Radenkovic, Silvia

Background: ALG13-CDG is an X-linked N-linked glycosylation disorder caused by pathogenic variants in the glycosyltransferase ALG13, leading to severe neurological manifestations. Despite the clear CNS involvement, the impact of ALG13 dysfunction on human brain glycosylation and neurodevelopment remains unknown. We hypothesize that ALG13-CDG causes brain-specific hypoglycosylation that disrupts neurodevelopmental pathways and contributes directly to cortical network dysfunction. Methods: We generated iPSC-derived human cortical organoids (hCOs) from individuals with ALG13-CDG to define the impact of hypoglycosylation on cortical development and function. Electrophysiological activity was assessed using MEA recordings and integrated with multiomic profiling, including scRNA-seq, proteomics, glycoproteomics, N-glycan imaging, lipidomics, and metabolomics. X-inactivation status was evaluated in both iPSCs and hCOs. Results: ALG13-CDG hCOs showed reduced glycosylation of proteins involved in ECM organization, neuronal migration, lipid metabolism, calcium homeostasis, and neuronal excitability. These pathway disruptions were supported by proteomic and scRNA-seq data and included altered intercellular communication. Trajectory analyses revealed mistimed neuronal maturation with early inhibitory and delayed excitatory development, indicating an E/I imbalance. MEA recordings demonstrated early network hypoactivity with reduced firing rates, immature burst structure, and shortened axonal projections, while transcriptomic and proteomic signatures suggested emerging hyperexcitability. Altered lipid and GlcNAc metabolism, along with skewed X-inactivation, were also observed. Conclusions: Our study reveals that ALG13-CDG is a disorder of brain-specific hypoglycosylation that disrupts key neurodevelopmental pathways and destabilizes cortical network function. Through integrated multiomic and functional analyses, we identify early network hypoactivity, mistimed neuronal maturation, and evolving E/I imbalance that progresses to compensatory hyperexcitability, providing a mechanistic basis for seizure vulnerability. These findings redefine ALG13-CDG as disorders of cortical network instability, offering a new framework for targeted therapeutic intervention.

Abstract Congenital disorders of glycosylation (CDG) are a group of neurogenetic conditions resulting from disruptions in the cellular glycosylation machinery. The majority of CDG patients have compound heterozygous pathogenic variants in the phosphomannomutase 2 ( PMM2) gene. Individuals with PMM2-CDG exhibit multi-systemic symptoms, prominently featuring neurological deficits with nearly all patients exhibiting cerebellar hypoplasia and ataxia. To overcome embryonic lethality caused by whole body knock-out of Pmm2 and mimic patient-related compound heterozygous pathogenic variants, we paired a Pmm2 flox allele ( Pmm2 fl ) with a catalytically inactive knock-in allele ( Pmm2 R137H ), commonly present in PMM2-CDG patients. Mice with post-mitotic loss of PMM2 from neurons or astrocytes are indistinguishable from unaffected littermates, including in a broad battery of neurological assessments. In contrast, removal of PMM2 from embryonic neural precursor cells leads to cerebellar hypoplasia, ataxia, seizures, and early lethality. Comprehensive multi-omics profiling, including metabolomics, glycomics, single-cell transcriptomics, proteomics, and glycoproteomics, reveal widespread molecular disturbances throughout the brain, with the cerebellum showing the most pronounced disruption. These findings highlight the heightened dependency of the developing cerebellum on intact N-glycosylation, aligning with clinical observations in PMM2-CDG patients. Importantly, glycoproteomic alterations identified in our mouse model are corroborated in PMM2-CDG patient post-mortem cerebellar tissue, underscoring the translational relevance of our findings and implicating impaired synaptic transmission as a key pathogenic mechanism.

Inherited metabolic disorders (IMDs) are genetic disorders that occur in as many as 1:2500 births worldwide. Nevertheless, they are quite rare individually and even more rare is the co-occurrence of two IMDs in one individual. To better understand the metabolic cross-talk between glycosylation changes and deficient energy metabolism, and its potential effect on outcomes, we evaluated patient fibroblasts with likely pathogenic variants in PGM1 and pathogenic variants in NDUFA13 derived from a patient who passed away at 16 years of age. The patient presented with characteristic of PGM1-CDG including bifid uvula, muscle involvement, abnormal glycosylation in blood, and elevated liver transaminases. In addition, hearing loss, seizures, elevated plasma and CSF lactate and a Leigh-like MRI brain pattern were present, which are commonly associated with Leigh syndrome. PGM1-CDG has been reported in about 70 individuals, while NDUFA13 deficiency has so far only been reported in 13 patients. As abundant energy is essential for glycosylation, and both PGM1 and NDUFA13 are linked to energy metabolism, we sought to better understand the underlying biochemical cause of the patient’s clinical presentation. To do so, we performed extensive investigations including tracer metabolomics, lipidomics and enzymatic studies on the patient’s fibroblasts. We found a profound depletion of UDP-hexoses, consistent with PGM1-CDG. Complex I enzyme activity and mitochondrial function were also impaired, corroborating complex I deficiency and Leigh syndrome. Further, lipidomics analysis showed similarities with both PGM1-CDG and OXPHOS-deficient patients. Based on our results, the patient was diagnosed with both PGM1-CDG and Leigh syndrome. In summary, we present the first case of combined CDG and Leigh syndrome, caused by (likely) pathogenic variants in PGM1 and NDUFA13, and underline the importance of considering the synergistic effects of multiple disease-causing variants in patients with complex clinical presentation, leading to the patient’s early demise.

Background and Aims: Acute-on-chronic liver failure (ACLF) is a complication of cirrhosis characterized by multiple organ failure and high short-term mortality. The pathophysiology of ACLF involves elevated systemic inflammation leading to organ failure, along with immune dysfunction that heightens susceptibility to bacterial infections. However, it is unclear how these aspects are associated with recovery and nonrecovery in ACLF. Approach and Results: Here, we mapped the single-cell transcriptome of circulating immune cells from patients with ACLF and acute decompensated (AD) cirrhosis and healthy individuals. We further interrogate how these findings, as well as immunometabolic and functional profiles, associate with ACLF-recovery (ACLF-R) or nonrecovery (ACLF-NR). Our analysis unveiled 2 distinct states of classical monocytes (cMons). Hereto, ACLF-R cMons were characterized by transcripts associated with immune and stress tolerance, including anti-inflammatory genes such as RETN and LGALS1. Additional metabolomic and functional validation experiments implicated an elevated oxidative phosphorylation metabolic program as well as an impaired ACLF-R cMon functionality. Interestingly, we observed a common stress-induced tolerant state, oxidative phosphorylation program, and blunted activation among lymphoid populations in patients with ACLF-R. Conversely, ACLF-NR cMon featured elevated expression of inflammatory and stress response genes such as VIM, LGALS2, and TREM1, along with blunted metabolic activity and increased functionality. Conclusions: This study identifies distinct immunometabolic cellular states that contribute to disease outcomes in patients with ACLF. Our findings provide valuable insights into the pathogenesis of ACLF, shedding light on factors driving either recovery or nonrecovery phenotypes, which may be harnessed as potential therapeutic targets in the future.

BACKGROUND Diagnosis of PMM2-CDG, the most common congenital disorder of glycosylation (CDG), relies on measuring carbohydrate-deficient transferrin (CDT) and genetic testing. CDT tests have false negatives and may normalize with age. Site-specific changes in protein N-glycosylation have not been reported in sera in PMM2-CDG. METHODS Using multistep mass spectrometry–based N-glycoproteomics, we analyzed sera from 72 individuals to discover and validate glycopeptide alterations. We performed comprehensive tandem mass tag–based discovery experiments in well-characterized patients and controls. Next, we developed a method for rapid profiling of additional samples. Finally, targeted mass spectrometry was used for validation in an independent set of samples in a blinded fashion. RESULTS Of the 3,342 N-glycopeptides identified, patients exhibited decrease in complex-type N-glycans and increase in truncated, mannose-rich, and hybrid species. We identified a glycopeptide from complement C4 carrying the glycan Man 5 GlcNAc 2 , which was not detected in controls, in 5 patients with normal CDT results, including 1 after liver transplant and 2 with a known genetic variant associated with mild disease, indicating greater sensitivity than CDT. It was detected by targeted analysis in 2 individuals with variants of uncertain significance in PMM2 . CONCLUSION Complement C4–derived Man 5 GlcNAc 2 glycopeptide could be a biomarker for accurate diagnosis and therapeutic monitoring of patients with PMM2-CDG and other CDGs. FUNDING U54NS115198 (Frontiers in Congenital Disorders of Glycosylation: NINDS; NCATS; Eunice Kennedy Shriver NICHD; Rare Disorders Consortium Disease Network); K08NS118119 (NINDS); Minnesota Partnership for Biotechnology and Medical Genomics; Rocket Fund; R01DK099551 (NIDDK); Mayo Clinic DERIVE Office; Mayo Clinic Center for Biomedical Discovery; IA/CRC/20/1/600002 (Center for Rare Disease Diagnosis, Research and Training; DBT/Wellcome Trust India Alliance)

AbstractAsparagine‐linked glycosylation 1 protein is a β‐1,4‐mannosyltransferase, is encoded by the ALG1 gene, which catalyzes the first step of mannosylation in N‐glycosylation. Pathogenic variants in ALG1 cause a rare autosomal recessive disorder termed as ALG1‐CDG. We performed a quantitative proteomics and N‐glycoproteomics study in fibroblasts derived from patients with one homozygous and two compound heterozygous pathogenic variants in ALG1. Several proteins that exhibited significant upregulation included insulin‐like growth factor II and pleckstrin, whereas hyaluronan and proteoglycan link protein 1 was downregulated. These proteins are crucial for cell growth, survival and differentiation. Additionally, we observed a decrease in the expression of mitochondrial proteins and an increase in autophagy‐related proteins, suggesting mitochondrial and cellular stress. N‐glycoproteomics revealed the reduction in high‐mannose and complex/hybrid glycopeptides derived from numerous proteins in patients explaining that defect in ALG1 has broad effects on glycosylation. Further, we detected an increase in several short oligosaccharides, including chitobiose (HexNAc2) trisaccharides (Hex‐HexNAc2) and novel tetrasaccharides (NeuAc‐Hex‐HexNAc2) derived from essential proteins including LAMP1, CD44 and integrin. These changes in glycosylation were observed in all patients irrespective of their gene variants. Overall, our findings not only provide novel molecular insights into understanding ALG1‐CDG but also offer short oligosaccharide‐bearing peptides as potential biomarkers.

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Phosphoglucomutase-1-congenital disorder of glycosylation (PGM1-CDG) (OMIM: 614921) is a rare autosomal recessive inherited metabolic disease caused by the deficiency of the PGM1 enzyme. Like other CDGs, PGM1-CDG has a multisystemic presentation. The most common clinical findings include liver involvement, rhabdomyolysis, hypoglycemia, and cardiac involvement. Phenotypic severity can vary, though cardiac presentation is usually part of the most severe phenotype, often resulting in early death. Unlike the majority of CDGs, PGM1-CDG has a treatment: oral D-galactose (D-gal) supplementation, which significantly improves many aspects of the disorder. Here, we describe five PGM1-CDG patients treated with D-gal and report both on novel clinical symptoms in PGM1-CDG as well as the effects of the D-gal treatment. D-gal resulted in notable clinical improvement in four patients, though the efficacy of treatment varied between the patients. Furthermore, there was a significant improvement or normalization in transferrin glycosylation, liver transaminases and coagulation factors in three patients, creatine kinase (CK) levels in two, while hypoglycemia resolved in two patients. One patient discontinued the treatment due to urinary frequency and lack of clinical improvement. Furthermore, one patient experienced recurrent episodes of rhabdomyolysis and tachycardia even on higher doses of therapy. D-gal also failed to improve the cardiac function, which was initially abnormal in three patients, and remains the biggest challenge in treating PGM1-CDG. Together, our findings expand the phenotype of PGM1-CDG and underline the importance of developing novel therapies that would specifically treat the cardiac phenotype in PGM1-CDG.

ObjectiveEpalrestat, an aldose reductase inhibitor increases phosphomannomutase (PMM) enzyme activity in a PMM2‐congenital disorders of glycosylation (CDG) worm model. Epalrestat also decreases sorbitol level in diabetic neuropathy. We evaluated the genetic, biochemical, and clinical characteristics, including the Nijmegen Progression CDG Rating Scale (NPCRS), urine polyol levels and fibroblast glycoproteomics in patients with PMM2‐CDG.MethodsWe performed PMM enzyme measurements, multiplexed proteomics, and glycoproteomics in PMM2‐deficient fibroblasts before and after epalrestat treatment. Safety and efficacy of 0.8 mg/kg/day oral epalrestat were studied in a child with PMM2‐CDG for 12 months.ResultsPMM enzyme activity increased post‐epalrestat treatment. Compared with controls, 24% of glycopeptides had reduced abundance in PMM2‐deficient fibroblasts, 46% of which improved upon treatment. Total protein N‐glycosylation improved upon epalrestat treatment bringing overall glycosylation toward the control fibroblasts’ glycosylation profile. Sorbitol levels were increased in the urine of 74% of patients with PMM2‐CDG and correlated with the presence of peripheral neuropathy, and CDG severity rating scale. In the child with PMM2‐CDG on epalrestat treatment, ataxia scores improved together with significant growth improvement. Urinary sorbitol levels nearly normalized in 3 months and blood transferrin glycosylation normalized in 6 months.InterpretationEpalrestat improved PMM enzyme activity, N‐glycosylation, and glycosylation biomarkers in vitro. Leveraging cellular glycoproteome assessment, we provided a systems‐level view of treatment efficacy and discovered potential novel biosignatures of therapy response. Epalrestat was well‐tolerated and led to significant clinical improvements in the first pediatric patient with PMM2‐CDG treated with epalrestat. We also propose urinary sorbitol as a novel biomarker for disease severity and treatment response in future clinical trials in PMM2‐CDG. ANN NEUROL 20219999:n/a–n/a

AbstractPhosphoglucomutase 1 (PGM1) catalyzes the interconversion of glucose‐6‐phosphate to glucose‐1‐phosphate and is a key enzyme of glycolysis, glycogenesis, and glycogenolysis. PGM1 deficiency (OMIM: 614921) was initially defined as a glycogen storage disorder (type XIV), and later re‐classified as a PGM1‐congenital disorder of glycosylation (PGM1‐CDG). Serum transferrin (Tf) glycan isoform analysis by liquid chromatography‐mass spectrometry (LC‐MS) is used as a primary diagnostic screen tool, and reveals a very unique CDG profile described as a mixture of CDG‐type I and CDG‐type II patterns. Oral d‐galactose supplementation shows significant clinical and metabolic improvements, which are indicated by the Tf glycan isoform normalization over time in patients with PGM1‐CDG. Thus, there is a need for biomarkers to guide d‐galactose dosage in patients in order to maintain effective and safe drug levels. Here, we present a simplified algorithm called PGM1‐CDG Treatment Monitoring Index (PGM1‐TMI) for assessing the response of PGM1‐CDG patients to d‐galactose supplementation. For our single‐center cohort of 16 PGM1‐CDG patients, the Tf glycan profile analysis provided the biochemical diagnosis in all of them. In addition, the PGM1‐TMI was reduced in PGM1‐CDG patients under d‐galactose supplementation as compared with their corresponding values before treatment, indicating that glycosylation proceeds towards normalization. PGM1‐TMI allows tracking Tf glycan isoform normalization over time when the patients are on d‐galactose supplementation.