Interpretation of pharmacometabolomics results, aiming particularly at biomarker (sets) discovery for drug exposure, remains a major challenge. The metabotyping of drug exposure depends on resolution of specific metabolomics techniques and comprises individual metabolic phenotypes ("metabotypes"), disease-, drug- and microbiome-specific patterns, as well as conditional metabolic states (e. g. fasting). In this clinical trial with 16 healthy participants, an exploratory objective was to evaluate the untargeted urinary metabolomics of voriconazole, administered in four single doses, using proton nuclear magnetic resonance (1H-NMR) spectroscopy. Voriconazole is a second-generation triazole and a potent inhibitor of drug-metabolizing enzymes such as cytochrome P450 (CYP) isozymes CYP3A4 and CYP2C19. Therefore, identification of metabolites reflecting acute CYP3A4 inhibition was of particular interest. On two treatment days without and with voriconazole (with background microdosed midazolam and omeprazole administration for CYP3A4 and CYP2C19 phenotyping, respectively), spot urine was collected after overnight fasting (predose) and 4 h later (postdose fasting). In the postdose versus predose fingerprints, most changes at the annotated metabolite level were attributable to fasting metabolomics or potential confounders. 1H-NMR spectroscopy identified neither a short-term voriconazole-specific signature nor patterns or metabolites potentially reflecting acute CYP3A4 inhibition. Our study emphasizes crucial significance of strict standardization of fasting time and minimization of confounder influences by clinical trial design as well as selection of adequate baselines and high-resolution analytical techniques in pharmacometabolomics research, especially for biomarker discovery.

Very preterm infants with immature kidneys exhibit high vulnerability to acute kidney injury (AKI). While AKI is associated with adverse outcomes, serum-creatinine-based diagnostics prove unreliable in early risk assessment of kidney damage. This pilot study investigated 1H-NMR spectroscopy-based metabolomics for the identification of very-low-birth-weight (VLBW < 1500 g) infants at risk of AKI before and during indomethacin treatment for patent ductus arteriosus (PDA). Longitudinal urine samples (0 h, 12 h, 36 h, 84 h, 120 h, 14 d, 28 d) from 12 VLBW infants receiving indomethacin for hemodynamically significant PDA were analyzed by 1H-NMR spectroscopy. In total, 150 urinary metabolites were annotated and single-metabolite and multivariate analyses were performed. At 36 h after treatment initiation, three patients (25%) developed AKI (KDIGO criteria). Principal component analysis (PCA) revealed significant differences in urinary metabolic profiles between the AKI and non-AKI groups 12 h after indomethacin initiation. Before treatment, five metabolites were significantly lower in the AKI group: adenine, creatine, dimethylglycine, 1-methylnicotinamide, and methylmalonic acid. Urinary creatine/creatinine (AUC 0.97) and 1-methylnicotinamide/creatinine (AUC 0.93) exhibited promising prognostic accuracy for the prediction of AKI. 1-methylnicotinamide/creatinine concentrations remained persistently reduced during the study. In conclusion, urinary metabolomics, particularly creatine and 1-methylnicotinamide levels, may serve as valuable non-invasive biomarkers for identifying VLBW infants at risk of AKI.

Transaldolase deficiency is a rare metabolic disease caused by pathogenic variants in the TALDO1 gene. Transaldolase plays an important role in the ribose-5-phosphate production, maintaining the NADPH-dependent lipid biosynthesis and cellular redox homeostasis. A small number of patients, predominantly children, have been reported, with a wide range of phenotypic presentations, including liver and kidney disease, involvement of the hematopoietic and endocrine systems, as well as possible early death. We aim to provide further insight into the clinical progression of transaldolase deficiency in adolescence and adulthood. We report on three adult patients with genetically confirmed transaldolase deficiency, including two novel genetic variants in TALDO1. Although the patients have been symptomatic since newborn age, initially with hepatomegaly and cytopenias, they were only diagnosed during adolescence or adulthood. Genetic analysis was performed only at 17, 26, and 32 years, respectively, which, however, did not reveal any genetic variants that would be expected to cause a milder disease course. In adulthood, the dominant clinical features were hypergonadotropic hypogonadism, osteopenia, renal and hepatic involvement. In conclusion, when reporting three new adult cases and comparing them with 47 accessible cases from the literature, our findings suggest that, even if clinical manifestations begin in the neonatal period, the overall phenotype may remain relatively mild, with gradual progression. This means that patients presenting with otherwise unexplained progressive liver disease, kidney dysfunction, cytopenia, and hypergonadotropic hypogonadism should be tested for transaldolase deficiency. We recommend closely monitoring patients with known transaldolase deficiency regarding the above-mentioned problems.

Phenylketonuria (PKU) is a rare metabolic disorder causing elevated phenylalanine (PHE) levels requiring lifelong dietary or pharmacological management and regular monitoring. Current PHE monitoring methods, such as tandem mass spectrometry (FIA-MS/MS), are laboratory and sample transport-dependent, leading to delays in obtaining results. This highlights the need for accessible point-of-care (PoC) solutions. Building on a previously developed bioluminescence-based PHE quantification method, we created a PoC PHE sensor integrated with a smartphone app (phenyx app) for real-time monitoring and data sharing. In a clinical study with PKU patients, we evaluated the reliability of the sensor and the app by comparing at-home PHE measurements with dried blood samples (DBS) analyzed using the gold standard FIA-MS/MS. We also assessed time differences between sampling and results, sensor practicality, and user experience. Forty-seven PKU patients (median age 10.0 years, range 0.7-53.4) performed one supervised and four at-home measurements using the PHE sensor, alongside DBS sampling for FIA-MS/MS. The sensor showed higher PHE values than FIA-MS/MS, with a mean difference of 84.8 μmol/L (p = 0.0017). Variability was influenced by test supervision (clinic vs. home) and test kit age (greater difference in older batches, p = 0.001). Usability of the PHE sensor and phenyx app was rated favorably; all patients preferred the PHE sensor over laboratory testing. PoC measurement using the PHE sensor and phenyx app is feasible, reliable, and provides rapid results comparable to FIA-MS/MS, representing a key step toward patient self-testing. Trial Registration: NCT05998109, NCT06940193, DRKS00031972.

Zusammenfassung Die Deutsche Diabetes Gesellschaft (DDG) bewertet im vorliegenden Positionspapier Chancen und Risiken eines bevölkerungsbasierten Screenings auf Typ‑1‑Diabetes mittels Insel‑Autoantikörpern in verschiedenen Kontexten. Der Nachweis multipler Antikörper erlaubt die frühe Identifikation präsymptomatischer Stadien mit hohem Progressionsrisiko, bei weiterhin begrenzter individueller Vorhersagbarkeit von Manifestationszeitpunkt und ‑verlauf. Ein potenzieller Nutzen besteht in der Reduktion diabetischer Ketoazidosen und einer möglichen Progressionsverzögerung durch immunmodulatorische Therapien, ist jedoch abhängig von strukturierter Nachsorge und hoher Versorgungsqualität. Dem stehen Einschränkungen durch unzureichend standardisierte Testverfahren, variable Verläufe, fehlende Langzeitdaten sowie psychologische und ethische Aspekte gegenüber. Zudem zeigen gesundheitsökonomische Analysen eine hohe Ressourcenbindung bei unklarer Kosteneffektivität. Insgesamt sieht die DDG derzeit keine Grundlage für ein populationsweites Screening; erforderlich sind bessere Evidenz, validierte Testverfahren und gesicherte Versorgungsstrukturen.

Principles of long-term medical management in individuals with urea cycle disorders (UCDs) encompass (1) a low protein diet, (2) supplementation of arginine and/or citrulline along with essential amino acids, nutrients, vitamins and trace elements, and (3) use of nitrogen scavenging agents to reduce recurrent hyperammonemic events (HAEs). These principles aim at providing metabolic stability, elimimation of chronic complications, and achievement of normal development as well as growth. A retrospective comparative analysis was performed by studying 138 individuals with male ornithine transcarbamylase deficiency (mOTC-D), citrullinemia type 1 (CTLN1) and argininosuccinic aciduria (ASA) based on in vitro residual enzymatic activity for severity-adjustment. Results show that individuals with mOTC-D, CTLN1 and ASA are at risk of progressive linear growth impairment, recurrent annual HAEs and an unfavorable neurocognitive outcome despite being under long-term nitrogen scavenging pharmacotherapy. No overall superiority among existing nitrogen scavenging agents with regard to the individual’s metabolic stability, linear growth impairment and poor neurocognitive outcome was observed. Novel therapeutic strategies are urgently needed to ultimately improve health outcomes in individuals with UCDs in order to sufficiently meet guideline-specific goals.

OBJECTIVE:To evaluate feasibility and diagnostic and process qualities as well as the clinical benefit of newborn screening (NBS) for urea cycle disorders (UCDs). METHODS:Between 2016 and 2023, 624 480 neonates were enrolled in the German NBS pilot study, including NBS for UCDs by tandem mass spectrometry. In addition, patients with confirmed cases were enrolled in an observational, multicenter outcome study assessing clinical and neurodevelopmental outcomes. RESULTS:NBS for UCDs demonstrated high sensitivity (100%) and specificity (99.988%) with a positive predictive value (PPV) of 0.2. Nineteen cases (7 ornithine transcarbamoylase deficiency, 7 argininosuccinate synthetase 1 deficiency, 4 argininosuccinate lyase deficiency, and 1 carbamoylphosphate synthetase 1 deficiency) were confirmed, yielding a cumulative birth prevalence of 1:32 867 newborns. Despite short process times, more than 50% of screened individuals with a UCD were already symptomatic at first NBS report. Retrospective analyses indicated that integration of metabolite ratios (eg, ornithine/citrulline) may further enhance PPV. Outcome data from 25 individuals revealed higher rates of metabolic decompensations and hospitalizations in mitochondrial UCDs vs cytosolic forms. At last follow-up, 55% showed disease-related symptoms, and mean IQ was reduced in symptomatic individuals, but mortality was lower than previously reported. CONCLUSION:NBS for UCDs is technically feasible and enables early diagnosis. Although early-onset decompensations remain difficult to prevent, screening reduces mortality. Refinement of the screening algorithm may further improve specificity and clinical benefit.

INTRODUCTION:The novel biomarker proenkephalin A 119-159 (PENK) has emerged as a promising marker of kidney function, with the potential to perform comparably to invasive gold-standard methods. This study provides the first reference values for PENK in a German pediatric and young adult cohort, with a particular focus on age-specific values. MATERIALS AND METHODS:PENK was measured in serum using a double-monoclonal sandwich immunoassay in a cohort of individuals aged 1 month-40 years. The study included 221 participants, comprising 188 pediatric participants (< 18 years) and 33 adults (≥ 18 years). The upper reference limit was defined as the estimated 97.5th percentile derived using quantile regression with lambda-mu-sigma (LMS). RESULTS:Serum PENK concentrations declined with age during the first 20 years of life. In the combined-sex cohort, the estimated 97.5th percentile decreased from 618 pmol/L in the first month of life to 103 pmol/L at age 20 years. Thereafter, the 97.5th percentile remained stable in young adults (>20 years), ranging from 86 to 93 pmol/L. Similarly, the estimated 50th percentile declined from 346 pmol/L in the first month of life to 58 pmol/L at age 20 years, remaining stable thereafter at 48-52 pmol/L. No statistically significant differences in PENK concentrations were observed between females and males within the respective age groups. CONCLUSIONS:This study provides the first age-dependent reference values for PENK. These reference values are a prerequisite for future studies evaluating the precision and clinical utility of PENK as a diagnostic biomarker across all age groups, particularly in pediatric populations.

BACKGROUND AND AIMS:Pathogenic variants in neuroblastoma amplified sequence (NBAS) gene causes infantile liver failure type 2 (IFLS2; MIM 616483), characterised by recurrent episodes of liver failure triggered by febrile infections. The underlying pathophysiological mechanisms remain incompletely understood. With this work we try to shed light on the pathomechanism and propose a potential therapeutic option. METHODS:For in vitro analyses, human skin fibroblasts were obtained from three individuals with ILFS2 and one healthy control. Cells were cultivated at 37°C or 40°C. Western blots were performed to assess NBAS protein and its interaction partners. Furthermore, immunofluorescence and electron microscopy were used to examine morphological changes associated with endoplasmic reticulum (ER) stress. Apoptosis was measured using flow cytometry. The effects of N-acetylcysteine (NAC) treatment were analysed not only in cultured fibroblasts but also in vivo retrospectively in 16 affected individuals. RESULTS:We demonstrate that elevated temperature induces ER stress in fibroblasts from individuals with NBAS variants, leading to increased reactive oxygen species (ROS) production and apoptosis. Treatment with the antioxidant NAC, an established therapeutic in acetaminophen-induced liver failure, effectively mitigated oxidative stress and reduced apoptosis in vitro. In a retrospective clinical analysis, there was a trend that NAC treatment was associated with reduced severity of hepatic crises, suggesting a potential therapeutic benefit. CONCLUSIONS:Our findings link fever-induced ER stress, ROS accumulation, and apoptosis to the pathogenesis of liver failure in NBAS deficiency. NAC attenuates these cellular stress responses and may represent a promising supportive treatment option in NBAS-associated liver failure.

The research challenges associated with rare diseases is characterized by a scarcity of information as well as reliable data due to their low prevalence. The problem of the “underpowered studies” is stemmed from a small research community, limited study participants and scarce data. The German project “Collaboration on Rare Diseases – Medical Informatics” (CORD-MI), tackles these problems by improving research opportunities and patient care by employing innovative IT solutions for collaborative data use across 20 German university hospitals. One possibility was to conduct decentralized studies based on secondary data. Three studies based on four rare diseases served as examples: (1) Cystic Fibrosis (CF), (2) Phenylketonuria (PKU), and (3) Kawasaki Disease and Multisystem Inflammatory Syndrome in Children (MIS-C). All three decentralized studies were conducted using routine inpatient data from German university hospitals. For each use case, an interdisciplinary team defined research questions, created analysis scripts based on the Core Data Set of the Medical Informatics Initiative (MII), executed these locally at participating sites, and aggregated anonymized results for descriptive statistical analysis. A frequency threshold rule was applied to protect patient privacy. Study protocols and analysis scripts for data extraction and evaluation are publicly available, and the studies were reported in detail in accordance with the Reporting of Studies Conducted Using Observational Routinely-Collected Health Data (RECORD) Statement. Results from up to 17 German university hospitals were achieved for all three decentralized studies. The challenges in the areas of health care process bias, inaccurate coding of rare diseases, difficult verification of study results, and loss of information due to masking of small study case numbers as well as imprecise definition of the cohorts are discussed. Naming the hurdles enables the identification of areas for improvements, which will be the base for the development of new approaches or adaptations of existing tools and methodologies for the future. Although adaptation would make an impact, the initial results already show that decentralized analyses based on secondary use of patient data can improve research and thus also the care for people with rare diseases. Not applicable

This retrospective study on X-linked PDHA1-related pyruvate dehydrogenase complex (PDHc) deficiency combined a systematic literature review with a multicentre survey exploring genotypes, phenotypes and survival. Data from 891 individuals (45% unpublished) were included. Of note, 53% of cases were females. Median age at last assessment was 6 years (range 0-80 years, n = 622). We detected 331 different (118 unpublished) PDHA1 variants, of which 75% (305/405) had occurred de novo. Variants in this study were uploaded to ClinVar (SCV006297015-SCV006297345). The 10 most frequent variants accounted for 36% of the diagnoses. Sixty-nine per cent of the variants were private; missense (50%) and frameshift (20%) variants were most common. Frameshift/nonsense (FS/N) variants in males (44/401, 11%) were confined to regions escaping nonsense-mediated decay (NMD) and were significantly less frequent than in females (151/461, 33%). Neonatal or infantile (405/529, 77%) presentations were most frequent, with pre/perinatal abnormalities reported in 47% (159/342). FS/N variants in the NMD-predicted region 3.9 [95% confidence interval (CI) 1.54-11.04] times increased the odds of fetal findings. Females presented significantly earlier [2 months, interquartile range (IQR) 7.0, n = 224] than males (8 months, IQR 16.6, n = 233), with increased risk of neonatal presentation [odds ratio (OR) 3.01 (95% CI 1.279-7.616)] when harbouring FS/N variants in the NMD-predicted region. The overall (n = 242) mean survival time was 10.9 (95% CI 9.9-11.9) years. On average, females survived 4.5 (95% CI 2.62-6.40) years longer than males despite presenting more severe phenotypes. Poor survival was associated with male sex [hazard ratio (HR) 3.3 (95% CI 1.95-5.62)], neonatal presentation [HR 5.5 (95% CI 2.17-14.09)], FS/N variants in the NMD-predicted region [HR 4.0 (95% CI 1.78, 9.16)] and splice variants [HR 2.3 (95% CI 1.15, 4.59)]. More severe clinical phenotypes were predicted by neonatal or infantile presentations and by female sex. Developmental delay (DD), intellectual disability (ID), muscle hypotonia, abnormal movements, seizures, feeding difficulties and microcephaly were the most frequent phenotypes, all occurring in more than half. Corpus callosum or basal ganglia alterations and cerebral atrophy were common. Four per cent (36/891) were reported to have mild phenotypes with no DD nor ID (25/36 males). This is the largest dataset on a nuclear-encoded defect of mitochondrial energy metabolism. The genotypic and phenotypic details further defines the disease landscape and can be used for variant interpretation. The correlations between genotypes, sex, phenotypes and survival, adds substantial improvement to counselling.

Arginase 1 deficiency (ARG1-D) is an ultra-rare urea cycle disorder characterized by progressive spastic paraplegia, developmental delay, epilepsy, and episodic hyperammonemia. Evidence on prevalence and clinical presentation is scarce. Therefore, epidemiology and the phenotypical spectrum were assessed in Germany, Austria, and Switzerland (DACH region). We conducted a questionnaire-based, cross-sectional study of confirmed ARG1-D patients in the DACH region. Patients were stratified into early-diagnosed (newborn screening [NBS] or high-risk family screening [HR]) and diagnosed after symptom onset. We evaluated clinical, biochemical, and therapeutic characteristics of individuals with confirmed ARG1-D. Epidemiological prevalence estimates were derived using national population data. A total of 20 patients were identified (Germany: 12, Austria: 7, Switzerland: 1). Eight were diagnosed early (NBS: 4, HR: 4) and 12 after symptom onset. Symptomatically diagnosed patients (median age 11 years) presented with a broad range of clinical manifestations, specifically progressive spastic paraplegia (67%), epilepsy (58%), dystonia (46%), developmental delay (58%), and hepatopathy (50%). Median age at diagnosis was 35 months in symptomatic patients versus 1 month in early-diagnosed patients (p = 0.03). Estimated pediatric prevalence was 1:1042080 in the DACH region, with high regional differences. Hyperammonemia was reported in 72%. Enzyme therapy had been initiated in 21%; 2 patients underwent liver transplantation. ARG1-D is a rare disease with a prevalence of approximately 1:1000000 individuals, and a complex and progressive clinical phenotype. Detection via NBS or HR allows early diagnosis and treatment initiation, potentially altering the clinical outcome.

Dystonia is a rare disease trait for which large-scale genomic investigations are still underrepresented. Genetic heterogeneity among patients with unexplained dystonia warrants interrogation of entire genome sequences, but this has not yet been systematically evaluated.To significantly enhance our understanding of the genetic contribution to dystonia, we (re)analysed 2874 whole-exome sequencing (WES), 564 whole-genome sequencing (WGS), as well as 80 fibroblast-derived proteomics datasets, representing the output of high-throughput analyses in 1990 patients and 973 unaffected relatives from 1877 families. Recruitment and precision-phenotyping procedures were driven by long-term collaborations of international experts with access to overlooked populations.By exploring WES data, we found that continuous scaling of sample sizes resulted in steady gains in the number of associated disease genes without plateauing. On average, every second diagnosis involved a gene not previously implicated in our cohort. Second-line WGS focused on a subcohort of undiagnosed individuals with high likelihood of having monogenic forms of dystonia, comprising large proportions of patients with early onset (81.3%), generalized symptom distribution (50.8%) and/or coexisting features (68.9%). We undertook extensive searches for variants in nuclear and mitochondrial genomes to uncover 38 (ultra)rare diagnostic-grade findings in 37 of 305 index patients (12.1%), many of which had remained undetected due to methodological inferiority of WES or pipeline limitations. WGS-identified elusive variations included alterations in exons poorly covered by WES, RNA-gene variants, mitochondrial-DNA mutations, small copy-number variants, complex rearranged genome structure and short tandem repeats. For improved variant interpretation in WGS-inconclusive cases, we employed systematic integration of quantitative proteomics. This aided in verifying diagnoses related to technically challenging variants and in upgrading a variant of uncertain significance (3 of 70 WGS-inconclusive index patients, 4.3%). Further, unsupervised proteomic outlier analysis supplemented with transcriptome sequencing revealed pathological gene underexpression induced by transcript disruptions in three more index patients with underlying (deep) intronic variants (3/70, 4.3%), highlighting the potential for targeted antisense-oligonucleotide therapy development. Finally, trio-WGS prioritized a de novo missense change in the candidate PRMT1, encoding a histone methyltransferase. Data-sharing strategies supported the discovery of three distinct PRMT1 de novo variants in four phenotypically similar patients, associated with loss-of-function effects in in vitro assays.This work underscores the importance of continually expanding sequencing cohorts to characterize the extensive spectrum of gene aberrations in dystonia. We show that a pool of unresolved cases is amenable to WGS and complementary multi-omic studies, directing advanced aetiopathological concepts and future diagnostic-practice workflows for dystonia. Using whole-genome sequencing and proteomics, Zech et al. have shown that many patients with suspected monogenic dystonia carry mutations that are undetectable with exome analysis alone. Complementary approaches, such as RNA sequencing and functional studies, can improve rates of diagnosis.

CALFAN syndrome is a rare genetic disorder affecting the nervous system and liver, with skeletal abnormalities also reported. It is caused by mutations in SCYL1, a gene encoding a ubiquitously expressed protein localized to the secretory pathway. SCYL1 interacts with trafficking components, including ARF GTPases and the COPI vesicle coat complex, and appears to function in retrograde secretory trafficking. Despite this knowledge, the mechanisms that underlie CALFAN pathology remain poorly understood. Here, using fibroblasts obtained from patients diagnosed with CALFAN syndrome and from SCYL1 knockout fibroblasts, we reveal an accumulation of the abundant secretory cargo procollagen type I in the endoplasmic reticulum (ER) upon SCYL1 deficiency. Surprisingly, we failed to observe procollagen-I-trafficking defects in the SCYL1-deficient cells. Nevertheless, ER accumulation of procollagen-I correlated with ER distension and induction of ER stress in the patient fibroblasts, which also underwent increased cell death. The phenotypes were observed at elevated temperatures, mimicking the induction of pathology under febrile conditions in patients with CALFAN syndrome. Our data suggest that ER stress induction is a pathological mechanism in CALFAN syndrome and that targeting this process may represent a therapeutic strategy.

Classic isovaleric aciduria (cIVA) is a rare inherited metabolic disorder characterized by recurrent life-threatening metabolic decompensations and neurocognitive impairment in untreated patients. This meta-analysis aims to assess the impact of early diagnosis by newborn screening (NBS) on mortality and neurocognitive outcome. A systematic literature search for articles published until 2022 was conducted following PRISMA protocol guidelines. We investigated effects on clinical outcomes and survival, analyzing outcome parameters using meta-analytical measures and estimating effect sizes with a random-effects model. Overall, 20 studies were included, reporting on 240 individuals with cIVA. Individuals identified by NBS presented with a lower frequency of neurological symptoms (13.0% vs. 44.9%; p = 0.0040) and developmental delay (6.1% vs. 51.2%; p < 0.0001), and had a lower mortality rate (1.1% vs. 10.9%; p = 0.0320). The quality of healthcare systems did not have a measurable impact on neurocognitive outcome and mortality. Despite the beneficial effect of NBS on clinical outcome and mortality, it could not reliably prevent the manifestation of neonatal decompensation in all individuals with cIVA identified by NBS. Early diagnosis through NBS is essential for the timely initiation of therapy and for improving outcomes and survival rates in individuals with cIVA.

PURPOSE. Primary mitochondrial disorders (PMDs) are a clinically heterogeneous group of genetic disorders that can affect many tissues, with a broad phenotypic spectrum ranging from isolated organ involvement to severe early-onset multisystem disease. Visual loss from optic atrophy is a frequent clinical manifestation of mitochondrial cytopathies. This study aimed to identify the missing heritability in previously unsolved cases of suspected isolated or syndromic optic neuropathy. Based on three recent reports on biallelic NSUN3 variants causing early-onset PMD, we explored in detail the genetic and clinical spectrum of NSUN3-associated disease. METHODS. Affected individuals were analyzed by exome or genome sequencing. In silico variant analysis and functional assays were performed to investigate the consequences of the identified variants. Detailed phenotyping data were collected from medical records and direct questioning after the identification of candidate-likely pathogenic variants. RESULTS. Interrogation of exome and genome sequencing data led to the identification of six candidate NSUN3 variants in eight affected individuals from five unrelated families (including a previously reported case). A broad phenotypic spectrum was observed ranging from isolated optic atrophy to severe early-onset PMD. Identified NSUN3 variants impairing NSUN3 activity are located within the S-adenosylmethionine-dependent methyltransferases domain and loss of function variants were associated with a more severe phenotype. Remarkably, bilateral optic atrophy was a unifying clinical feature observed in almost all affected individuals. CONCLUSIONS. Pathogenic or likely pathogenic biallelic variants in NSUN3 disrupt mt-tRNAMet methylation and mitochondrial translation leading to mitochondrial disease ranging from mild isolated optic atrophy to a severe multisystemic phenotype with possible limited life expectancy.