pathology, University of Washington, Seattle, USA, 4 College of Medicine, University of Cincinnati, USA * [email protected] Abstract: Maple Syrup Urine Disease (MSUD) is an inherited mitochondrial disorder caused by a deficiency in the branched-chain α-keto acid dehydrogenase (BCKDH) enzyme resulting in the accumulation of branched-chain amino acids (BCAAs) and their corresponding keto acids (BCKAs) in body fluids. This accumulation leads to a range of clinical features, including poorly understood dysfunctions in skeletal muscle and brain. Current treatment options involve life-long dietary restriction and expensive liver transplantation. Due to limited understanding of the disease’s pathophysiology, MSUD remains an orphan disease in urgent need of novel therapeutic interventions. In our study, we identified that the FDA-approved mTOR inhibitor, rapamycin, significantly improved disease symptoms and extended survival in MSUD mouse models through tissue-specific mechanisms. Our data suggests the existence of both mTOR-sensitive and mTOR-insensitive pathogenic pathways corresponding with brain and skeletal muscle degeneration. Using high-performance liquid chromatography (HPLC), we demonstrated that rapamycin reduces the levels of BCAAs and BCKAs in iMSUD mouse model. Our data reflected that mice showing severe muscle defects such as hindlimb weakness and muscle atrophy were unaffected by rapamycin, but it reduced neurodegeneration in iMSUD mice with mild muscle defects. Furthermore, we applied rapamycin treatment to the most prevalent and severe subtype classic MSUD (cMSUD) mice model, typically die within three days of birth. Remarkably, rapamycin significantly extended the lifespan of these mice. Overall, our findings support rapamycin as a promising therapeutic candidate for MSUD. Abstract #: 2025PA-0000000154 Presenter: Tarun Yadav

Rahaman, I. 1 , Flickinger, J. 1,2 , Santos, J.D. 1 , Ly A. 1 , Kaushal, P. 1 , Stanley, K. 1 , Macmullen, L. 1 , Peterson, J. 1 , Martin, I. 1 , O’Leary, S. 1,2 , Fogliano, J. 1,2 , Ballance, E., Xiao, R. 3,4 , Zolkipli-Cunningham, Z. 1,3* 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, USA, 2 Division of Rehabilitation, Children’s Hospital of Philadelphia, USA, 3 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA, 4 Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania Perelman School of Medicine, USA. * [email protected] Abstract: We utilized the Zeno TM Walkway Gait Analysis System 1 (Zeno walkway) and the mSway TM inertial sensor 2 (mSway) to characterize gait and balance in genetically confirmed and verified subjects with primary mitochondrial disease (PMD) and individuals with self-reported PMD (SR-PMD), compared to healthy volunteers at the 2023 and 2024 United Mitochondrial Disease Foundation (UMDF) Symposium Clinical Research Pavilion. To demonstrate clinical meaning, digital wearables results were correlated to Mitochondrial Myopathy Composite Assessment Tool (MM-COAST) assessments 3 previously validated in Mitochondrial Myopathy patients. Subjects completed a six-minute walk test (6MWT) incorporating the Zeno walkway, and MM-COAST static balance assessments of single leg stance with eyes closed (SLEC), and tandem stance with eyes closed (TSEC) and eyes open (TSEO) incorporating mSway sensors. Gait assessments were performed in 51 PMD (mean age ± SD, 28.3 ± 15.4 years, 49.0% female), 14 SR-PMD (45.2 ± 17.9 years, 92.8% female), and 35 controls (42.3 ± 15.9 years, 65.7% female). Balance assessments were completed in 18 PMD (22.3 ± 10.8 years, 77.8% female), 17 SR-PMD (37.1 ± 16.4 years, 82.4% female), and 14 controls (34.6 ± 10.9 years, 78.6% female). Subjects were asked to hold a stance for as long as possible (maximum duration of 20 seconds) with an mSway sensor secured to their lower back and right lower extremity. Gait characteristics, including stride width (cm), length (cm), velocity (cm/sec), cadence (steps/min), stance percentage (%), single support percentage (SSP%), and single support center of pressure distance percentage 1 (SSCOPD%), were significantly different between PMD (n=51) and controls (n=35), p<0.05. Longitudinal analysis by linear mixed-effects models (LMMs) for repeated measures revealed significant decrease over 6 minutes (slope) in PMD subjects in stride velocity (-1.99 cm/s/min, p<0.001), cadence (-1.29 steps/min 2 , p<0.001), and SSCOPD% (-0.54 %/min, p0.05. The SR-PMD group also demonstrated significant decline in stride velocity (-1.44 cm/s/min, p<0.001), cadence (-0.99 steps/min 2 , p<0.001), and SSCOPD% (-0.3 %/min, p<0.001) compared to controls. For PMD, we identified significant correlations between all gait parameters with MM-COAST measures including composite score ([min, max]), r= [-0.68,0.52]) and SLEC z-score (r=[-0.33,0.5]), p<0.05. For PMD (n=18), analysis of mSway balance parameters including change in mean sway velocity in the vertical (V), anterior-posterior (AP), and medial-lateral (ML) direction (m/s 2 ) and normalized vertical jerk index 4 were observed to be significantly different compared to controls (n=14), p<0.05, demonstrating higher and more variable sway while standing tandem stance or on one leg. Longitudinal analysis by LMM revealed a significant decrease over 20s (slope) for TSEO sway (-22.4 mm 2 /s 2 , p<0.001), demonstrating a larger range of movement in PMD compared to controls, that did not change over time (-7.36 mm 2 /s 2 , p=0.3). Our results demonstrate the utility of digital assessments in PMD for enhancing MM-COAST clinical assessments through quantification of specific gait impairments that could be measured in future natural history studies and clinical trials. References 1. Lynall RC, Zukowski LA, Plummer P, Mihalik JP. Reliability and validity of the Protokinetics movement analysis software in measuring center of pressure during walking. Gait Posture. 2017;52:308–311 2. Palmerini L, Rocchi L, Mellone S, Valzania F, Chiari L. Feature selection for accelerometer-based posture analysis in Parkinson's disease. IEEE Trans Inf Technol Biomed. 2011 May;15(3):481-90. doi: 10.1109/TITB.2011.2107916. Epub 2011 Feb 24. PMID: 21349795, 3. Flickinger, J., Fan, J., Wellik, A., Ganetzky, R., Goldstein, A., Muraresku, C. C., Glanzman, A. M., Ballance, E., Leonhardt, K., McCormick, E. M., Soreth, B., Nguyen, S., Gornish, J., George-Sankoh, I., Peterson, J., MacMullen, L. E., Vishnubhatt, S., McBride, M., Haas, R., Falk, M. J., Xiao, R., and Zolkipli-Cunningham, Z. (2021) Development of a Mitochondrial Myopathy-Composite Assessment Tool. JCSM Clinical Reports, 6: 109– 127. https://doi.org/10.1002/crt2.41 4. Baudendistel ST, Schmitt AC, Balthaser KC, Wade FE, Hass CJ. The effect of limb selection methods on gait analysis in Parkinson's disease. Parkinsonism & Related Disorders 2022; published online 12 October 2022. Abstract #: 2025PA-0000000073 Presenter: Asha Anand, MD

Matei Ionita 1,2 , Richard Schretzenmair 1 , Derek Jones 1 , Kelsey Keith 3# , Matthew Sullenberger 4 , Benjamin R. Fischer 4 , Hakon Hakonarson 3,5 , Jonni Moore 1 , Marni J. Falk 4,5* 1 Penn Cytomics & Cell Sorting Shared Resource Laboratory, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 2 Institute for Immunology and Immune Health, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 3 Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA, 4 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, 5 Center for Applied Genomics, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, 6 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, *Corresponding author [email protected] # Presenting author Abstract: Primary mitochondrial diseases (PMDs) are a highly heterogenous group of genetic disorders, with multi-system symptoms ranging in severity and timing of clinical progression. Immune system dysfunction is common in PMD, where patients are often vulnerable to disease progression at the time of viral infections and may not completely respond to vaccination. PMD patients with pathogenic mtDNA variants often show evidence of purifying selection against the variant in blood cells, indicative of mitochondrial dysfunction directly harming immune function. Immune system dysfunction characterization in PMD patients has been limited, with existing work largely limited to case reports or examination of a single cell type in a single PMD disorder. To systematically characterize the immune system in diverse PMD patients, deep cell immunophenotyping of peripheral blood mononuclear cells (PBMCs) was performed on a spectrally enhanced BD Symphony X50 instrument using a customized 41 parameter flow cytometry panel designed for simultaneous broad typing of lymphocytes and monocytes, with particular focus on T cell activation, memory, and senescence markers. After data acquisition, analysis was performed with an automated computational pipeline, classifying cell types in a hierarchical fashion using multinomial logistic regression to assign each cell as debris or to one of the 8 major cell types. Major cell types were then divided into predetermined phenotypes using relevant markers, ending with 56 immune sub-features, represented as proportions of the major cell populations characterized. Testing for association between PMD and immune features in pediatric subjects, defined as less than 18 years old (27 cases, 16 controls), and adult subjects (18 cases, 14 controls) were tested separately due to immune system maturation in adults. In the pediatric cohort, 19 of 56 features were significantly different in PMD patients, with differential features associated with more mature or more exhausted T cells (fewer Naïve and CD28+ T cells, more EMRA, CD57+, and KLRG1+ T cells). PMD patients also had more NK cells (CD57+). No significant differences were found in PMD adults, suggestive of age-dependent immune effects in PMD. Principal component analysis (PCA) also revealed an immune maturity axis, progressing from pediatric controls to pediatric cases, then to all adults irrespective of disease status. This study delineates a unique immune signature in pediatric PMD, characterized by heightened immune cell maturity and senescence, which may contribute to immune dysregulation and increased susceptibility to infections in affected individuals. Furthermore, the novel 41-parameter immunophenotyping panel and computational methodology established in this work offer broad applicability to human immunology research. Abstract #: 2025PA-0000000080 Presenter: Herodes Guzman

Gordon-Lipkin, EM 1 , Hesselgesser, MM 1 , Kruk, S 1 , McGuire, PJ 1 1 Metabolism, Infection and Immunity Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD * [email protected] Abstract: Background: Leigh Syndrome (LS) is subtype of Mitochondrial Disease (MtD) that classically presents as a progressive neurologic disease with developmental regression. In its initial descriptions, the diagnosis occurred postmortem by identification of basal ganglia lesions on autopsy. However, modern neuroimaging has facilitated the diagnosis in life with the use of MRI. Now, modern genomics has allowed physicians to noninvasively identify pathogenic variants that are biochemically associated with the LS phenotype. Frequently, when patients are diagnosed with LS, the history may reveal early concerns that occurred prior to their first metabolic decompensation or developmental regression. The goal of this study is to identify the earliest symptoms of LS to facilitate earlier diagnosis and opportunities for intervention. Methods: Patients examined were enrolled on the NIH MINI Study and were identified as LS and Leigh Like Syndrome (LLS) per the criteria in Lake et al 2016.1,2 Clinical data was extracted retrospectively from medical records supplied by patient families. Results: 29 patients (37.9% female and 62.1% male) were included. All patients had molecular diagnosis with a gene associated with LS (48.3% LS and 51.7% LLS). The average age of first parental concerns within our initial cohort was 1.4 (SD: 1.8) years with the range from prenatal to 7 years. The average age of MRI imaging for our cohort was 3.6 (SD: 4.1) years with a range of 0 to 13 years. The average interval between first parental concerns and neuroimaging was 2.4 (SD: 3.9) years with a range of 0 to 13 years. The average age of genetic diagnosis with LS or LSS was 4.0 (SD: 3.5) years with the range being 0.4 to 12.7 years. The average interval between first parental concerns and genetic diagnosis was 2.9 (SD: 3.1) years with the range being 0.16 to 10.7 years. Out of the initial cohort, the most common first concerns were failure to meet milestones (37.9%), movement disorders (37.9%), and failure to thrive (20.7%). 6 (20.7%) patients had developmental regression. 6 (20.7%) of these issues occurred in the context of infection. 5 (17.2%) patients had a history of lactic acidosis and 2/5 of had lactic acidosis in the context of acute metabolic crisis requiring hospitalization. An expanded cohort will be included at the time of presentation. Conclusion: Developmental regression or acute metabolic crisis in LS/LLS occurred in the minority of patients. The most common presenting symptom for patients with LS or LLS was developmental delay. The average diagnostic odyssey was over two years. An absence of acute metabolic crisis or developmental decline should not exclude a clinician from considering MtD in the differential diagnosis. Longitudinal studies can help us better understand genotype-phenotype interactions. References: Lake, Nicole J et al. “Leigh syndrome: One disorder, more than 75 monogenic causes.” Annals of neurology vol. 79,2 (2016): 190-203. doi:10.1002/ana.24551 Rahman, S et al. “Leigh syndrome: clinical features and biochemical and DNA abnormalities.” Annals of neurology vol. 39,3 (1996): 343-51. doi:10.1002/ana.410390311 Abstract #: 2025PA-0000000160 Presenter: Julia Grafstein

Min-Kyung Nam 1 , Youngmo Seong 1 , Gi Heon Jeong 1 , Seung-Ah Yoo 1 , Hyangshuk Rhim 1 * 1 Department of Biomedicine & Health Sciences, Department of Medical Life Sciences, College of Medicine, The Catholic University of Korea, Seoul, South Korea *(Corresponding author’s email [email protected] ) Abstract: Mitochondrial dysfunction represents a critical pathological mechanism in Parkinson's disease (PD), where α-synuclein (α-Syn) accumulation and oxidative stress converge to promote neurodegeneration. This study elucidates the pivotal role of HtrA2 serine protease in maintaining mitochondrial homeostasis through selective α-Syn regulation. HtrA2 specifically targets the NAC region of α-Syn for proteolytic degradation, preventing pathological α-Syn accumulation within mitochondria. This mitochondria-protective mechanism significantly reduces reactive oxygen species (ROS) production, preserving mitochondrial integrity and cellular viability. Conversely, HtrA2 deficiency exacerbates α-Syn-mediated mitochondrial oxidative stress, triggering downstream microglial activation and neuroinflammation. Our findings demonstrate that HtrA2 functions as a crucial mitochondrial guardian, maintaining the delicate balance between protein quality control and organellar function. The HtrA2/α-Syn axis represents a novel therapeutic target for preserving mitochondrial health in PD, offering insights into neuroprotective strategies that address fundamental mitochondrial pathology underlying neurodegeneration. Abstract #: 2025PA-0000000171 Presenter: Santiago Restrepo Castillo

Ruth F. Hailemeskel 1 , Laryssa A. Huryn 2 , Ellen Macnamara 1 , Johan L.K. Van Hove 3,4 , Marisa W. Friederich 3,4 , David Adams 1,5 , William Gahl 1 , Lynne Wolfe 1 1 National Institute of Health, Undiagnosed Diseases Program, Bethesda, MD, 2 National Eye Institute, Bethesda, MD, 3 Department of Pediatrics, Section of Clinical Genetics and Metabolism, University of Colorado, Aurora, CO, USA, 4 Department of Pathology and Laboratory Medicine, Children’s Hospital Colorado, Aurora, CO, 5 National Office of the Clinical Director, National Human Genome Research Institute, Bethesda, MD * [email protected] Abstract: Complex I deficiency is a mitochondrial disease that results from genetic mutations that interfere with the oxidative phosphorylation system, the primary mechanism of energy production. Dysfunctional mitochondria can lead to heterogenous symptomatology and severity in clinical presentation, as exemplified by Leigh Syndrome, Leber’s hereditary optic neuropathy (LHON), myopathy, and other disorders with neurological involvement. We report two siblings of Kyrgyzstan and Turkish descent who presented to the NIH Undiagnosed Disease Program for evaluation. Our proband is a 14-year-old female who presented with bilateral optic atrophy, nystagmus, and a history of surgically corrected strabismus. Our second proband is a 9-year-old male with bilateral optic atrophy, nystagmus, and bilateral ankle clonus. The female proband was born by an uncomplicated cesarean-section to non-consanguineous parents. Perinatal and early life were unremarkable until visual concerns began at 2-years-old prompting brain imaging. She was noted to have strabismus at 3-years-old and underwent corrective surgery at 5-years-old in Turkey. The following year, she developed nystagmus and sequential vision loss. Further evaluation for cause of optic atrophy included another unremarkable MRI. Prior to their NIH evaluation, extensive genetic testing was completed, revealing many benign or likely benign variants. A non-diagnostic muscle biopsy was also completed in 2020. By 12-years-old, she experienced further vision loss and was diagnosed as legally blind. In 2024, the siblings were evaluated at the NIH Undiagnosed Disease Program. Family genome sequencing revealed compound heterozygous variants of uncertain significance (VUS) in the NDUFS1 gene, intronic variant c.1885-18G>A and missense variant c.412G>A (p.Asp138Asn). To provide biochemical confirmation, mitochondrial functional studies were completed. A muscle biopsy revealed normal activity of complexes I-V and assembly of complex V on blue native polyacrylamide gel electrophoresis (BN-PAGE) with in-gel activity staining. Respiratory chain enzyme activities showed isolated low activity of complex I at 28% of mean controls and -3.4 SD, as well as -3.6 SD for the ratio over citrate synthase activity. In fibroblasts, the BN-PAGE was normal, and respiratory chain enzymes showed a complex I enzyme activity at 68% (-1.2 SD). The low complex I activity validates the functional impact of the intronic and missense compound heterozygous mutations in NDUFS1 . This disrupts the function of complex I in the electron transport chain. This case reveals the value of repeating and expanding inconclusive testing to investigate a diagnosis associated with a VUS, missense variant, and using functional studies to validate findings. NDUFS1 joins an expanding list of variants associated with complex I deficiency due to the presence of deep intronic variants in complex I subunits and assembly proteins. The muscle biopsy results served as a multipurpose diagnostic tool for the sibling probands. Furthermore, the probands have expanded the clinical phenotype of Complex I deficiency by presenting with isolated bilateral optic atrophy caused by compound heterozygous missense and intronic mutations in the NDUFS1 gene. Abstract #: 2025PA-0000000168 Presenter: Kyrie Wilson 1, 7

Stacpoole P 1 , Abdenur J 2 , Bedoyan JK 3 , Botto L 4 , Enns G 5 , Falk MJ 6 , Ganetzky R 6 , Garganta C 7 , Glinton KE 14 , Gropman A 8 , Hamm S 9 , Henry E 10 , Longo N 11 , Neiberger R 12 , Saneto R 13 , Scaglia F 14 , Subramony SH 15 , Vockley J 3 , Wagner R 16, 1 Department of Medicine, Department of Biochemistry & Molecular Biology, College of Medicine, University of Florida Gainesville, FL, USA, 2 Rady Children’s Health, CA, USA, 3 Department of Pediatrics and UPMC Children’s Hospital University of Pittsburgh School of Medicine, Pittsburgh, PA, USA, 4 Department of Pediatrics, School of Medicine, University of Utah, Salt Lake City, UT, USA, 5 Department of Pediatrics, School of Medicine, Stanford University, Stanford, CA, USA, 6 Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia and University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA, 7 Department of Pediatrics, College of Medicine, University of Florida Gainesville, FL, USA, 8 St. Jude Children’s Research Hospital, Memphis, TN, USA, 9 Saol Therapeutics, Roswell, GA: 10 Firma Clinical Research, Elk Grove Village, IL, USA, 11 University of California Los Angeles (UCLA), Los Angeles, CA, USA, 12 Department of Pediatrics, College of Medicine, University of Florida, Gainesville, FL, USA, 13 Division of Pediatric Neurology, Neuroscience Institute, Norcliffe Foundation for Integrative Brain Research, Seattle Children’s Hospital/University of Washington, Seattle, WA, USA, 14 Department of Molecular and Human Genetics, Texas Children’s Hospital and Baylor College of Medicine, Houston, TX, USA, 15 Joint Baylor College of Medicine-Chinese University of Hong, Center of Medical Genetics, Prince of Wales Hospital, Hong Kong SAR, China, 16 Department of Neurology, College of Medicine, University of Florida, Gainesville, FL, USA, 17 Medosome Biotec, LLC, Alachua, FL, USA Corresponding Author: Stacpoole PW [email protected] Abstract: Pyruvate dehydrogenase complex deficiency (PDCD) typically results in progressive neurological and neuromuscular degeneration and early childhood death. It is a common cause of congenital lactic acidosis, with high morbidity and mortality rates. Oral dichloroacetate (DCA) is an investigational drug that stimulates residual PDC activity throughout the body by inhibiting the activity of pyruvate dehydrogenase kinases, which phosphorylate and inhibit PDC activity. DCA has previously shown benefit in treating congenital lactic acidosis. 1 This study reports the first Phase 3 trial of DCA for PDCD. The study was conducted by nine academic health centers across the United States, and was funded by the FDA (R01FD005407), the National Institutes of Health (R42HD089804), and Saol Therapeutics. Enrolled subjects continued their ketogenic diets and other supplements. Thirty-four subjects, aged 6 months to 17 years, were randomized to DCA or placebo in a 9-month crossover study, followed by open label DCA of indefinite duration. DCA dosing was based on haplotype variations in the glutathione transferase zeta family 1 ( GSTZ1 ) isoform, which dechlorinates DCA to inactive glyoxylate and dichotomizes subjects into ‘fast’ or ‘slow’ DCA metabolizers. DCA was well-tolerated and safe, even upon chronic administration of over three years. The original primary efficacy endpoint, a novel Observer Reported Outcome (ObsRO) survey 2 , was not met. Additional analysis suggested by the FDA showed a clinically meaningful, but not statistically significant (p=0.66), improvement in motor function among the most seriously affected subjects, based on ObsRO data. DCA also significantly (p=0.006) decreased plasma lactate concentrations and improved survival (p=0.027), compared to appropriately matched controls who were treated with a ketogenic diet. 3 Given that plasma lactate elevations are correlated with increased mortality in many acquired diseases (e.g. cancer, sepsis), the marked and sustained reduction in lactate and increased survival in our patients may also be mechanistically linked. Our results show that DCA is safe and represents the first potential therapy for PDCD. Abstract #: 2025PA-0000000125 Presenter: Vijay Modur, MD

S. Jordan Kerns 1 , Xie Xie 2 , Carol Geukens 2 , Anja V. Gruszczyk 2 , Sebastian Valenzuela 3 , Emily Hoberg 3 , Louise Jenninger 3 , Javier Miralles Fuste 2 , Mattias Stamgren 2 , Yonghong Shi 2 , Laleh Arabanian 2 , Sofie Ekström 2 , Andrew M. Griffin 1 , Gunther Kern 1 , Paul S. Charifson 1 , Jeremy Green 1 , Carlos Pardo-Hernandez 2 , Barbara Küppers-Munther 2 , Cindy Phan 2 , Simon Giroux 1 , Juli E. Jones 1* , Gabriel Martinez Botella 1 , Thomas A. Keating 1 , Claes M. Gustafsson 3 & Maria Falkenberg 3 1 Pretzel Therapeutics, Waltham, MA, USA, 2 Pretzel Therapeutics, Mölndal, Sweden, 3 Department of Medical Biochemistry and Cell Biology, University of Gothenburg, Gothenburg, Sweden, (corresponding author [email protected] ) Abstract: Mitochondrial DNA depletion syndromes (MDDS), a subset of primary mitochondrial diseases (PMDs), are characterized by mitochondrial DNA (mtDNA) depletion and/or mtDNA deletions/duplications, which compromise normal mitochondrial function. DNA polymerase γ (POLγ) mutations have emerged as one of the most common genetic causes of MDDS given the critical role of POLγ in mtDNA replication. To date, over 300 POLγ mutations have been identified in MDDS patients, underscoring the complexity of disease mechanisms and the urgent need for targeted therapeutic interventions. We have discovered a series of small molecules that bind specifically to POLγ and increase enzyme activity and processivity in a mutation-agnostic manner. The molecule presented here, PZL-A, exerts its action by binding to an allosteric site at the interface between the catalytic POLγA subunit and the proximal POLγB subunit, a region that is preserved in most mutant POLγ examples, including A467T, G848S and W748S. Additionally, PZL-A activates various mutant POLγ enzyme variants resulting in an increase in mtDNA synthesis in POLG patient cells, concomitant with a restoration of cellular respiration. Abstract #: 2025PA-0000000163 Presenter: Islam Alshamleh

Karaa A 1 *, Hareendran A 2 , Lambert J 3 , Morrison A 4 , Ross M 4 , Waller K 5 , Yeske P 6 and Balcells C 7 1 Massachusetts General Hospital, Genetics Division, Harvard Medical School, Boston, MA, USA, 2 UCB, Slough, UK, 3 UCB, Colombes, France, 4 Rare Disease Research Partners, Amersham, UK, 5 The Lily Foundation, Warlingham, UK, 6 United Mitochondrial Disease Foundation, Pittsburgh, PA, USA, 7 UCB, Smyrna, GA, USA. * [email protected] Abstract: Thymidine kinase 2 deficiency (TK2d) is an ultra-rare, genetic, mitochondrial disease, associated with progressive, life-threatening myopathy that affects motor function, breathing and feeding. The aim of this study was to characterize the experiences of patients with TK2d and their caregivers. Caregiver findings are presented here. Caregivers of patients with genetically confirmed TK2d were invited by global mitochondrial disease patient groups, to complete an online mixed-methods survey (between September 2023 and February 2024). The survey comprised multiple-choice and open-text questions exploring caregivers’ experiences and how caregiving affects their quality of life (QoL). Sixteen caregivers (11 parents) from nine countries participated; most (n=15) cared for patients with age of TK2d symptom onset of ⩽12 years (13 patients were living; median [range] age of TK2d symptom onset was 2.0 [1.0–14.0] years and age at time of survey was 13.0 [2.0–48.0] years; three patients were deceased, all aged ⩽12 years at death). Most caregivers (n=11) spent ⩾75 hours per week caregiving (n=7, ⩾100 hours per week), including those employed full time (n=5); others spent <12 to 74 hours per week caregiving (n=5). Although most caregivers were employed full or part time (part time, n=8), adjustments to working patterns or role were common. Four caregivers received professional in-home support. The QoL domains that were most negatively affected were social and leisure activities (n=13), employment and education (n=12) and finances (n=12). Physical health impacts (n=10), including reduced self-care activities, low energy, lack of sleep, and injuries, aches and pains due to caregiving demands, were reported. Mood (n=9) and relationships with friends (n=8) and partners and family (n=7) were also negatively affected. Caregivers reported that the constant demands of caregiving and minimal support or respite caused persistent stress and emotional burnout. Witnessing loved ones experiencing pain, undergoing invasive procedures and losing independence was distressing. Financial and caregiving strains were compounded by limited funding for care and equipment. In conclusion, this survey study supports prior research on the extensive burden and impact of rare diseases on caregivers, helps to characterize the lived experience unique to caregivers supporting individuals with TK2d and highlights the need for support considerations to improve health-related QoL for patients and their caregivers alike. The study was funded by UCB. Abstract #: 2025PA-0000000096 Presenter: Amel Karaa, MD

Dirain, C.O. 1 , Sambasivan, P. 1 , Shori, P. 1 , Liyanarachchi, H. 1 ; Fisher, J. 2 ; Iacoboni, M. 2 , Holdhoff, M. 2 , Schreck, K. 3 , Tatter, S. B. 4 , Strowd, R. 5 , Grossman, S. 2 , Stacpoole, P. W. 1,6 1 Department of Medicine, University of Florida College of Medicine, Gainesville, FL, USA, 2 Department of Oncology, Sidney Kimmel Cancer Center, Johns Hopkins University School of Medicine, Baltimore, MD, USA, 3 Departments of Neurology and Oncology, Johns Hopkins University School of Medicine, Baltimore, MD, USA, 4 Department of Neurosurgery, Comprehensive Cancer Center, Wake Forest School of Medicine, Winston-Salem, NC, 5 Departments of Neurology, Internal Medicine, and Translational Sciences Institute, Wake Forest School of Medicine, Winston-Salem, NC, 6 Department of Biochemistry and Molecular Biology, University of Florida College of Medicine Corresponding author: Dirain, C.O. Abstract: Glioblastoma (GBM) is the most common brain cancer in adults, with few treatment options. Characterized by the Warburg effect, GBM cells favor glycolysis over oxidative phosphorylation, driven by upregulation of pyruvate dehydrogenase kinase (PDK), which phosphorylates, and inhibits, the pyruvate dehydrogenase complex (PDC) E1a (PDHA1). Dichloroacetate (DCA), a PDK inhibitor, restores PDC activity, reverses the Warburg effect, and induces selective apoptosis in tumor cells. DCA may be a safe and effective metabolic therapy for GBM. This study is a multicenter, open label Phase IIA trial of oral DCA in patients (18-80 years) with recurrent GBM scheduled for debulking surgery. Patients were genotyped for GSTZ1 haplotype to establish safe dosing, then sequentially allocated to receive DCA (N=20) or no DCA (N=20) for one week prior to surgery. After surgery recovery, all patients received DCA. The primary objectives were to assess: 1) safety of chronic DCA treatment, and 2) tumor levels of phosphorylated PDHA1 (p-PDHA1). Plasma samples were collected before, during and after surgery to measure DCA and lactate levels. Tumor tissues (T1, contrast-enhanced and T2, non-contrast-enhanced) obtained at surgery were analyzed for expression of PDHA1, PDK isoforms1-4 and proliferation markers, VEGF-α, PCNA, ERK1/2, and PGK1. Data were analyzed with t-test. This update reports on 24 enrolled patients, aged 43 to 73 years (mean age 60). The cohort was 62% male and 96% were IDH-wildtype. Only one patient experienced drug-related adverse effects (peripheral neuropathy). Lactate levels at baseline were similar between the two arms (1.91 and 1.90 mmol/l, p=0.47). At surgery, DCA-treated patients had a mean plasma DCA level of 38μg/ml and lower plasma lactate, (1.46 vs 2.54 mmol/l; p=0.01) compared to those who did not receive DCA. After 8 weeks of DCA post-surgery, plasma lactate decreased 50% from baseline (0.95 vs 1.90 mmol/l, p=0.003). Biochemical analysis showed lower p-PDHA1 (p=0.002) and PDK4 (p=0.001) in the more necrotic (contrast-enhancing) tumors of patients who received DCA pre-surgery (N=9), compared to patients without DCA (N=8). Our findings are consistent with the known action of DCA and suggest a shift towards aerobic cellular respiration and a decrease in tumor cell proliferation. Increased expression of PDKs and increased circulating lactate levels have frequently been reported to inversely correlate with clinical outcome in cancer. No differences in p-PDHA1 and PDK 1-4 expression were found in non-enhancing tumors. In DCA-treated patients, VEGF-α is reduced (p=0.001) in contrast-enhancing tissue. In the non-enhancing tumors, PCNA and PGK1 levels were reduced and ERK1/2 was increased in patients who received DCA pre-surgery (p=0.02, p=0.004, p=0.03, respectively), all consistent with decrease tumor proliferation. In summary, initial results suggest DCA is well-tolerated and safe. It inhibited PDK-induced PDC phosphorylation in contrast-enhancing tumors, decreased tumor proliferation markers and lowered plasma lactate levels. DCA may be combined with other therapies and improve outcome in GBM patients. This study is funded by FDA R01FD007271. Abstract #: 2025PA-0000000122 Presenter: Marni Falk

McCormick EM 1 , Lott MT 2 , Muraresku CC 1 , Sheta L 3 , Wong S 3 , Procaccio V 4 , Wallace DC 2,5 , GaiX 6,7 , Falk MJ 1,5 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, USA, 2 Center for Mitochondrial and Epigenomic Medicine, Children’s Hospital of Philadelphia, USA, 3 Ambry Genetics, USA, 4 Genetics Department, MitoVasc Institute, Angers Hospital University, France, 5 Perelman School of Medicine, University of Pennsylvania, USA, 6 Mellowes Center for Genomic Sciences and Precision Medicine, Medical College of Wisconsin, USA, 7 Department of Pediatrics, Medical College of Wisconsin, USA * [email protected] Abstract: Special consideration is required when classifying the pathogenicity of mitochondrial DNA (mtDNA) variants owing to unique features of this genome, which include its composition and structure, evolutionarily conserved haplogroups and phylogeny, maternal inheritance, heteroplasmy, specific databases and computational algorithms, and unique functional analyses. The Clinical Genome Resource (ClinGen) Mitochondrial Disease Variant Curation Expert Panel (Mito-VCEP) has engaged over 50 international experts for a highly productive effort over 9 years ( https://clinicalgenome.org/affiliation/50027 ). Funded by the National Institutes of Health (NIH) first in 2016 by the National Human Genome Research Institute (NHGRI) via ClinGen to establish the Mito-VCEP, grant funding was obtained in 2017 through the National Institute of Child Health and Human Development (NICHD) U24 grant program to curate variant pathogenicity in association with Leigh syndrome spectrum. This initiative was then co-funded in 2021 by the NICHD and National Institute of Neurological Disorders and Stroke (NINDS) to classify mtDNA variants in the context of Primary Mitochondrial Disease (PMD). Through this work, we developed and published further specifications and guidance for mtDNA variant classification to the Richards et al. 2015 American College of Medical Genetics (ACMG) and Association of Molecular Pathology (AMP) standards and guidelines, which has now become widely used for clinical interpretation of DNA sequence variants (McCormick et al., 2020). The Mito-VCEP has now completed mtDNA variant expert curation according to the specifications published by this group for prioritized mtDNA variants as follows: (1) “Confirmed” in MITOMAP as of 2022 (n=97); (2) conflicting pathogenicity assertions in ClinVar (n=36); (3) entered as pathogenic (P) in ClinVar (n=103); (4) entered as likely pathogenic (LP) in ClinVar (n=49); and (5) requested by Mito-VCEP members (n=5). Curation and expert panel consensus have been reached to date for all of these 290 mtDNA variants. Among these, 23 variants reached a P classification, including 4 that initially met criteria for LP but were reclassified as P by the Mito-VCEP; 85 reached a classification of LP, including 17 that required Mito-VCEP modification; 172 were classified as VUS, including 1 that required Mito-VCEP modification; 5 were classified as likely benign (LB), including 1 that required Mito-VCEP modification, and 5 were classified as benign. Importantly, the 23 variants that did not reach the appropriate classification upon Mito-VCEP consensus review highlight the limitations to the current ACMG/AMP classification system specified for mtDNA variants. To further optimize mtDNA variant classification and interpretation guidelines, the Mito-VCEP is developing a second specification version which will bolster mtDNA variant classification guidelines around functional studies, in silico predictor informatics tools, proband counting, and beyond. In addition, MITOMAP now includes the Mito-VCEP’s assessed P and LP variants as “Confirmed,” where a total of 130 mtDNA variants now have this categorization. Abstract #: 2025PA-0000000037 Presenters: Quynh Kieu, MD; Betty Nguyen, BS

Iness AN 1 *, Strouphauer E 1 , Hirano M 2 , Iglesias A 3 , Emmanuele V 4 , Kaplan S 5 , Scaglia F 1 1 Department of Molecular and Human Genetics, Baylor College of Medicine, Texas Children's Hospital, Houston, TX, USA, 2 Neuromuscular Medicine Division, Columbia University Irving Medical Center, H. Houston Merritt Center for Muscular Dystrophy and Related Diseases, New York, NY, USA, 3 Division of Medical Genetics, Department of Pediatrics at New York Presbyterian Hospital/Columbia University Medical Center, 4 Department of Neurology, Columbia University Irving Medical Center, New York, USA, 5 Division of Infectious Diseases, Department of Pediatrics, Baylor College of Medicine, Texas Children's Hospital, Houston, TX, USA * [email protected] Abstract: Dengue virus (DENV) infects approximately 400 million people globally each year, with 450,000 cases progressing to dengue hemorrhagic fever (DHF). Emerging evidence suggests that DENV exploits host cell mitochondria during replication, leading to mitochondrial dysfunction and dysregulated innate immune response. These effects may be particularly consequential in individuals with pre-existing mitochondrial disorders, yet little is known about their clinical course and optimal management in the setting of severe DHF. We report a 16-year-old male with mitochondrial complex I deficiency (homozygous c. 733G>A, p. V245M variant in NDUFV1 ) who developed DHF with severe neurological involvement. His clinical course was characterized by rapidly worsening weakness, respiratory distress, seizures, and intracranial hemorrhage. Initial magnetic resonance imaging of the brain revealed multifocal intracranial hemorrhages, encephalitis, myelitis, and arachnoiditis, features consistent with expanded dengue syndrome, likely exacerbated by his underlying mitochondrial disorder. Treatment included empiric antibiotics, seizure control with levetiracetam, and immune modulation with dexamethasone (10mg IV bolus followed by 2mg every six hours for three days). N-acetylcysteine (100mg/kg/day divided TID) was initiated to help protect against oxidative stress and cellular damage, mitigating mitochondrial dysfunction and inflammation. The patient was previously noted to have lower extremity weakness and myelopathy with hyperreflexia at baseline but despite initial stabilization, had persistent neurological deficits beyond his baseline prompting escalation of therapy to intravenous methylprednisolone (1g/day for five days) followed by intravenous immunoglobulin (IVIG 2g/kg/day for five days). Due to refractory weakness, plasma exchange (PLEX) was introduced using a “zipper method,” alternating IVIG and PLEX over five days. Following multimodal treatment, the patient’s motor function gradually improved, correlating with resolving encephalitis and myelitis on imaging. He was transitioned to inpatient rehabilitation for weakness and cognitive deficits limiting his ability to perform activities of daily living. He made continued improvement but was discharged on baclofen for lower extremity dystonia and spasticity. This case highlights the profound impact of DENV infection in patients with mitochondrial dysfunction, emphasizing the need for early recognition and tailored immunomodulatory strategies. Mitochondrial disorders predispose patients to dysregulated immune responses, particularly to pathogens like DENV that target mitochondria. The patient’s pro-inflammatory phenotype and prolonged recovery underscore the importance of integrating mitochondrial-targeted therapies with standard dengue management. Given the risk of severe neurological and systemic complications, further research is needed to define evidence-based treatment guidelines for this vulnerable population. Abstract #: 2025PA-0000000085 Presenter : Audra Iness

Qvit N The Azrieli Faculty of Medicine in the Galilee, Bar-Ilan University, Safed, Israel * [email protected] Abstract: Excessive mitochondrial fission impairs cellular function and energy production, contributing to the pathogenesis of neurodegenerative, cardiovascular, and oncological diseases. The interaction between dynamin-related protein 1 (Drp1) and fission protein 1 (Fis1) plays a pivotal role in this process. A decade ago, we developed P110, a linear peptide that selectively inhibits Drp1/Fis1 signaling, demonstrating neuroprotective effects in Parkinson’s disease models and cardioprotective benefits in ischemia/reperfusion (I/R) injury. However, the clinical utility of P110 is limited due to its susceptibility to proteolytic degradation and suboptimal pharmacokinetics. In this study, we present CVP-350, a macrocyclic (cyclic small molecule) analog of P110, engineered to improve its pharmacological properties through structure-activity optimization. CVP-350 shows a six-fold increase in selective Drp1/Fis1 inhibition and a significantly extended half-life in protease-rich environments. It binds Drp1 with a 282 nM affinity and an IC50 of 480 nM. In cardiomyocyte stress models, CVP-350 outperforms P110 by effectively preventing mitochondrial swelling and fragmentation. Furthermore, in both cellular and in vivo animal models of I/R injury, CVP-350 provides more than twice the myocardial protection compared to P110. These findings highlight CVP-350 as a promising therapeutic candidate for mitochondrial dysfunction, with enhanced pharmacological properties and potential clinical applications in the treatment of cardiovascular and neurodegenerative diseases. Abstract #: 2025PA-0000000027 Presenter: Tsering Yangzom *

Diaz V 1 , Morisue J 2 , Mota M 1 , Klein J 1 , Savage K 3 , Hernandez-Herrera G 3 , Schimmenti L 3 , Ogiso E 2 , Ekker SC 1,3 , Sabharwal A 1,3* 1 Department of Pediatrics, Dell Medical School, The University of Texas at Austin, USA, 2 Children’s Hospital of Philadelphia, Philadelphia, Pennsylvania, USA; 3 Mayo Clinic, Rochester, Minnesota, USA * Corresponding author’s email: [email protected] Abstract : Mitochondrial DNA (mtDNA) pathogenic variants underlie diverse mitochondrial disorders, impacting multiple organ systems and posing significant clinical challenges. Historically, the absence of precise animal models has limited mechanistic understanding and therapeutic development. The mitochondrial genome represents one of the most highly conserved known stretches of vertebrate DNA, with all 37 genes found in the same order from zebrafish (Danio rerio) to humans. Leveraging recent advances in mitochondria-targeted base editing technologies, we have successfully established zebrafish (Danio rerio) models using via FusX TALE Base Editors (FusXTBE) to recapitulate human mtDNA disorders, providing potential for unprecedented opportunities for mechanistic insights and therapeutic exploration. First, we explored germline transmission dynamics and systemic consequences of near-complete (>80% heteroplasmy) mt-tl1 mutations in zebrafish. Embryos harboring these edits exhibited impaired mitochondrial bioenergetics and altered transcriptomic signatures involving respiration and cholesterol synthesis pathways. Intriguingly, differential segregation patterns across generations correlated with distinct transcriptomic profiles and hearing impairments analogous to clinical mitochondrial disorders, highlighting the utility of this model for studying inheritance and systemic manifestations of mtDNA mutations. Second, we introduced a novel premature stop codon mutation in number of mitochondrial protein coding genes achieving stable heteroplasmy levels between 40-70%. One of these mutants in mt-nd5 m.13311C>T mutation mirrors biochemical and behavioral phenotypes observed in primary mitochondrial diseases (PMDs), facilitating translational studies aimed at therapeutic intervention. Finally, we generated zebrafish harboring the prevalent LHON-associated m.11778G>A mutation using DddA-derived cytosine base editors (DdCBEs). These mutants displayed stable retinal heteroplasmy exceeding 90%, with heteroplasmy also observed in liver and heart tissues across successive generations. Current studies focus on retinal ganglion cell-specific degeneration characteristic of LHON, bioenergetic profiling, and cellular responses under stress conditions to elucidate tissue-specific vulnerability mechanisms and potential moonlighting roles beyond bioenergetics. Collectively, these zebrafish avatars offer potential to be used as platforms to dissect mitochondrial pathophysiology in vivo, significantly advancing mechanistic understanding and targeted therapeutic strategies for mitochondrial diseases. Abstract #: 2025PA-0000000173 Presenter: Daniel Mendez, MS

Traschütz A 1,2 , Kern J 3 , Cox MK 4 , Nguyen SA 5 , Sharma S 5 , Demczko M 5 , Ly A 5 , MacMullen, L 5 , Muraresku C 5 , Pantano C 5 , Balance, L 5 , Flickinger J 5,6 , George-Sankoh I 5 , Serai S 7 , Van Cura D 4 , Narain NR 4,8 , Henao J 4 , Kiebish MA 4 , Modur V 4 , Zolkipli Cunningham Z 5,9 *, Synofzik M 1 1 Center of Neurology and Hertie-Institute for Clinical Brain Research, Tübingen, Germany, 2 German Center for Neurodegenerative Diseases (DZNE), Tübingen, Germany, 3 Children's Hospital, University of Tübingen, Germany, 4 BPGbio, USA, 5 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia (CHOP), USA, 6 Department of Rehabilitation, CHOP, USA, 7 Department of Radiology, CHOP, USA, 8 University of Miami, USA, 9 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA, * [email protected] , [email protected] Abstract: Primary CoQ10 deficiency (PCD) is a multisystem primary mitochondrial disease that leads to neurologic manifestations (particularly cerebellar ataxia), steroid-resistant nephrotic syndrome, hypertrophic cardiomyopathy, retinopathy or optic atrophy, and sensorineural hearing loss, caused by mutations in any of 9 genes involved in CoQ10 synthesis. The current standard of care for PCD is oral nutritional CoQ10 replacement therapy resulting in varying results likely related to lack of standardized formulations, limited gastrointestinal absorption, as well as low tissue uptake and blood brain barrier penetrance. BPM31510IV is a novel pharmaceutical grade formulation of intravenously administered drug-lipid conjugated ubidecarenone (oxidized Coenzyme Q10, also known as CoQ10) that addresses the challenges of delivering CoQ10 into tissues (including the brain), cells, and mitochondria as well as the inconsistencies due to lack of standardization. IV administration of BPM31510IV bypasses the inconsistent absorption in the gastrointestinal tract and the nanolipid suspension bypasses the accumulation of CoQ10 in the LDL particle which are hypothesized as key contributors to variable clinical response to oral CoQ10 therapy. Three patients with PCD due to mutations in COQ8A are currently being treated with BPM31510IV under named-patient and compassionate use requests (a 9-year-old [c.812G>A; p.Arg271His; c.1821C>A; p.Tyr607Ter] and a 16-year-old [c.1042C>T; p.Arg348* homozygous] at the University of Tübingen, and a 10-year-old at CHOP [1q42.13 28kb deletion (maternally inherited pathogenic variant; c.1390 C>T; p.Arg464Trp (paternally inherited likely pathogenic missense variant)]. A second patient receiving treatment at CHOP is underway. In the two Tübingen patients, treatment with 30 mg/kg of OTC CoQ10 supplements for several years had minimal benefit on ataxia or cognitive impairment and baseline plasma levels of CoQ10 varied significantly between them. A 4-to-5-week dose titration was initiated to reach a maintenance dose of 50 mg/kg weekly IV infusion. Prior oral CoQ10 supplements were continued alongside IV treatment. Both Tubingen patients showed improvements in the Scale for the Assessment and Rating of Ataxia (SARA), the Friedreich’s Ataxia Rating Scale Activities of Daily Living, 9-Hole Peg Test, and patient- and/or caregiver-reported global impression of change and Goal Attainment scales (particularly writing speed) observed within 4 weeks of treatment initiation. At 16 weeks of IV treatment, these patients are showing continued improvement. The patient at CHOP has been receiving 50 mg/kg/day of OTC CoQ10 supplements with minimal benefit. This patient has recently initiated BPM31510IV treatment and will be evaluated using the MM - COAST (Mitochondrial Myopathy Composite Assessment Tool) 1 which includes some of the clinical assessments evaluated in the first 2 patients, as well as muscle CrCest MRI measurement of in vivo muscle mitochondrial function 2 and biochemical measurements including plasma and urine amino acids, hsCRP and ketone bodies panel. In addition, parents, schoolteachers, and/or parents of peers have reported substantial clinical improvement in all 3 patients. None of the patients have experienced any clinically significant adverse events from the BPM31510IV infusions with concurrent vitamin K supplementation. This preliminary evidence suggests that BPM31510IV can be safely administered, is well tolerated, and may have a substantial effect on PCD disease progression. Abstract #: 2025PA-0000000126 Presenter: Zimu Cen

Dana V. Mitchell 1* , Donna M. Iadarola 2 , Neal D. Mathew 2 , Kelsey Keith 1 , Christoph Seiler 3 , Sanghyeon Yu 4 , Man S. Kim 4 , Niki Woodard 2 , Vernon E. Anderson 2 , Eiko Nakamaru-Ogiso 2,5 , Deanne M. Taylor 1,5 , and Marni J. Falk 2,5 1 Department of Biomedical and Health Informatics, The Children's Hospital of Philadelphia, Philadelphia, PA, 2 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA, 3 Zebrafish Core, The Children's Hospital of Philadelphia, Philadelphia, PA, 4 Translational-Transdisciplinary Research Center, Clinical Research Institute, Kyung Hee University Hospital at Gangdong, Kyung Hee University College of Medicine, Seoul, Republic of Korea, 5 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA* [email protected] Abstract: Mitochondrial complex I deficiency manifests with complex multi-system dysfunction, commonly in the Leigh syndrome spectrum (LSS), with early-onset metabolic strokes, lactic acidemia, and early mortality. To facilitate pre-clinical screening of novel therapeutic candidates for complex I diseases including LSS disorder, we developed a stable genetic knockout zebrafish animal model for a highly conserved nuclear-encoded complex I subunit, Ndufs2. CRISPR/Cas9 technology was used to generate a 16 base pair deletion in ndufs2 , which are maintained as heterozygotes and in-crossed to generate homozygous ndufs2 -/- mutants and phenotypically wild-type (WT), ndufs2 +/+ and ndufs2 +/- siblings for larval stage analyses. Larvae were phenotypically characterized for survival, neuromuscular swimming activity, and gross morphologic defects. RNA-Seq transcriptome profiling was performed to evaluate pathway adaptations in ndufs2 -/- larvae, utilizing traditional enrichment analysis as well as a novel metabolic modeling approach that simulates potential metabolic flux capacity through a genome-scale metabolic model reconstructed from biochemical reactions comprising cellular processes of interest. RNA-Seq zebrafish results were compared to previous transcriptome profiling datasets from ndufs2 -/- missense mutant C. elegans ( gas-1(fc21) ) and complex I disease patient fibroblasts. Therapies previously identified in the C. elegans ndufs2 -/- missense model were screened for their ability to rescue morphological malformations in the zebrafish ndufs2 -/- knockout model. ndufs2 -/- zebrafish had severely reduced survival to a median of 11 days post fertilization (dpf) relative to wild-type (WT) animals that live greater than 2 years. ndufs2 -/- zebrafish had marked neuromuscular dysfunction with ~50% decreased swimming activity and showed 80% reduced CI enzyme activity. Morphological analysis showed an uninflated swim bladder, decreased yolk absorption, an enlarged and dark liver phenotype, and small eyes. Transcriptome profiling of ndufs2 -/- larvae revealed dysregulation of the electron transport chain, TCA cycle, fatty acid beta-oxidation, and one-carbon metabolism. Interestingly, pathways associated with eye development were downregulated in mutant larvae. Similar transcriptomic profiles were observed in ndufs2 -/- missense mutant C. elegans ( gas-1(fc21) ) and two human CI-disease fibroblast cell lines stressed in galactose media. Metabolic modeling revealed specific dysregulation of pentose phosphate pathway reactions. One-carbon metabolism associated pathway alterations appeared to contribute to CI disease pathophysiology, as folic acid treatment rescued the growth defect and hepatomegaly in ndufs2 -/- larvae. We have established and validated a novel vertebrate animal model of severe complex I deficiency using CRISPR/Cas9 technology to knock-out ndufs2 in zebrafish. This preclinical model recapitulated many phenotypes associated with mitochondrial disease and LSS, provides novel insight into the unique gene expression profiles of LSS, and objectively demonstrates the therapeutic value of folic acid to replete one carbon metabolism in mitochondrial complex I disease. This translational model of PMD will enable future investigations of organ specific mechanisms of disease and high-throughput treatment studies. Abstract #: 2025PA-0000000145 Presenter: Kelsey Keith

O’Hara TG 1 , Lu A 1 , Tara Z 1 , Haus E 1 , Campbell C 1 , Wei S 1 , Mendel R 1 , Mathew N 1 , Keith K 2 Seiler C 3 , Chen S 4 , Nakamaru-Ogiso E 1 , Falk MJ 1,5 , and Haroon S 1,5 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, 2 Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA, 3 Aquatics Core Facility, The Children’s Hospital of Philadelphia, Philadelphia, PA, 4 Medical University of South Carolina, Charleston, South Carolina, 5 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA Abstract: Background : Pathogenic OPA1 variants lead to progressive vision loss, with 20% of OPA1 +/- patients developing additional symptoms such as motor and/or sensory neuropathy, ataxia, myopathy, and sensorineural hearing loss. OPA1 is a mitochondrial GTPase, where pathogenic OPA1 variants induce fragmented mitochondria, mitochondrial dysfunction, and mtDNA depletion that lead to bioenergetic dysfunction and subsequent mitochondrial degradation via mitophagy. One approach to prevent the continual cycling of mitochondrial degradation and biogenesis is to modulate mitophagy, which we postulate may reduce ATP consumption, preserve mitochondrial mass, and stabilize mtDNA content. Our long-term goal is to utilize mitophagy modulator screening approaches to develop OPA1 +/- disease therapies. Here, we report the development of a humanized worm model for OPA1 +/- disease in which to screen candidate therapies, with lead candidates then validated across 3 evolutionarily distinct species, including 2 worms, a zebrafish, and various patient cell line models. Methods : Using CRISPR/Cas9, we generated the R289Q mutant worm strain of eat-3 , which is orthologous to the pathogenic R345Q (previously R290Q) OPA1 variant and obtained a V328I missense mutant eat-3 strain. Both mutant worm strains showed defects in mtDNA content, mitochondrial respiration, fecundity, animal development, and neuromuscular function. Substantially increased mitochondrial unfolded protein stress response (UPR mt ) induction in R289Q worms was used to screen (i) potential therapies identified for complex I disease worm models, (ii) 62 mitophagy modulating drugs, and (iii) a library of 2,560 FDA-approved and natural product compounds. Results : Using the UPR mt fluorescence screen, Thiamine, Lipoic Acid, Celastrol and Hemin were identified from the first two compound sets, and the library screen identified 16 compounds as potential therapeutic candidates. Among these, Thiamine, Celastrol, and Bromindione reproducibly reduced UPR mt , increased neuromuscular activity, and improved development in the OPA1 mutant ( eat-3 ) worm strains. OPA1 -/- zebrafish larvae have been found to have quantified impaired visual function by optokinetic assay, which will be used to screen candidate therapies identified in the worm models on visual function. In OPA1 -/- R345Q patient fibroblast cells, Thiamine, Celastrol, and Bromindione rescued cell death (CellTox) to healthy patient cell levels. Additional validation studies are underway on multiple fitness outcomes in eat-3 mutant worms and in human fibroblast cell lines (R345Q +/- and I458T +/- ) on cell death (CellTox) and mitochondrial physiology. We have also derived R345Q +/- induced-Pluripotent Stem Cells (iPSC) from OPA1 -/- patient peripheral blood mononuclear cells and are using CRISPR-Cas9 technology to generate the revertant wild-type cells. iPSC-derived retinal ganglion cells (RGC) are being generated to better characterize OPA1 -/- disease and validate lead therapies. Work is ongoing to evaluate whether Red Light Therapy shows preclinical efficacy in these translational models of OPA1 -/- disease. Conclusion : Overall, we describe several novel OPA1 disease models across multiple species that manifest key aspects of human disease. Candidate therapy screens have identified 20+ possible therapeutic leads, with validation studies showing preclinical efficacy of Thiamine, Celastrol, and Bromindione on multiple outcomes in OPA1 worm models and a human patient fibroblast cell line. Abstract #: 2025PA-0000000130 Presenter: Alexander Stover, MS

Aoyama YJ 1* , Grafstein J 1 , Houston P 1 , Friederich MW 2,3 , Van Hove J 2,3 , Macnamara E 1 , Wolfe L 1 , Gahl WA 1,4 , and Adams DR 1 1 Undiagnosed Diseases Program, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA, 2 Section of Clinical Genetics and Metabolism, Department of Pediatrics, University of Colorado Anschutz Medical Campus, Aurora, CO 80045, USA, 3 Department of Pathology and Laboratory Medicine, Children’s Hospital Colorado, Aurora, CO 80045, USA, 4 Human Biochemical Genetics Section, National Human Genome Research Institute, National Institutes of Health, Bethesda, MD 20892, USA * [email protected] Abstract: We present a 6-year-old female patient evaluated in the National Institutes of Health Undiagnosed Diseases Program who presented with global developmental delay, chronic kidney disease (stage III), and renal tubular dysfunction. Genome sequencing revealed two variants in NDUFAF6 : a paternally inherited intronic variant NM_152416.3 :c.298-768T>C and a maternally inherited 1.6 kb deletion NC_000008.11 :g.95044573_95046180del, spanning exons 5 and 6. NDUFAF6 plays an important role in mitochondrial complex I assembly by regulating ND1 biogenesis and facilitating the incorporation of NDAFS8. Variants in NDUFAF6 are associated with two OMIM disorders: Fanconi renotubular syndrome 5 (618913) and Mitochondrial complex I deficiency, nuclear type 17 (618239). The associated phenotype includes degeneration of the central nervous system and proximal tubule dysfunction. Studies revealed the role of NDUFAF6 in complex I deficiency as well as in the manifestation of Leigh syndrome, a progressive neurodegenerative disease. The intronic single nucleotide variant in this case has been previously reported to cause aberrant splicing, manifesting as renal Fanconi syndrome, further expanding the phenotypes associated with variants in NDUFAF6. This case expands our understanding of this gene and its associated phenotypes and suggests that specific variants may result in more complex phenotypes. This case also highlights the need to consider atypical variants in mitochondrial disease, potentially missed by exome sequencing methods, during the planning of comprehensive diagnostic strategy. Abstract #: 2025PA-0000000167 Presenter: Ruth F. Hailemeskel

Aristizabal-Henao JJ 1 , Kiebish MA 1 , Stopka S 1 , Gesta S 1 , Van Cura D 1 , Karmacharya S 1 , Wessel S 1 , Zavidij O 1 , Cox MK 1 , Pesini A 2 , Barriocanal Casado E 2 , Narain NR 1,3 , Quinzii CM 2 , Modur V 1 1 BPGbio, USA, 2 Columbia University, USA, 3 Department of Molecular Biology and Department of Dermatology and Cutaneous Surgery, University of Miami Miller School of Medicine, USA Abstract: Coenzyme Q10 (CoQ10) plays an essential role in mitochondrial energy production and antioxidant defense. Dysregulated CoQ10 biosynthesis and metabolism contributes to mitochondrial diseases and deficiency syndromes, with oral supplementation leading to variable and limited efficacy due to unpredictable bioavailability of CoQ10. To address this issue, we developed BPM31510, a lipid nanoparticle containing oxidized CoQ10, which has demonstrated the ability to restore CoQ10 levels in tissue and mitochondria. To assess efficiency of delivery of CoQ10 (BPM31510) in cells and tissues in in vivo models of CoQ10 deficiency, we developed a novel LC-MS/MS and MALDI-MSI quinomics workflow to comprehensively profile CoQ10 and related metabolites along with assessing the metabolome. This approach uncovers novel quinone regulation, its impact on cell metabolism and potential for therapeutic application in mitochondrial diseases. In vivo experiments: COQ4KI mice (carrying a point mutation in exon 5 of Coq4 gene; c[440T>G], p.F147C, homologue of the human mutation 437T>G; F146C, exhibiting a CoQ deficiency) were administered BPM31510 (200 mg/kg CoQ10) via intraperitoneal injection every other day over two weeks. Serum was collected throughout the study and at termination liver, brain, muscle, kidney, heart, adipose, and ear (skin) tissues were harvested. Additionally, we performed in vitro experiments and assessed control, CoQ2 mutant, and PDSS2 mutant patient fibroblasts, along with PABA treated SH-SY5Y cells dose dependently treated with BPM31510 and measured CoQ10 content, ATP, and mitochondrial ROS. Quinones were extracted from cells, serum, and tissues using isopropanol and deuterium-labeled CoQ10 as an internal standard. Samples were analyzed by LC MS/MS using a Thermo Q-Exactive Plus mass spectrometer. Data were acquired using scheduled parallel reaction monitoring (PRM) to semi-quantify ~30 quinones. MALDI MSI was performed on tissue sections to map the spatial distribution of CoQ10, related quinones, and metabolites using a Bruker TIMSTOF Flex system with microgrid technology. BPM31510 treatment effectively restored and significantly increased the CoQ pool in brain (cerebellum), kidney, heart, muscle as well as other tissues in treated CoQ4KI mice compared to untreated CoQ4KI and corresponding WT mouse controls. Additionally, fibroblast and chemically induced CoQ10 deficiency in vitro models demonstrated a dose dependent increase in CoQ10 levels, restoration of ATP content, and attenuation of mitochondrial ROS upon BPM31510 treatment. CoQ10 deficiency represents a heterogenous metabolic disorder in which oral CoQ10 provides sub-optimal clinical benefit due to poor absorption and biodistribution. Treatment with BPM31510 restored the CoQ pool impacting metabolic adaptation in a CoQ deficient mouse model, demonstrating its therapeutic potential, by overcoming oral CoQ10 bioactivity and biodistribution challenges. These results may have therapeutic benefit in other mitochondrial disorders where CoQ10 replacement can improve mitochondrial function. Abstract #: 2025PA-0000000136 Presenter: Elizabeth M. McCormick, MS, LCGC

Friederich MW 1,2 , Van Hove, JLK 1 , 2* 1 Department of Pediatrics, Section of Clinical Genetics and Metabolism, University of Colorado, Aurora, CO, USA, 2 Department of Pathology and Laboratory Medicine, Children’s Hospital Colorado, Aurora, CO 80045, USA, Abstract: Complex V, also known as ATP synthase, generates ATP through the phosphorylation of ADP using the energy derived from the electrochemical proton gradient established during the process of cellular respiration. The enzymatic activity is reversible also allowing for hydrolysis of ATP to ADP and inorganic phosphate (Pi). Complex V consists of 15 structural subunits of which two are mitochondrial DNA encoded (MT-ATP6 and MT-ATP8) and the remaining are nuclear DNA encoded and uses two assembly factors during its genesis. Whereas most complex V disorders are caused by mutations in its mtDNA-encoded subunits, nuclear DNA-encoded defects are becoming more commonly recognized. The ATP -synthesis enzymatic activity of Complex V is located in the F1 domain in the catalytic head. A kinetics spectrophotometric assay of the enzymatic hydrolysis activity has been reported, but not the clinical utility for the diagnosis of complex V deficiency, which is the goal of this study. In this assay, ATP is converted to ADP and P i by ATP synthase which through linked enzymatic assays over PEP kinase and lactate dehydrogenase is coupled to the oxidation of NADH to NAD + , which is followed at 340 nm. The Complex V specific activity is identified as the oligomycin sensitive rate. The assay was optimized in fibroblasts, showed more than 70% oligomycin sensitivity, robust reproducibility. A control range was developed from 30 control cell lines. All nuclear encoded complex V deficiencies (N=6) showed a clear decrease in complex V activity (-3.98 to -9.18 SD), including for autosomal dominant ATP5F1A cases. Several patient cell lines with combined OXPHOS deficiencies also had decreased complex V activity. This assay however only identified a small proportion of the MT-ATP6 and MT-ATP8 variants, and most showed borderline activity. This distinction most likely stems from mutations that affect the stability of complex V resulting in decreased hydrolysis activity as opposed to mutations that impair the proton flow without affecting the enzymatic activity. This assay shows excellent functionality to identify patients with nuclear complex V defects and to functionally validate VUS in these genes including for autosomal dominant inheritance but has limited utility to evaluate MT-ATP6 variants. Abstract #: 2025PA-0000000094 Presenter: Alejandra Romero

Melis Kose 1 , Suraiya Haroon 1,2 , Elizabeth McCormick 1 , Eiko Nakamaru-Ogiso 1,2 , Marni J. Falk 1,2 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, 2 Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Email: [email protected] Abstract: Cockayne syndrome (CS) is a rare autosomal recessive genetic disorder caused by pathogenic variants in ERCC6 (CSB), leading to transcription-coupled DNA repair defects and progressive neurodegeneration 1 . Given the overlap in clinical features with primary mitochondrial disease (PMD), accumulating evidence suggests mitochondrial dysfunction plays a key role in CS pathophysiology 2 . However, no mitochondrial-targeted therapies are available. Here, we report characterization of mitochondrial physiology in CSB⁻/⁻ human patient fibroblasts and C. elegans models and the effects of potential therapeutic interventions. Patient-derived CSB⁻/⁻ human fibroblast cell lines from two affected siblings carrying a compound heterozygous mutation in ERCC6 (c.1526+1G>T and c.2800C>A, p.P934T) exhibited significant mitochondrial dysregulation, including altered oxidative phosphorylation (OXPHOS) capacity. Basal respiration and extracellular acidification rates were increased in CSB⁻/⁻ cells compared to controls, while maximal respiration was significantly reduced, indicative of mitochondrial inefficiency. CSB⁻/⁻ fibroblasts displayed a marked reduction in mitochondrial DNA content, retaining only 47% of control levels (p<0.05). Patient platelet enzymatic analyses revealed reduced complex I activity and increased citrate synthase levels, suggesting altered mitochondrial biogenesis. Cell survival analysis was performed under metabolic stress conditions in glucose-free media containing galactose (10 mM), glutamine (0.5 mM), and L-buthionine (S, R)-sulfoximine (BSO, 50 µM) to acutely induce oxidative stress by depleting glutathione. CSB⁻/⁻ fibroblasts were highly sensitive to oxidative stress, displaying severe viability loss (10-15% of control mean). Coenzyme Q10 (CoQ10, 50 µM) supplementation partially rescued cell survival, increasing viability by 45% to approximately 50-55% of control level. Taurine treatment (1 mM and 2 mM) effectively rescued CSB⁻/⁻ cell survival under oxidative stress conditions, while N-acetylcysteine (NAC, 5 mM) also provided significant protection from cell death, together highlighting the therapeutic potential of antioxidant strategies in CS. To explore mitochondrial dysfunction in a whole-organism model, we utilized the C. elegans ok2335 strain, which carries a 1.6 kb deletion in csb-1. c sb-1 -/- worms exhibited developmental delay and growth deficiency, with 46% shorter worm length at the L4 larval stage than wild-type worms (p<0.05). Progeny count was also significantly reduced, indicating a defect in reproductive fitness. Furthermore, mitochondrial DNA (mtDNA) copy number was reduced by 62% (p<0.05) and mitochondrial unfolded protein response (UPR mt ) induction was increased in c sb-1 -/- worms, supporting the direct role of mitochondrial dysfunction in CS pathogenesis. While neuromuscular thrashing assays at the L4 stage showed no statistically significant difference between c sb-1 -/- a nd wild-type worms, it remains possible this would develop with age and/or be influenced by their smaller body size on the microscopy analysis performed. Collectively, these preclinical modeling findings demonstrate that CSB deficiency leads to profound mitochondrial dysfunction across evolutionary distinct species, with mtDNA depletion, impaired oxidative phosphorylation complex I capacity, mitochondrial unfolded protein response stress, developmental delay, and growth deficiency in worms, recapitulating key aspects of human CS phenotypes. The significant rescue effects of CoQ10, NAC, and taurine support the pursuit of rigorous clinical trials to evaluate mitochondrial-targeted therapies for CS. References 1. Scheibye-Knudsen M, Croteau DL, Bohr VA. Mitochondrial deficiency in Cockayne syndrome. Mech Ageing Dev. 2013 May-Jun;134(5-6):275-83. doi: 10.1016/j.mad.2013.02.007. Epub 2013 Feb 19. PMID: 23435289; PMCID: PMC3663877. 2. Aamann MD, Sorensen MM, Hvitby C, Berquist BR, Muftuoglu M, Tian J, de Souza-Pinto NC, Scheibye-Knudsen M, Wilson DM 3rd, Stevnsner T, Bohr VA. Cockayne syndrome group B protein promotes mitochondrial DNA stability by supporting the DNA repair association with the mitochondrial membrane. FASEB J. 2010 Jul;24(7):2334-46. doi: 10.1096/fj.09-147991. Epub 2010 Feb 24. PMID: 20181933; PMCID: PMC2887265 Abstract #: 2025PA-0000000051 Presenter: Keri-Lyn Kozul, PhD.

Restrepo Castillo S. 1 , Sahu A. 1 , Ali R. 1 , Sabharwal A. 1 , Ekker S.C. 1 1 Department of Pediatrics, Dell Medical School, The University of Texas at Austin, USA (Corresponding author’s email [email protected] ) (Presenting author’s email [email protected] ) Abstract: Sequence differences as little as a single nucleotide constitute a critical component in precision medicine. The development of tools for disease modeling and gene correction with such accuracy has been hampered by sequence-context constraints around the most common systems, such as PAM sequences for CRISPR-derived technologies (which are impractical for mitochondrial gene editing), and the 5’-T base requirement for tools based on transcription activator-like effectors (TALEs). Notably, the latter have been successfully applied for both nuclear and organellar genome editing across many species. In line with this, seeking to address the sequence-context constraint limitation of TALE-based tools and further expand their utility, we have recently characterized sequence-context flexible TALEs in a mitochondrial cytosine base editing format both in vitro and in vivo. These efforts led to the establishment of unconstrained mitochondrial cytosine base editors, with the potential to access virtually all cytosines in mitochondrial DNA (mtDNA) at single-nucleotide resolution. These results suggested that unconstrained TALEs can be leveraged for the development of novel and flexible TALE-based technologies, such as distinct nuclear or organellar cytosine or adenine base editors, or entirely new applications. Consequently, we have continued to expand our genome engineering toolbox by including unconstrained TALEs in emerging tools for precise genetic manipulations, such as nuclear or mitochondrial strand-selective cytosine or adenine base editing. In addition, we are currently exploring the generation of pioneering TALE-based technologies beyond base editing, such as tools for the installation of targeted insertions in mtDNA. Furthermore, leveraging our growing repertoire of enhanced TALE-based technologies, we are designing reverse genetics approaches to evaluate the phenotypic outcomes of otherwise inaccessible human mitochondrial variants of unknown significance. Finally, predicting whether a TALE-based technology will be effective based on its structure remains impractical beyond the TALE–DNA recognition cipher. Aiming to address this design bottleneck, we are establishing an in silico structure-driven pipeline for the development of enhanced TALE-based technologies. Using deep learning-based structure prediction tools (such as AlphaFold 3) to model complex biomolecular assemblies (including protein-DNA interactions) we are systematically sampling the design space of TALE-based tools, such as unconstrained mitochondrial cytosine base editors. Altogether, we are continuing to expand our CRISPR-free precision genome engineering capabilities, exploring mitochondrial reverse genetics approaches, and building a structure-driven design pipeline for the generation of enhanced TALE-based technologies. These efforts will further facilitate the creation of designer genes for discovery science, disease modeling, and mitochondrial gene therapy. Abstract #: 2025PA-0000000172 Presenter: Ankit Sabharwal

Stover A 1 , Huang W 1 , Vo U 1 , Russell B 2 , Barrick R 1 , Schwartz PH 1 , Latini A 1,d , Abdenur JE 1,3 1 Laboratory for Energy Metabolism, Metabolic Disorders, Rady Children's Health, USA, 2 Human Genetics Department, University of California, Los Angeles, USA, 3 Pediatrics Department, University of California, Irvine, California, USA, d LABOX, Biochemistry Department, Universidade Federal de Santa Catarina, Brazil Abstract: FBXL4 deficiency is a mitochondrial disorder characterized by reduced mitochondrial DNA (mtDNA) content, decreased steady-state levels of mtDNA-encoded respiratory chain enzyme subunits, reduced mitochondrial mass, and a fragmented mitochondrial network. Loss-of-function mutations in the FBXL4 gene cause encephalomyopathic mtDNA depletion syndrome 13 (MTDPS13; OMIM #615471), which typically manifests in early infancy with failure to thrive, severe neurodevelopmental delays, encephalopathy, hypotonia, and persistent lactic acidosis. Currently, only supportive treatments are available, and most affected individuals do not survive beyond infancy. Given our recent findings that ALA/Fe exposure can restore mtDNA levels, bioenergetics, and oxidative balance in a mitochondrial disorder caused by DARS2 deficiency [1], we sought to investigate whether a similar approach could be beneficial in FBXL4 deficiency. Thus, this study aimed (A) to determine whether fibroblasts from three individuals with FBXL4 deficiency exhibit the characteristic energy impairment phenotype, validating them as a model for testing new pharmacological approaches, and (B) to assess the potential therapeutic effects of ALA/Fe. Fibroblast cell lines were established from skin biopsies after informed consents were obtained (CHOC IRB 130990). The three patients had a severe neonatal clinical presentation. Patient 1 [c.1304G>A (p.Arg435Gln) and c.1232G>A (p.Cys411Tyr)] and Patient 2 [c.1641_1642del (p.Cys547Ter) and c.1442T>C (p.Leu481Pro)] were compound heterozygous, while Patient 3 [c.1303 C>T (p.R435*)] was homozygous. All these variants have been previously reported as pathogenic. Bioenergetics was assessed by measuring the cellular oxygen consumption rate using a Seahorse XFe96 bioanalyzer (Agilent, USA) and lactate production with a YSI Bioanalyzer (YSI, USA). The levels of mtDNA were assessed by qPCR, both under basal conditions and after 14 days of exposure to ALA/Fe (100 µM/50 µM). The three patient-derived cell lines exhibited bioenergetic deficiencies, characterized by significantly reduced basal cellular oxygen consumption rates, maximal respiration, mitochondrial spare capacity, and ATP synthesis efficiency. Additionally, the FBXL4-deficient fibroblasts showed increased extracellular acidification and elevated lactic acid production. A two-week exposure to ALA/Fe significantly rescued these deficiencies, improving bioenergetic parameters and mtDNA content. This study demonstrates that fibroblasts derived from individuals with FBXL4 deficiency provide a relevant model for testing new pharmacological approaches, particularly through the use of compounds already approved by the Food and Drug Administration (FDA), which simplifies regulatory processes and addresses the urgent need for treatment options in mitochondrial disorders. 1. Huang et al. Aminolevulinate/iron exposure elicited Nrf-2-mediated cytoprotection in human DARS2 deficient fibroblasts with impaired energy and antioxidant metabolisms. BBA - Molecular Basis of Disease, In press. 2025 Financial support: This work was supported by the Emery Kline Family Grant (16984023). Abstract #: 2025PA-0000000131 Presenter: Arlene Garcia

Fatima Alkhyeli MD 1 , Mallory Owen MBBCH 3 , Tyler Struver 3 , Richard Haas MB, BChir, MRCP 2 , Jennifer H. Yang, MD 2 , Kennedy Krieger 1 1 Johns Hopkins University, 2 University of California San Diego, Rady Children’s Hospital San Diego, 3 University of Arizona Abstract: Objective: To understand the neurodevelopmental trajectories and associations with clinical characteristics in pediatric mitochondrial diseases. Background: Developmental delays are common in mitochondrial diseases. However, risk factors associated with poor developmental outcomes are poorly understood. Methods: We conducted a single-center cohort study of pediatric participants (⩽18 years) with a clinical and/or molecular diagnosis of mitochondrial disease. Developmental severity was defined as those with ⩾2 domains of developmental delay. We employed descriptive analyses for clinical characteristics and multivariable logistic regression to assess the association between clinical characteristics at disease onset and developmental severity. Results: We identified 108 participants in this cohort: female 53%, median age of onset of 1.16 years (IQR 0.55 – 2.52), 82% with a confirmed molecular diagnosis. 60 participants presented critically ill; 33 (47%) in NICU, 27 (41 %) in PICU. Seizures at disease onset (58%) and MRI brain abnormality (79%) were common. Half had EEGs at onset, 83% of which were abnormal, and 62% showed epileptiform discharges. The median follow-up time was 4.11 years (IQR 1.54 – 7.87). Long-term developmental outcomes included gross motor delay (69%), fine motor delay (46%), speech delay (67%), ⩾2 domains of developmental delay (66%). Age of onset was associated with ⩾2 delays (OR 0.7, 95% CI 0.6 – 0.9, p=0.0001). After adjusting for sex and age of onset, ⩾2 delays was associated with abnormal EEG (OR 6.9, 95% CI 1.2 - 43.5, p=0.04), seizures at onset (OR 3.6, 95% CI 1.0 - 18.9, p=0.04), and ICU admission (NICU: OR 4.4, 95% CI 1.0 - 18.9, p=0.04; PICU: OR 4.3, 95% CI 1.0 - 18.0, p=0.046). Conclusions: Younger age of onset is a major predictor of developmental outcomes in mitochondrial disease. Nevertheless, there is higher odds of having poorer developmental outcomes with abnormal EEG, seizures, and ICU admission even after adjusting for age of onset and sex. Funding: This work was supported by the National Institute of Health (Award Number 5K12NS098482-08) Abstract #: 2025PA-0000000020 Presenter: Estefania Nova-Lamperti

McCormick EM 1 , Blakely EL 2 , Berger D 3 , Hyslop L 2 , Stewart J 2 , Feeney C 2 , Gorman G 2 , McFarland R 2 , Dokras A 3 , Falk MJ 1,4 . 1 Mitochondrial Medicine Frontier Program (MMFP), Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, USA, 2 Highly Specialised Service for Rare Mitochondrial Disorders, Newcastle upon Tyne Hospitals NHS Foundation Trust, UK, 3 Penn Fertility Care, USA, 4 Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, USA. Abstract: Primary mitochondrial disorders (PMD), caused by pathogenic variants in genes located in nuclear (nDNA) or mitochondrial DNA (mtDNA) genomes, are often progressive, have limited treatment options, and may be present early in life. Reproductive options for families wishing to have a genetic relationship to their child depend on the molecular etiology of PMD in their family. Those with an nDNA disorder have the same options as other single gene disorders, namely preimplantation genetic testing for monogenic disorders (PGT-M), in the setting of in vitro fertilization (IVF). In contrast, PGT for mtDNA disorders is complicated by unique biologic features of mtDNA however has now been successfully performed by several centers globally. Despite these advances, PGT for mtDNA disorders is not offered in the United States (US) due to inexperience with specialized techniques for accurately assessing mtDNA heteroplasmy in embryo biopsies. To address this gap in options for US families with mtDNA disorders and the cost of traveling overseas, we, by collaboration between the Mitochondrial Medicine Frontier Program at the Children’s Hospital of Philadelphia (CHOP), Penn Fertility Care at the Hospital of the University of Pennsylvania (Penn), and the Newcastle Rare Mitochondrial Disorders Service (Newcastle, UK), established a process to facilitate PGT for mtDNA disorders for families living in the US. The PGT for mtDNA disorders process for US patients includes (1) patient and family counseling, clinical evaluation, and necessary specialist referrals at CHOP; (2) baseline fertility evaluation, IVF cycle planning and monitoring, oocyte retrieval, fertilization, and day 3 single cell biopsy at Penn, followed by biopsy shipment from Penn to Newcastle for mtDNA PGT; (3) fitness for fertility assessment, blastomere heteroplasmy analysis, and telehealth consultation for embryo biopsy heteroplasmy analysis and discussion of PGT outcomes by the Newcastle team; (4) selected embryo transfer based on embryo biopsy mtDNA heteroplasmy analysis at Penn; with (5) follow-up of resultant children born after successful life birth at CHOP. A female patient aged 29 years with m.3243A>G (5-11% heteroplasmy in blood, buccal, and urine) pursued IVF and PGT for the m.3243A>G variant at Penn Fertility Care, with consultation at CHOP, IVF cycle planning and care at Penn, and telehealth consultation and samples sent to Newcastle for mtDNA variant heteroplasmy determination. One IVF cycle yielding 13 oocytes and biopsy of 7 embryos was completed. Embryos had low to undetectable heteroplasmy, and one with undetectable heteroplasmy was transferred resulting in a successful live birth. Post-natal follow-up at age 20 months has confirmed the female child’s normal health and development, with post-natal heteroplasmy level testing actively being coordinated. Families with mtDNA disorders living in the US have had limited family planning options given the unique nature of mtDNA variants, despite the potentially devastating nature of these conditions. Families carrying a known pathogenic mtDNA disorder with a desire for a genetic relationship to their child can now remain in the US while undergoing IVF and PGT for mtDNA disorders. Formal establishment of this process was completed to help other individuals with mtDNA disorders achieve their family planning goals. Abstract #: 2025PA-0000000137 Presenter: Jean Flickinger

Awad EK 1 , Waskow E 2 , Tunuguntla H 3 , Spinner J 3 , Emrick LT 2 , Calame DG 2 , Scaglia F 2 1 Department of Human Genetics and Genomics, Fellow, Baylor College of Medicine, USA, 2 Department of Human Genetics and Genomics, Faculty, Baylor College of Medicine, USA, 3 Department of Cardiology, Faculty, Baylor College of Medicine, USA, 4 Department of Neurology, Faculty, Baylor College of Medicine, USA Corresponding Author: [email protected] Abstract: COX14 encodes a small single-pass transmembrane protein that plays a crucial role in the early stages of cytochrome C oxidase (COX) complex assembly, particularly in the synthesis and assembly of the COX I subunit. Biallelic pathogenic variants in COX14 are associated with mitochondrial complex IV deficiency nuclear type 10 (MC4DN10), an autosomal recessive mitochondrial disorder with multi-organ involvement (MIM #619053). There is scant information about this condition in the scientific literature. To date, there has been 1 published report of 3 children born to a consanguineous Portuguese couple who harbored a homozygous variant (c.88G>A; p.Met19Ile) in COX14 . These children developed severe infantile lactic acidosis that was ultimately lethal. Neurological findings on presentation were brain hypertrophy, cavitating lesions, and abnormal brain myelination. Other associated symptoms included respiratory distress, dysmorphic features, and findings consistent with multi-organ involvement such as hepatomegaly, hypertrophic cardiomyopathy, renal hypoplasia, and adrenal hyperplasia. Here we report a case of a 9-year-old male who initially presented at 6 months of age with hypotonia, feeding difficulties, poor growth, and global developmental delay. He was found to have growth hormone deficiency at 2 years of age and subsequently treated with growth hormone. Genetic evaluation was performed at 4 years of age. The patient was non-dysmorphic and did not have hepatomegaly. There was no evidence of hypertrophic cardiomyopathy on initial echocardiogram, but on follow up echocardiogram, a mildly dilated ascending aorta was found. Trio whole exome sequencing identified compound heterozygous variants of uncertain significance in COX14 (c.82delT, p.Tyr28Thrfs*83 and c.3G>A, p.Met1?), a gene implicated in mitochondrial complex IV assembly. At 8 years of age, he showed signs of developmental regression and ataxia. Brain MRI revealed characteristic deep gray matter abnormalities consistent with Leigh syndrome. MR spectroscopy of the brain demonstrated a lactate peak. Laboratory evaluation showed normal blood lactate level, normal plasma amino acids with no elevation of alanine, and a normal acylcarnitine profile. His GDF15 was normal at 544 pg/ml (Ref range: <750 pg/ml). The CK level was elevated at 512 U/L (Ref range: <177 U/L). A skeletal muscle biopsy was performed, and on skeletal muscle the activity of cytochrome C oxidase was reduced to 33% of the mean. Coenzyme Q10 supplementation was started at 8 years of age. This new case of MC4DN10 represents an attenuated encephalomyopathic phenotype that includes growth hormone deficiency, dilation of the ascending aorta, and longer survival expanding the clinical spectrum of MC4DN10. It emphasizes the importance of comprehensive genomic evaluation in the diagnostic workup of patients with suspected mitochondrial disorders. This presentation also illustrates the need for better plasma biomarkers for mitochondrial disease to facilitate early disease detection and provide a way to clinically monitor the progression of disease over the course of time. Abstract #: 2025PA-0000000141 Presenter : Graeme Preston

Ritchie J 1,2 , Marshall C 1,2 , Desaigoudar P 2 , Brock D 1,2 , Means M 1,2 1 Department of Pediatrics, Division of Child Neurology, University of Louisville School of Medicine, Louisville, KY, USA, 2 Norton Children’s Neuroscience Institute, Louisville, KY, USA Corresponding author’s email: [email protected] Abstract: Thymidine kinase 2 deficiency (TK2d) is a rare autosomal recessive disease caused by biallelic deleterious variants in the TK2 gene leading to mitochondrial DNA (mtDNA) depletion and inadequate cellular energy production. Patients typically present with myopathy starting in infancy, childhood, or adulthood, though infantile-onset disease has also been associated with developmental delay, microcephaly, lissencephaly, dysmyelination, encephalopathy, and seizures 1 . Here, we describe a case series of two brothers of Guatemalan parents with distantly shared ancestry (1.67% homozygosity of autosomal genes) with prenatal-onset TK2d with cerebral, cerebellar, and brainstem malformations resulting in death in the neonatal period. The first brother was born in 2020 with pregnancy complicated by ultrasound findings of cerebral ventriculomegaly, absent cerebellar vermis, and IUGR. He was born at term by emergency cesarean section due to non-reassuring fetal heart tones and required extensive resuscitation at birth. Exam was remarkable for diffuse hypotonia, multiple extremity contractures, hydrops fetalis, facial dysmorphisms, and apnea requiring intubation. Head ultrasound showed cerebral and cerebellar volume loss with ventriculomegaly and suggestion of cortical dysgenesis. Brain magnetic resonance imaging (MRI) was not able to be completed due to clinical instability and redirection of care. Chromosomal Microarray Analysis (CMA) demonstrated multiple regions of homozygosity. He had no spontaneous movements or respiratory effort over the ventilator, so was transitioned to comfort care and subsequently died on day of life 3. Research trio whole genome was posthumously performed and significant for homozygous pathogenic frame shift variants in TK2 (p.K43Nfs*9) resulting in premature truncation and loss-of-function (LoF). Both parents were confirmed carriers. The second brother was born in 2024 at term via repeat c-section, with prenatal scans concerning for cerebellar hypoplasia and microcephaly. Delivery was complicated by tight nuchal cord and shoulder dystocia, and he required extensive resuscitation including intubation for apnea. He had diffuse hypotonia, no spontaneous movements, no reaction to painful stimuli, and nonreactive and dilated pupils, so was started on therapeutic hypothermia protocol for presumed hypoxic ischemic encephalopathy. Electroencephalogram (EEG) was notable for focal seizures which responded to phenobarbital and a background with burst suppression. Brain MRI was notable for micrencephaly with extensive hypoplasia and dysgenesis of the cortex, deep grey structures, brainstem, and cerebellum. Rapid trio exome revealed the same homozygous pathogenic variant in TK2 (p.K43Nfs*9). Compassionate use of deoxyribonucleoside therapy (dC/dT) was considered, however was not pursued due to presumed lack of benefit given structural brain abnormalities and lack of brainstem reflexes or responsiveness on exam. He was compassionately extubated and died shortly thereafter on day of life 16. The pathogenic frameshift mutation identified in our patients has previously been described in association with myopathic phenotypes when in trans (compound heterozygotes) with missense variants 2,3 , though no previous homozygotes have been reported. Moreover, no cases of biallelic LoF variants in TK2 have been reported to date. We propose that this homozygous LoF variant in TK2 resulted in our patients’ severe neurologic phenotype characterized by extensive cerebral, cerebellar, and brainstem dysgenesis and dysfunction. References 1. Garone C, Taylor RW, Nascimento A, Poulton J, Fratter C, Domínguez-González C, Evans JC, Loos M, Isohanni P, Suomalainen A, Ram D, Hughes MI, McFarland R, Barca E, Lopez Gomez C, Jayawant S, Thomas ND, Manzur AY, Kleinsteuber K, Martin MA, Kerr T, Gorman GS, Sommerville EW, Chinnery PF, Hofer M, Karch C, Ralph J, Cámara Y, Madruga-Garrido M, Domínguez-Carral J, Ortez C, Emperador S, Montoya J, Chakrapani A, Kriger JF, Schoenaker R, Levin B, Thompson JLP, Long Y, Rahman S, Donati MA, DiMauro S, Hirano M. Retrospective natural history of thymidine kinase 2 deficiency. J Med Genet. 2018 Aug;55(8):515-521. doi: 10.1136/jmedgenet-2017-105012. Epub 2018 Mar 30. PMID: 29602790; PMCID: PMC6073909. 2. Collins J, Bove KE, Dimmock D, Morehart P, Wong LJ, Wong B. Progressive myofiber loss with extensive fibro-fatty replacement in a child with mitochondrial DNA depletion syndrome and novel thymidine kinase 2 gene mutations. Neuromuscul Disord. 2009 Nov;19(11):784-7. doi: 10.1016/j.nmd.2009.08.002. Epub 2009 Sep 6. PMID: 19736010. 3. Chanprasert S, Wang J, Weng SW, Enns GM, Boué DR, Wong BL, Mendell JR, Perry DA, Sahenk Z, Craigen WJ, Alcala FJ, Pascual JM, Melancon S, Zhang VW, Scaglia F, Wong LJ. Molecular and clinical characterization of the myopathic form of mitochondrial DNA depletion syndrome caused by mutations in the thymidine kinase (TK2) gene. Mol Genet Metab. 2013 Sep-Oct;110(1-2):153-61. doi: 10.1016/j.ymgme.2013.07.009. Epub 2013 Jul 17. PMID: 23932787. Abstract #: 2025PA-0000000100 Presenter: Katelynn Stanley

Scheller, SB Bioethics Center for the Practices of Maternal, Family, and Mitochondrial Health, USA [email protected] Abstract: With the identification of mitochondrial disease or dysfunction, decisions regarding fertility take on profound, new significance. In one context, parents who discover they are carriers, after suffering the loss of their child due to mitochondrial disease, may wish to initially delay a subsequent pregnancy and also seek ways to lower the risk of a recurrence. While the Creighton Model Fertlity Care TM System (CrMS) offers superior advantages in fertility effectiveness, it is not usually included in options presented to families who face these challenges in their fertility journey. This study analyses responses from patient interviews about fertility-related needs and the fertility-options offered in clinical settings in 4 nations. A finding is that a very low percentage of family members were informed about fertility-options by their mitochondrial healthcare professionals. Answers also revealed higher than average comorbidities with certain women’s health conditions like endometriosis, polycystic ovarian syndrome, and anomalies during the perinatal period. A literature review highlights connections between these fertility-related patterns, which can be studied through CrMS. In this effective system, the female cycle is read as a vital sign, providing woman-specific feedback relating to endometrial health with epigenetic implications for mother and child. Therefore, this article presents a framework for improving the health of the mother and positive outcomes of pregnancy, centering on interdisciplinary work, leading to improved protocol for informed consent in the mitochondrial disease community around fertility-related concerns. It is argued that it is a professional obligation to provide family members with this effective option in their fertility odyssey. Abstract #: 2025PA-0000000117 Presenter : Jeffrey A. Haltom

Wang Y 1,2 , Pan X 1,2 , Liu N 1,2 , Sutton VR 1,2,3 , Craigen WJ 1,3 , Sun Q 1,2* 1 Department of Molecular & Human Genetics, Baylor College of Medicine, USA, 2 Baylor Genetics Laboratory, USA, 3 Texas Children’s Hospital, USA * [email protected] Abstract: Large deletions in the mitochondrial genome are significant contributors to mitochondrial disorders. Sporadic single large mitochondrial DNA (mtDNA) deletions are associated with chronic progressive external ophthalmoplegia (CPEO), Kearns-Sayre syndrome (KSS), and Pearson syndrome (PS), collectively termed single large-scale mtDNA deletion syndromes (SLSMDSs). CPEO and KSS present with a spectrum of symptoms including ptosis, external ophthalmoplegia, and mitochondrial myopathy, while PS is characterized by early-onset anemia or pancytopenia in infancy with multisystem involvement. In addition to sporadic mtDNA deletions, pathogenic variants in nuclear genes can lead to multiple mtDNA deletions, resulting in overlapping clinical features with CPEO and KSS. Unlike mitochondrial disorders caused by single gene variants, where heteroplasmy levels often correlate with disease severity and age of onset, such correlations remain unclear in SLSMDSs. We previously showed that low-level large mtDNA deletions were observed in patients with mitochondrial disorders. To investigate the functional impact of low-level mtDNA deletions in muscle, we retrospectively analyzed consecutive clinical cases from the past decade, each with both mtDNA genome sequencing and electron transport chain (ETC) functional testing performed on the same muscle specimens. Of these, 3.6% cases had single large mtDNA deletions, and 16.5% cases exhibited multiple deletions. Within 7.9% cases presenting with clinical features consistent with the CPEO/KSS spectrum, 18.2% were diagnosed with SLSMDSs, while 36.4% had multiple mtDNA deletions. No PS cases were identified in the cohort, consistent with its lack of primary muscle involvement. Among the 16 CPEO/KSS cases with SLSMDSs, 7 demonstrated deficient ETC complex activity (<30% of normal, per modified Walker minor criteria). Notably, all 7 cases displayed low heteroplasmy levels (<10%) for the mtDNA deletions. ETC deficiencies in these 7 cases included single complex deficiency in 4 samples and multi-complex defects in 3 samples. Complex IV was most commonly impacted, being reduced to 4 samples, whereas the nuclear-encoded complex II was consistently unaffected. No clear correlation emerged between the deleted mitochondrial genes and the ETC deficiencies. In summary, our study highlights that significant mitochondrial dysfunction can occur even when mtDNA deletion heteroplasmy level is well below the expected deficient thresholds and underscores the importance of integrating molecular and functional evaluations for accurate diagnosis of SLSMDSs. Abstract #: 2025PA-0000000025 Presenter: Norman Liu

Alexander J Sercel 1 , Douglas C Wallace 2 , Anu Suomalainen 3 , Michael P Murphy 4 , Navdeep Chandel 5,6 , and Gordon Freedman 1 1 MitoWorld, National Laboratory for Education Transformation, Oakland, CA, USA, Department of Ophthalmology, 2 Center for Mitochondrial and Epigenomic Medicine, The Children’s Hospital of Philadelphia, Philadelphia, PA, USA, 3 Stem Cells and Metabolism, Faculty of Medicine, University of Helsinki, Helsinki, Finland, 4 MRC-Mitochondrial Biology Unit, The Keith Peters Building, University of Cambridge, 5 Division of Pulmonary and Critical Care Medicine, Department of Medicine, Northwestern University, Chicago, IL, USA, 6 Biochemistry and Molecular Genetics, Northwestern University, Chicago, IL, USA Cambridge, CB2 0XY, UK, 7 Medical University of South Carolina, Charleston, SC, USA Abstract: MitoWorld.org is a dynamic, web-based platform designed to accelerate progress in mitochondrial biology, enhance patient care, and raise public awareness of mitochondria’s vital role in health and disease. By connecting researchers, clinicians, patients, and advocacy networks, MitoWorld fosters a global community dedicated to collaboration, knowledge-sharing, and the advancement of mitochondrial science. The platform’s central mission is to streamline the translation of mitochondrial research into clinical practice by serving as a hub for organizing research priorities, coordinating large-scale collaborations, and unifying efforts across broad cross-disciplinary fields. Despite rapid advances in the field and growing recognition of mitochondria’s involvement in a wide array of diseases, these organelles remain underappreciated and misunderstood by much of the biomedical community and the public. Known primarily as the cell’s “powerhouses,” mitochondria are becoming recognized as key regulators of cellular health, with diverse organ and disease-specific functions in metabolism, signaling, disease mechanisms, and even aging. As mitochondrial discoveries expand across disciplines, the need to effectively organize and integrate this knowledge has become urgent. MitoWorld aims to meet this challenge by providing a centralized resource for data, collaboration, and communication—bridging research gaps and promoting cross-disciplinary insights that ultimately drive innovation in medicine. Equally vital is the platform’s role in public engagement. By highlighting progress in mitochondrial research and its clinical impact, MitoWorld seeks to build broader awareness, inspire support, and attract sustained investment in discovering mitochondria’s full role in human health and developing therapies to support mitochondrial function in diseases. Through community-building and coordinated action, MitoWorld is accelerating the path from discovery to comprehension to therapy. Abstract #: 2025PA-0000000169 Presenter : Min-kyung Nam

Baker ZN 1 , Zhu Y 2,3 , Guerra RM 1 , Smith AJ 1 , Arra A 4 , Serrano LR 2,3 , Overmyer KA 2,3,6 , Mukherji S 4 , Craig EA 5 , Coon JJ 2,3,6,7 , and Pagliarini DJ 1,8,9,10* 1 Department of Cell Biology and Physiology, Washington University School of Medicine, St. Louis, Missouri, USA, 2 Department of Chemistry, University of Wisconsin–Madison, Madison, WI, USA, 3 National Center for Quantitative Biology of Complex Systems, Madison, WI, USA, 4 Department of Physics, Washington University, St. Louis, Missouri, USA, 5 Department of Biochemistry, University of Wisconsin–Madison, Madison, WI, USA, 6 Morgridge Institute for Research, Madison, WI, USA, 7 Department of Biomolecular Chemistry, University of Wisconsin–Madison, Madison, WI, USA, 8 Department of Biochemistry and Molecular Biophysics, Washington University School of Medicine, St. Louis, Missouri, USA, 9 Department of Genetics, Washington University School of Medicine, St. Louis, Missouri, USA, 10 Howard Hughes Medical Institute, Washington University School of Medicine, St. Louis, Missouri, USA * e-mail: [email protected] Abstract: Mitochondria are a central hub for cellular metabolism and thus even moderate mitochondrial dysfunction can lead to a host of severe pathological conditions. This importance highlights a need for understanding how cells adapt to and overcome mitochondrial dysfunction, however, previous efforts to characterize the global responses to mitochondrial dysfunction have often used models with insurmountable stressors. Furthermore, these studies have mostly focused on the role of protein effectors, marginalizing the role of metabolites and lipids in these pathways. To address these gaps in knowledge, we leveraged the power of high-throughput mass spectrometry to generate multiomics datasets and determine responses involved in overcoming moderate-to-severe mitochondrial dysfunction. Using a novel extraction method that allowed us to measure proteins, lipids, and metabolites from a single sample, we screened multiple strains of yeast grown with a diverse array of pharmacological and genetic perturbations eliciting mitochondrial dysfunction. We found that strains undergoing moderate mitochondrial dysfunction exhibited a distinct molecular profile characterized by a widespread loss of mitochondrial proteins and an increased mobilization of triacylglycerols (TAGs). Over time, these strains were able to adapt to mitochondrial dysfunction by increasing their mitochondrial content, thus resuming growth to near the wild-type rate. This recovery to normal growth was dependent on the mobilization of TAGs but independent of fatty acid oxidation, with the acyl-groups generated from TAG mobilization being shuttled into nascent cardiolipin biosynthesis required for mitochondrial membrane expansion. Correlation analysis linked the partially uncharacterized perilipin protein Pln1p to this phenomenon, and overexpression of Pln1p prevented the lipolysis of TAGs required for recovery. Finally, we found that using TAG lipolysis as a mechanism for mitochondrial dysfunction recovery was conserved in mammalian cells as knockout of the lipase ATGL resulted in increased sensitivity to doxycycline treatment. In summary, our work suggests a model in which moderate mitochondrial dysfunction is overcome with the biogenesis of new organelles, at least in part, through the mobilization of TAG stores for membrane generation. This work not only expands our understanding of a fundamental biological response but also potentially provides a novel approach for treatment of patients with mitochondrial dysfunction such as that seen in mitochondrial disease. Abstract #: 2025PA-0000000133 Presenter: Suraiya Haroon

Romero A 1,2 , Timón-Gómez A 2 , Campagnol S 1 , Madonia D 1,2 , Willis J 2 , Gnaiger E 2 1 VASCage- Centre on Clinical Stroke Research, Innsbruck, Austria, 2 Oroboros Instruments, Innsbruck, Austria. * [email protected] Abstract: Bioenergetic assessment of peripheral blood mononuclear cells (PBMCs) is emerging as a promising minimally invasive tool for diagnosing mitochondrial diseases. Prior studies have linked PBMC mitochondrial dysfunction to primary mitochondrial diseases, such as Leber Hereditary Optic Neuropathy (LHON), highlighting their potential as systemic biomarkers 1,2 . However, reliance on freshly isolated cells restricts their clinical and research applicability. To address this challenge, standardization of cryopreservation protocols that maintain mitochondrial function is essential to enable wider diagnostic implementation. We optimized protocols for PBMC isolation from human liquid biopsies, cryopreservation, and thawing, aiming to preserve mitochondrial function 3 . Mitochondrial respiration was assessed by high-resolution respirometry in freshly isolated PBMCs and after 1 and 4 weeks of storage at -80 °C. Three substrate-uncoupler-inhibitor titration (SUIT) protocols were applied in parallel to both living and permeabilized PBMCs, to assess 25 mitochondrial pathway and coupling control states, generating high-resolution bioenergetic profiles 4 . Coupling-control analysis in living cells demonstrated stable routine respiration ( R ) and leak respiration ( L ) after 4 weeks of cryopreservation. Over 92 % of electron transfer (ET) capacity was preserved. Reserve capacity and routine coupling control ratios remained consistent between freshly isolated and cryopreserved samples. Precision analysis of oxidative phosphorylation (OXPHOS) in permeabilized PBMCs showed no significant differences between fresh and cryopreserved cells across major mitochondrial pathways, including NADH-linked respiration through Complex I (N), succinate-linked respiration through Complex II (S), fatty acid oxidation (F), glycerophosphate oxidation (Gp), and Complex IV activity. A decline in ET capacity by 8 % was observed only in the combined substrate condition (FNSGp) after 4 weeks, consistent with the trend in ET capacity of living cells. In conclusion, we provide an optimized protocol for PBMC isolation and cryopreservation that preserves mitochondrial bioenergetic function for at least 4 weeks. Evaluation of longer storage times is ongoing. This approach enables robust and reproducible profiling of mitochondrial function from stored PBMCs, supporting broader diagnostic applications and potential biomarker development for mitochondrial diseases. References 1. Petrovic´ P et al. Atypical Leber Hereditary Optic Neuropathy (LHON) associated with a novel MT-CYB:m.15309T>C (Ile188Thr) variant. Genes. 2025;16(1):108. doi: 10.3390/genes16010108. 2. Stenlid R et al. Altered mitochondrial metabolism in peripheral blood cells from patients with inborn errors of β-oxidation. Clin Transl Sci . 2022; 15: 182–194. https://doi.org/10.1111/cts.13133 3. Gumpp AM et al. Investigating mitochondrial bioenergetics in peripheral blood mononuclear cells of women with childhood maltreatment from post-parturition period to one-year follow-up. Psychol. Med. 2023;53(9):3793–3804. doi: 10.1017/S0033291722000411. 4. Timón-Gómez A et al. Analysis of mitochondrial respiratory pathway and coupling control by substrate-uncoupler-inhibitor titration reference protocols. MitoFit Preprints. 2024;8. doi: 10.26124/mitofit:2024-0008. Abstract #: 2025PA-0000000095 Presenter: Amel Karaa

Pérez MJ 1,2 , Colombo RB 1 , Real SM 1,3 , Branham MT 1,4 , Laurito SR 1,5 , Moraes CT 6 , Mayorga L 1 1 Instituto de Histología y Embriología de Mendoza (IHEM, Universidad Nacional de Cuyo, CONICET)- Mendoza, Argentina, 2 Facultad de Ciencias de la Nutrición. Universidad Juan Agustín Maza. Mendoza-Argentina, 3 Instituto de Fisiología, Facultad de Ciencias Médicas. Universidad Nacional de Cuyo. Mendoza, Argentina, 4 Facultad de Ciencias Médicas. Universidad de Mendoza. Mendoza-Argentina, 5 Facultad de Ciencias Exactas y Naturales. Universidad Nacional de Cuyo. Mendoza, Argentina, 6 Department of Neurology, University of Miami Miller School of Medicine, Miami, Florida, USA. Abstract : Mitochondrial disorders (MD) arise from mutations in either nuclear or mitochondrial DNA (mtDNA). For the latter, the coexistence of mutant and wild-type mtDNA molecules, known as heteroplasmy, determines disease onset when the mutant load surpasses a critical threshold. Thus, reducing the proportion of mutant mtDNA, termed "heteroplasmy shift," represents a promising therapeutic strategy. While gene therapy targeting nuclear DNA is well-established and expanding to many rare diseases, editing mtDNA remains challenging. Mitochondrial stress has been shown to trigger nuclear epigenetic modifications, and we previously proposed that nuclear DNA methylation serves as an adaptive response to chronic mitochondrial dysfunction. Since high heteroplasmy is associated with severe mitochondrial impairment, we hypothesized that cells experiencing greater dysfunction rely on this epigenetic mechanism for survival. We aimed to disrupt this adaptation, selectively compromising high-heteroplasmy cells while favoring those with lower heteroplasmy, ultimately shifting the mutant load toward a healthier balance. Using cybrid cells carrying the m.13513G>A and m.8344A>G mtDNA mutations at different heteroplasmy levels, we characterized nuclear DNA methylation profiles with the Infinium 850k Methylation EPIC array®. We found distinct epigenetic patterns shaped by both mutation type and load, with gene enrichment analysis revealing differential methylation in pathways associated with cell survival. Functionally, high-heteroplasmy cybrids displayed increased proliferation (pA cybrids, high-heteroplasmy cells exhibited reduced apoptosis (p<0.05), a phenotype reversed by DMI treatment or DNMT1 knockdown (pG cybrids were also sensitive to DMIs, while low-heteroplasmy counterparts for both mutations remained unaffected by treatment. In patient tissues, cells with high and low heteroplasmy often coexist, forming a mosaic of mutant loads. To model this scenario, we tested our approach in mixed heteroplasmy cultures and a xenograft model where both high- and low-heteroplasmy m.13513G>A cybrids were present. Treatment with the DMI decitabine significantly reduced heteroplasmy (~20% in cell culture, ~40% in vivo), suggesting a selective advantage for low-heteroplasmy cells upon treatment with DMIs. Importantly, direct analysis of mtDNA methylation in xenograft tumors confirmed that mtDNA methylation levels were minimal and unaffected by decitabine. In contrast, nuclear DNA methylation patterns were shaped by heteroplasmy levels and altered by decitabine treatment, highlighting the role of nuclear epigenetics as a central hub for sensing mitochondrial dysfunction and triggering adaptive responses. This bidirectional cross-talk suggests that nuclear epigenetic modifications not only detect and respond to varying degrees of mitochondrial impairment but can also play an active role in regulating heteroplasmy. These findings demonstrate that mitochondrial dysfunction drives nuclear epigenetic modifications, which in turn regulate cellular responses which can ultimately influence mtDNA heteroplasmy. While further research is needed, these results raise the possibility that epigenetic interventions could be explored as a strategy to modulate heteroplasmy in mitochondrial disorders. Abstract #: 2025PA-0000000044 Presenter: Charles DiFalco, MD

Brooks D. 1,2 , Koh H.Y. 3,4 , Kerrins T. 1,2 , Lang S. 1,2 , Magness E. 1,2 , Yang R. 5,6 , Nicholas S. 5,6 , Sikkink S. 9 , Eng C. 7 , Murali C.N. 1,2 , Lalani S. 1,2 , Magoulas P. 1,2 , and Scaglia F. 1,2,8 1Department of Molecular and Human Genetics, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA, 2 Department of Molecular and Human Genetics, Texas Children's Hospital, Houston, Texas, USA, 3 Division of Neurology, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA, 4 Division of Neurology, Texas Children's Hospital, Houston, Texas, USA, 5 Division of Allergy and Immunology, Department of Pediatrics, Baylor College of Medicine, Houston, Texas, USA, 6 Division of Allergy and Immunology, Texas Children's Hospital, Houston, Texas, USA, 7 Baylor Genetics, Houston, Texas, USA, 8 Joint BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, ShaTin, Hong Kong SAR, China, 9 Geisenger Medical Center, Department of Pediatrics and Department of Internal Medicine Corresponding Author: Dr. Fernando Scaglia, [email protected] Abstract: Mitochondrial diseases are a molecularly and clinically heterogeneous group of inborn errors of energy metabolism caused by pathogenic variants in nuclear or mitochondrial DNA (mtDNA), impairing oxidative phosphorylation and leading to multi-system dysfunction. While the metabolic consequences of mitochondrial disease are well recognized, emerging evidence suggests that mitochondrial dysfunction can also trigger immune dysregulation and chronic inflammation. Mitochondrial damage-associated molecular patterns (DAMPs), including mtDNA and mitochondrial double-stranded RNA (mt-dsRNA), activate innate immune pathways such as the cyclic GMP-AMP synthase–stimulator of interferon genes (cGAS-STING) and the Retinoic acid-inducible gene I/Melanoma Differentiation-Associated protein 5 (RIG-I/MDA5) pathways, resulting in type I interferon-driven inflammation. PNPT1 encodes polyribonucleotide nucleotidyltransferase 1 (PNPase), a critical enzyme that degrades mitochondrial RNA to prevent its cytosolic escape, thereby regulating the above pathways. Thus, in addition to primary mitochondrial disease, biallelic PNPT1 variants have also been associated with excessive interferon signaling and autoinflammatory phenotypes. We describe a 4-month-old female initially suspected to have a mitochondrial disorder. She was born at 37 weeks with symmetric intrauterine growth restriction (IUGR) and experienced a prolonged NICU stay due to hypotonia, decerebrate posturing, and persistent apnea. Additional findings included failure to thrive and severe gastroesophageal reflux disease requiring Nissen fundoplication and gastrostomy tube placement. After discharge, she suffered a life-threatening apneic event requiring CPR, intubation, and ICU admission. Brain MRI with MR spectroscopy revealed cerebellar hypoplasia, immature brain development, and elevated lactate, consistent with mitochondrial dysfunction. Laboratory testing showed an elevated serum lactate of 7.9 mmol/L (normal: 0.5–2.2 mmol/L), elevated ALT/AST of 73/88 U/L (normal: ALT 14–45 U/L; AST 20–60 U/L), and blood gas with pH 7.25/pCO₂ 63, indicating respiratory acidosis. Genetic testing identified compound heterozygous variants in PNPT1 : a maternally inherited likely pathogenic intronic variant ( NM_033109.5 : c.680-3T>A) and a paternally inherited missense variant of uncertain significance ( NM_033109.5 : c.1297G>A) with high pathogenicity prediction scores (CADD 26.9, REVEL 0.723). A targeted immune workup identified a mild type I interferon signature in classical monocytes (82%) with modestly expanded non-classical monocytes (13%), consistent with an underlying interferonopathy. Given her clinical presentation and laboratory findings consistent with a type 1 interferonopathy, tofacitinib, a Janus kinase (JAK) inhibitor that blocks JAK-STAT pathway activation and subsequent cytokine release downstream of the abnormal interferon signaling, was initiated. Following treatment initiation, type I interferon markers and AST/ALT levels normalized. Blood gas values improved (pH 7.38/pCO₂ 46), and serum lactate declined to 2.6 mmol/L. The patient also demonstrated notable clinical improvements. Although she required tracheostomy placement due to frequent apneas, ventilator settings were weaned to minimal levels. From a nutritional perspective, she had improved weight gain with her weight-for-age Z-score increasing from –3.36 to –2.18. Additionally, her dystonic posturing movements decreased. This case highlights the intersection between mitochondrial pathology and immune activation, suggesting that some mitochondrial disorders may have an autoinflammatory component. These findings underscore the therapeutic potential of JAK inhibition in certain mitochondrial disorders. Targeted immunomodulatory therapies may mitigate excessive type I interferon signaling and improve clinical outcomes. However, longer-term monitoring will be essential to evaluate the impact on developmental and neurologic trajectories. Further studies are warranted to elucidate the molecular mechanisms linking mitochondrial dysfunction and innate immunity, and to explore precision therapies in mitochondrial disease with immune dysregulation. Abstract #: 2025PA-0000000112 Presenter : Elizabeth McCormick

Mitchell DV 1* , Zhang Y 1 , Keith K 1 , McCormick EM 1,2 , George-Sankoh I 1 , Kim MS 3 , Zhang Z 1 , Falk MJ 2,4 , Taylor D 1,4 1 Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, Philadelphia, PA, 2 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, 3 School of Medicine, Kyung Hee University, Dongdaemun-gu, Seoul, South Korea, 4 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA* [email protected] Abstract: Primary mitochondrial disease (PMD) cellular and animal models with impaired aerobic energy production have improved health and viability when given glucose, exploiting their upregulated glycolytic anaerobic energy production capacity. However, our previous preclinical modeling studies showed that glucose administration masks mechanistic investigation into metabolic stress adaptations that occur in PMD cells. We hypothesized that culturing PMD fibroblast cells in galactose medium would unmask underlying cellular adaptations, enabling evaluation of mitochondrial disorders’ effects on intermediary metabolic processes and physiologic functions. 12 human fibroblast cell lines molecularly confirmed (n=2) or clinically suspected (n=10) PMD patients, and 2 healthy controls were cultured at ~80% confluence in glucose (10 mM) or galactose (10 mM) media for 24 hours, after which RNA-Seq based transcriptome profiling was performed. Pairwise comparisons identified differentially expressed genes and enriched pathways. In addition, a novel metabolic modeling approach was performed to discern variations in metabolic potential between healthy and diseased cells. This systems biology focused method uses genome-scale metabolic models reconstructed from biochemical reactions comprising cellular processes of interest, which, when constrained by the RNA-seq data, simulate metabolic capacity and potential metabolic flux alterations. Having established the utility of galactose media to elucidate cellular metabolic adaptations in mitochondrial disease cells, our methodology and analysis was validated in a larger dataset of 92 additional samples comprising 12 healthy controls and 45 definite PMD patients cohorted by molecularly-related etiologies, including 6 complex I deficiency, 2 complex III deficiency, 5 complex IV or V deficiency, 2 fission/fusion defects, 9 mtDNA maintenance disorders, 1 single large-scale mtDNA deletion (SLSMD), 2 nDNA gene disorders, 5 pyruvate dehydrogenase complex disorders, and 13 mitochondrial translation disorders. This expanded investigation sought to elucidate discrepancies in the potential activity of biochemical reactions between healthy controls and PMD patients with varying subgroups of genetic etiologies. Significant differences were identified in gene expression, pathway enrichment, and biochemical regulation between PMD cells grown in galactose versus glucose media. For multiple patients, significant downregulation in galactose media were observed of pathways associated with cell cycle activities, chromosomal organization, and translational regulation. By contrast, few differences were identified in glucose vs galactose exposed healthy control cells. Across the larger cohort, metabolic flux capacity modeling unveiled significant differences between PMD patients and healthy controls in a number of biochemical subsystems (including glycolysis and the TCA cycle), after only subtle differences in transcriptomic profiles could be elucidated through differential expression and pathway enrichment analyses. In vitro transcriptome analyses in PMD demonstrated that significant differences occurred in gene expression of cell growth related pathways and potential metabolic flux capacity across numerous metabolic reactions in PMD patient cells grown in galactose compared to glucose media. This study underscores the critical importance of carefully controlling for cellular growth conditions when studying PMD and utilizing transcriptome profiling to investigate cellular mechanisms underlying complex metabolic disorders. Further, metabolic flux modeling offers a novel approach to unveil previously unrecognized systemic biochemical disparities that occur among different classes of mitochondrial pathology that may be overlooked with canonical RNA-seq analyses. Abstract #: 2025PA-0000000146 Presenter: Elizabeth Mizerik, MS, CGC

Alshamleh, I 1 , Tippetts T. S 1 , Mishra P 1 and DeBerardinis R 1 1 Children’s Medical Center Research Institute, University of Texas Southwestern Medical Center *(Corresponding author’s email [email protected] ) Abstract: Mitochondrial diseases are caused by mutations in oxidative mitochondrial pathways, such as the tricarboxylic acid (TCA) cycle and electron transport chain (ETC). The extreme phenotypic and genetic heterogeneity of mitochondrial diseases, coupled with challenges in interpreting genomic variants and obtaining tissue for definitive biochemical analysis, make diagnosing these diseases difficult. We also lack techniques to monitor mitochondrial function over time in affected tissues, particularly the brain and muscle, and thus to detect therapeutic responses. We propose to use deuterium metabolic imaging (DMI) to close this gap. DMI enables real-time monitoring of tissue metabolism, including in brain and muscle, and is well-suited to assess fuel oxidation in the mitochondria. Deuterium is a stable isotope of hydrogen that can be used to label nutrients such as glucose and lactate. Tissue metabolism transfers deuterium from these nutrients to downstream products, whose appearance is detected by MRI. To develop DMI for mitochondrial diseases, we will use mouse models of TCA cycle and ETC defects manifesting in brain and muscle. Deuterium-labeled nutrients will be infused into wild-type and mutant mice, during real-time acquisition of magnetic resonance spectra with a deuterium coil positioned over the affected tissue. Mathematical models will convert resulting spectra into key metabolic fluxes, including glucose uptake, glycolysis and the TCA cycle. We hypothesize that mitochondrial defects will produce quantitative differences in these pathways that could be used diagnostically. Given that deuterium MRI has been used in humans, this project will pave the way clinical imaging tests to monitor and diagnose mitochondrial diseases. Abstract #: 2025PA-0000000165 Presenter: Yuka Aoyama

Anand A 1* , Goldstein A 2 , Ganetzky R 2 , Waldman A 3 , Morava E 1 , Elsharkawi I 1 1 Department of Genetics and Genomics, Icahn School of Medicine, USA, 2 Division of Human Genetics, Children’s Hospital of Philadelphia, USA, 3 Division of Neurology, Children’s Hospital of Philadelphia, USA [email protected] Abstract: Mitochondrial leukodystrophies comprise 5-10 % of all leukodystrophies. Nuclear gene NDUFV1 encodes NADH-Ubiquinone Oxidoreductase Flavoprotein I, an important subunit of Complex I of the mitochondrial respiratory chain. NDUFV1-related mitochondrial disease presents on a clinical spectrum of disease severity including Leigh Syndrome, leukodystrophy, or an overlapping phenotype, with variable age of onset, symptoms, and neuroimaging features. Here we describe two siblings, one presenting with regression and leukodystrophy in infancy and an older asymptomatic brother, both found to harbor biallelic pathogenic and likely pathogenic variants in NDUFV1 . The brother remains symptom free at 2.5 years of age; however, serum ketone body analysis for both patients revealed elevated beta-hydroxybutyrate to acetoacetate ratio and elevated alpha-hydroxybutyrate level. This reflects an abnormal mitochondrial redox state consistent with Complex 1 deficiency and suggests a potential benefit of repleting NAD+, for instance, via niacin/niacinamide treatment. Moreover, we incorporate existing literature for NDUFV1 to help guide prognostic counseling for the family for both children and discuss potential benefits and drawbacks of prophylactic brain imaging and targeted mitochondrial cocktail for the asymptomatic sibling. The sibling cases described reinforce the phenotypic heterogeneity that has previously been described with NDUFV1-related mitochondrial leukodystrophy and the absence of a genotype-phenotype correlation. We demonstrate the utility of ketone body analysis as a non-invasive functional study tool to augment a molecular diagnosis and guide treatment, although prognostic challenges remain. Abstract #: 2025PA-0000000075 Presenter: Stephen C. Ekker, PhD

Fibi Meshrkey 1,2,3#, Kelly M. Scheulin 4,5,6 #, Bibhuti Saikia 1 , Edward J. Lesnefsky 7,8,9,10 , Raj R. Rao 2,11 , Franklin D. West 4,5,6 , Shilpa Iyer 1,2* 1 Department of Biological Sciences, Fulbright College of Arts and Sciences, University of Arkansas, Fayetteville, AR, USA, 2 Cell and Molecular Biology Program, University of Arkansas, Fayetteville, AR, USA, 3 Department of Histology and Cell Biology, Faculty of Medicine, Alexandria University, Egypt, 4 Regenerative Bioscience Center, University of Georgia, Athens, GA, USA, 5 Department of Animal and Dairy Science, University of Georgia, Athens, GA, USA, 6 Biomedical and Health Sciences Institute, Neuroscience Program, University of Georgia, Athens, GA, USA, 7 Department of Physiology and Biophysics, Virginia Commonwealth University, Richmond, VA, USA, 8 Cardiology Section Medical Service, McGuire Veterans Affairs Medical Center, Richmond, VA, USA, 9 Department of Biochemistry and Molecular Biology, Virginia Commonwealth University, Richmond, VA, USA, 10 Pauley Heart Center, Division of Cardiology, Department of Internal Medicine, Virginia Commonwealth University, Richmond, VA, USA, 11 Department of Biomedical Engineering, College of Engineering, University of Arkansas, Fayetteville, AR, USA Abstract: Introduction: Mitochondrial DNA (mtDNA) mutations contribute to respiratory dysfunction and cause mitochondrial diseases. The pathologies of these multisystemic inherited diseases are poorly understood. Mutations in the mitochondrial tRNA gene are one of the most frequent mtDNA mutations and associated with various clinical symptoms such as diabetes mellitus, hearing loss, cardiomyopathy, exercise intolerance, and found in patients with mitochondrial disorders. Human induced pluripotent stem cells (hiPSCs), generated by reprogramming patient-specific somatic cells are recognized as useful tools for disease modeling and serve to better understand the multisystemic pathologies associated with mitochondrial tRNA mutations. Results: Reprogrammed hiPSCs expressed pluripotent stem cell markers including transcription factors POU5F1, NANOG, and SOX2 and cell surface markers SSEA4, TRA-1-60, and TRA-1-81 at the protein level. Sanger sequencing analysis confirmed presence of mutations in both hiPSCs. Next-generation sequencing demonstrated the variable presence of mutant mtDNA in hiPSCs. Cytogenetic analyses confirmed presence of normal karyotype in both hiPSCs. Mitochondrial morphological analysis indicates presence of hyperfused mitochondria in both diseased hiPSC lines. The composite BHI values, a measure of mitochondrial dysfunction that was based on comprehensive bioenergetics analysis of OXPHOS and glycolysis demonstrated the mitochondrial functional defects were severe in both the hiPSC lines exhibiting tRNA mutations. Conclusion : Overall, the hiPSCs exhibited variable mitochondrial morphology and respiratory dysfunction that has the potential to alter hiPSC differentiation potential, cell fate and tissue development. These results indicate the potential significance of using hiPSCs and their derivatives to assess mitochondrial morphology and key bioenergetics parameters as a means to better understand early developmental defects in mitochondrial disorders in children. Note: Only major formatting alterations have been made and abstract content remains consistent with what was entered at time of submission by the author(s).

Schecter D 1 , Pang J 2 , Kozicz T 1 , Morava E 1 , Hirano M 3 , Itan Y 1 , Naini A 2 , Ganesh J 1 1 Department of Medical Genetics and Genomics, Icahn School of Medicine at Mount Sinai, USA, 2 Department of Pathology and Cell Biology, Columbia University Irving Medical Center, USA, 3 Division of Neuromuscular Medicine, Department of Neurology, Columbia University Irving Medical Center, USA *(Corresponding author’s email [email protected] ) Abstract: The prevalence of primary mitochondrial disease is approximately 1 in 5,000; however, this is likely an underestimate due to the difficulty in diagnosis, the wide variability in age of onset, and the broad clinical spectrum of symptoms. 1 Mitochondrial disease can affect almost any organ individually or present as a multisystem disorder at any age. The challenge of diagnosis is further exacerbated by the unique properties of mitochondrial DNA, such as heteroplasmy. While the neurological and cardiovascular effects of mitochondrial disease are well documented, less is known about its renal manifestations and prevalence. When adjusted for weight, the kidney is one of the highest energy-demanding organs, rich in mitochondria, due to its roles in maintaining electrolyte balance, regulating blood pressure, and filtering waste products. Mitochondrial dysfunction is known to cause proximal tubular dysfunction, distal tubular dysfunction, nephrotic syndrome, and tubulointerstitial nephritis. 1 However, the disease mechanisms and genetic causes of mitochondrial disorders affecting the kidney remain poorly understood. Publications or review articles cataloging the mitochondrial and nuclear genes responsible for primary mitochondrial disorder-associated renal disease are scarce. A recent article investigating the use of exome sequencing to determine the genetic etiology of chronic kidney disease, one of the most extensive studies on this topic, excluded mitochondrial genome analysis. 2 This results in gaps in our understanding of the etiology of renal dysfunction in affected patients, including children. Given the concerns regarding the underestimation of the prevalence of primary mitochondrial disorders and the lack of investigation into mitochondrial-associated renal disease, especially in the pediatric population, we have analyzed the mitochondrial genome, using whole genome sequencing (WGS) data from the Nephrotic Syndrome Study Network (NEPTUNE). NEPTUNE is a multicenter cohort study for children and adults with minimal change disease (MCD), focal and segmental glomerulosclerosis (FSGS), and membranous nephropathy (MN), to identify if any individuals enrolled have variants in the mitochondrial genome associated with renal disease secondary to a primary mitochondrial disorder. 3 Analysis of the mitochondrial genome of 600 samples from NEPTUNE is being completed using NextGENe, a next-generation sequencing software that incorporates the alignment and analysis of the mitochondrial genome. Additionally, the molecular results will be compared to the clinical information provided by NEPTUNE, which included age, sex, race, diagnosis, GFR, creatinine, APOL1, urine, and histopathology. References 1. Govers LP, Toka HR, Hariri A, Walsh SB, Bockenhauer D. Mitochondrial DNA mutaLons in renal disease: an overview. Pediatr Nephrol. 2021 Jan;36(1):9-17. doi: 10.1007/s00467-019-04404-6. Epub 2020 Jan 10. PMID: 31925537; PMCID: PMC7701126 2. Groopman EE, Marasa M, Cameron-Christie S, Petrovski S, Aggarwal VS, Milo-Rasouly H, Li Y, Zhang J, Nestor J, Krithivasan P, Lam WY, Mitrotti A, Piva S, Kil BH, Chatterjee D, Reingold R, Bradbury D, DiVecchia M, Snyder H, Mu X, Mehl K, Balderes O, Fasel DA, Weng C, Radhakrishnan J, Canetta P, Appel GB, Bomback AS, Ahn W, Uy NS, Alam S, Cohen DJ, Crew RJ, Dube GK, Rao MK, Kamalakaran S, Copeland B, Ren Z, Bridgers J, Malone CD, Mebane CM, Dagaonkar N, Fellström BC, Haefliger C, Mohan S, Sanna-Cherchi S, Kiryluk K, Fleckner J, March R, Platt A, Goldstein DB, Gharavi AG. Diagnostic Utility of Exome Sequencing for Kidney Disease. N Engl J Med. 2019 Jan 10;380(2):142-151. doi: 10.1056/NEJMoa1806891. Epub 2018 Dec 26. PMID: 30586318; PMCID: PMC6510541 3. Gadegbeku CA, Gipson DS, Holzman LB, Ojo AO, Song PX, Barisoni L, Sampson MG, Kopp JB, Lemley KV, Nelson PJ, Lienczewski CC, Adler SG, Appel GB, Cattran DC, Choi MJ, Contreras G, Dell KM, Fervenza FC, Gibson KL, Greenbaum LA, Hernandez JD, Hewitt SM, Hingorani SR, Hladunewich M, Hogan MC, Hogan SL, Kaskel FJ, Lieske JC, Meyers KE, Nachman PH, Nast CC, Neu AM, Reich HN, Sedor JR, Sethna CB, Trachtman H, Tuttle KR, Zhdanova O, Zilleruelo GE, Kretzler M. Design of the Nephrotic Syndrome Study Network (NEPTUNE) to evaluate primary glomerular nephropathy by a multidisciplinary approach. Kidney Int. 2013 Apr;83(4):749-56. doi: 10.1038/ki.2012.428. Epub 2013 Jan 16. PMID: 23325076; PMCID: PMC3612359. Abstract #: 2025PA-0000000078 Presenter: Kelsey Keith

Sahu A 1 , Castillo S 2 , Kar B 2 , Nanayakkara K 2 , Simone BW 2 , Mota M 1 , Ogiso E 3 , Clark KJ 3 , Sabharwal A 1,3 , Ekker SC 1,3 1 Department of Pediatrics, Dell Medical School, The University of Texas at Austin, USA, 2 Children’s Hospital of Philadelphia, Pennsylvania, USA, 3 Mayo Clinic, Rochester, Minnesota, USA. (Corresponding author’s email [email protected] ) Abstract: The mitochondrial genome represents one of the most highly conserved known stretches of vertebrate DNA, with all 37 genes found in the same order from zebrafish ( Danio rerio ) to humans. Recently CRISPR-free, DddA-derived mitochondrial TALE base editors have enabled the precise edits of mitochondrial DNA (mtDNA). Deploying an enhanced version of this tool that can induce near-complete levels of heteroplasmy enabled the molecular tagging, biochemical testing, functional genetic assessments and the establishment of stable, multi-generation novel in vivo disease models using zebrafish. To further expand the utility of mtDNA base editors, we deployed a TALE architecture that does not have any sequence constraint. We show this unconstrained scaffold enables enhanced editing activity and improved precision in nearly all loci tested, and this approach has enabled new mtDNA mutant zebrafish not possible using prior editors. Further, we identified and then screened a family of active DNA interacting proteins enabling DNA base editing on double-stranded DNA substrates. We used enhanced in silico biochemistry approaches with these diverse proteins to map protein/DNA/cofactor complexes, resolving discrepancies between static structures derived from X-ray crystals and base editing activities observed from biochemical reaction kinetics. This mechanistic understanding will open-up the targeting of new pathogenic edits and corresponding biology. Further, this system also enabled the establishment of human cell lines with sufficiently high heteroplasmy levels to induce key biochemical deficits in vitro . Together, we are triangulating between zebrafish in vivo testing with human in vitro models to understand the molecular nature of genes in this fully syntenic circular chromosome to better understand the normal functions of mitochondria and their role, when altered, in disease. Abstract #: 2025PA-0000000076 Presenter: Kaushal Prajapati

MacMullen, LE 1* , Stanley KD 1 , Tormey, C 1 , Demczko, M 1 , Goldstein, A 1,2 , and Ganetzky, R 1,2,3 1 Division of Human Genetics, The Children’s Hospital of Philadelphia, USA, 2 Department of Pediatrics, The Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA, 3 Center for Computational Medical Genomics, Children’s Hospital of Philadelphia, Philadelphia, PA 19104, USA * [email protected] Abstract: Primary lactic acidosis (PLA) is a phenotype caused by a broad range of primary mitochondrial diseases. PLA typically presents in the neonatal period and is fatal in as many as 88% of cases. Most fatalities are due to acidosis that overwhelms existing clinical buffering options and causes cardiovascular sequelae such as cardiogenic shock and pulmonary hypertension. No proven treatment for PLA exists. Sodium Dichloroacetate (DCA) is an investigational medication that increases pyruvate dehydrogenase complex activity by inhibiting the activity of the inactivating enzymes and by stabilizing the complex and decreasing its rate of turnover. At the Children’s Hospital of Philadelphia (CHOP), in collaboration with Saol Therapeutics and TCI America, we have treated 6 patients with DCA under single-patient investigator-initiated emergency treatment protocols since 2018. Emergency use DCA was considered for patients with life-threatening PLA, typically corresponding to a persistent elevation of lactic acid > 12 mmol (reference range 0-2). Treatment was initiated at an average age of 38 days of life (range: 4-105) for a peak lactate average of 21 mmol (range: 17.4->25 mmol). Two had Pearson syndrome, 2 had pyruvate dehydrogenase deficiency ( PDHA1 ), and one each had primary mitochondrial disease due to pathogenic variants in NDUFA10 and SUCLG1. 5/6 patients had a biochemical improvement of their lactate, which decreased by an average of 13.4 mmol (range: 3.9-19.9 mmol) to a nadir of 7.8 mmol (range 1.7-16.7 mmol). Decrease in lactate was steady and sustained in most cases (although two cases had increases from co-morbid sepsis) and the nadir was reached in an average of 9 days (range 3-19). Those 5 patients also had clinical improvements including the ability to be extubated, decreased seizure response and increased alertness, although DCA was given in parallel to other clinical therapies. Patients have been followed for an average of 110 days post-treatment (14-340). 4/6 patients survived to be discharged from the intensive care unit and one is currently admitted. Three patients ultimately passed away at an average of 111 days of age. Four patients were continued life-long on DCA. In one patient, DCA was stopped after 7 days because of lack of response and transition to palliative care; that patient passed away 7 days later. In a second case, DCA was stopped after 30 days due to what we believed to be disease-related cholestasis. He was able to sustain a lower lactate level, cholestasis was unchanged, and he was discharged from the NICU. He survived for 9 months, before being readmitted for severe lactic acidosis in the setting of pseudomonas sepsis. DCA was reinitiated, but unfortunately, the patient died four days later. Here, we treated 6 patients with life-threatening PLA with DCA. 83% responded as characterized by improved clinical status, substantial decline of lactate and survival to ICU discharge. No definite complications of DCA were seen in any patients. In contrast, in historical cohorts of PLA, only 12% survived in the published literature and 25% in CHOP historic controls. These results suggest that DCA has significant promise for patients with PLA. Abstract #: 2025PA-0000000110 Presenter: Fernando Scaglia, MD

Cristina Remes 1 , Neal D. Matthew 1 , Maia Perrault 1 , Shannon Schrope 1 , Eiko Nakamaru-Ogiso 1,2 , Marni J. Falk 1,2 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA 19104, 2 Department of Pediatrics, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104 Abstract: Defects in mitochondrial translation cause severe impaired respiratory chain deficiencies, variably affecting the brain, liver, skeletal muscle, heart, and other organs. Mitochondrial aminoacyl-tRNA synthetases (mtARS, generally encoded by ARS2 genes) are enzymes that charge mitochondrial tRNAs with their cognate amino acid. Although mtARS share a common biochemical function, patients with mtARS disorders develop variable, severe, neurological dysfunction and multi-system problems. Here, we report characterization of mtARS deficiency in C. elegans by (1) using feeding RNAi interference to individually knock down expression of the full set of 19 conserved mtARS genes; and (2) using CRISPR/Cas9 technology to generate 4 stable worms strains harboring patient specific pathogenic variants in: aars-2 -/- , dars-2 -/- , ears- 2 -/- , and vars- 2 -/- . In addition, cognate amino acids were modeled as a possible therapy for ARS2 deficiencies. Individual ars-2 gene expression was knocked down by feeding RNAi clones to wild-type (N2 Bristol) worms for one or two generations. Four CRISPR stable mutant strains were generated by InVivo Biosystems. Worm linear growth was quantified in larval stage L4+1 Day adults. To determine in vivo mitochondrial stress (UPR mt ) induction, RNAi for each ARS2 gene was performed in a . C. elegans transgenic strain carrying both hsp-6p::GFP and myo-2p::mCherry reporters. Worm neuromuscular activity in liquid media was quantified by thrashing assay. C. elegans lifespan was analyzed with a semi-automated image acquisition system (WormScan). Worm fecundity was analyzed by progeny count assay. Egg hatch rate and larval development were also studied by microscopy. Steady-state OXPHOS subunit levels were quantified by western immunoblot analysis. Cognate amino acid treatment specifically for each ARS2 gene was performed on solid media from the embryo phase, with analyses performed in stage L4 + 1 Day adult worms. ars-2 knockdown in C. elegans was associated with significant decrease in worm length in all strains, and with variable reduction of neuromuscular activity, variable impact on lifespan, and increased mitochondrial stress relative to wild-type worms. First generation RNAi knockdown of cars-2, fars-2 , hars-1 , kars-1,and mars-2 resulted in a near-complete sterile phenotype in C. elegans . The 4 C. elegans ars-2 stable mutant strains each had significantly decreased worm linear growth and neuromuscular activity in liquid media. A consistent developmental defect was present, with dars-2 -/- mutant worms showing the most severe developmental delay relative to wild-type worms. Fecundity analysis showed aars-2 -/- and dars-2 -/- mutants had significant egg hatching defects, while aars-2 -/- , ears-2 -/- and vars-2 -/- mutants had significantly reduced numbers of progeny. aars-2 -/- , dars-2 -/- and ears-2 -/- mutants each had significantly increased mitochondrial stress induction. Treatment of the first- and/or second-generation RNAi knockdown worms with their cognate amino acid significantly increased worm length and neuromuscular activity and reduced mitochondrial stress in a dose-dependent fashion. Additionally, fecundity of hars-1 and fars-2 knockdown strains were rescued with 100 µM treatment of histidine and phenylalanine, respectively. Steady-state OXPHOS protein levels in dars-2 RNAi knockdown worms showed decrease complex I and III levels, which were significantly increased upon treatment with 100 µM aspartate. Similarly, 100 µM aspartate treatment significantly increased worm linear growth and neuromuscular activity and significantly decreased mitochondrial stress in dars-2 -/- mutant worms. A comprehensive study of ars-2 gene inhibition was performed by RNAi knock-down in C. elegans of all 19 conserved human ARS2 genes that replicated the major but variable neurologic, survival, growth, and mitochondrial phenotypes of human mtARS deficiencies, with subsequent CRISPR/Cas9 creation of 4 stable genetic lines harboring ARS2 patient mutations. Importantly, cognate amino acids significantly rescued multi-dimensional disease phenotypes in the mtARS deficiency knockdown models and in the dars-2 -/- stable genetic mutation worms model. These preclinical studies provide compelling evidence that treatment with cognate amino acids could be considered a potential therapy to be studied in rigorous clinical trials for human ARS2 deficiencies. Abstract #: 2025PA-0000000071 Presenter: Michio Hirano

Walimbe Ameya S 1, 2 , Yishay Ben Moshe 2, 3 , Andres Caceres Salgado 2, 3 , Christine M. Eng 3,4 , Elizabeth Mizerik 2, 3 , Lindsay Burrage 2,3 , Keren Machol 2,3 , Claudia Soler Alfonso 2,3 , Lisa T Emrick 1,2 , Scaglia F 2,3,5 * 1 Division of Pediatric Neurology and Developmental Neuroscience, Department of Pediatrics, Baylor College of Medicine, Houston, TX, 2 Texas Children’s Hospital, Houston, TX, 3 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX, 4 Baylor Genetics, Houston, TX, 5 BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, Hong Kong SAR, China * [email protected] Abstract: EARS2 deficiency (MIM #614924) is an autosomal recessive mitochondrial encephalopathy caused by biallelic pathogenic variants in the gene encoding the mitochondrial glutamyl-tRNA synthetase ( EARS2 , NM_001083614.2 ). EARS2 catalyzes the transfer of glutamate to its cognate tRNA during mitochondrial protein translation. The clinical spectrum of EARS2 deficiency can range from a severe neonatal epileptic encephalopathy with profound developmental delay, rapidly progressive neurodegeneration followed by stagnation, leukoencephalopathy with thalamus and brainstem involvement (LTBL) on brain MRI, and lactate peak on magnetic resonance spectroscopy (MRS) to late infantile neurologic regression with partial recovery or an adult-onset disorder with mild neurological and biochemical involvement. In severe cases, patients experience lactic acidosis, failure to thrive (FTT), feeding difficulties, hypoglycemia during acute illness, hepatomegaly, and liver failure. Here, we describe a twenty-month-old girl who presented at two months of age with poor oral intake, daily emesis, and FTT, leading to persistent hypoglycemia (glucose: 35 mg/dL, ref: 74-127 mg/dL), requiring intravenous dextrose and nasogastric feeds. The physical exam was notable for jaundice, scleral icterus, and hepatomegaly. Initial laboratory work-up was notable for elevated lactate (5.7 mmol/L, ref: 0.8-2.0 mmol/L) and liver dysfunction, including elevated AST (127 IU/L, ref: 10-80 IU/L), ALT (140 IU/L, ref: 0-68 IU/L), and reduced albumin (2.8 g/dL, ref: 3.5-4.8 g/dL); abnormal coagulation profile including prolonged international normalized ratio (INR) (1.6, ref: 0.9-1.1), prothrombin time (18.2 seconds, ref: 9.4-12.5 sec), and partial thromboplastin time (57.3 seconds, ref: 25.1-36.5 sec), mildly positive d-dimer (0.74 µg/mL, ref: 6000 pg/mL, reference: <750 pg/mL). A right upper quadrant ultrasound revealed hepatomegaly, and an abdominal MRI showed near-diffuse fatty infiltration of the liver. Liver biopsy revealed macrovascular and microvascular steatosis, iron deposition in peripheral and pericentral hepatocytes, canalicular and hepatocyte cholestasis, and glycogen accumulation, which has not been previously reported. MRI brain and MRS were normal. Hypoglycemia persisted, prompting the use of uncooked cornstarch, and vitamin K was required to normalize the coagulation abnormalities. Exome sequencing revealed compound heterozygous variants in EARS2 : c.328G>A, (p.Gly110Ser), a pathogenic variant inherited from her mother, and c.1015G>T, (p.Val339Phe), a variant of uncertain significance inherited from the patient’s father. Testing of the mitochondrial enzymes of the electron transport chain in cultured fibroblasts was normal. She restarted enteral feeds with formula and gained weight appropriately. Her persistent hypoglycemia was resolved, only rarely occurring during viral illnesses, and cornstarch was eventually discontinued. Her liver dysfunction and hepatomegaly subsided, and a liver ultrasound demonstrated nonuniform hepatic steatosis. She achieved normal developmental milestones. Our patient’s clinical course does not fit previously described patterns of EARS2 deficiency. Her early symptom onset, before six months of age, was like other severe cases, but her phenotype of isolated liver dysfunction, which subsided, highlights a milder presentation. Collectively, our work expands the clinical spectrum of EARS2 deficiency and adds to the limited number of cases. It may help identify and manage patients with this condition. Abstract #: 2025PA-0000000083 Presenter: Xiaowu Gai, PhD

Purandare N 1 , Gomez-Lopez N 2 ,Grossman LI 1 , Aras S 1,3,4* 1 Center for Molecular Medicine and Genetics, Wayne State University School of Medicine, Detroit, MI, USA 2 Department of Obstetrics and Gynecology & Department of Pathology and Immunology, Center for Reproductive Health Sciences, Washington University School of Medicine, St. Louis, MO, USA, 3 Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, USA, . 4 Department of Oncology, Wayne State University School of Medicine, Detroit, MI, USA * [email protected] (corresponding author) Abstract: The bi-organellar protein MNRR1 (CHCHD2) is a key regulator of mitochondrial and cellular function. In the mitochondria, MNRR1 functions towards activating OxPhos and also as an anti-apoptotic. In the nucleus, MNRR1 functions as a transcriptional regulator of stress-responsive genes. Reduced levels of MNRR1 are commonly associated with a multitude of pathologies harboring a phenotype of mitochondrial dysfunction. The reduction depending on the underlying disorder can be either at the transcriptional or post-translational level. Using cells heteroplasmic for the A3243G MELAS mutation, we have identified a HIF2α-dependent transcriptional inhibition of the MNRR1 promoter. Similarly, in the placental inflammation-induced pre-term birth model, where mitochondrial dysfunction has been shown to play a vital role in the pathogenesis, MNRR1 levels are post-translationally reduced as a result of enhanced activity of the mitochondrial protease YME1L1. Overexpression of MNRR1 in vitro resulted in a complete rescue of the phenotype in both the models. As a next step, we sought to identify specific activators of MNRR1 from and FDA-approved compound library with the potential to repurpose existing clinical drugs. One of the compounds identified on the screen and tested using orthogonal assays was Nitazoxanide, an anti-parasitic drug. Nitazoxanide treatment enhanced MNRR1 levels and mitochondrial function and induced the mitochondrial unfolded protein response (UPRmt), both in cell lines and primary cells from MELAS patients and also prevented pre-term birth in mice. In MELAS cells, increasing MNRR1 levels either genetically or by activator compounds shifted the mitochondrial DNA heteroplasmy to levels below biochemical threshold. Mechanistically, we have identified the nuclear function of MNRR1 to be sufficient to rescue the defective phenotype. We therefore designed minimal peptides that could activate the nuclear function of MNRR1. We show that a 20-amino acid peptide can rescue the MELAS phenotype. MNRR1 activation also rescues mitochondrial function in secondary mitochondrial disorders such as NPC1 and ALS. Taken together, our results suggest MNRR1 can be an attractive therapeutic target for some mitochondrial syndromes by boosting cellular bioenergetics and maintaining homeostasis. Abstract #: 2025PA-0000000036 Presenter: Elizabeth M. McCormick, MS, LCGC

Sharma S 1-3 , Catenaccio E 1,2 , Ganetzky R 2,3 , Goldstein A 1-3 1 Division of Neurology, Department of Pediatrics, Children’s Hospital of Philadelphia, PA, USA 2 Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA, 3 Mitochondrial Medicine Frontier Program, Division of Genetics, Children’s Hospital of Philadelphia, PA, USA [email protected] Abstract: Epilepsy is a common manifestation in primary mitochondrial disease (PMD), with a prevalence of approximately 23% in adults and 40-60% in pediatric cohorts. In this study, we reviewed 17 patients with genetically confirmed mitochondrial disease who developed infantile epileptic spasms syndrome (IESS). In a cohort of 17 patients, 10 (59%) were male. A few recurrent genetic causes were seen: MT-ATP6 in 6, PDHA1 and NDUFAF8 in 2 patients, with single cases of FBXL4, MT-ND5, NDUFS8, ATP5O, RARS2, FARS2 and POLG variants. PMD was caused by pathogenic variants in nuclear DNA genes in 10 (59%) patients. Age of onset of spasms ranged from 2 months to 2 years, with a median of 7 months. Age of diagnosis of PMD ranged from 2 months to 34 yrs with a median of 14 months. Epileptic spasms were the initial type of seizure in all patients except one, a patient with PDHA1 -related disease who presented with focal seizures at 5 weeks and developed spasms at 3 months. EEG findings showed hypsarrhythmia in 9 (53%) and modified hypsarrhythmia in 2 (12%). MRI brain around age of spasm onset was normal in 2 (12%) and abnormal in 11 (65%). Findings included bilateral basal ganglia and brainstem T2/FLAIR hyperintensities (typical of Leigh syndrome), agenesis or thinning of corpus callosum, volume loss and ventriculomegaly. Polymicrogyria was noted in 1 patient with NDUFAF8 - related disease. First medication to treat spasms was ACTH in 9 (53%), oral steroids in 3 (18%) and vigabatrin in 3 (18%) patients. Four (23.5%) patients required one, 2 (12%) required two and 11 (65%) required three or more medications for treatment of spasms. Therapies resulted in resolution of spasms in 9 patients (53%) within 1 to 24 months. Spasms remained uncontrolled in 8 (47%) patients. Nine (53%) patients eventually developed different types of seizures with 3 meeting criteria for Lennox-Gastaut syndrome. All 9 patients who received ACTH responded well except one who developed worsening acidosis. Oral steroids were the second medication of choice in one, Vigabatrin in 3 and Topamax in 2 of these 9 patients as spasms returned after ACTH was weaned. 7 out of these 9 patients eventually had resolution of spasms. Ketogenic diet was used in 5 (29%) including the two PDHA1 patients. Vigabatrin toxicity was reported in one patient with MT-ATP6 related disease, this was identified as restricted diffusion in bilateral basal ganglia and brainstem. However, this impression was prior to establishing the genetic diagnosis. IESS can be the presenting seizure type in patients with PMD, highlighting the need for comprehensive metabolic and genetic evaluation in cryptogenic cases. It is possible for PMD to present with epileptic spasms with normal neuroimaging. We did not identify any significant side effects with ACTH, oral steroids or vigabatrin except one patient who developed worsening acidosis. We believe ACTH or oral steroids are a safe treatment option. Majority of patients who experience resolution of spasms go on to develop other types of seizures including generalized, focal, atonic or myoclonic seizures and therefore need ongoing surveillance. Abstract #: 2025PA-0000000063 Presenter: Cristina Remes

Lang S.H., 1,2 Caceres Salgado A.E., 1,2 Rawls-Castillo B., 1,2 Gijavanekar C., 1 Elsea S., 1 Soler-Alfonso C. 1,2 and Scaglia F. 1,2,3,* 1 Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, TX, USA, 2 Texas Children's Hospital, Houston, TX, USA, 3 Joint BCM-CUHK Center of Medical Genetics, Prince of Wales Hospital, Sha Tin, Hong Kong SAR, China *Corresponding author; [email protected] Abstract: Ethylmalonic encephalopathy (MIM # 602473) is an invariably fatal autosomal recessive inborn error of energy metabolism caused by biallelic variants in the ETHE1 gene leading to impaired detoxification of hydrogen sulfide (H 2 S). Secondary mitochondrial dysfunction is due to inhibition of cytochrome c oxidase (COX) activity by H 2 S. H 2 S is produced both exogenously by anerobic intestinal bacteria as well as by the endogenous catabolism of the sulfur containing amino acids methionine and cysteine. Existing therapies including metronidazole, N-acetylcysteine, and liver transplantation have been pursued with the objective of reducing or detoxifying exogenously produced H 2 S, however, strategies to reduce endogenously produced H 2 S using methionine and cysteine free medical foods are an understudied therapeutic strategy. We performed an open label, single-arm study to evaluate the effects of dietary intervention with a methionine and cysteine restricted diet on biochemical parameters and overall clinical trajectory in three patients with molecularly confirmed ethylmalonic encephalopathy. All three patients were status-post liver transplant at the time of diet initiation and on medical therapy with metronidazole and N-acetylcysteine. Biochemical parameters including creatine corrected urine thiosulfate levels, ethylmalonic acid, and methylsuccinic acid levels in dried blood spots, and plasma butyrylcarnitine levels were measured at baseline prior to dietary initiation, and again every three months up to 30 months on dietary intervention. Additionally, we obtained untargeted metabolomic studies and directly evaluated ethylmalonate, butyrylcarnitine, isobutyrylcarnitine, isovalerylcarnitine, 2-methylbutyrylcarnitine, glutarylcarnitine, and methylsuccinate levels in the pre- liver transplant, post- liver transplant, and post- dietary intervention states. We did not observe a robust change in the biochemical profile of any of the three subjects during the 30-month study period. The lack of biochemical response to a low sulfur diet in our cohort likely reflects the overall low contribution of endogenous sulfur containing amino acid catabolism to overall H 2 S production. Abstract #: 2025PA-0000000111 Presenter: Dan Brooks

Jiani Chen 1 , Juliana Troiani 1 , Heather E Pearce 1 , Nicole E Blake 1 , Sarah Pascual 1 , Kathleen H Wood 1 , Matthew C Dulik 1,2 , Jing Wang 1,2,* 1 Division of Genomic Diagnostics, Children’s Hospital of Philadelphia, Philadelphia, PA, USA, 2 Clinical Pathology and Laboratory Medicine, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA, USA.* [email protected] Abstract: Introduction: Mitochondrial DNA (mtDNA) depletion syndromes (MDS) are a group of genetically heterogenous disorders characterized by significant reduction in mtDNA within cells, leading to impaired energy production. Pathogenic variants in nuclear genes involving in mtDNA biosynthesis and maintenance can cause MDS. Reduced mtDNA content has also been observed in individuals with neurodegenerative disorders, associated with aging. In this study, we correlated genetic testing results with mtDNA content and characterized the clinical features of each category of mtDNA content result. Methods: We reviewed the medical records of individuals who underwent muscle mtDNA content analysis in the Division of Genomic Diagnostics at the Children’s Hospital of Philadelphia. For each case, we collected results from mtDNA content analysis and genetic testing. The genetic diagnoses were evaluated based on genotype-phenotype correlation. The mtDNA content results were calculated by comparing with age- and tissue- matched controls. MtDNA values between -1 and +1 standard deviation (SD) from the mean were considered as normal. Values  +2 SD as proliferated. Genetic diagnostic yield was assessed for each category based on mtDNA sequencing, exome sequencing (ES), and/or genome sequencing (GS). Results: A total of 90 muscle samples underwent mtDNA content analysis. Patient ages (at sample collection) ranged from 10 months to 71 years, with 11 (12.2%) under 3 years and 79 (87.8%) older than 3 years, which are the two age groups for mtDNA content evaluation. The distribution of mtDNA content results was as follows: depleted (8.9%, 8/90), reduced (18.9%, 17/90), proliferated (13.3%, 12/90), increased (16.7%, 15/90), and normal (42.2%, 38/90). Among the 27 individuals with mtDNA content in depleted or reduced categories, 5 (out of 27) had diagnostic or likely diagnostic WS or GS results, and two individuals also had multiple large-scale mtDNA deletions (>15% heteroplasmy). The percentage of individuals with diagnostic ES or GS in each category is 50% (4/8, depleted), 5.9% (1/17, reduced), 10.5% (4/38, normal), 26.7% (4/15, increased), and 16.7% (2/12, proliferated) respectively. Diagnoses associated with depleted mtDNA content included two cases of autosomal recessive POLG -related MDS, one case of MECR -related neurological disorder. Additionally, one case with confirmed SMC3 -related Cornelia de Lange syndrome was also had mtDNA depletion, but this condition is not known for MDS. The reduced mtDNA category included one case with HCCS -related primary mitochondrial disorder. Conclusion: Both the depleted and reduced mtDNA content categories contained diagnoses of primary mtDNA disorder, while the depleted mtDNA category having the highest diagnosis yield, although sample size (n=8) was small. Diagnoses in the normal mtDNA content category were generally unrelated to primary mtDNA disorders. MtDNA content analysis is a valuable tool for screening MDS and has proven effective in identifying cases for further genetic work-up. Abstract #: 2025PA-0000000049 Presenter: Melis Kose

Prajapati, K. 1 , Rahaman, I. 1 , Flickinger, J. 1,2 , Santos, J.D. 1 , Ly, A. 1 , MacMullen, L. 1 , Stanley, K. 1 , Hill, D 5 , Chinwalla, A. 5 , Peterson, J.T. 1 , Lazariu, V. 6 , Xiao, R. 3,4 , Zolkipli-Cunningham, Z. 1,3 * 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Children’s Hospital of Philadelphia, USA, 2 Division of Rehabilitation, Children’s Hospital of Philadelphia, USA, 3 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA, 4 Department of Biostatistics, Epidemiology and Informatics, University of Pennsylvania Perelman School of Medicine, USA, 5 Department of Biomedical and Health Informatics, Children’s Hospital of Philadelphia, USA, 6 Biostatistics and Data Management Core, Children’s Hospital of Philadelphia, USA. * [email protected] Abstract: Our Mitochondrial Myopathy-Specific Global Impression Change (MM-GIC) scale, modified from Duong et al. 1 , assesses change in the Mitochondrial Myopathy-Composite Assessment Tool (MM-COAST) 2 . The MM-GIC scale facilitates an anchor-based approach to determine the Minimally Clinically Important Difference (MCID) of the MM-COAST. This seven-point global scale integrates the patient/caregiver perspective on i) magnitude of perceived change and ii) whether this change is considered meaningful. Results will be compared to the distribution-based statistical equation estimates of MCID. In our MM natural history study (NIAMS 1R01AR083552-01A1), we obtained data from 35 paired patient visits in a subset of MM patients, including MM-GIC and MM-COAST assessments. Subjects rated their progression on a 7-point scale ("very much improved" to "very much worse") alongside whether the change was meaningful compared to the previous visit, with interim periods of 14.7 ± 9.7 months. We collapsed GIC scores into “improved” (scores 1-3), “no change” (score 4) and “worse” (scores 5-7) categories. Caregivers completed 8/35 (23%) MM-GIC assessments. Least squares (LS) mean change in MM-COAST scores were estimated using linear regression. The MM-COAST includes 30-second sit-to-stand (30s STS), assessing exercise intolerance and 9-hole peg test (9HPT), assessing dexterity, used in recent mitochondrial disease trials 3,4,5,6 , and considered 'gold standard’. Analyses across our natural history MM cohort showed that MM-COAST correlated with both 30sSTSs (r = -0.56, p<0.0001, n=97) and 9HPT (r = 0.63, p<0.0001, n=134). Deming regression further demonstrated narrow confidence intervals (CI) to support 30s STS (slope -0.092; 95% CI: -0.12, -0.07; n=97) and 9HPT (slope 0.027; 95% CI: 0.021, 0.033; n=134) being significantly predictive of MM-COAST scores, confirming MM-COAST clinical relevance. We then compared change in the MM-GIC to corresponding MM-COAST scores across 2 visits in 15 pediatric (13.5 ± 2.6 years; 66.7% male) and 18 adult (40.4 ± 13.5 years; 44.4% male) subjects. Across the MM-GIC cohort (n=33), mean patient-reported score was 3.44 (SD = 2.5), falling between “little improvement” and “no change”, and reported as meaningful. A significant positive correlation (r = +0.54, p = 0.04, n=14) between MM-GIC scores and change (δ) in MM-COAST scores was observed, demonstrating subject perception of worsening symptoms with higher MM-COAST scores that indicate greater disease severity 2 . Subjects reporting “improved” show no significant change in MM-COAST score (-0.004, p = 0.97, n = 9). However, subjects who reported “no change” and “worse” showed significant change (+0.62, p = 0.012, n=2; +0.90, p = 0.001, n=3) respectively. This preliminary data suggests that a LS mean change of +0.90 (raw score change of +0.5) in MM-COAST scores is considered clinically meaningful. Distribution-based MCID estimates were variable depending on the equation, including Standard Error of Mean (SEM = +0.2) and Minimal Detectable Change with 95% CI (MDC95 = +0.57). Further study is needed in a larger MM cohort to identify the MCID of MM-COAST using anchor-based MM-GIC. Despite preliminary results showing MM-GIC estimates are concordant with statistically-derived distribution-based estimates, there remains a critical need for MCID qualitative interviews in addition to GIC assessment to ensure that MM-COAST MCID estimates truly represent the patient voice. References 1. Duong T, Staunton H, Braid J, Barriere A, Trzaskoma B, Gao L, Willgoss T, Cruz R, Gusset N, Gorni K, Randhawa S, Yang L, Vuillerot C. A Patient-Centered Evaluation of Meaningful Change on the 32-Item Motor Function Measure in Spinal Muscular Atrophy Using Qualitative and Quantitative Data. Front Neurol. 2022 Jan 17; 12:770423. doi: 10.3389/fneur.2021.770423 2. Flickinger, J., Fan, J., Wellik, A., Ganetzky, R., Goldstein, A., Muraresku, C. C., Glanzman, A. M., Ballance, E., Leonhardt, K., McCormick, E. M., Soreth, B., Nguyen, S., Gornish, J., George-Sankoh, I., Peterson, J., MacMullen, L. E., Vishnubhatt, S., McBride, M., Haas, R., Falk, M. J., Xiao, R., and Zolkipli-Cunningham, Z. (2021) Development of a Mitochondrial Myopathy-Composite Assessment Tool. JCSM Clinical Reports, 6: 109– 127. https://doi.org/10.1002/crt2.41 3. Pizzamiglio C, Stefanetti RJ, McFarland R, Thomas N, Ransley G, Hugerth M, Grönberg A, Serrano SS, Elmér E, Hanna MG, Hansson MJ, Gorman GS, Pitceathly RDS. Optimizing rare disorder trials: a phase 1a/1b randomized study of KL1333 in adults with mitochondrial disease. Brain. 2025 Jan 7;148(1):39-46. doi: 10.1093/brain/awae308 4. Janssen MCH, Koene S, de Laat P, Hemelaar P, Pickkers P, Spaans E, Beukema R, Beyrath J, Groothuis J, Verhaak C, Smeitink J. The KHENERGY Study: Safety and Efficacy of KH176 in Mitochondrial m.3243A>G Spectrum Disorders. Clin Pharmacol Ther. 2019 Jan;105(1):101-111 5. Lynch DR, Farmer JM, Tsou AY, Perlman S, Subramony SH, Gomez CM, Ashizawa T, Wilmot GR, Wilson RB, Balcer LJ. Measuring Friedreich ataxia: complementary features of examination and performance measures. Neurology. 2006 Jun 13;66(11):1711-6 6. Saccà F, Puorro G, Marsili A, Antenora A, Pane C, Casali C, Marcotulli C, Defazio G, Liuzzi D, Tatillo C, Cambriglia DM, Schiano di Cola G, Giuliani L, Guardasole V, Salzano A, Ruvolo A, De Rosa A, Cittadini A, De Michele G, Filla A. Long-term effect of epoetin alfa on clinical and biochemical markers in friedreich ataxia. Mov Disord. 2016 May;31(5):734-41. doi: 10.1002/mds.26552. Abstract #: 2025PA-0000000077 Presenter : Daniel Schecter

Holjencin C 2 , Lee H 3 , Annamalai B 1 , Ishii M 1 , Gilbert J L 3 , Jakymiw A 2,4 , Rohrer B* 1 1 Department of Ophthalmology, College of Medicine, Medical University of South Carolina (MUSC), Charleston, SC 29425, USA, 2 Division of Basic Science Research, Department of Biomedical & Community Health Sciences, James B. Edwards College of Dental Medicine, MUSC, Charleston, SC 29425, USA, 3 Department of Bioengineering, Clemson University, Clemson - MUSC Bioengineering Program, MUSC, Charleston, SC 29425, USA. 4 Department of Biochemistry & Molecular Biology, College of Medicine, Hollings Cancer Center, MUSC, Charleston, SC 29425, USA * [email protected] Abstract: Mitochondrial dysfunction, characterized by the accumulation of mitochondrial DNA (mtDNA) damage, is a key factor in the development of a wide range of diseases, including inherited mitochondrial diseases, neurodegenerative disorders, ocular pathologies, and even some cancers. Developing therapies that address the underlying mechanisms of mitochondrial dysfunction has the potential to benefit diverse patient populations. Despite progress in single-gene therapies for diseases with specific mtDNA mutations and advancements in mitochondrial transplantation, no method currently exists for restoring the entire mtDNA genome in a clinically translatable manner. Existing gene therapy approaches face challenges due to the heteroplasmic nature of mtDNA mutations within and between patients, while mitochondrial transplantation lacks long-term mtDNA retention. In nature, mtDNA can be transferred between cells via extracellular vesicles, but this process is not clinically controllable. To address these limitations, we developed a novel nanoparticle-based platform capable of delivering exogenous, intact, and healthy mtDNA templates directly into cells, which we hypothesized would enable the replication of undamaged mtDNA and synthesis of functional mitochondrial proteins. mtDNA nanoparticles were generated by complexing intact mtDNA (isolated from ARPE-19 cell mitochondria) with a cell penetrating peptide (CPP; RD3AD) and a mitochondrial targeting compound (Rho123). Polarized monolayers of mtDNA depleted ARPE-19 cells, which provide a blank background against which to examine mtDNA transfer, were treated with nanoparticles, and mtDNA retention was analyzed over four weeks using PCR. Functionality of delivered mtDNA was assessed with qPCR and western blotting. Cells treated with the RD3AD/mtDNA/Rho123 demonstrated mtDNA persistence, along with increased production of mitochondrial RNA and protein, at four+ weeks post-treatment. These findings demonstrate early feasibility of restoring mtDNA in cells using a CPP-based delivery, presenting a promising therapeutic strategy for correcting mtDNA damage, regardless of the number or type of gene mutations present. Abstract #: 2025PA-0000000129 Presenter: Suraiya Haroon

Garcia A 1* , Patel MR 2 1 Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN 2 Department of Biological Sciences, Department of Cell and Developmental Biology, Vanderbilt University, Nashville, TN Abstract: Mitochondria are semi-autonomous organelles that have a central role in energy production. In animal cells, mitochondria are the only source of DNA outside of the nucleus. The mitochondrial genome (mtDNA) is a small genome measuring 16 kB in humans but encodes essential components of the electron transport chain. mtDNA is replicated independent of the cell cycle permitting mtDNA to reach levels of hundreds to thousands of copies per cell. Cells of high energy demand, such as skeletal muscle cells and neurons, require thousands of copies of mtDNA to generate sufficient cellular energy. Dysregulation of mtDNA copy number in these cells severely impairs energy production and hence cellular function. Therefore, mtDNA copy number must be tightly regulated yet the mechanisms controlling mtDNA copy number remain unclear. Using the metazoan model organism Caenorhabditis elegans and precise quantification of mtDNA copy number using droplet digital PCR (ddPCR), we have found a precipitous age-dependent decline in somatic mtDNA copy number. Surprisingly, this loss of mtDNA is not associated with a decline in mitochondrial organellar content, suggesting that there is a sub-organellar mechanism selectively driving the loss of mtDNA. Additionally, C. elegans has a stress-resistant alternative developmental stage called dauer induced by unfavorable environmental conditions, such as starvation or overcrowding. Many aging phenomena, including age-dependent senescence, are suspended during dauer, permitting animals to survive for several months compared to the average C. elegans lifespan of 2 to 3 weeks. We have found that dauers maintain mtDNA copy number and this maintenance is promoted by reduced insulin signaling. Further, reduced insulin signaling is sufficient to promote mtDNA maintenance during regular development. The results of this work identify reduced insulin signaling as a novel mechanism of mtDNA copy number regulation. Abstract #: 2025PA-0000000132 Presenter : Zakery Baker

Casey E 1* , Kong N 1 , Hoke A 3 , Milbrandt J 1 , Lake N 2 , Jin SC 1 and Peripheral Neuropathy Research Registry 1 Department of Genetics, Washington University School of Medicine, USA, 2 Department of Genetics, Yale School of Medicine, USA, 3 Department of Neuroscience, Johns Hopkins University School of Medicine, USA * [email protected] Abstract: Peripheral neuropathy (PN) is a common neurodegenerative disease affecting more than 30 million people in the United States. PN encompasses a spectrum of disorders affecting the peripheral nervous system, characterized by pain, sensory loss, and weakness. Despite targeted genetic testing using inherited neuropathy panels, about 30% of neuropathy patients present slowly progressive, distal axonal polyneuropathy, with no known environmental or genetic cause, termed idiopathic peripheral neuropathy (iPN). Given the critical role of mitochondrial axonal transport, bioenergetics, and dynamics in maintaining neuronal homeostasis, as well as the susceptibility of mitochondrial DNA (mtDNA) to mutation, we hypothesized that pathogenic variants in mtDNA or nuclear genes encoding mitochondrial proteins could explain a proportion of iPN cases. To investigate this, we conducted whole-genome sequencing on 710 IPN patients from the Peripheral Neuropathy Research registry (PNRR) cohort. Variants calling was performed using GATK Haplotype Caller and Mutect2 mitochondrial mode with subsequent bioinformatics analyses integrating functional annotations via MitoCarta3.0 and stringent filtering criteria to reduce false positives. Variant pathogenicity was classified according to the American College of Medical Genetics and Genomics (ACMG) guidelines. Our analyses identified two likely pathogenic mtDNA variants in MT - ND6 and MT - CYB , one pathogenic nuclear variant in SDHA , and five likely pathogenic nuclear variants in NDUFS4 , SURF1 , MMUT , MTO1 , and AARS2 . While these genes are established in mitochondrial disorders, their association with iPN remains understudied, offering potential new insights into disease mechanisms. Currently, we are focusing on validating these candidate variants using targeted biochemical assays to confirm impact on mitochondrial processes. Our integrative approach could pave the way for the development of targeted therapies for patients suffering from iPN, enhancing their quality of life and advancing neurogenetic research. Abstract #: 2025PA-0000000139 Presenter : Elias Awad, MD

Viktoria Szeifert 2‡ , Jacob S. Bedia 1 , Frederik Denorme 3 , Azusa Terasaki 2 , Wentong Jia 1 , Rocky Giwa 1 , Stella Varnum 1 , Sarah R. Coffey 1 , Jasmine M. Wright 1 , John R. Moley 1 , Keshav Bhatnagar 2 , Hawa R. Thiam 4 , Theodore L. Roth 2 , Cara C. Rada 5 , Rachael L. Field 1 , Nicholas Borcherding 1 , Monika Bambouskova 7 , Robert A. Campbell 3 , Irfan J. Lodhi 7 , Christina L. Stallings 8 , Derick Okwan-Duodu 2,6◊ *, Jonathan R. Brestoff1◊* 1 Department of Pathology and Immunology, Washington University School of Medicine, St Louis, MO, USA; 2 Department of Pathology, Stanford University, Palo Alto, CA, USA; 3 Department of Emergency Medicine, Washington University School of Medicine, St Louis, MO, USA; 4 Department of Bioengineering, Stanford University, Palo Alto, CA, USA; 5 Department of Medicine, Division of Hematology, Stanford University School of Medicine, Stanford, CA, USA; 6 Department of Medicine, Stanford University, Palo Alto, CA, USA; 7 Department of Medicine, Washington University School of Medicine, St Louis, MO, USA; 8 Department of Molecular Microbiology, Washington University School of Medicine, St Louis, MO, USA; ‡ These authors contributed equally. ◊ These authors contributed equally. Abstract: Brown adipose tissue (BAT) is a thermogenic fat depot in both mice and humans and is believed to maintain metabolic homeostasis by increasing energy expenditure and secreting hormones that regulate glucose homeostasis. BAT is activated primarily by sympathetic neurons that release catecholamines to induce expression of Uncoupling protein 1 (UCP1), and this sympathetic input is dampened by macrophages that import and degrade catecholamines. However, the functional roles of other innate immune cells in regulating BAT metabolism are poorly understood. Here, we made the unexpected observation that neutrophils are the most abundant immune cell population in BAT, where they undergo dramatic metabolic and transcriptional reprogramming to acquire a BAT-specific functional state and are required for optimal adaptive thermogenesis. As neutrophils infiltrate into BAT, they capture mitochondria released by thermogenically stressed brown adipocytes, a process that directly triggers neutrophils to transfer their own mitochondria to brown adipocytes in a bidirectional exchange. The transfer of neutrophil mitochondria to brown adipocytes is mediated by the formation of neutrophil extracellular traps (NETs). Disruption of neutrophil mitochondrial metabolism or inhibition of NET formation severely compromises adaptive thermogenesis in BAT and leads to cold-induced hypothermia. These findings reveal that neutrophils participate in bidirectional intercellular mitochondrial exchange with brown adipocytes to meet the metabolic demands of BAT and are critical for defending against changes in environmental temperature. Abstract #: 2025PA-0000000149 Presenter: Fernando Scaglia, MD

Seminotti B 1,2* , Ortmann K 2 , Vockley J 1,2 , Dobrowolski SF 2,3 , and Bedoyan JK 1,2 1 Division of Genetic and Genomic Medicine, Department of Pediatrics, University of Pittsburgh School of Medicine, Pittsburgh, USA, 2 UPMC Children's Hospital of Pittsburgh, Pittsburgh, USA, 3 Division of Clinical Chemistry, Department of Pathology, University of Pittsburgh School of Medicine, Pittsburgh, USA.* [email protected] Abstract: Mitochondrial disorders (MtDs) encompass a diverse group of diseases caused by mitochondrial dysfunction and are among the most common inborn errors of metabolism (IEM). Pyruvate dehydrogenase complex deficiency (PDCD), a specific MtD, results from impaired carbohydrate oxidation, primarily affecting the brain and leading to an energy deficit. PDCD is a leading cause of primary lactic acidemia, contributing to high morbidity and mortality. The incidence of PDCD is approximately 1 in 40,000 live births annually in North America. PDCD is the second most common genetically resolved MtD in the North American Mitochondrial Disease Consortium (NAMDC) Registry, which includes over 2,200 participants. Lactic acidosis and elevated blood amino acids (AAs), such as alanine (Ala) and proline (Pro), are biomarkers commonly observed in PDCD and other MtDs. Specific combinations of AA ratios—Ala/Leucine (Ala/Leu, ⩾4.0) and Pro/Leu (⩾3.0)—have been identified as potential clinical biomarkers for use in newborn screening 1,2 . This study builds upon previous research. We prospectively reviewed and analyzed the medical records of 94 patients at UPMC between January 2023 and February 2025, evaluating them for conditions including PDCD, organic acidemias, primary MtDs, fatty acid oxidation disorders, and other IEMs. Patients ranged in age from one day to 46 years (median 3.7 ± SD 8.3 years), with 45% female and 55% male (male-to-female ratio of 1.24). All 94 patients underwent plasma amino acid testing and were initially screened for Ala/Leu ⩾ 4.0 and/or Ala/Lysine (Ala/Lys) ⩾ 3.0. They were then evaluated for three additional AA ratio combinations and cutoffs: a) Ala/Leu ⩾ 4.0 versus (Ala + Pro)/Leu ⩾ 6.5; b) Ala/Leu ⩾ 4.0 versus (Ala + Pro)/(Leu + Lys) ⩾ 2.5; and c) (Ala + Pro)/Leu ⩾ 6.5 versus (Ala + Pro)/(Leu + Lys) ⩾ 2.5. These AA ratio combinations and cutoffs were assessed for their clinical utility in differentiating PDCD from other MtDs and IEMs. Our analysis identified 18 patients (19%, 18/94) with a confirmed diagnosis following genetic testing. These included cases of PDCD (n=1), complex IV deficiency (n=1), complex V deficiency (n=1), methylmalonic acidemia (n=1), 3-methylglutaric aciduria (n=1), Williams syndrome (n=1), congenital contractures of the limbs and face, hypotonia, and developmental delay (CLIFAHDD) due to a NALCN variant (n=1), facioscapulohumeral dystrophy type 1 (FSHD1) (n=2), and urea cycle disorders (UCDs) (n=9), including ornithine transcarbamylase deficiency, carbamoyl phosphate synthetase 1 deficiency, citrullinemia type 1, and arginase deficiency. The correlation between Ala/Leu and Ala/Pro ratios effectively differentiated 16 additional patients (17%, 16/94) who exhibited symptoms overlapping those of PDCD (e.g., developmental delay, movement disorder, hypotonia, seizures, brain anomalies, and/or acidosis). Genetic testing of 10 (62%, 10/16) of these patients yielded negative or inconclusive results for PDCD, while 6 (38%, 6/16) patients remain under investigation for an underlying etiology. Among the AA ratios studied, the Ala/Leu versus Ala/Pro ratio showed higher sensitivity in distinguishing patients without a confirmed diagnosis. This work supports earlier studies emphasizing the clinical utility of specific AA ratios, and their combinations, as biomarkers for MtDs, including PDCD. 1 Bedoyan et al., JIMD Rep 2020; 2 Verma et al., MGGM 2024. Abstract #: 2025PA-0000000059 Presenter: Sonal Sharma, MD

Bogush E. 1 , Wilson N. 2 , McCormick E.M. 1 , Qunell E. 1 , Yeske P. 2 , Falk M.J. 1,3 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, 2 United Mitochondrial Disease Foundation (UMDF), Pittsburgh, PA, 3 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA. * [email protected] Abstract: Over 400 genes from both nuclear and mitochondrial genomes have been associated to primary mitochondrial disease (PMD), yet many individuals with a clinical presentation highly concerning for PMD do not have a confirmed genetic etiology. While clinical diagnostic genomic testing for PMD has become widely utilized, genomic data analysis is often restricted to clinical diagnostic laboratories, which typically report only variants in known disease-related genes. This presents an opportunity for novel gene discovery and/or more complex genomic analyses of existing genomic data from PMD subjects. We describe here a collaborative, community-driven approach to facilitate such analyses, involving the United Mitochondrial Disease Foundation (UMDF) worldwide patient registry, mitoSHARE, and the Mitochondrial Disease Sequence Data Resource (MSeqDR) Consortium. Participants enrolled in mitoSHARE are encouraged to share their diagnostic status during enrollment, enabling the identification of individuals who have undergone genetic testing. If a participant indicates interest in sharing their genomic data, they receive information on how to participate in the MSeqDR genomic data repository study, which is approved by the Children’s Hospital of Philadelphia (CHOP) Institutional Review Board (IRB). Once consented and enrolled, participants' genomic data is transferred from clinical diagnostic testing laboratories to a secure cloud-based server. This data may include exome, genome, and RNA-Seq transcriptome data to allow for a more extensive data analysis. Participants are assigned a global universal identifier (GUID), which they can then share with a medical or scientific professional of their choice to serve as a proxy. The proxy can then access the data through a secure, web-based user-friendly data query platform called Genesis, allowing for proxy’s direct analysis of the full dataset without needing bioinformatics expertise. 488 mitoSHARE participants have reported previous genetic testing, however, only 333 of these participants have indicated having a genetic diagnosis. To date, 83 participants have completed the informed consent process, and 25 genomic datasets have been transferred. The GUID-encoded datasets are made accessible to proxies within Genesis. Video tutorials created by the MSeqDR study team are available to proxies to learn how to self-navigate Genesis’ platform. Enrollment and genomic data collection continue, with opportunities for both those with known and unknown genetic causes of PMD to participate in research. This collaborative effort between mitoSHARE and MSeqDR offers PMD patients a chance to engage in clinical research, contributing to a more robust genomic analysis pipeline for mitochondrial disease research. Importantly, this project allows individuals and families with PMD, whether diagnosed or undiagnosed, to choose specific medical professionals to access and meaningfully analyze their complex genomic data. Abstract #: 2025PA-0000000113 Presenter: Leonard Burg, PhD

Chen A 1,2 , Yangzom T 1,3 , Hong Y 1 , Lundberg BC 1,4 , Sullivan GJ 5 , Tzoulis C 1,6 , Bindoff LA 1 , Liang KX 1 1 Department of Clinical Medicine (K1), University of Bergen, Norway, 2 Department of Neurosurgery, Xinhua Hospital Affiliated to Shanghai Jiaotong University School of Medicine, China, 3 Centre for International Health, University of Bergen, Bergen, Norway, 4 Department of Biomedicine, University of Bergen, Norway, 5 Department of Pediatric Research, Oslo University Hospital, Norway, 6 Neuro-SysMed, Center of Excellence for Clinical Research in Neurological Diseases, Haukeland University Hospital, Norway ( [email protected] ) Abstract: Mitochondrial diseases, particularly those resulting from mutations in the POLG gene, are among the most severe mitochondrial disorders and lack effective treatments, underscoring the need for novel therapeutic strategies. In this study, we generated 3D cortical organoids from POLG patient-derived induced pluripotent stem cells (iPSCs) to model key disease features, including neuronal loss, mitochondrial dysfunction, mtDNA depletion, and respiratory chain defects. Single-cell RNA sequencing (scRNA-seq) revealed significant downregulation of mtDNA-encoded genes essential for oxidative phosphorylation, including MT-ND5 (NADH dehydrogenase activity), MT-TL1 (mitochondrial protein synthesis), MT-TV (oxidative phosphorylation assembly), and MT-RNR1/MT-RNR2 (metabolic homeostasis and neuronal apoptosis regulation). POLG organoids exhibited NOTCH and JAK-STAT pathway upregulation, astrocytosis, aberrant cell populations (melanocytes, fibroblasts), and a reduction in dopaminergic and inhibitory dopamine-glutamate (DA-GLU) neurons. Metformin treatment over two months partially restored neuronal populations (notably dopaminergic and glutamatergic subtypes), reduced astrocytosis, and improved mitochondrial mass, complex I levels, and mtDNA content via MARK/MTOR activation. However, DA-GLU neurons remained unaffected, indicating an incomplete rescue. Pathway analysis highlighted metformin’s influence on neurodegenerative-associated genes, including HSP90B1, a molecular chaperone involved in cellular stress response and oncogenic signaling, suggesting its dual role in neuronal survival and disease progression. These findings establish POLG organoids as a robust platform for disease modeling and drug screening. While metformin improved mitochondrial function and neuronal survival, its limited impact on DA-GLU neurons highlights therapeutic gaps. This study provides crucial insights into mtDNA stability, energy pathway disruptions, and neuron-glia interactions in disease progression, advancing targeted therapy development for POLG-related and broader mitochondrial neurodegenerative disorders. Abstract #: 2025PA-0000000032 Presenter: Siddhesh Aras

Almannai M 1 , Duong J 2 , El-Hattab AW 3 , Ali M 4 , Bauri D 4 , Soler-Alfonso C 4,5 , Gijavanekar C 4 , Elsea SH 4 , Delay L 6 , Chu Z 7 , Kralik SP 7 , Lee S 2 , Scaglia F 4,5,8 1 Genetics and Precision Medicine Department, King Abdullah Specialized Children’s Hospital, King Abdulaziz Medical City, Riyadh, Saudi Arabia, 2 Columbia University Mailman School of Public Health, New York, New York, USA, 3 College of Medicine, University of Sharjah, Sharjah, United Arab Emirates, 4 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, Texas, USA, 5 Texas Children's Hospital, Houston, Texas, USA, 6 Core for Advanced MRI, Baylor College of Medicine, Houston, Texas, USA, 7 Department of Radiology, Baylor College of Medicine, Houston, Texas, USA, 8 Joint BCM-CUHK Center of Medical, Genetics, Prince of Wales Hospital, Hong Kong SAR, China (corresponding author [email protected] ) Abstract: There is growing evidence that nitric oxide (NO) deficiency occurs in MELAS and results in impaired blood perfusion in small blood vessels contributing to stroke-like episodes (SLEs). The primary aim of this phase 1 dose-finding and safety study was to estimate the maximum tolerated dose (MTD) of citrulline in adults with MELAS, using Time-To-Event (TITE) Continual Reassessment Method (CRM) design. Citrulline was provided at one of the following doses: 10 g/day, 20 g/day, 30 g/day, or 40 g/daily (divided four times a day) for one month with safety visits scheduled for weeks 6 and 8 of the trial. The primary safety outcomes were orthostatic hypotension, and adverse events at grade 3 or higher or an increase from grade 0 to 2 based on the CTCAE Version 4.03 in the first 8 weeks after treatment initiation. The secondary aims were to study changes in cerebral blood flow (CBF) using arterial spin labeling-MRI, and cerebrovascular reactivity (CVR) by measuring the increase in CBF after a vasodilatory stimulus with a breath holding task used in conjunction with an MR-compatible system for measuring end-tidal CO2. Other secondary aims were the measurement of citrulline and arginine levels from baseline to end of treatment (week 4), the evaluation of plasma guanidino compounds from baseline to week 1 to assess for potential arginine toxicity, and the assessment of plasma lactate and alanine from baseline to end of treatment as pharmacodynamic biomarkers. Five males and five females (age range: 19-57 years) were screened, enrolled and completed the eight weeks of the study. There were no severe adverse events observed with any of the used L-citrulline doses. Analysis of plasma guanidino compounds by untargeted metabolomics did not show analyte outliers with Z scores >2. Plasma arginine and citrulline levels were higher at 4 weeks when compared to baseline. Mean CBF (ml/100 grams of tissue/minute) was decreased at baseline in all study participants in the whole brain and in all brain regions when compared to a normal population. Citrulline supplementation increased CBF in all study participants. In six research subjects on the highest dose, the major CBF increase was observed in the occipital region. CVR was increased in these participants and it followed different patterns: parietal-occipital, temporo-parietal, and diffuse changes. There was no correlation between increased CVR and the degree of neurologic disease severity or site of anatomical stroke-like lesion. With the highest dose of citrulline, a substantial decrease in plasma lactate and a decrease in plasma alanine were observed from baseline to 4 weeks. In summary, there were no SAEs observed with any of the used L-citrulline doses. Untargeted metabolomics analysis did not show evidence of citrulline toxicity. Subjects on 40 grams of daily citrulline had the highest increase in CBF in the occipital region. CVR was increased in these participants and with no correlation to degree of neurologic disease severity or site of anatomical stroke-like lesion. These results suggest that CBF and CVR may be useful endpoints in a future trial that could evaluate the efficacy of citrulline. Abstract #: 2025PA-0000000150 Presenter: Swankita Godara*

Varnum S 1* , Nakai R 1,2 , Shi H 2 , Field RL 1 , Giwa R 1 , Jia W 1 , Cohen EF 1 , Krysa SJ 1 , Borcherding N 1 , Yokota T 2 , and Brestoff JR 1 1 Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO, USA, 2 Department of Hematology and Oncology, Graduate School of Medicine, Osaka University, Suita, Japan Abstract: Mitochondria transfer is a recently described phenomenon in which donor cells deliver mitochondria to acceptor cells. One possible consequence of mitochondria transfer is energetic support of neighboring cells; for example, exogenous healthy mitochondria can rescue cell-intrinsic defects in mitochondrial metabolism in cultured ρ0 cells or Ndufs4-/- peritoneal macrophages. Exposing hematopoietic stem cells to purified mitochondria before autologous hematopoietic stem cell transplantation allowed for treatment of anemia in patients with large-scale mitochondrial DNA mutations, and mitochondria transplantation was shown to minimize ischemic damage to the heart, brain, and limbs. However, the therapeutic potential of using mitochondria transfer-based therapies to treat inherited mitochondrial diseases is unclear. Here, we demonstrate improved morbidity and mortality of the Ndufs4-/- mouse model of Leigh syndrome (LS) in multiple treatment paradigms associated with mitochondria transfer. Transplantation of bone marrow from wild-type mice, which is associated with release of hematopoietic cell-derived extracellular mitochondria into circulation and transfer of mitochondria to host cells in multiple organs, ameliorates LS in mice. Furthermore, administering isolated mitochondria from wild-type mice extends lifespan, improves neurological function and increases energy expenditure of Ndufs4-/- mice, whereas mitochondria from Ndufs4-/- mice did not improve neurological function. Finally, we demonstrate that cross-species administration of human mitochondria to Ndufs4-/- mice also improves LS. These data suggest that mitochondria transfer-related approaches can be harnessed to treat mitochondrial diseases, such as LS. Abstract #: 2025PA–0000000116 Presenter: Suzanne B. Scheller

Weisshappel, KM 1,* , Souder, JP 1 , Moore Burk, M 2 , Van Hove, JLK 1 1 Department of Pediatrics, Section of Clinical Genetics and Metabolism, University of Colorado, Aurora, CO, USA, 2 Department of Physical Medicine and Rehabilitation, Children’s Hospital Colorado, Aurora, CO, USA Abstract: Methionyl-tRNA formyltransferase (MTFMT) deficiency is a rare autosomal recessive mitochondrial disorder, causing Leigh syndrome phenotype, involving biallelic variants in MTFMT. Affected individuals exhibit developmental delay, cognitive impairment, ataxia, optic atrophy, cardiomyopathy, and lactic acidosis. The disease mechanism is through impaired initiation of mitochondrial translation, leading to dysfunction of oxidative phosphorylation. A common pathogenic variant present in most patients impairs splicing leaving only approximately 12% normally spliced isoform. Nicotinamide riboside is a NAD+ precursor that acts as a mitochondrial biogenesis up-regulator with good cerebral distribution, and proven safety. Mitochondrial biogenesis upregulation could increase the transcription of the normally spliced isoform, thus improving translation. Increasing the enzymatic substrate formyltetrahydrofolate can further enhance enzymatic activity, with L-serine being the best substrate donor. In this study, three young adults with genetically confirmed MTFMT deficiency, each harboring at least one copy of the common variant, were clinically treated with nicotinamide riboside. Two of the three patients were also treated in adjunct with L-serine. Objective measures were utilized to measure efficacy, which included clinical exam, the Scale for Assessment and Rating of Ataxia (SARA) score, the 6-minute walk test, and hand grip strength. All three patients showed marked improvements in these measures. One patient improved from barely ambulating with supportive aid, to independently running without support. Treatment was well-tolerated without side effects; however, the large serine dose was difficult to take. This study provides the first etiologic treatment for MTFMT deficiency, is widely available, and was effective even when initiated years after symptom onset. Abstract #: 2025PA-0000000092 Presenter: Ritsuko Nakai

McCormick EM 1 , Peterson JT 1 , Taylor JP 2 , Ertmanska I 3 , Bluske K 3 , Clause AR 4 , Chandrasekhar A 3 , Lowry J 3 , Coffey AJ 3 , Gai X 5,6 , Falk MJ 1,7 , Zolkipli-Cunningham Z 1,7 , Rahman S 8 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, USA, 2 Blueprint Genetics, a Quest Diagnostics Company, Espoo, Finland, 3 Illumina Laboratory Services, Illumina Inc., USA, 4 Department of Neurology, Washington University in St. Louis, St. Louis, USA, 5 Mellowes Center for Genomic Sciences and Precision Medicine, Medical College of Wisconsin, USA, 6 Department of Pediatrics, Medical College of Wisconsin, USA, 7 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, USA, 8 Genetics and Genomic Medicine, UCL Great Ormond Street Institute of Child Health, UK * [email protected] Abstract: Primary mitochondrial diseases (PMD) are caused by pathogenic variants in either mitochondrial DNA (mtDNA) or more than 350 genes in nuclear DNA (nDNA). The ClinGen Mitochondrial Diseases Gene Curation Expert Panel (Mito-GCEP, https://clinicalgenome.org/affiliation/40027/ ), funded since 2017 through the National Institutes of Health (NIH) National Institute of Child Health and Human Development (NICHD) U24-HD093483 grant program (Falk and Gai, Multi-PIs) and re-funded in 2021 jointly by NICHD and the National Institute of Neurological Disorders and Stroke (NINDS), has been a highly productive effort engaging more than 50 international PMD experts. The first 3-year project period focused on systematic expert panel evaluation of the strength of evidence between select nuclear and mtDNA genes and Leigh syndrome spectrum (LSS; McCormick et al., 2023). The current project period has expanded to perform rigorous expert panel curation of all genes with published associations with PMD. After completing curation of the 37 mtDNA genes for their association with PMD (October 2022 – March 2024), nDNA genes were prioritized for curation based on disease mechanism. Although the ClinGen Gene-Disease Validity Curation Process was developed for curation of nuclear genes, there is an opportunity to optimize the process for characteristics of specific classes of disorders. Specific curation approaches for nDNA genes associated with PMD were then established, including defining baseline inclusion criteria for scoring variants in reported cases and a unified approach to scoring variants with varying levels of functional validation published (e.g., biochemical evidence, mtDNA depletion, and founder variants). This new standardized framework allowed for systematic and rigorous review of published literature by biocurators to reach Mito-GCEP consensus on the strength of the relationship between each nDNA gene and PMD. Genes were prioritized for curation based on known mechanisms that include roles in specific mitochondrial processes, including OXPHOS subunits and assembly factors, mtDNA gene expression, mtDNA maintenance, mitochondrial protein import and processing, mitochondrial dynamics, iron-sulfur cluster biogenesis, metabolite transport, mitochondrial toxicity, and cofactor biosynthesis. To date, the Mito-GCEP has completed curation of 184 nDNA genes in association with PMD. Among these curations, 111/184 (60%) reached a “Definitive” classification, 3/184 (2%) reached a “Strong” classification, 33/184 (18%) reached a “Moderate” classification, 33/184 (18%) reached a “Limited” classification, 2/184 (1%) were classified as “Disputed,” and 2/184 (1%) had no known human disease relationship. Overall, this Mito-GCEP curation work has rigorously evaluated the varied strength of relationship of many nDNA genes to PMD based on standardized review of evidence available in published literature. Collaborative international effort to reach consensus on gene-disease relationships for PMD is critical for accurate variant interpretation and confirmation of genetic diagnoses necessary in individual cases, to optimize medication management, tailored multi-system organ screening, accurate recurrence risk counseling and prevention, and clinical trial inclusion. Abstract #: 2025PA-0000000040 Presenter: Yijen L. Wu

Hong-Anh T. Pham 1 , Min Jung Kwon 1 , Hua Bai 1 1 Genetics, Development, and Cell Biology, Iowa State University, Ames, IA, USA [email protected] Abstract : Mitochondrial cristae are intricate folds of the inner membrane that optimize oxidative phosphorylation (OXPHOS) by compartmentalizing respiratory complexes. Their architecture is shaped by both protein complexes, such as ATP synthase dimers, MICOS complex proteins, and OPA1, and the biophysical properties of membrane lipids. Cone-shaped phospholipids have been proposed to support the membrane curvature characteristic of cristae, but the functional roles of specific lipid species remain incompletely understood. Plasmalogens are cone-shaped ether phospholipids present in mitochondrial membranes, enriched in high-energy-demanding tissues and linked to aging, neurodegeneration, and cardiac pathologies. Although previous studies have shown that disrupting plasmalogen biosynthesis impairs mitochondrial function and morphology, their specific role in shaping cristae architecture remains unclear. In this study, we investigated how plasmalogens influence mitochondrial membrane organization through interactions with inner membrane proteins. Using a biotinylated plasmalogen pulldown assay in mouse cardiac tissue, followed by TMT-based mass spectrometry, we identified 150 plasmalogen-interacting proteins. Among these, 86 (~57%) localized to mitochondrial membranes, with OXPHOS subunits, particularly from ATP synthase (Complex V), being highly enriched. Western blotting validated the selective enrichment of ATP synthase subunits in the plasmalogen-bound fraction, supporting the proteomic findings. To explore the impact of plasmalogen deficiency, we used a PEDS1-knockout (KO) cell line, which lacks the final enzyme in plasmalogen biosynthesis. Transmission electron microscopy revealed that KO mitochondria exhibited altered cristae morphology, appearing more rounded and disorganized compared to wild-type. Blue Native-PAGE further showed reduced ATP synthase dimerization in KO cells, consistent with our proteomic data. Given that previous studies propose ATP synthase dimerization helps form the high-curvature ridges of cristae, these results suggest that plasmalogens contribute to maintaining cristae architecture by supporting ATP synthase organization, either through direct lipid–protein interactions or by modulating membrane biophysics. Altogether, our findings reveal a new lipid–protein interaction network linking plasmalogens to mitochondrial inner membrane structure and OXPHOS organization. This work provides mechanistic insight into how lipid dysregulation may contribute to mitochondrial dysfunction, with potential relevance to aging, neurodegenerative diseases, and cardiomyopathies, core concerns of the mitochondrial medicine field. Abstract #: 2025PA-0000000162 Presenter: S. Jordan Kerns

Mendez, D.C. 1,2* , Carlson, L.H. 1 , Ali, S.I. 1 , Christensen, T.A. 3 , Salisbury, J.L. 3 Georgieff, M.K. 1 and Bastian, T.W. 1 1 Department of Pediatrics, School of Medicine, University of Minnesota, USA, 2 Department of Neuroscience, University of Minnesota, USA, 3 Mayo Clinic, Microscopy and Cell Analysis Core, USA *( Corresponding author’s email [email protected] ) Abstract: Iron deficiency (ID) is the most common micronutrient deficiency in the world causing deficits in neurobehavioral outcomes (e.g., hippocampal-mediated learning and memory) when acquired in early life (fetal through toddler stages). These deficits persist into adulthood despite prompt iron repletion. Human neonatal brain development is metabolically demanding, consuming 60% of the body’s oxygen and iron is necessary for mitochondrial enzymes involved in cellular energy (i.e., ATP) production through oxidative phosphorylation (OXPHOS). Thus, ID blunts high energy demanding neurodevelopmental processes (e.g., dendritic growth and branching), as shown in a reversible transgenic dominant negative-transferrin receptor 1 (DNTfR1) mouse model of hippocampal neuron ID at postnatal day 21 (p21), leading to impaired long-term learning/memory behavior. With iron’s critical role in mitochondrial functions and vulnerability of hippocampus to ID, we hypothesized that hippocampal mitochondria in untreated DNTfR1 mice would have impaired respiratory capacity and ultrastructure at juvenile (p21) and mature adult (37-week) stage, contributing to impaired hippocampal function. Unexpectedly, mitochondrial respiratory capacity at p21 showed no significant difference across respiration states between wild-type (WT) (n=8) and DNTfR1 (n=7). The electron flow across protein complexes of the electron transport chain (ETC) was also unaffected in hippocampal mitochondria of DNTfR1 mice (n=5) compared to WT (n=6). Initial data on isolated hippocampal mitochondria from 33–42-week-old WT (n=5) and DNTfR1 (n=3) mice showed a significant ID-induced decrease in oxygen consumption rate for ADP to ATP conversion (p=0.0159), and maximal respiration (p=0.0266) with a trending decrease in basal respiration (p=0.0584). The respiratory control ratio, a mitochondrial coupling measure, revealed no impairment in coupling between the electron transport chain and OXPHOS. To understand potential structural effects of fetal-neonatal ID on long-term mitochondrial and neuronal functional capacity, we used serial block face-scanning electron microscopy. Comparisons between 37-week-old WT (n=3) and DNTfR1 (n=3) suggest a 14% increase in mitochondrial number in the stratum radiatum (SR) of the ID hippocampus. Furthermore, 3D reconstructions of dendritic processes reveal reduced dendritic spine density of DNTfR1 CA1-SR hippocampal neurons. The data supports that chronic iron deficiency causes impaired mitochondrial respiratory capacity in terms of ATP synthesis and reduction in maximal respiration under metabolic stress. An increase in mitochondrial density in the DNTfR1 CA1-SR hippocampus may signify a compensatory response due to mitochondria’s impaired function. Altogether, the data supports that mitochondrial dysregulation due to chronic iron deficiency can be a potential mechanistic driver of long-term neurodevelopmental and behavior deficits reported over time. Abstract #: 2025PA-0000000174 Presenter: Shilpa Iyer

Goldstein AC 1,2 * , Weis M 1 , George-Sankoh I 1 , Sharma S 1,2 , Peterson J 1 , Falk MJ 1,2 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children's Hospital of Philadelphia, Philadelphia, PA, 2 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA * [email protected] Abstract: Polymerase gamma ( POLG ) is the most common nuclear gene cause of primary mitochondrial disease. Clinical symptoms may range broadly and affect all ages, with higher morbidity and mortality in younger patients due to refractory epilepsy and fulminant liver failure. Once thought to cause clinically distinct phenotype syndromes, POLG -related mitochondrial disease has been reclassified, where age of symptom debut and epilepsy status are recognized as the two sentinel features defining clinical outcome. Here, we report a semi-automated retrospective data query performed using MMFP-Tableau under CHOP IRB 08-6177, with support of a POLG Foundation sponsored research award, of the Children’s Hospital of Philadelphia (CHOP) Mitochondrial Medicine Frontier Program (MMFP) POLG clinical cohort since 2003, describing the age of onset, debut symptom, genotype, epilepsy status, electrophysiology results, neuroimaging findings, biomarker data, medications, and survival outcomes. Retrospective data query identified 40 CHOP patients with molecularly confirmed POLG disease; 95% (38/40) had autosomal recessive disease. 70% (28/40) had epilepsy, of whom 82% (23/28) were under age 12 years at symptom debut and 52% (12/23) are now deceased; of the18% (5/28) of epilepsy patients who had symptom debut between ages 12 and 40 years, none are deceased; all are female. 25% (10/40) autosomal recessive POLG patients never had epilepsy, of whom 60% (6/10) had symptom debut under age 12 years (33% (2/6) are now deceased) and 40% (4/10) had symptom debut between ages 12 and 40 years. The remaining 5% (2/40) POLG patients had autosomal dominant disease with symptom debut between ages 12 and 40 years and no history of epilepsy. Debut symptoms in epilepsy patients presenting under age 12 years included 2 with febrile status epilepticus, 2 with unprovoked status epilepticus, 10 with global developmental delay, 3 with unprovoked seizures, 4 with refractory focal motor status and 1 with feeding intolerance and hepatopathy. In all 5 female patients who presented with epilepsy between ages 12 and 40 years, the debut symptom was status epilepticus with cortical focal lesions on brain MRI. Among patients without epilepsy, debut symptom in those who presented under age 12 years were motor delay in 67% (4/6) and those who presented between ages 12 and 40 years had neuropathy in 100% (6/6 patients, including 2 with autosomal dominant POLG ). No single biomarker was diagnostic or predictive of POLG disease severity or prognosis. The observed 52% mortality rate in the younger epilepsy cohort was consistent with other reported cohorts. Notably, early mortality (between ages 1.2 and 11 years, median 4.3 years) was 100% in POLG premature truncation and missense variant compound heterozygotes (n=5/5), all of whom had epilepsy. The underlying cause leading up to death was variable, including respiratory failure (n=5), status epilepticus (n=2), liver failure (n=2), and sepsis (n=2), although compassionate withdrawal was the most common cause. This single-site retrospective natural history highlights important clinical insights into POLG disease, which will guide prospective natural history study design and serve as a baseline by which to compare future interventional clinical trials designed to reduce the pronounced morbidity and mortality of POLG disease. Abstract #: 2025PA-0000000098 Presenter: Melissa Walker

Iness AN 1,2 , Bachireddy P 1 1 Department of Hematopoietic Biology and Malignancy, Division of Cancer Medicine, The University of Texas MD Anderson Cancer Center, Houston, TX, 2 Department of Molecular and Human Genetics, Baylor College of Medicine, Houston, TX * [email protected] Abstract: Nuclear-embedded mitochondrial DNA sequences (NUMTs) may act as insertional mutagenesis events. We hypothesize that cells with less stable genomes (i.e. cancer), likely harbor more de novo NUMTs than healthy cells, which could enable their use as cell lineage markers. To this end, we sought to develop a bioinformatics pipeline for reliably detect NUMTs in whole genome and single-cell sequencing data for cell lineage tracing. Publicly available reference genomes (GRCh38/Gencode v24) and single cell DNA sequencing (LAND-treated BJ-hTERT nuclei processed via 10x Genomics scATAC-seq kit) data were analyzed. The NUMT_finder pipeline ( https://github.com/balintbiro/ ) was employed to identify NUMTs via LAST alignment of nuclear and mitochondrial genomes. NUMT_finder was then modified to process single-cell DNA sequencing data via two workflows. For single-cell specific analysis, the pipeline processed individual FASTQ files independently to identify NUMTs at the single-cell level. In contrast, pseudobulk analysis aggregated reads into a single, combined FASTQ file to identify NUMTs for cross-referencing whole genome sequencing data. Shared steps between the workflows include reference genome alignment, BAM conversion, sorting, and NUMT identification, ensuring consistency and comparability. Comparison to mitoblacklist ( https://github.com/caleblareau/mitoblacklist ), a standard NUMT masking tool, was conducted using BLAST to verify overlapping regions. The modified NUMT_finder pipeline identified 2679 NUMTs on pseudobulk analysis, with 1653 detected in the reference genome analysis and 886 unique overlaps between them. Single-cell sequencing analysis on 50 cells revealed NUMTs in 3 cells. NUMT_finder detected 1651 NUMTs compared to 820 from mitoblacklist, demonstrating greater sensitivity that conventional NUMT reference lists. While the analysis is currently limited by low read depth in available data, this work demonstrates that single-cell sequencing of LAND-treated nuclei enables access to both nuclear and mitochondrial DNA for NUMT detection. Future directions include integrating machine learning models to identify tissue-specific de novo NUMTs in larger datasets, such as those from the Somatic Mosaicism Across Human Tissues (SMaHT) initiative. These findings lay the groundwork for using NUMTs as novel lineage-tracing tools and as indicators of genomic instability. Abstract #: 2025PA-0000000091 Presenter: Kaitlin Weisshappel, MD

Liu NN 1 , Houten SM, PhD 2 , and Webb BD*, MD 1 1 Division of Genetics and Metabolism – Department of Pediatrics, Center of Human Genomics and Precision Medicine, University of Wisconsin – School of Medicine and Public Health, 2 Department of Genetics and Genomic Sciences, Icahn School of Medicine at Mount Sinai *(Corresponding author’s email [email protected] ) Abstract: Disorders caused by mutations in mitochondrial aminoacyl-tRNA synthetases (mt-ARSs) are now recognized as a distinct subclass of mitochondrial disease. These enzymes play a fundamental role in mitochondrial protein synthesis by charging mitochondrial tRNA molecules with their cognate amino acid, and therefore, enable translation of mitochondrial-encoded proteins essential for oxidative phosphorylation (OXPHOS). Pathogenic variants in mt-ARS genes result in a wide spectrum of clinical manifestations, including encephalopathy, stroke-like episodes, ataxia, myopathy, cardiomyopathy, sensorineural hearing loss, optic neuropathy, premature ovarian failure, renal failure, and sideroblastic anemia. Methionyl-tRNA synthetase 2 ( MARS2 ) is an mt-ARS responsible for charging mt-tRNA MET with methionine. We identified that pathogenic, biallelic, single nucleotide variants in MARS2 cause combined oxidative phosphorylation deficiency, 25 (COXPD25), which presents with intellectual disability, growth failure, and sensorineural hearing loss. In patient-derived fibroblasts , we observed decreased enzymatic activity of Complex I (CI) and Complex IV (CIV), and in both fibroblasts and lymphoblasts, we observed reduced expression of NDUFB8 (CI) and COXII (CIV) by immunoblotting . To investigate the molecular mechanisms underlying COXPD25, we generated a Mars2 R135W/R135W mouse model using CRISPR-Cas9 to introduce the p.R135W variant, homologous to the pathogenic p.R142W mutation seen in patients. Our mouse model is homozygous for this hypomorphic allele (R135W) as this allele in trans with a null allele is embryonic lethal. Previously we have shown by Western blotting that liver and kidney tissue from Mars2 R135W/R135W mice have decreased expression of NDUFB8. Here, we report our analysis of heart tissue. Although heart disease has not been reported in COXPD25 patients, MARS2 is ubiquitously expressed, and the heart is highly dependent on mitochondrial metabolism. We collected heart tissue from Mars2 R135W/R135W and Mars2 +/+ mice at 3 months old for immunoblotting and RNA sequencing (RNA-seq) to assess alterations in mitochondrial respiratory chain protein expression and global gene expression profiles. Using a mitochondrial OXPHOS antibody cocktail, we did not observe differences in OXPHOS protein subunit expression in Mars2 R135W/R135W and Mars2 +/+ heart. However, RNA-seq analysis revealed a distinct transcriptomic profile in Mars2 R135W/R135W mice marked by significantly reduced expression of multiple OXPHOS genes, including Ndufa11 , Ndufb7 , Ndufs8 , Ndufv3 , and Ndufa9 (CI); Uqcr11 , Uqcrc1 , and Cyc1 (CIII); Cox6b1 (CIV); and Atp5g1 (CV). Gene Set Enrichment Analysis (GSEA) confirmed downregulation of both mitochondrial Complex I and Complex IV gene sets in Mars2 R135W/R135W heart tissue. Our data reveal transcriptional evidence of mitochondrial dysfunction in the heart, suggesting that this tissue may be more susceptible to MARS2 deficiency than previously appreciated. Ongoing work will investigate whether these molecular findings translate into functional impairment of cardiac tissue and whether the heart may represent a therapeutic target in MARS2 -related disease. Abstract #: 2025PA-0000000026 Presenter : Nir Qvit

Mendel R 1 , Cheng Y 1 , Tara Z 1 , Schrope S 1 , Keith K 3 , Matsuno S 1 , O’Hara TO 1 , Mathew N 1 , Nakamaru-Ogiso E 1 , Falk MJ 1,2 , and Haroon S 1,2 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, The Children’s Hospital of Philadelphia, Philadelphia, PA, 2 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, 3 Department of Biomedical and Health Informatics (DBHi), Children’s Hospital of Philadelphia, Philadelphia, PA Abstract: Introduction: Kearns-Sayre syndrome (KSS) and Pearson Syndrome (PS) are primary mitochondrial diseases caused by single large-scale mitochondrial DNA (mtDNA) deletions (SLSMD). SLSMD syndromes, as with most mitochondrial diseases, have no effective treatments or FDA-approved therapies. One major roadblock to therapeutic development is the lack of tools to genetically engineer models harboring mtDNA deletions that are stable and transgenerational. As such, studying naturally occurring animal models helps to advance understanding of disease progression and enable therapeutic development. Specifically, the uaDf5 C. elegans model harbors a heteroplasmic 3.1 kilobase mtDNA deletion of 11 genes that encode 7 MT-tRNAs and 4 mitochondrial proteins. Methods: Here, we use a mitochondrial stress phenotype in the uaDf5 animals to screen for potential therapeutic leads from two libraries, consisting of 62 mitophagy modulators and 2,560 FDA-approved and natural compounds. To conduct the screen, uaDf5 animals were crossed with the myo2::mcherry reporter, a red fluorescent pharyngeal bulb marker for automated animal count, and an hsp6p::GFP reporter that fluoresces green upon mitochondrial stress induction. These triple transgenic animals were used to screen potential therapies using the CX5 high content imager (Thermo Fisher). We use mtDNA heteroplasmy analysis (qPCR), mitochondrial fitness (mitochondrial stress reporter), fecundity (hatch rate and progeny count), and organismal fitness (development) in worms, and cell survival (Cell Tox) in patient fibroblast cells to assess disease progression and therapeutic treatment efficacy. Results: Two drugs, Thiamine and Lipoic Acid, were identified as positive controls for the library screen based on their ability to rescue mitochondrial stress in uaDf5 animals. Thiamine reduced mtDNA heteroplasmy in uaDf5 animals and increased fitness their fitness. Thimaine also increase survival in patient cells. Hemin and Celastrol were identified in the mitophagy modulator screen to rescue mitochondrial stress in uaDf5 mutant animals. Furthermore, Celastrol reduced mtDNA heteroplasmy and improved organismal fitness in uaDf5 animals. Celastrol also improved patient cell survival. Finally, multiple repeat experiments have validated 4 new potential therapeutic candidates that were identified from a screen of 2,560 FDA-approved and natural compounds in the uaDf5 animals. Conclusions: The uaDf5 worm model of SLSMD Syndromes enable mechanistic and therapeutic modeling insights, as well as high throughput screening of drug libraries to identify novel therapeutic leads for SLSMD diseases. Therapies identified to ameliorate disease in uaDf5 worm model can also rescue patient cell death, showing remarkable cross-species conservation of therapy efficacy. We are currently developing patient-derived neuronal cells and zebrafish as additional models for better understanding of disease progression in SLSMD Syndromes and validating therapies identified in the uaDf5 worms. Abstract #: 2025PA-0000000134 Presenter: Jean Flickinger

London Health Sciences Centre Research Institute Abstract: Background: Mitochondrial dysfunction often caused by hereditary mutation impairs genes responsible for mitochondrial ATP production. Globally, one in 5000 people are affected by this heterogenous group of mitochondrial disorders for which there is no cure. A symbiotic relationship between mitochondrial diseases and systemic inflammation exists. Mitochondrial dysfunction activates inflammatory pathways (e.g., Toll-Like receptors or TLRs) which can then reciprocally contribute to the pathogenesis of mitochondrial diseases. Research studies support a complex association between inflammation and defective ATP production as main contributors of neurological diseases. Inflammatory cytokines produced by activated microglia and infiltrated immune cells impede mitochondrial function and metabolism. For example, cytokine TNF hinders oxidative phosphorylation, ATP production and provoke intra-mitochondrial production of reactive oxygen species (ROS). This results in cell death. Subsequently, cellular debris released into the cytosol and extracellular environment can further amplify the inflammatory milieu. Here, we provide a small-scale drug repositioning approach to identify anti-inflammatory target(s) by utilizing a model human immune cell line (THP1) as well as an ischemic injury tissue culture model (sIRI). These two in vitro models can serve as handy and inexpensive models for studying inflammation and mitochondrial function simultaneously while explaining their mechanism of actions. Objectives: Our primary objective is to identify drug target(s) to neutralize TLR-induced inflammation to optimize mitochondrial function in an immune model THP1 Blue cells through large-scale drug repurposing method. Aims: 1) To incubate bacterial LPS and viral RNA with THP1 cells as a positive immune response 2) to measure proinflammatory markers including ROS in THP1 cell supernatant 2) to incubate sIRI and THP1 cells with or without the presence of drugs obtained from an optimized and commercially-sourced drug library 3) to list the most efficacious drugs (10-20) for the evaluation of inflammation markers 4) to score impaired mitochondrial function by measuring iROS, apoptosis and ATP production. Methods/Approaches: THP1 model cell line has already been established in the laboratory. Briefly, for aim 1, THP1 cells are incubated with several TLR ligands (both viral and Bacterial PAMPs). Viability was tested using live-cell imaging and flow cytometry. Cell supernatants are analyzed for injury markers (HMGB1, KIM1, NGAL), and inflammation (IL-6, TNF-α, ROS, iROS etc.) by Multiplex ELISA Eve Technologies, Alberta, Canada. Drug screening: THP1-Blue™ cells were incubated with biological DAMPs (e.g., HMGB1) with or without drug for 18h. NF-κB-inducible alkaline phosphatase reporter gene product will be measured by Quanti-Blue. Drug(s) that yield no color possess anti-inflammatory/anti-TLR/anti-NF-κB activity. Results: we have found that THP1 and sIRI model are perfect models to study inflammation. We have identified overwhelming inflammation markers in these cells. Initially, we have used 20 randomly chosen existing off-patent drug to see if any of them can effectively interact with TLRs and reduce inflammation. Based on NF-kB induced gene expression, we found that anti-diabetic drugs Metformin and Glyburide can significantly decrease ischemic injury and increase ATP. We plan to test more than 700 therapeutic drugs. Significance: We hope to identify drug target(s) that can attenuate inflammation and improve mitochondrial function in mitochondrial diseases. Abstract #: 2025PA-0000000056 Presenter: Bianca Seminotti MSc, PhD

McGinn DE 1* , McCormick EM 1 , Shen L 2 , Wilson N 3, Gai X 4 , Falk MJ 1,5 , Yeske P 3 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children's Hospital of Philadelphia, Philadelphia, PA, USA, 2 Center for Personalized Medicine, Department of Pathology & Laboratory Medicine, Children's Hospital Los Angeles, Los Angeles, CA, USA, 3 United Mitochondrial Disease Foundation, Pittsburgh, PA, USA, 4 Division of Bioinformatics and Quantitative Child Health, Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA, 5 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA, * [email protected] Abstract: Primary mitochondrial diseases (PMD) are caused by pathogenic variants in more than 400 genes encoded by both nuclear (nDNA) and mitochondrial DNA (mtDNA) genomes. Still, many individuals with medical concerns for suspected PMD lack a confirmed genetic etiology, often without prior comprehensive genetic diagnostic testing. The lack of a confirmed genetic etiology is a significant hindrance, decreasing treatment and research opportunities. To address this, the United Mitochondrial Disease Foundation (UMDF), in collaboration with Medical Neurogenetics (MNG) Laboratories, launched a no-cost pilot genetic testing program in 2022. The aim was to aid clinicians in obtaining genetic diagnoses for patients with suspected PMD, where participating clinicians could order next-generation sequencing (NGS) testing of 320 PMD nDNA genes on an exome backbone and the mtDNA genome (Comprehensive Cellular Energetics Defects, NGS301). For patient privacy, the program-wide diagnostic yield was not disclosed by MNG Laboratories, requiring secondary dataset reanalysis. Proband-only NGS sequencing, raw genomic sequencing data quality control, and variant calling were conducted by MNG Laboratories for 344 participants without prior genetic testing. Genetic diagnostic testing reports were shared with ordering providers by MNG Laboratories, limiting understanding of the overall impact of the program. To facilitate cohort analysis, de-identified Variant Call Format (VCF) files were securely transferred from MNG Laboratories to UMDF. De-identified VCF files were then transferred to the Children’s Hospital of Philadelphia’s Mitochondrial Medicine Frontier Program (MMFP) research group for systematic reanalysis using the Mitochondrial Disease Sequence Data Resource Quick-Mitome Web resource 1 , followed by MMFP Genetic Counselor expert review. A genetic diagnosis was made in 34 individuals, as evidenced by pathogenic and/or likely pathogenic variants found in genes associated with their phenotype. A mtDNA etiology was found in 26 individuals, including 13 with m.3243A>G, 5 with m.11778G>A, 4 with m.8344A>G, and 4 with other mtDNA variants. Five had pathogenic variants confirming an autosomal recessive (AR) condition (2 SURF1 -related PMD, 1 POLG -related PMD, 1 PYGM -related Glycogen Storage Disorder V, and 1 SLC22A5 -related Carnitine Deficiency). One had an X-linked condition ( PHKA1 -related Glycogen Storage Disorder IX). Two had autosomal dominant conditions ( OPA1 -related Optic Atrophy, CPT2 -related Carnitine Deficiency). Ten individuals had compelling variants but lacked information, such as segregation, age and tissue type tested, necessary to confirm whether these variants were causal of symptoms. 19 individuals had one pathogenic variant for an AR disease gene, interpreted as being asymptomatic carriers. An additional 202 participants had variant(s) of uncertain significance, which did not appear to be associated with their reported phenotype. Significant barriers to accessing genetic diagnostic testing exist in individuals with clinical features highly concerning for PMD. Secondary data analysis confirmed at least a 10% diagnostic yield of this industry sponsored, no-cost pilot genetic testing program, demonstrating the utility of providing patients and clinicians with direct access to cutting-edge NGS genetic testing. Confirming a precise genetic etiology is critical for optimizing medication management, tailoring multi-system organ screening, providing accurate recurrence risk counseling and prevention, and enabling clinical trial inclusion for PMD. Data from this pilot program will inform future UMDF strategies for genetic diagnostic testing initiatives. Reference 1. Shen L, Falk MJ, Gai X. MSeqDR Quick-Mitome (QM): Combining Phenotype-Guided Variant Interpretation and Machine Learning Classifiers to Aid Primary Mitochondrial Disease Genetic Diagnosis. Curr Protoc. 2024 Jan;4(1):e955. doi: 10.1002/cpz1.955. PMID: 38284225. Abstract #: 2025PA-0000000108 Presenter: Katelynn Stanley

Preston G 1 , Jacobs N 1 , Elsharkawi I 1 , Morava E 1,2 , and Kozicz T 1,3 1 Department of Genetics & Genomic Sciences, Icahn School of Medicine at Mount Sinai, USA, 2 Department of Medical Genetics, University of Pecs Medical School, Hungary, 3 Department of Anatomy, University of Pecs Medical School, Hungary Abstract: Primary mitochondrial diseases (PMD) are a heterogeneous group of disorders caused by impaired mitochondrial respiratory chain function due to pathogenic variants in nuclear or mitochondrial DNA. These variants disrupt enzyme activity, membrane integrity, or mitochondrial genome maintenance. Phosphodiesterase type 5 (PDE5) inhibitors have recently emerged as potential modulators of mitochondrial function. Prompted by self-reported symptom improvement in an individual with mitochondrial taking tadalafil, we investigated the effects of PDE5 inhibitors in several individuals with PMD. Using high-resolution respirometry, we analyzed mitochondrial metabolic flux in fibroblasts derived from six individuals with primary mitochondrial disease following treatment with sildenafil or tadalafil. Clinical outcomes were also assessed in three individuals receiving off-label tadalafil therapy. We hypothesized that PDE5 inhibition would improve mitochondrial respiratory function in vitro and alleviate clinical symptoms in individuals with PMD. While patient-derived fibroblasts displayed reduced maximal respiration, consistent with reduced mitochondrial respiratory chain flux, these cells also showed elevated basal and non-mitochondrial respiration, along with increased glycolytic flux, consistent with a so called “hypermetabolic” phenotype. Treatment with PDE5 inhibitors, particularly tadalafil, reduced proton leak-associated OCR, improved coupling efficiency, and normalized metabolic profiles in vitro . Off-label tadalafil use was associated with acute, dose-dependent, and sustained symptom improvements in all three individuals, with no adverse effects reported. These findings suggest PDE5 inhibitors may offer safe, accessible, and personalized therapeutic options for individuals with a diverse range of mitochondrial diseases. Abstract #: 2025PA-0000000144 Presenter: Kelsey Keith

Burg L 1 , O’Hara T 1 , Haroon S 1 , Reesey Gretzmacher E 1 , Magnitsky S 2 , Seiler C 3 , Nakamaru-Ogiso E 1 , Falk MJ 1,4 1 Mitochondrial Medicine Frontier Program, Division of Human Genetics, Department of Pediatrics, Children’s Hospital of Philadelphia, 2 Small Animal Imaging Core, Children's Hospital of Philadelphia, 3 Zebrafish Core Facility, Children's Hospital of Philadelphia, 4 Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA. [email protected] Abstract: Introduction: Mitochondrial respiratory chain defects caused by pathogenic variants in both nuclear and mitochondrial DNA genes often result in impaired exercise capacity and reduced oxygen utilization by muscles. Zebrafish ( Danio rerio ) offer an increasingly robust translational model to investigate human mitochondrial respiratory chain disease pathophysiology, owing to an array of tractable forward and reverse genetic approaches and their relative ease of use for large-scale pharmacologic treatment screens. We have used CRISRP/Cas9 technology to generate genetic knockout zebrafish with surf1 -/- deficiency as a model of Leigh Syndrome spectrum 1 (LSS), which have reduced whole body oxygen consumption and swimming speed as adults. Development of zebrafish MRI methods further identified growth abnormalities in these animals and their organs over time 2 . Here, we describe ongoing investigations in this model including (1) brain cell death mechanistic studies to understand the pathophysiology of metabolic stroke in LSS, (2) high-throughput drug library screening efforts to identify therapeutics that will rescue the neurologic manifestations of SURF1 disease, and (3) novel live-animal metabolic imaging techniques we have developed to advance understanding of organ-specific metabolic adaptations that occur in LSS disorders. Methods: (1) Using our surf1 -/- zebrafish model, we have evaluated whether specific cell death pathway inhibitors prevent brain cell death upon exposure to low-dose sodium azide in this stressor-hypersensitive LSS model. (2) We are pursuing a high-throughput screen to identify candidate therapies from a 2,600 FDA approved drug compound and natural products library in a larval surf1 -/- zebrafish swimming-based neuromuscular activity assay. (3) We have developed a novel live-animal imaging protocol to analyze adult zebrafish by PET/CT to assess their in vivo glycolytic activity, as well as a protocol for MRI/NMR to evaluate multiple metabolites in distinct organs of adult surf1 -/- and wild-type zebrafish. Results: Interestingly, pharmacologic inhibitors of 5 major cell death pathways in surf1 -/- zebrafish larvae exposed to low dose sodium azide exposure have failed to prevent acute brain cell death. Reduced swimming activity at 7 days post fertilization in surf1 -/- zebrafish larvae exposed to low-dose azide can be prevented by pretreatment with N -acetylcysteine (NAC), which is being used as a positive control to complete a high-throughput screen to identify additional therapeutic leads that improve health and survival in SURF1 disease. A novel protocol has been effectively established to perform PET/CT scans using 18-FDG tracer in living adult zebrafish, which we have applied to identify surf1 -/- adult zebrafish having increased uptake of 18-FDG in the brain relative to wild-type animals, suggestive of 2-fold higher glycolytic activity in surf1 -/- disease. A novel MRI/ NMR protocol has been developed in live adult zebrafish, with ongoing work to characterize their organ-specific metabolic adaptations at baseline or upon candidate treatments in living s surf1 -/- adult zebrafish. Conclusion: surf1 -/- zebrafish offer an important animal model that faithfully replicates key aspects, and thereby facilitates the preclinical translational research study, of human mitochondrial respiratory chain disease including neurologic acute metabolic stroke and exercise intolerance manifestations commonly seen in LSS. References 1. Haroon et al, 2023 PMID: 36795052. 2. Sharm et al, 2024 PMID: 37603286 Abstract #: 2025PA-0000000114 Presenter: Deborah G. Murdock

Amin* ST and Wolfe* JH Department of Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA, USA [email protected] Abstract: The lysosomal enzyme ß-glucuronidase (GUSB) is an acid-hydrolase that catabolizes glycosaminoglycans (GAGs). A homozygous single gene defect in GUSB causes Sly disease (mucopolysaccharidosis type VII) in humans and animals, in which excess GAGs accumulate and alter numerous pathways in diseased cells. A major feature of MPS VII is progressive, degenerative neurodevelopment. Our previous mass spectroscopy-based proteomic and next-generation genetic screening of the MPS VII mouse hippocampus showed significant alterations in mitochondrial pathway proteins. 1 These altered expressions included both nuclear and mitochondrial DNA-encoded mitochondrial proteins. Examination of mitochondria and GUSB in fibroblasts from human MPS VII patients identified novel interactions between mitochondrial proteins with those in the nucleus and lysosome. Mitochondria-lysosome inter-organelle contact modulates mitochondrial dynamics and aberrant mitochondria-lysosome crosstalk pathways impair mitochondrial turnover. Altered expression was found in intrinsic lysosomal proteins involved in mitophagy, proteins dually expressed in lysosome and mitochondria, and notably intrinsic mitochondrial proteins (both nuclear and mtDNA encoded). Thus, alterations were evaluated in the interactions of functional mitochondrial and lysosomal protein complex assemblies involved in cell trafficking, ROS metabolism, and DNA repair in MPS VII cells. GUSB physical interactions were detected with sub-cellular trafficking protein 14-3-3 and peroxiredoxin isoforms. Altered subcellular accumulation of 14-3-3 and PRDX isoforms occurred in mitochondria and lysosomes. Alteration of PRDX6 interaction with SOD1 and PRDX5 interaction with SOD2 have both been implicated in neurodevelopment and accumulation of ROS. In addition, GUSB interaction with DNA SSB repair proteins activates DNA SSB repair involving both mtDNA and ncDNA proteins. Finally, GUSB deficiency in Sly disease cells or deletion in LN229 cells resulted in inefficient basal mitophagy and altered mitochondrial dynamics following altered calcium signalling. This was due to altered mitochondrial and lysosomal calcium signalling protein interactions between SCARB2 or LIMP2 and GUSB. The data show mitophagy aberrations in Sly disease cells, altered recruitment of cytosolic proteins to mitochondria, and variation of intrinsic mitochondrial proteins in the cellular bio-energetic processes and apoptosis. The alteration in basal mitophagy is a result of an altered TBK1 signalling pathway, which is associated with increased interaction of pTBK1 with PINK1, leading to increased MAPLC3A/B and TBK1 interaction and inefficient translocation of proteins in mitochondria. The data demonstrates that GUSB is involved in inter-organelle cross-talks, both between the mitochondria and lysosomes and between the mitochondria and nucleus. The next step will therefore involve understanding the role of these cross-talks in primary differentiated neurons of MPSVII patient fibroblasts, unearthing MPSVII-specific neuronal phenotype targets which can be reinstated with gene therapy. Reference 1. Parente et al (2016) Integrated analysis of proteome and transcriptome changes in the mucopolysaccharidosis type VII mouse hippocampus. Mol Genet Metab . 118(1): 41-54. Abstract #: 2025PA-0000000104 Presenter: Wanqing Xie