METHYLATION-ASSOCIATED PATHWAYS IN MACULAR TELANGIECTASIA TYPE 2 AND OPHTHALMOLOGIC FINDINGS IN PATIENTS WITH GENETIC METHYLATION DISORDERS.

PURPOSE: Serine (Ser) and glycine (Gly) levels were reported to differ between patients with macular telangiectasia type 2 (MacTel) compared with healthy controls. Because they are closely related to methylation metabolism, this report investigates methylation-associated metabolite levels in patients with MacTel and retinal changes in monogenetic methylation disorders. METHODS: Prospective, monocentric study on patients with MacTel and healthy controls underwent a standardized protocol including a blood draw. Methylation-associated metabolite levels in plasma were determined using targeted quantitative metabolomics. Furthermore, patient records of cystathionine beta-synthase, methylenetetrahydrofolate reductase, and methylmalonic aciduria and homocystinuria type C protein (MMACHC) deficiency were screened for reported retinal changes. RESULTS: In total, 29 patients with MacTel and 27 healthy controls were included. Patients with MacTel showed lower plasma Ser ( P = 0.02 and P = 0.01) and Gly ( P = 0.11 and P = 0.11) levels than controls. Principal component analyses revealed that methylation-associated metabolite, especially homocysteine, contributed to a distinct clustering of patients with MacTel. No retinal changes were seen in cystathionine beta-synthase (n = 1) and methylenetetrahydrofolate reductase (n = 2) deficiency, while two patients with MMACHC (n = 4) deficiency displayed extensive macular dystrophy. CONCLUSION: Patients with MacTel show distinct clustering of methylation-associated metabolite compared with controls. Of the three homocystinurias, only MMACHC resulted in macular dystrophy, possibly due to distinct compensatory pathways.

Spic regulates one-carbon metabolism and histone methylation in ground-state pluripotency.

Understanding mechanisms of epigenetic regulation in embryonic stem cells (ESCs) is of fundamental importance for stem cell and developmental biology. Here, we identify Spic, a member of the ETS family of transcription factors (TFs), as a marker of ground state pluripotency. We show that Spic is rapidly induced in ground state ESCs and in response to extracellular signal-regulated kinase (ERK) inhibition. We find that SPIC binds to enhancer elements and stabilizes NANOG binding to chromatin, particularly at genes involved in choline/one-carbon (1C) metabolism such as Bhmt, Bhmt2, and Dmgdh. Gain-of-function and loss-of-function experiments revealed that Spic controls 1C metabolism and the flux of S-adenosyl methionine to S-adenosyl-L-homocysteine (SAM-to-SAH), thereby, modulating the levels of H3R17me2 and H3K4me3 histone marks in ESCs. Our findings highlight betaine-dependent 1C metabolism as a hallmark of ground state pluripotency primarily activated by SPIC. These findings underscore the role of uncharacterized auxiliary TFs in linking cellular metabolism to epigenetic regulation in ESCs.

Analysis of S-Adenosylmethionine and S-Adenosylhomocysteine: Method Optimisation and Profiling in Healthy Adults upon Short-Term Dietary Intervention.

S-adenosylmethionine (SAM) is essential for methyl transfer reactions. All SAM is produced de novo via the methionine cycle. The demethylation of SAM produces S-adenosylhomocysteine (SAH), an inhibitor of methyltransferases and the precursor of homocysteine (Hcy). The measurement of SAM and SAH in plasma has value in the diagnosis of inborn errors of metabolism (IEM) and in research to assess methyl group homeostasis. The determination of SAM and SAH is complicated by the instability of SAM under neutral and alkaline conditions and the naturally low concentration of both SAM and SAH in plasma (nM range). Herein, we describe an optimised LC-MS/MS method for the determination of SAM and SAH in plasma, urine, and cells. The method is based on isotopic dilution and employs 20 µL of plasma or urine, or 500,000 cells, and has an instrumental running time of 5 min. The reference ranges for plasma SAM and SAH in a cohort of 33 healthy individuals (age: 19-60 years old; mean ± 2 SD) were 120 ± 36 nM and 21.5 ± 6.5 nM, respectively, in accordance with independent studies and diagnostic determinations. The method detected abnormal concentrations of SAM and SAH in patients with inborn errors of methyl group metabolism. Plasma and urinary SAM and SAH concentrations were determined for the first time in a randomised controlled trial of 53 healthy adult omnivores (age: 18-60 years old), before and after a 4 week intervention with a vegan or meat-rich diet, and revealed preserved variations of both metabolites and the SAM/SAH index.

Thiolatocobalamins repair the activity of pathogenic variants of the human cobalamin processing enzyme CblC.

Thiolatocobalamins are a class of cobalamins comprised of naturally occurring and synthetic ligands. Glutathionylcobalamin (GSCbl) occurs naturally in mammalian cells, and also as an intermediate in the glutathione-dependent dealkylation of methylcobalamin (MeCbl) to form cob(I)alamin by pure recombinant CblC from C. elegans. Glutathione-driven deglutathionylation of GSCbl was demonstrated both in mammalian as well as in C. elegans CblC. Dethiolation is orders of magnitude faster than dealkylation of Co-C bonded cobalamins, which motivated us to investigate two synthetic thiolatocobalamins as substrates to repair the enzymatic activity of pathogenic CblC variants in humans. We report the synthesis and kinetic characterization of cysteaminylcobalamin (CyaCbl) and 2-mercaptopropionylglycinocobalamin (MpgCbl). Both CyaCbl and MpgCbl were obtained in high purity (90-95%) and yield (78-85%). UV-visible spectral properties agreed with those reported for other thiolatocobalamins with absorbance maxima observed at 372 nm and 532 nm. Both CyaCbl and MpgCbl bound to wild type human recombinant CblC inducing spectral blue-shifts characteristic of the respective base-on to base-off transitions. Addition of excess glutathione (GSH) resulted in rapid elimination of the ß-ligand to give aquacobalamin (H2OCbl) as the reaction product under aerobic conditions. Further, CyaCbl and MpgCbl underwent spontaneous dethiolation thereby repairing the loss of activity of pathogenic variants of human CblC, namely R161G and R161Q. We posit that thiolatocobalamins could be exploited therapeutically for the treatment of inborn errors of metabolism that impair processing of dietary and supplemental cobalamin forms. While these disorders are targets for newborn screening in some countries, there is currently no effective treatment available to patients.

Metabolic Profiling in Human Fibroblasts Enables Subtype Clustering in Glycogen Storage Disease.

Glycogen storage disease subtypes I and III (GSD I and GSD III) are monogenic inherited disorders of metabolism that disrupt glycogen metabolism. Unavailability of glucose in GSD I and induction of gluconeogenesis in GSD III modify energy sources and possibly, mitochondrial function. Abnormal mitochondrial structure and function were described in mice with GSD Ia, yet significantly less research is available in human cells and ketotic forms of the disease. We hypothesized that impaired glycogen storage results in distinct metabolic phenotypes in the extra- and intracellular compartments that may contribute to pathogenesis. Herein, we examined mitochondrial organization in live cells by spinning-disk confocal microscopy and profiled extra- and intracellular metabolites by targeted LC-MS/MS in cultured fibroblasts from healthy controls and from patients with GSD Ia, GSD Ib, and GSD III. Results from live imaging revealed that mitochondrial content and network morphology of GSD cells are comparable to that of healthy controls. Likewise, healthy controls and GSD cells exhibited comparable basal oxygen consumption rates. Targeted metabolomics followed by principal component analysis (PCA) and hierarchical clustering (HC) uncovered metabolically distinct poises of healthy controls and GSD subtypes. Assessment of individual metabolites recapitulated dysfunctional energy production (glycolysis, Krebs cycle, succinate), reduced creatinine export in GSD Ia and GSD III, and reduced antioxidant defense of the cysteine and glutathione systems. Our study serves as proof-of-concept that extra- and intracellular metabolite profiles distinguish glycogen storage disease subtypes from healthy controls. We posit that metabolite profiles provide hints to disease mechanisms as well as to nutritional and pharmacological elements that may optimize current treatment strategies.

Targeted Metabolic Profiling of Methionine Cycle Metabolites and Redox Thiol Pools in Mammalian Plasma, Cells and Urine.

The concentration of thiol and thioether metabolites in plasma has diagnostic value in genetic diseases of B-vitamin metabolism linked to methionine utilization. Among these, cysteine/cystine (Cys/CSSC) and glutathione/oxidized glutathione (GSH/GSSG) act as cellular redox buffers. A new LC-MS/MS method was developed for the simultaneous detection of cystathionine (Cysta), methionine (Met), methionine sulfoxide (MSO), creatinine and the reduced and oxidized pairs of homocysteine (Hcy/HSSH), cysteine (Cys/CSSC) and glutathione (GSH/GSSG). A one-step thiol-blocking protocol with minimal sample preparation was established to determine redox thiol pairs in plasma and cells. The concentrations of diagnostic biomarkers Hcy, Met, Cysta, and Cys in a cohort of healthy adults (n = 53) agreed with reference ranges and published values. Metabolite concentrations were also validated in commercial samples of human, mouse, rat and Beagle dog plasma and by the use of a standardized ERNDIM quality control. Analysis of fibroblasts, endothelial and epithelial cells, human embryonic stem cells, and cancer cell lines showed cell specificity for both the speciation and concentration of thiol and thioether metabolites. This LC-MS/MS platform permits the fast and simultaneous quantification of 10 thiol and thioether metabolites and creatinine using 40 µL plasma, urine or culture medium, or 500,000 cells. The sample preparation protocols are directly transferable to automated metabolomic platforms.