He, Miao

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)

AbstractATP6AP1‐CDG is an X‐linked disorder typically characterized by hepatopathy, immunodeficiency, and an abnormal type II transferrin glycosylation pattern. Here, we present 11 new patients and clinical updates with biochemical characterization on one previously reported patient. We also document intrafamilial phenotypic variability and atypical presentations, expanding the symptomatology of ATP6AP1‐CDG to include dystonia, hepatocellular carcinoma, and lysosomal abnormalities on hepatic histology. Three of our subjects received successful liver transplantation. We performed N‐glycan profiling of total and fractionated plasma proteins for six patients and show associations with varying phenotypes, demonstrating potential diagnostic and prognostic value of fractionated N‐glycan profiles. The aberrant N‐linked glycosylation in purified transferrin and remaining plasma glycoprotein fractions normalized in one patient post hepatic transplant, while the increases of Man4GlcNAc2 and Man5GlcNAc2 in purified immunoglobulins persisted. Interestingly, in the single patient with isolated immune deficiency phenotype, elevated high‐mannose glycans were detected on purified immunoglobulins without glycosylation abnormalities on transferrin or the remaining plasma glycoprotein fractions. Given the diverse and often tissue specific clinical presentations and the need of clinical management post hepatic transplant in ATP6AP1‐CDG patients, these results demonstrate that fractionated plasma N‐glycan profiling could be a valuable tool in diagnosis and disease monitoring.

Purpose To summarise the clinical, molecular and biochemical phenotype of mannosyl-oligosaccharide glucosidase-related congenital disorders of glycosylation (MOGS-CDG), which presents with variable clinical manifestations, and to analyse which clinical biochemical assay consistently supports diagnosis in individuals with bi-allelic variants in MOGS. Methods Phenotypic characterisation was performed through an international and multicentre collaboration. Genetic testing was done by exome sequencing and targeted arrays. Biochemical assays on serum and urine were performed to delineate the biochemical signature of MOGS-CDG. Results Clinical phenotyping revealed heterogeneity in MOGS-CDG, including neurological, immunological and skeletal phenotypes. Bi-allelic variants in MOGS were identified in 12 individuals from 11 families. The severity in each organ system was variable, without definite genotype correlation. Urine oligosaccharide analysis was consistently abnormal for all affected probands, whereas other biochemical analyses such as serum transferrin analysis was not consistently abnormal. Conclusion The clinical phenotype of MOGS-CDG includes multisystemic involvement with variable severity. Molecular analysis, combined with biochemical testing, is important for diagnosis. In MOGS-CDG, urine oligosaccharide analysis via matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry can be used as a reliable biochemical test for screening and confirmation of disease.

Abstract Pathogenic variants in ALG13 ( ALG13 UDP‐N‐acetylglucosaminyltransferase subunit ) cause an X‐linked congenital disorder of glycosylation (ALG13‐CDG) where individuals have variable clinical phenotypes that include developmental delay, intellectual disability, infantile spasms, and epileptic encephalopathy. Girls with a recurrent de novo c.3013C>T; p.(Asn107Ser) variant have normal transferrin glycosylation. Using a highly sensitive, semi‐quantitative flow injection‐electrospray ionization‐quadrupole time‐of‐flight mass spectrometry (ESI‐QTOF/MS) N‐glycan assay, we report subtle abnormalities in N‐glycans that normally account for <0.3% of the total plasma glycans that may increase up to 0.5% in females with the p.(Asn107Ser) variant. Among our 11 unrelated ALG13‐CDG individuals, one male had abnormal serum transferrin glycosylation. We describe seven previously unreported subjects including three novel variants in ALG13 and report a milder neurodevelopmental course. We also summarize the molecular, biochemical, and clinical data for the 53 previously reported ALG13‐CDG individuals. We provide evidence that ALG13 pathogenic variants may mildly alter N‐linked protein glycosylation in both female and male subjects, but the underlying mechanism remains unclear.

Background and Aims Vacuolar H+‐ATP complex (V‐ATPase) is a multisubunit protein complex required for acidification of intracellular compartments. At least five different factors are known to be essential for its assembly in the endoplasmic reticulum (ER). Genetic defects in four of these V‐ATPase assembly factors show overlapping clinical features, including steatotic liver disease and mild hypercholesterolemia. An exception is the assembly factor vacuolar ATPase assembly integral membrane protein (VMA21), whose X‐linked mutations lead to autophagic myopathy. Approach and Results Here, we report pathogenic variants in VMA21 in male patients with abnormal protein glycosylation that result in mild cholestasis, chronic elevation of aminotransferases, elevation of (low‐density lipoprotein) cholesterol and steatosis in hepatocytes. We also show that the VMA21 variants lead to V‐ATPase misassembly and dysfunction. As a consequence, lysosomal acidification and degradation of phagocytosed materials are impaired, causing lipid droplet (LD) accumulation in autolysosomes. Moreover, VMA21 deficiency triggers ER stress and sequestration of unesterified cholesterol in lysosomes, thereby activating the sterol response element‐binding protein–mediated cholesterol synthesis pathways. Conclusions Together, our data suggest that impaired lipophagy, ER stress, and increased cholesterol synthesis lead to LD accumulation and hepatic steatosis. V‐ATPase assembly defects are thus a form of hereditary liver disease with implications for the pathogenesis of nonalcoholic fatty liver disease.