Renae J. Stefanetti

Rare diseases affect over 400 million people worldwide and present a major opportunity for health system reform. Primary mitochondrial disease comprises progressive, genetically heterogeneous disorders caused by pathogenic variants in mitochondrial or nuclear DNA, with an estimated prevalence of 1 in 4300 and likely underdiagnosis due to unequal access to genomic testing. As a complex, multisystem condition requiring coordinated, lifelong care, mitochondrial disease serves as a sentinel for structural challenges in rare disease services, including diagnostic delay, fragmented care and inequitable access to expertise. Methods: This study synthesises findings from Project PEARL, integrating a UK-wide survey (n=1000), in-depth interviews with patients and caregivers (n=21), policy analysis and existing evidence on rare disease care to identify system-level implications for healthcare leadership and service design. Results: Four system-level insights emerge. Rare disease services provide a platform to design and test integrated, equity-focused care models. Digital innovation requires transparent and accountable data governance to sustain trust. High-quality care depends on empathy, communication and meaningful patient partnership. Coordinated cross-sector leadership is required to translate innovation into service redesign, workforce development and policy implementation. Conclusions: Mitochondrial disease acts as both an exemplar and a strategic lever for rare disease-driven health system innovation, functioning as a sentinel of system weakness and a testbed for scalable solutions. This approach enables the design, evaluation and scaling of integrated care pathways and ethically governed data infrastructures, providing a practical route to more equitable, coordinated and sustainable healthcare systems.

Background: Quantifying mitochondrial DNA (mtDNA) heteroplasmy is critical for diagnosis, prognosis, and monitoring of mitochondrial disease, particularly that caused by the m.3243 A > G pathogenic variant, where tissue-specific heteroplasmy contributes to disease expression. However, current methods often rely on invasive or clinic-based sampling, limiting accessibility and repeat testing. We validate a non-invasive, decentralised approach using faecal-derived mtDNA to quantify m.3243 A > G heteroplasmy. Methods: Fifty participants with genetically confirmed m.3243 A > G mitochondrial disease were recruited remotely and sent a postal stool collection kit. DNA was extracted from returned stool samples and heteroplasmy at nucleotide position m.3243 A > G within MT-TL1 was quantified using pyrosequencing. Where available, stool mtDNA heteroplasmy was compared with previously collected measurements from muscle, blood, urine, and buccal swabs. Associations were assessed using Pearson correlation, agreement between measurements was evaluated using Bland-Altman analysis, and diagnostic performance for identifying high muscle heteroplasmy was assessed using receiver operating characteristic (ROC) analysis. Results: Here we show that 47 of 50 participants return a sample and 44 of 47 yield an analysable result. Faecal m.3243 A > G and heteroplasmy correlates with muscle heteroplasmy ( r = 0.638, R ² = 0.407, p = 0.0141). For identifying individuals with a high muscle heteroplasmy ( ≥ 60%), a faecal cut-off of ≥56.4% provides 83.3% sensitivity, 50% specificity, and a positive predictive value (PPV) of 90.9%, with high overall accuracy (area under the curve (AUC) 0.92). Conclusions: We demonstrate that faecal mtDNA heteroplasmy is a promising biomarker and validate the feasibility of remote, postal home sample collection with central laboratory analysis aligned with decentralised precision medicine. This approach may reduce reliance on clinic-based sampling, broaden access to laboratory testing, and enable longitudinal monitoring and remote data collection in decentralised clinical trials.

Primary mitochondrial diseases (PMDs) are one of the most common genetic disorders with an estimated prevalence of 1 in 4300. This review article summarises the latest updates in the field of mitochondrial medicine over the last decade. The availability of exome and genome sequencing in clinical practice has empowered clinicians to unravel the phenotypic heterogeneity of PMD and to end the diagnostic odyssey experienced by many patients and families. In unresolved cases, the detection of variant(s) of unknown significance by next-generation sequencing creates diagnostic and clinical uncertainties, and integrating a multi-omics approach can improve diagnostic yield. Alongside breakthroughs in genomic technologies, there is growing interest in using fluid biomarkers to guide diagnosis, monitor disease progression, and potentially serve as clinical trial endpoints. However, the clinical application of these fluid biomarkers in unselected patient cohorts with different disease onset and phenotypes would require more robust evidence. Natural history studies derived from national and international collaborations have provided insights into genotype–phenotype relationships and prognostic factors across several genotypes, including m.3243A>G, MT-ATP6, POLG, and TK2. Advances in therapeutic discoveries and clinical trials are challenging the obsolete dogma that PMDs are untreatable and bringing hope to patients; four compounds have been licensed, and many trials are in progress. Many barriers and challenges to translating laboratory discoveries into clinical therapy in PMD remain, including preclinical models for efficacy and safety testing, sample size, trial design, and the selection of outcome measures and trial endpoints.

Primary mitochondrial myopathies (PMM) are rare, genetically-defined disorders characterised by defects of oxidative phosphorylation, predominantly affecting skeletal muscle. This Phase 1b open-label trial evaluated mavodelpar, a selective peroxisome proliferator-activated receptor delta (PPARδ) agonist, over 12 weeks (Part A), with an optional 36 week extension (Part B) in adults with PMM. The primary objective was to assess safety and tolerability, with secondary assessments of pharmacokinetics, pharmacodynamics, and exploratory performance, patient-reported, and muscle biopsy outcomes. Of the 23 participants who received mavodelpar, 17 completed Part A; none completed Part B due to premature study termination during the COVID-19 pandemic. Adverse events were mild-moderate severity, with headache and constipation most common (4/23 participants; 17.4% each). Exploratory measures showed a mean increase of 104 m in the twelve minute walk test (95% CI: 53 to 156) and a mean reduction of -10.5 points in patient-reported fatigue (95% CI: -16.3 to -4.6). No consistent changes in mitochondrial function were detected in muscle biopsies (n = 10), while transcriptomic profiling (n = 6) revealed modest upregulation of fatty acid–metabolism pathways. Although findings from this Phase 1b trial supported progression to later-phase evaluation, the subsequent Phase 2b trial did not demonstrate clinical efficacy for mavodelpar. The results reported here should be interpreted as exploratory and not indicative of therapeutic benefit. Nevertheless, this Phase 1b trial provides important methodological insights to inform future PMM clinical trial design and outcome measure development.

Over 1 million people in the UK live with heart failure (HF), of which ∼50% are women. Older women are underrepresented in HF research. There is insufficient literature on how women’s experiences of how their new HF diagnosis may impact their physical activity (PA), sedentary behaviour, sleep, quality of life and social support. Seventeen women newly diagnosed with HF (73 (65-81) years old) were recruited from HF diagnostic clinics and participated in a semi-structured interview. Data were analysed using inductive thematic analysis. Four themes were identified: (1) Changes in behaviours and quality of life post-diagnosis, (2) PA: perceived barriers and future intentions (3) Mood and emotions; (4) Social support. Participants reported a decrease in PA and an increase in sedentary behaviour. Barriers to PA included low energy, breathlessness, and safety concerns. Feelings of isolation, worry, and tiredness were reported by some whilst others highlighted a renewed appreciation for life and better access to treatment. Our findings highlight the complex, multifactorial challenges faced by women at the time of their HF diagnosis, with PA emerging as a central theme influencing and being influenced by physical symptoms, emotional well-being, and social support.

Gliomas are the most common type of malignant brain tumour and are frequently associated with seizures. Seizures associated with gliomas are frequently refractory to conventional antiseizure medications and constitute a significant cause of morbidity. There is a clear need for a biomarker that will guide resection of the ictogenic areas during neurosurgical procedures to improve seizure control, limit morbidity and achieve better tumour clearance. Several studies have investigated glutamate in the peri-tumoural region using magnetic resonance spectroscopy (MRS), but findings remain inconclusive regarding the association among elevated glutamate, tumour type and seizure history. Furthermore, it is not known if high cortical glutamate would lead to spontaneous interictal discharges (IIDs) ex vivo. In this prospective cross-sectional observational study of patients undergoing surgery for supratentorial gliomas, pre-operative 1D MRS imaging using an echo time (TE) averaged Point Resolved Spectroscopy (PRESS) sequence was performed to quantify glutamate levels in the peri-tumoural region. Ex vivo human cortical extracellular local field potential (LFP) recordings from the cortical tissue were used to record the presence or absence of spontaneous IIDs. Metabolite data were compared between cortical regions with spontaneous IIDs (n = 6 patients), versus regions with no evidence of spontaneous IIDs (n = 14 patients). Our findings provide new metabolic evidence that peri-tumoural glutamate accumulation is strongly linked to spontaneous IIDs (P = 0.006), reinforcing its role in glioma-associated seizures. The lack of significant differences in choline to creatine and N-acetyl aspartate to creatine ratios between IID and non-IID regions suggests seizure susceptibility is independent of tumour infiltration or neuronal degeneration. These results highlight MRS as a promising non-invasive tool for identifying metabolically active, seizure-prone cortical regions, which could aid in refining surgical planning. However, further validation is required to determine its utility in predicting clinical seizure occurrence and guiding post-operative seizure management.

The m.3243A>G variant in the MT-TL1 gene is the most prevalent pathogenic variant in mitochondrial DNA in adults, associated with a wide clinical spectrum from asymptomatic individuals to mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome. Although pharmacological trials in mitochondrial disorders are increasing, the lack of validated endpoints remains a significant barrier to therapeutic development. This cross-sectional observational study aimed to evaluate patients with and without mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome to identify factors associated with disease burden. Seventeen individuals genetically confirmed to harbour the heteroplasmic m.3243A>G pathogenic variant were enrolled: six who met the consensus-based diagnostic criteria for mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome (median age: 30.0 (inter-quartile range: 29.3–45.0) years). Ten patients who did not have a previous history of stroke-like episodes were assigned as ‘non-mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes’ (age: 37.5 (32.8–48.3) years). Of these patients in the non-mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes group, seven exhibited variable features of mitochondrial disease, including hearing loss, diabetes mellitus, migraine and gastrointestinal involvement, while the remaining three were asymptomatic. One patient was excluded from analysis due to a confirmed ischaemic stroke unrelated to mitochondrial disease. Assessments included disease severity (Newcastle mitochondrial disease adult scale) and patient-reported outcomes of fatigue (fatigue impact scale), health-related quality of life (Newcastle Mitochondrial-QoL), mental well-being (Warwick–Edinburgh mental wellbeing scale), autonomic symptoms (the composite autonomic symptom) and physical activity (The International Physical Activity Questionnaire). Performance outcomes included timed-up and go, handgrip strength, cardiopulmonary exercise testing and accelerometry. Age- and sex-matched healthy controls were included for comparison of accelerometry data (age: 35.5 (28.8–50.5) years). Despite comparable age and mitochondrial DNA heteroplasmy, patients with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome had significantly higher disease burden, reduced exercise capacity and lower levels of objectively measured physical activity compared to non-mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes and controls (P < 0.05–0.001). Patient-reported outcomes did not significantly differ between mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome/non-mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes. While non-mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes patients showed expected alignment between perceived and objective measures, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome patients demonstrated weak, absent, or paradoxical associations. This mismatch may reflect altered symptom perception, cognitive impairment, or disease-related adaptation. These findings underscore the complexity of disease expression in mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes syndrome. Regulatory agencies encourage the inclusion of patient-centred endpoints; however, this study highlights the potential limitations of relying solely on patient-reported outcomes. The divergence between subjective and objective assessments supports the need for multi-dimensional outcomes that integrate both patient perspectives and objective measures to enhance the reliability and interpretability of clinical trials in primary mitochondrial disease.

Muscle twitch dynamics and fatigability change in response to muscle disease. In this study, we developed an imaging paradigm to measure muscle twitch dynamics, and the response of the muscle to voluntary fatiguing contractions. We used a novel imaging technique called motor unit magnetic resonance imaging (MUMRI). MUMRI allows visualisation of muscle and motor unit activity by combining in‐scanner electrical stimulation with dynamic pulsed gradient spin echo (twitch dynamics, PGSE‐MUMRI) and phase contrast (fatigue, PC‐MUMRI) imaging. In Part I of this study, we scanned 10 healthy controls, we measured the muscle rise (T_rise), contraction (T_contract) and half‐relaxation time (T_half‐relax) of the tibialis anterior (TA) muscle on a voxel‐wise basis using PGSE‐MUMRI. Five controls were scanned twice to assess reproducibility; PGSE‐MUMRI demonstrated reproducible results, with low variation between scans 3.4% for T_rise, 6.4% for T_contract and 7.1% for T_half‐relax. We then developed a PC‐MUMRI paradigm to measure the recovery of the TA in response to a fatiguing voluntary exercise. In Part II of the study, we applied these two novel imaging paradigms in a cohort study of nine patients with single large‐scale mtDNA deletion primary mitochondrial myopathy (PMM). Patients underwent a 12‐week resistance exercise programme and baseline, and follow‐up MRI was performed. PGSE‐MUMRI detected a significantly longer muscle contraction time between baseline and follow‐up in PMM patients 108.7 ± 7.9 vs. post‐119.3 ± 10.4 ms; p = 0.018. There was no statistical difference in the recovery half maximum measured using PC‐MUMRI in PMM patients between baseline and follow‐up 254 ± 109 vs. 137 ± 41 s; p = 0.074. In conclusion, PGSE‐MUMRI has detected differences in muscle twitch dynamics between controls and PMM following an exercise programme, and we can visualise differences in twitch dynamics subregions of muscle using this technique. The PC‐MUMRI technique has shown promise as a novel measure of muscle fatigue.

Introduction Pathogenic variants in the gene encoding the catalytic subunit of DNA polymerase gamma ( POLG ), comprise an important single-gene cause of inherited mitochondrial disorders. Clinical manifestations are now recognised as an array of overlapping clinical features rather than discrete syndromes as originally conceptualised. Animal and cellular models have been used to address numerous scientific questions, from basic science to the development and assessment of novel therapies. Here, we sought to employ systematic approaches, wherever possible, to investigate the cellular and animal models used in POLG -related research and assess how well they help us understand disease mechanisms in patients. Methods Four databases were searched from inception to May 31 st , 2022: MEDLINE, Scopus, Web of Science, and Cochrane Review. Original articles available in English, reporting the use of a model system designed to recapitulate POLG -related disease, or related pathogenicity, were eligible for inclusion. Risk of bias and the methodological quality of articles were assessed by an adapted version of the Cochrane Risk of Bias Tool, with the quality of evidence synthesized across each model. Results A total of 55 articles, including seven model organisms (Human, yeast [ Saccharomyces cerevisiae and Schizosaccharomyces pombe ], Drosophila , Mouse, Caenorhabditis elegans , and Zebrafish) with 258 distinct variants were included. Of these, 69% (N=38/55) of articles recapitulated mitochondrial DNA (mtDNA) depletion, 33% (N=18/55) utilised tissue-specific models of POLG -related dysfunction, while 13% (N=7/55) investigated the effect of potential therapeutics in POLG -related mitochondrial disorders. Discussion While some evidence is available to support the ability of POLG -related disease models to recapitulate molecular mechanisms and phenotypes, much is of limited quality, with inconsistencies evident across the literature. Further success in examining and translating novel therapies into effective treatments will be enhanced by the availability of more robust models that better recapitulate the entire spectrum of POLG -related disease. PROSPERO registration CRD42021234883

Over the past two decades there has been increased interest in orphan drug development for rare diseases. However, hurdles to clinical trial design for these disorders remain. This phase 1a/1b study addressed several challenges, while evaluating the safety and tolerability of the novel oral molecule KL1333 in healthy volunteers and subjects with primary mitochondrial disease. KL1333 aims to normalize the NAD+:NADH ratio that is critical for ATP production. The trial incorporated innovative design elements with potential translatability to other rare diseases including patient involvement, adaptive design and exploratory objectives, all of which have subsequently informed the protocol of an ongoing phase 2, pivotal efficacy study of KL1333. Results indicate KL1333 is safe and well tolerated, with dose-dependent gastrointestinal side effects, and validate potential novel outcome measures in primary mitochondrial disease including the 30-s Sit to Stand, and the patient-reported fatigue scales. Importantly, the data from the trial support efficacy of KL1333 based on improvements in fatigue and functional strength and endurance. Furthermore, the study highlights the value in using phase 1 studies to capture data that helps optimize later phase efficacy trial design.