Impaired catabolism of free oligosaccharides due to MAN2C1 variants causes a neurodevelopmental disorder.

Free oligosaccharides (fOSs) are soluble oligosaccharide species generated during N-glycosylation of proteins. Although little is known about fOS metabolism, the recent identification of NGLY1 deficiency, a congenital disorder of deglycosylation (CDDG) caused by loss of function of an enzyme involved in fOS metabolism, has elicited increased interest in fOS processing. The catabolism of fOSs has been linked to the activity of a specific cytosolic mannosidase, MAN2C1, which cleaves α1,2-, α1,3-, and α1,6-mannose residues. In this study, we report the clinical, biochemical, and molecular features of six individuals, including two fetuses, with bi-allelic pathogenic variants in MAN2C1; the individuals are from four different families. These individuals exhibit dysmorphic facial features, congenital anomalies such as tongue hamartoma, variable degrees of intellectual disability, and brain anomalies including polymicrogyria, interhemispheric cysts, hypothalamic hamartoma, callosal anomalies, and hypoplasia of brainstem and cerebellar vermis. Complementation experiments with isogenic MAN2C1-KO HAP1 cells confirm the pathogenicity of three of the identified MAN2C1 variants. We further demonstrate that MAN2C1 variants lead to accumulation and delay in the processing of fOSs in proband-derived cells. These results emphasize the involvement of MAN2C1 in human neurodevelopmental disease and the importance of fOS catabolism.

Psychiatric assessment and therapy in an adolescent with ALG6-CDG: a six-month follow-up case report.

ALG6-congenital disorder of glycosylation (ALG6-CDG) is a complex of rare inherited disorders caused by mutations in the ALG6 gene, which encodes the α-1,3-glucosyltransferase enzyme required for N-glycosylation. ALG6-CDG affects multiple systems and exhibits clinical heterogeneity. Besides developmental delays and neurological signs and symptoms, behavioral and psychological symptoms are also an important group of clinical features of ALG6-CDG. Here, we present the case of a 17-year-old Chinese girl with ALG6-CDG who first visited the psychiatric department with apathy, language reduction, and substupor symptoms. The psychiatric assessments and treatment processes performed are described and discussed in this report. During diagnostic process, we found a novel mutation, c.849delT, in ALG6 by whole-exome sequencing. The patient's symptoms improved with escitalopram and risperidone treatment. However, above a certain dosage, she was sensitive to extrapyramidal side effects. This study accumulates clinical experience for diagnosing and treating ALG6-CDG and improves our understanding of this rare genetic disorder.

Congenital diaphragmatic hernia in siblings with PIGA-related congenital disorder of glycosylation.

There are over 150 proteins involved in glycosylphosphatidylinositol (GPI)-anchored protein biosynthesis, a class within the larger category of congenital disorders of glycosylation (CDG). Pathogenic variants identified in phosphatidylinositol glycan class A protein (PIGA) are associated with X-linked PIGA-CDG, a GPI-anchor defect. The disease has primarily been characterized by hypotonia, epilepsy, and global developmental delay; however, only 89 known cases are reported, so the phenotypic spectrum has likely not yet been fully delineated. Congenital diaphragmatic hernia (CDH) has been reported in patients with various GPI-anchor related defects but has only been described in one prior individual with PIGA-CDG. Here, we describe the second and third reported cases of CDH in two brothers with PIGA-CDG caused by a pathogenic missense variant in PIGA: c.355C > T, p.R119W. Chromosomal microarray and whole exome sequencing did not reveal another plausible explanation for the CDH. We relate our patients' clinical features to the single previously reported individual with CDH and PIGA-CDG. We then compare this case series with the subset of individuals with CDH and other GPI-anchor defects. These findings suggest that CDH should be considered in the phenotypic disease spectrum of PIGA-CDG.

N-glycoproteomics reveals distinct glycosylation alterations in NGLY1-deficient patient-derived dermal fibroblasts.

Congenital disorders of glycosylation are genetic disorders that occur due to defects in protein and lipid glycosylation pathways. A deficiency of N-glycanase 1, encoded by the NGLY1 gene, results in a congenital disorder of deglycosylation. The NGLY1 enzyme is mainly involved in cleaving N-glycans from misfolded, retro-translocated glycoproteins in the cytosol from the endoplasmic reticulum before their proteasomal degradation or activation. Despite the essential role of NGLY1 in deglycosylation pathways, the exact consequences of NGLY1 deficiency on global cellular protein glycosylation have not yet been investigated. We undertook a multiplexed tandem mass tags-labeling-based quantitative glycoproteomics and proteomics analysis of fibroblasts from NGLY1-deficient individuals carrying different biallelic pathogenic variants in NGLY1. This quantitative mass spectrometric analysis detected 8041 proteins and defined a proteomic signature of differential expression across affected individuals and controls. Proteins that showed significant differential expression included phospholipid phosphatase 3, stromal cell-derived factor 1, collagen alpha-1 (IV) chain, hyaluronan and proteoglycan link protein 1, and thrombospondin-1. We further detected a total of 3255 N-glycopeptides derived from 550 glycosylation sites of 407 glycoproteins by multiplexed N-glycoproteomics. Several extracellular matrix glycoproteins and adhesion molecules showed altered abundance of N-glycopeptides. Overall, we observed distinct alterations in specific glycoproteins, but our data revealed no global accumulation of glycopeptides in the patient-derived fibroblasts, despite the genetic defect in NGLY1. Our findings highlight new molecular and system-level insights for understanding NGLY1-CDDG.

Fractionated plasma N-glycan profiling of novel cohort of ATP6AP1-CDG subjects identifies phenotypic association.

ATP6AP1-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.

Oral sialic acid supplementation in NANS-CDG: Results of a single center, open-label, observational pilot study.

NANS-CDG is a congenital disorder of glycosylation (CDG) caused by biallelic variants in NANS, encoding an essential enzyme in de novo sialic acid synthesis. It presents with intellectual developmental disorder (IDD), skeletal dysplasia, neurologic impairment, and gastrointestinal dysfunction. Some patients suffer progressive intellectual neurologic deterioration (PIND), emphasizing the need for a therapy. In a previous study, sialic acid supplementation in knockout nansa zebrafish partially rescued skeletal abnormalities. Here, we performed the first in-human pre- and postnatal sialic-acid study in NANS-CDG. In this open-label observational study, 5 patients with NANS-CDG (range 0-28 years) were treated with oral sialic acid for 15 months. The primary outcome was safety. Secondary outcomes were psychomotor/cognitive testing, height and weight, seizure control, bone health, gastrointestinal symptoms, and biochemical and hematological parameters. Sialic acid was well tolerated. In postnatally treated patients, there was no significant improvement. For the prenatally treated patient, psychomotor and neurologic development was better than two other genotypically identical patients (one treated postnatally, one untreated). The effect of sialic acid treatment may depend on the timing, with prenatal treatment potentially benefiting neurodevelopmental outcomes. Evidence is limited, however, and longer-term follow-up in a larger number of prenatally treated patients is required.

An oligogenic case of severe neonatal thrombocytopenia and a purportedly benign variant in GFI1B requiring reinterpretation.

Although thrombocytopenia in neonatal intensive care patients is rarely due to inherited disorders, the number of genetic variants implicated in platelet defects has grown dramatically with increasing genome-wide sequencing. Here we describe a case of severe, oligogenic neonatal thrombocytopenia and reinterpret a reportedly benign mutation that is likely pathogenic. Despite this patient's synonymous mutation (GFI1B 576 C>T, Phe192=) being annotated as benign, GFI1B is a well-known regulator of megakaryopoiesis, this variant alters splicing and megakaryocyte maturation, and our analysis of existing genome-wide associated studies demonstrates that it likely causes gray platelet syndrome. This variant has not been reported in a case of life-threatening thrombocytopenia. We propose that the severity of this patient's phenotype is due to synergistic epistasis between the intrinsic platelet defect caused by this mutation and her concomitant inherited PMM2 congenital glycosylation disorder neither of which have been associated with such a severe phenotype. This case highlights the importance of whole-exome/genome sequencing for critically ill patients, reexamining variant interpretation when clinically indicated, and the need to study diverse genetic variation in hematopoiesis.

What is the context? Low platelets (thrombocytopenia) in the neonatal population is not frequently inherited. As we perform unbiased DNA sequencing in more patients, the number of inherited platelet disorders and implicated variants is growing.The gene GFI1B encodes for a transcription factor that regulates megakaryocytes, the cell type that produces platelets. A synonymous substitution in GFI1B (576 C>T, Phe192=) is annotated as benign; however, experimental studies have shown that it inhibits megakaryocyte production.There is growing appreciation for oligogenic inheritance, where multiple causal variants contribute to clinical phenotypes.What is new? We present a case of life-threatening neonatal macrothrombocytopenia (large, hypogranulated sparse platelets) that has an oligogenic cause. We reinterpret the synonymous substitution GFI1B 576 C>T as pathogenic.This patient's severe phenotype was likely due to the combined effect of GFI1B 576 C>T and her inherited glycosylation disorder (PMM2-CDG). Neither variant alone causes severe thrombocytopenia, but the combined intrinsic platelet defect (GFI1B mutation) and consumption (PMM2-CDG) likely produced her life-threatening phenotype.What is the impact? GFI1B is a critical regulator of megakaryocyte production. The purportedly benign mutation 576 C>T is likely pathogenic causing thrombocytopenia by impairing megakaryocyte maturation.As more patients have unbiased genome sequencing, oligogenic and polygenic inheritance will become increasingly appreciated as causes of platelet disorders.NICU providers should consider whole genome or exome sequencing of neonates with severe thrombocytopenia after reversible causes are ruled out.

In Vitro Skeletal Muscle Model of PGM1 Deficiency Reveals Altered Energy Homeostasis.

Phosphoglucomutase 1 (PGM1) is a key enzyme for the regulation of energy metabolism from glycogen and glycolysis, as it catalyzes the interconversion of glucose 1-phosphate and glucose 6-phosphate. PGM1 deficiency is an autosomal recessive disorder characterized by a highly heterogenous clinical spectrum, including hypoglycemia, cleft palate, liver dysfunction, growth delay, exercise intolerance, and dilated cardiomyopathy. Abnormal protein glycosylation has been observed in this disease. Oral supplementation with D-galactose efficiently restores protein glycosylation by replenishing the lacking pool of UDP-galactose, and rescues some symptoms, such as hypoglycemia, hepatopathy, and growth delay. However, D-galactose effects on skeletal muscle and heart symptoms remain unclear. In this study, we established an in vitro muscle model for PGM1 deficiency to investigate the role of PGM1 and the effect of D-galactose on nucleotide sugars and energy metabolism. Genome-editing of C2C12 myoblasts via CRISPR/Cas9 resulted in Pgm1 (mouse homologue of human PGM1, according to updated nomenclature) knockout clones, which showed impaired maturation to myotubes. No difference was found for steady-state levels of nucleotide sugars, while dynamic flux analysis based on 13C6-galactose suggested a block in the use of galactose for energy production in knockout myoblasts. Subsequent analyses revealed a lower basal respiration and mitochondrial ATP production capacity in the knockout myoblasts and myotubes, which were not restored by D-galactose. In conclusion, an in vitro mouse muscle cell model has been established to study the muscle-specific metabolic mechanisms in PGM1 deficiency, which suggested that galactose was unable to restore the reduced energy production capacity.

Mannose: a potential saccharide candidate in disease management.

There are a plethora of antibiotic resistance cases and humans are marching towards another big survival test of evolution along with drastic climate change and infectious diseases. Ever since the first antibiotic [penicillin], and the myriad of vaccines, we were privileged to escape many infectious disease threats. The survival technique of pathogens seems rapidly changing and sometimes mimicking our own systems in such a perfect manner that we are left unarmed against them. Apart from searching for natural alternatives, repurposing existing drugs more effectively is becoming a familiar approach to new therapeutic opportunities. The ingenious use of revolutionary artificial intelligence-enabled drug discovery techniques is coping with the speed of such alterations. D-Mannose is a great hope as a nutraceutical in drug discovery, against CDG, diabetes, obesity, lung disease, and autoimmune diseases and recent findings of anti-tumor activity make it interesting along with its role in drug delivery enhancing techniques. A very unique work done in the present investigation is the collection of data from the ChEMBL database and presenting the targetable proteins on pathogens as well as on humans. It shows Mannose has 50 targets and the majority of them are on human beings. The structure and conformation of certain monosaccharides have a decisive role in receptor pathogen interactions and here we attempt to review the multifaceted roles of Mannose sugar, its targets associated with different diseases, as a natural molecule having many success stories as a drug and future hope for disease management.

[Advances in the diagnosis and treatment of phosphomannomutase 2 deficiency]. / ç£·é ¸ç”˜éœ²ç³–å˜ä½é ¶2缺乏症的诊治进展.

Phosphomannomutase 2 deficiency is the most common form of N-glycosylation disorders and is also known as phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG). It is an autosomal recessive disease with multi-system involvements and is caused by mutations in the PMM2 gene (OMIM: 601785), with varying severities in individuals. At present, there is still no specific therapy for PMM2-CDG, and early identification, early diagnosis, and early treatment can effectively prolong the life span of pediatric patients. This article reviews the advances in the diagnosis and treatment of PMM2-CDG.

A Double-Blind, Placebo-Controlled, Crossover Study of Magnesium Supplementation in Patients with XMEN Disease.

X-linked MAGT1 deficiency with increased susceptibility to Epstein-Barr virus (EBV) infection and N-linked glycosylation defect (XMEN) disease is an inborn error of immunity caused by loss-of-function mutations in the magnesium transporter 1 (MAGT1) gene. The original studies of XMEN patients focused on impaired magnesium regulation, leading to decreased EBV-cytotoxicity and the loss of surface expression of the activating receptor "natural killer group 2D" (NKG2D) on CD8+ T cells and NK cells. In vitro studies showed that supraphysiological supplementation of magnesium rescued these defects. Observational studies in 2 patients suggested oral magnesium supplementation could decrease EBV viremia. Hence, we performed a randomized, double-blind, placebo-controlled, crossover study in 2 parts. In part 1, patients received either oral magnesium L-threonate (MLT) or placebo for 12 weeks followed by 12 weeks of the other treatment. Part 2 began with 3 days of high-dose intravenous (IV) magnesium sulfate (MgSO4) followed by open-label MLT for 24 weeks. One EBV-infected and 3 EBV-naïve patients completed part 1. One EBV-naïve patient was removed from part 2 of the study due to asymptomatic elevation of liver enzymes during IV MgSO4. No change in EBV or NKG2D status was observed. In vitro magnesium supplementation experiments in cells from 14 XMEN patients failed to significantly rescue NKG2D expression and the clinical trial was stopped. Although small, this study indicates magnesium supplementation is unlikely to be an effective therapeutic option in XMEN disease.

An interaction between SLC35A1 and ST3Gal4 is differentially affected by CDG-causing mutations in the SLC35A1 gene.

The sialylation of glycoconjugates is performed by a variety of sialyltransferases using CMP-sialic acid (CMP-Sia) as a substrate. Sialylation requires the translocation of CMP-Sia across the Golgi membranes. This function has been assigned to SLC35A1, the only CMP-Sia transporter identified to date. Mutations in the SLC35A1 gene cause a subtype of congenital disorder of glycosylation (CDG). Over the past several years, heterologous complexes formed in the Golgi membrane by some SLC35A subfamily members and functionally related glycosyltransferases have been reported. However, to date no interaction between SLC35A1 and a sialyltransferase has been identified. In this study we attempted to clarify the role of SLC35A1 in α2,3 sialylation of N-glycans. We showed that SLC35A1 associates with ST3Gal4, the main α2,3-sialyltransferase acting on N-glycans. This phenomenon is compromised by the E196K (but not T156R) mutation in the SLC35A1 gene. We also demonstrated that the E196K mutant is less efficient in restoring N-glycan sialylation upon expression in the SLC35A1 knockout cells. On the basis of our findings, we propose that the interaction between SLC35A1 and ST3Gal4 may be important for proper sialylation.

TRAPPC9-CDG: A novel congenital disorder of glycosylation with dysmorphic features and intellectual disability.

PURPOSE: TRAPPC9 deficiency is an autosomal recessive disorder mainly associated with intellectual disability (ID), microcephaly, and obesity. Previously, TRAPPC9 deficiency has not been associated with biochemical abnormalities. METHODS: Exome sequencing was performed in 3 individuals with ID and dysmorphic features. N-Glycosylation analyses were performed in the patients' blood samples to test for possible congenital disorder of glycosylation (CDG). TRAPPC9 gene, TRAPPC9 protein expression, and N-glycosylation markers were assessed in patient fibroblasts. Complementation with wild-type TRAPPC9 and immunofluorescence studies to assess TRAPPC9 expression and localization were performed. The metabolic consequences of TRAPPC9 deficiency were evaluated using tracer metabolomics. RESULTS: All 3 patients carried biallelic missense variants in TRAPPC9 and presented with an N-glycosylation defect in blood, consistent with CDG type I. Extensive investigations in patient fibroblasts corroborated TRAPPC9 deficiency and an N-glycosylation defect. Tracer metabolomics revealed global metabolic changes with several affected glycosylation-related metabolites. CONCLUSION: We identified 3 TRAPPC9 deficient patients presenting with ID, dysmorphic features, and abnormal glycosylation. On the basis of our findings, we propose that TRAPPC9 deficiency could lead to a CDG (TRAPPC9-CDG). The finding of abnormal glycosylation in these patients is highly relevant for diagnosis, further elucidation of the pathophysiology, and management of the disease.

A rare cause of infantile achalasia: GMPPA-congenital disorder of glycosylation with two novel compound heterozygous variants.

Achalasia is rare in the pediatric population and should prompt clinicians to consider genetic disorders associated with this condition. While AAA syndrome (also known as Allgrove or Triple A syndrome) is commonly considered, GMPPA-congenital disorder of glycosylation (CDG) should also be in the differential diagnosis. We report a 9-month-old female born to nonconsanguineous parents with achalasia and alacrima found to have two novel compound heterozygous variants in the GMPPA gene associated with GMPPA-CDG. This rare disorder is commonly associated with developmental delay and intellectual disability. We discuss management of this disorder including the importance of confirming a genetic diagnosis and summarize reported cases.

Defining the mild variant of leukocyte adhesion deficiency type II (SLC35C1-congenital disorder of glycosylation) and response to l-fucose therapy: Insights from two new families and review of the literature.

Leukocyte adhesion deficiency type II (LAD II, also known as SLC35C1-congenital disorder of glycosylation) is an autosomal recessive disorder characterized by growth and cognitive impairment, peripheral neutrophilia, recurrent infections, and the Bombay blood phenotype. A subset of patients with a milder presentation has been described with short stature and developmental delay but minimal immune and hematologic features. Some patients with LAD II benefit from oral fucose therapy, though this has not been previously studied in patients with milder disease. In this study, we describe two new patients from separate families with the milder variant of LAD II and review the published literature on this rare disorder. We demonstrate improvement in speech and cognition, CD15 expression, and core fucosylation of serum glycoproteins after 27 months of oral fucose supplementation in one patient. These patients further support the stratification of this disorder into distinct subtypes, a classical severe and an attenuated variant, and provide preliminary evidence of benefit of fucose therapy in the latter group.

PIGN encephalopathy: Characterizing the epileptology.

OBJECTIVE: Epilepsy is common in patients with PIGN diseases due to biallelic variants; however, limited epilepsy phenotyping data have been reported. We describe the epileptology of PIGN encephalopathy. METHODS: We recruited patients with epilepsy due to biallelic PIGN variants and obtained clinical data regarding age at seizure onset/offset and semiology, development, medical history, examination, electroencephalogram, neuroimaging, and treatment. Seizure and epilepsy types were classified. RESULTS: Twenty six patients (13 female) from 26 families were identified, with mean age 7 years (range = 1 month to 21 years; three deceased). Abnormal development at seizure onset was present in 25 of 26. Developmental outcome was most frequently profound (14/26) or severe (11/26). Patients presented with focal motor (12/26), unknown onset motor (5/26), focal impaired awareness (1/26), absence (2/26), myoclonic (2/26), myoclonic-atonic (1/26), and generalized tonic-clonic (2/26) seizures. Twenty of 26 were classified as developmental and epileptic encephalopathy (DEE): 55% (11/20) focal DEE, 30% (6/20) generalized DEE, and 15% (3/20) combined DEE. Six had intellectual disability and epilepsy (ID+E): two generalized and four focal epilepsy. Mean age at seizure onset was 13 months (birth to 10 years), with a lower mean onset in DEE (7 months) compared with ID+E (33 months). Patients with DEE had drug-resistant epilepsy, compared to 4/6 ID+E patients, who were seizure-free. Hyperkinetic movement disorder occurred in 13 of 26 patients. Twenty-seven of 34 variants were novel. Variants were truncating (n = 7), intronic and predicted to affect splicing (n = 7), and missense or inframe indels (n = 20, of which 11 were predicted to affect splicing). Seven variants were recurrent, including p.Leu311Trp in 10 unrelated patients, nine with generalized seizures, accounting for nine of the 11 patients in this cohort with generalized seizures. SIGNIFICANCE: PIGN encephalopathy is a complex autosomal recessive disorder associated with a wide spectrum of epilepsy phenotypes, typically with substantial profound to severe developmental impairment.

Insight on molecular pathogenesis and pharmacochaperoning potential in phosphomannomutase 2 deficiency, provided by novel human phosphomannomutase 2 structures.

Phosphomannomutase 2 (PMM2) deficiency, the most frequent congenital disorder of glycosylation (PMM2-CDG), is a severe condition, which has no cure. Due to the identification of destabilizing mutations, our group aims at increasing residual activity in PMM2-CDG patients, searching for pharmacochaperones. Detailed structural knowledge of hPMM2 might help identify variants amenable to pharmacochaperoning. hPMM2 structural information is limited to one incomplete structure deposited in the Protein Databank without associated publication, which lacked ligands and residues from a crucial loop. Here we report five complete crystal structures of hPMM2, three for wild-type and two for the p.Thr237Met variant frequently found among Spanish PMM2-CDG patients, free and bound to the essential activator glucose-1,6-bisphosphate (Glc-1,6-P2 ). In the hPMM2 homodimer, each subunit has a different conformation, reflecting movement of the distal core domain relative to the dimerization cap domain, supporting an opening/closing process during catalysis. Two Mg2+ ions bind to the core domain, one catalytic and one structural. In the cap domain, the site for Glc-1,6-P2 is well delineated, while a Cl- ion binding at the intersubunit interface is predicted to strengthen dimerization. Patient-found amino acid substitutions are nonhomogeneously distributed throughout hPMM2, reflecting differential functional or structural importance for various parts of the protein. We classify 93 of 101 patient-reported single amino acid variants according to five potential pathogenetic mechanism affecting folding of the core and cap domains, linker 2 flexibility, dimerization, activator binding, and catalysis. We propose that ~80% and ~50% of the respective core and cap domains substitutions are potential candidates for pharmacochaperoning treatment.

Leukocyte Phosphomannomutase and Phosphomannose Isomerase Activity in an Indian Cohort.

Advances in molecular sequencing technology has increased the diagnostic yield for Congenital disorder of glycosylation (CDG). However, novel variants or those of uncertain significance (vus) often pose a challenge and in such cases confirmed diagnosis can be warranted through enzyme analysis of these defects. We thus, aimed to optimize leukocyte-based enzyme assays for first two enzymes involved in N-glycosylation pathway i.e. Phosphomannomutase (PMM) and Phosphomannose isomerase (MPI). Study population comprised of 50 healthy non-alcoholic adults and 20 pediatric controls. Leukocyte enzyme activity was measured by monitoring the conversion of NADP to NADPH at 340 nm. The conditions were optimized and precision was assessed for both low and normal activity leukocyte controls. Enzyme activities for PMM and MPI in healthy individuals were measured in the range 1.6-3.9 and 7-20 nmol/min/mg protein respectively and did not vary with age and gender. The precision for both PMM and MPI showed %CV of 19.9 and 19.8 respectively. The enzyme activity in leukocyte pellet was found to be stable for up to 9 months when stored at -80 °C. The enzyme assays are optimized for PMM and MPI and can be used for evaluation of CDG patients in India.

Pathogenic Variants in Fucokinase Cause a Congenital Disorder of Glycosylation.

FUK encodes fucokinase, the only enzyme capable of converting L-fucose to fucose-1-phosphate, which will ultimately be used for synthesizing GDP-fucose, the donor substrate for all fucosyltransferases. Although it is essential for fucose salvage, this pathway is thought to make only a minor contribution to the total amount of GDP-fucose. A second pathway, the major de novo pathway, involves conversion of GDP-mannose to GDP-fucose. Here we describe two unrelated individuals who have pathogenic variants in FUK and who presented with severe developmental delays, encephalopathy, intractable seizures, and hypotonia. The first individual was compound heterozygous for c.667T>C (p.Ser223Pro) and c.2047C>T (p.Arg683Cys), and the second individual was homozygous for c.2980A>C (p.Lys994Gln). Skin fibroblasts from the first individual confirmed the variants as loss of function and showed significant decreases in total GDP-[3H] fucose and [3H] fucose-1-phosphate. There was also a decrease in the incorporation of [5,6-3H]-fucose into fucosylated glycoproteins. Lys994 has previously been shown to be an important site for ubiquitin conjugation. Here, we show that loss-of-function variants in FUK cause a congenital glycosylation disorder characterized by a defective fucose-salvage pathway.

International consensus guidelines for phosphoglucomutase 1 deficiency (PGM1-CDG): Diagnosis, follow-up, and management.

Phosphoglucomutase 1 (PGM1) deficiency is a rare genetic disorder that affects glycogen metabolism, glycolysis, and protein glycosylation. Previously known as GSD XIV, it was recently reclassified as a congenital disorder of glycosylation, PGM1-CDG. PGM1-CDG usually manifests as a multisystem disease. Most patients present as infants with cleft palate, liver function abnormalities and hypoglycemia, but some patients present in adulthood with isolated muscle involvement. Some patients develop life-threatening cardiomyopathy. Unlike most other CDG, PGM1-CDG has an effective treatment option, d-galactose, which has been shown to improve many of the patients' symptoms. Therefore, early diagnosis and initiation of treatment for PGM1-CDG patients are crucial decisions. In this article, our group of international experts suggests diagnostic, follow-up, and management guidelines for PGM1-CDG. These guidelines are based on the best available evidence-based data and experts' opinions aiming to provide a practical resource for health care providers to facilitate successful diagnosis and optimal management of PGM1-CDG patients.