This is the first large-scale metabolomic study in genetically confirmed autosomal dominant tubulointerstitial kidney disease (ADTKD), providing a new resource for rare kidney diseases. ADTKD-UMOD and ADTKD-MUC1 are metabolically indistinguishable across stages, supporting the development of unified monitoring strategies. The plasma kynurenine-to-tryptophan ratio increases with CKD progression, supporting its use as a noninvasive marker of inflammation in ADTKD. Metabolomic profiling has not yet been performed in autosomal dominant tubulointerstitial kidney disease (ADTKD) due to UMOD or MUC1 mutations and could provide valuable insights into the pathophysiology of these conditions and identify the biomarkers of disease activity. Untargeted metabolomic analysis of plasma samples was performed on a cohort comprising 40 controls, 51 individuals with ADTKD-UMOD, and 49 individuals with ADTKD-MUC1 with CKD stages ranging from 1 to 4. Principal component analysis and hierarchical clustering revealed that the metabolic profiles of controls and ADTKD-UMOD and ADTKD-MUC1 patients with CKD stages 1 and 2 were similar. The metabolome was also similar between patients with ADTKD-UMOD and ADTKD-MUC1 at each stage of CKD. Compared with stage 2 CKD, stage 3 CKD was characterized by an increased kynurenine-to-tryptophan ratio, indicating activation of indoleamine 2,3-dioxygenase, an inflammation-induced and rate-limiting enzyme of tryptophan metabolism, and increased levels of pseudouridine, 3-indoxylsulfate, N-formylmethione, N-acetylated amino acids, acylcarnitines, and several other metabolites. In total, 121 metabolites were identified as significantly altered in patients in stage 4 compared with controls. Enrichment analysis of this set revealed that the most significant alterations were in the biosynthesis of arginine and branched-chain amino acids, carnitine synthesis, transfer RNA metabolism, tryptophan catabolism, urea cycle, metabolism of amino acids, glucose homeostasis, and solute carrier-mediated transmembrane transport. Patients with ADTKD-UMOD and ADTKD-MUC1 had similar metabolomic profiles across CKD stages. Although ADTKD is a tubulointerstitial kidney disease rather than glomerular, the effects on the metabolomic pathways appear comparable with those of other forms of CKD. The kynurenine-to-tryptophan ratio appears to be a promising biomarker of ADTKD progression and will require additional study.

Primary systemic carnitine deficiency (CDSP) is a rare inherited metabolic disorder characterized by impaired carnitine transport due to mutations in the SLC22A5 gene, leading to impaired mitochondrial fatty acid oxidation. The aim of this retrospective, descriptive study was to investigate the clinical, biochemical, and molecular features of CDSP in Turkey, where the lack of a national expanded metabolic screening program contributes to delayed diagnosis and severe complications. The clinical, biochemical, and molecular profiles of 12 patients from eight families diagnosed between 2003 and 2025 were retrospectively analyzed. Data on family history, consanguinity, clinical manifestations-including cardiomyopathy, muscle weakness, neurological symptoms, and liver dysfunction-plasma free carnitine levels, and echocardiographic measurements were collected and analyzed. The majority of patients (92%) were from consanguineous families. Cardiomyopathy was the most common clinical feature (75%), followed by muscle weakness (50%), neurological symptoms (42%), and liver dysfunction (33%). A novel SLC22A5 variant (c.125T>C; p.Leu42Pro) was identified that expands the known genetic spectrum of CDSP. Oral carnitine supplementation significantly increased plasma free carnitine levels (p = 0.01) and improved long-term interventricular septal thickness Z-scores (p = 0.045). In addition, cholestasis was observed in two patients, suggesting an expanded disease phenotype. These results emphasize the crucial role of early detection and family screening in the prevention of life-threatening complications associated with CDSP. Long-term carnitine therapy improves metabolic and cardiac outcomes, underscoring the need for early intervention and inclusion of CDSP in national newborn screening programs.

The PNPLA3 rs738409 polymorphism is the most abundant genetic risk factor associated with progression of metabolic dysfunction-associated steatotic liver disease (MASLD) to steatohepatitis (MASH) and fibrosis. Although fasting and feeding affect PNPLA3 expression, molecular insights into the pathophysiological influence remain scarce. We analyzed 353 serum samples of patients with MASLD from two German tertiary centers using nuclear magnetic resonance (NMR)-proteometabolomics. Patients were stratified by PNPLA3 rs738409 C>G genotype: 'CC', 'CG', and 'GG'. Metabolites, lipoproteins, and glycoproteins were assessed based on fasting status. PNPLA3 GG displayed a distinct metabolic profile, with notable alterations between fasting and non-fasting states. During the latter, GG carriers showed lower levels of VLDL-1, reflecting impaired triglyceride (TG) efflux from hepatocytes. Following an overnight fast, GG carriers exhibited higher tricarboxylic acid cycle metabolites and ketone bodies, overall indicating increased β-oxidation likely attributed to lower PNPLA3 expression, facilitating unrestricted adipose triglyceride lipase activity and consecutive increased hepatic TG secretion. In addition, the ketogenic amino acid lysine, critical for mitochondrial carnitine transport, was significantly reduced (GG 0.14 ± 0.09 mM vs. CC 0.18 ± 0.08 mM, q = 0.015). Consistently, TGs were enriched in LDL and HDL particles, and an increased number of intermediate-density lipoproteins emerged as a distinct marker in fasted GG carriers (GG 202.9 ± 68.2 mg/dl vs. CC 160.8 ± 65.6 mg/dl, q = 0.007). These metabolic changes were enhanced in patients with type 2 diabetes mellitus and/or obesity. Our findings suggest a dichotomous pattern of increased hepatic lipid storage during feeding and excessive lipid oxidation during fasting, which exceeds metabolic capacity, inducing cellular toxicity in PNPLA3 GG carriers. This interplay fuels a detrimental fasting/non-fasting cycle, thus pointing to the need for preventive strategies. The PNPLA3 rs738409 (p.I148M) polymorphism is the most prevalent genetic risk factor for metabolic dysfunction-associated steatotic liver disease progression and is influenced by fasting and feeding cycles. However, the pathophysiological consequences of this regulation remain poorly understood. Nuclear magnetic resonance-proteometabolomics reveals a distinct signature in homozygous PNPLA3 GG carriers that changes significantly with fasting status, providing important implications for diagnosis and preventive strategies.

Carnitine transport and cycle disorders (CCD) are a group of metabolic disorders characterized by either carnitine depletion or dysfunction in the carnitine cycle, a critical process for the transport of fatty acids into the mitochondria and their subsequent β-oxidation. Clinically, CCD can manifest with a wide range of symptoms, including hypoketotic hypoglycemia, which may be accompanied by signs of liver dysfunction, hepatic steatosis, myopathy and cardiomyopathy. Biochemical diagnosis typically involves measuring carnitine and acylcarnitine levels in blood, alongside organic acid profiling in urine. However, due to phenotypic overlaps with other metabolic disorders, precise molecular diagnosis is essential for accurate disease classification and subtype determination. The present study aimed to develop and clinically validate a novel CCD panel, specifically designed for Oxford Nanopore Technologies (ONT) platform compatibility. The panel targeted four key CCD related genes (CPT-1, CPT-2, SLC22A5 and SLC25A20). An amplification-based library preparation method pooling 21 primers specific to the CCD-related genes into two tubes was optimized. The panel was then applied to screen 20 patients previously diagnosed with CCD via second-generation sequencing platform. Comparative analysis of results from both platforms revealed a 100% concordance in detecting pathogenic, likely pathogenic, and variants of unknown significance associated with CCD. In silico analysis was also performed to predict the pathogenic potential of the variants of unknown significance. Here we report the development and clinical validation of a multi-gene diagnostic panel for ONT platform. The results demonstrated the feasibility of ONT-based genetic testing for CCD and set the stage for the development of similar diagnostic panels for other genetic disorders, offering a streamlined and putatively cost-effective alternative to current sequencing methodologies.

Mitochondrial complex I (CI) deficiency represents a common biochemical pathophysiology underlying Leigh syndrome spectrum (LSS), manifesting with progressive multi-system dysfunction, lactic acidemia, and early mortality. To facilitate mechanistic studies and rigorous screening of therapeutic candidates for CI deficient LSS, we used CRISPR/Cas9 to generate an ndufs2 -/- 16 bp deletion zebrafish strain. ndufs2 -/- larvae exhibit markedly reduced survival, severe neuromuscular dysfunction including impaired swimming capacity, multiple morphologic malformations, reduced growth, hepatomegaly, uninflated swim bladder, yolk retention, small intestines, and small eyes and pupils with abnormal retinal ganglion cell layer. Transcriptome profiling of ndufs2 -/- larvae revealed dysregulation of the electron transport chain, TCA cycle, fatty acid beta-oxidation, and one-carbon metabolism. 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. ndufs2 -/- zebrafish had 80% reduced CI enzyme activity. Unbiased metabolomic profiling showed increased lactate, TCA cycle intermediates, and acyl-carnitine species. One-carbon metabolism associated pathway alterations appear to contribute to CI disease pathophysiology, as folic acid treatment rescued the growth defect and hepatomegaly in ndufs2 -/- larvae. Overall, ndufs2 -/- zebrafish recapitulate severe CI deficiency, complex metabolic pathophysiology, and relevant LSS neuromuscular and survival phenotypes, enabling future translational studies of therapeutic candidates.