A mutation in SLC37A4 causes a dominantly inherited congenital disorder of glycosylation characterized by liver dysfunction.

SLC37A4 encodes an endoplasmic reticulum (ER)-localized multitransmembrane protein required for transporting glucose-6-phosphate (Glc-6P) into the ER. Once transported into the ER, Glc-6P is subsequently hydrolyzed by tissue-specific phosphatases to glucose and inorganic phosphate during times of glucose depletion. Pathogenic variants in SLC37A4 cause an established recessive disorder known as glycogen storage disorder 1b characterized by liver and kidney dysfunction with neutropenia. We report seven individuals who presented with liver dysfunction multifactorial coagulation deficiency and cardiac issues and were heterozygous for the same variant, c.1267C>T (p.Arg423∗), in SLC37A4; the affected individuals were from four unrelated families. Serum samples from affected individuals showed profound accumulation of both high mannose and hybrid type N-glycans, while N-glycans in fibroblasts and undifferentiated iPSC were normal. Due to the liver-specific nature of this disorder, we generated a CRISPR base-edited hepatoma cell line harboring the c.1267C>T (p.Arg423∗) variant. These cells replicated the secreted abnormalities seen in serum N-glycosylation, and a portion of the mutant protein appears to relocate to a distinct, non-Golgi compartment, possibly ER exit sites. These cells also show a gene dosage-dependent alteration in the Golgi morphology and reduced intraluminal pH that may account for the altered glycosylation. In summary, we identify a recurrent mutation in SLC37A4 that causes a dominantly inherited congenital disorder of glycosylation characterized by coagulopathy and liver dysfunction with abnormal serum N-glycans.

Association between acute complications in PMM2-CDG patients and haemostasis anomalies: Data from a multicentric study and suggestions for acute management.

OBJECTIVES: Patients with PMM2-CDG develop acute events (stroke-like episodes (SLEs), thromboses, haemorrhages, seizures, migraines) associated with both clotting factors (factor XI) and coagulation inhibitors (antithrombin, protein C and protein S) deficiencies. The aim of the study was to correlate acute events to haemostasis and propose practical guidelines. METHODS: In this multicentric retrospective study, we evaluated clinical, radiological, haemostasis and electroencephalography data for PMM2-CDG patients hospitalized for acute events. Cerebral events were classified as thrombosis, haemorrhage, SLE, or "stroke mimic" (SM: normal brain imaging or evoking a migraine). RESULTS: Thirteen patients had a total of 31 acute episodes: 27 cerebral events with 7 SLEs, 4 venous thromboses, 4 haemorrhages (3 associated with thrombosis), 15 SMs at a mean age of 7.7 years; 4 non-cerebral thromboses, one of which included bleeding. A trigger was frequently involved (infection, head trauma). Although sometimes normal at baseline state, factor XI, antithrombin and protein C levels decreased during these episodes. No correlation between haemostasis anomalies and type of acute event was found. DISCUSSION: Acute events in PMM2-CDG are not negligible and are associated with haemostasis anomalies. An emergency protocol is proposed for their prevention and treatment (https://www.filiere-g2m.fr/urgences). For cerebral events, brain Magnetic Resonance Imaging with perfusion weight imaging and diffusion sequences, electroencephalogram and haemostasis protein levels guide the treatment: anticoagulation, antithrombin or fresh frozen plasma supplementation, antiepileptic therapy. Preventing bleeding and thrombosis is required in cases of surgery, prolonged immobilization, hormone replacement therapy. CONCLUSION: Acute events in PMM2-CDG are associated with abnormal haemostasis, requiring practical guidance.

SORD-related peripheral neuropathy in a French and Swiss cohort: Clinical features, genetic analyses, and sorbitol dosages.

BACKGROUND AND PURPOSE: Biallelic variants in SORD have been reported as one of the main recessive causes for hereditary peripheral neuropathies such as Charcot-Marie-Tooth disease type 2 (CMT2) and distal hereditary motor neuropathy (dHMN) resulting in lower limb (LL) weakness and muscular atrophy. In this study, phenotype and genotype landscapes of SORD-related peripheral neuropathies were described in a French and Swiss cohort. Serum sorbitol dosages were used to classify SORD variants. METHODS: Patients followed at neuromuscular reference centres in France and Switzerland were ascertained. Sanger sequencing and next generation sequencing were performed to sequence SORD, and mass spectrometry was used to measure patients' serum sorbitol. RESULTS: Thirty patients had SORD peripheral neuropathy associating LL weakness with muscular atrophy, foot deformities (87%), and sometimes proximal LL weakness (20%) or distal upper limb weakness (50%). Eighteen had dHMN, nine had CMT2, and three had intermediate CMT. Most of them had a mild or moderate disease severity. Sixteen carried a homozygous c.757delG (p.Ala253Glnfs*27) variant, and 11 carried compound heterozygous variants, among which four variants were not yet reported: c.403C > G, c.379G > A, c.68_100 + 1dup, and c.850dup. Two unrelated patients with different origins carried a homozygous c.458C > A variant, and one patient carried a new homozygous c.786 + 5G > A variant. Mean serum sorbitol levels were 17.01 mg/L ± 8.9 SD for patients carrying SORD variants. CONCLUSIONS: This SORD-inherited peripheral neuropathy cohort of 30 patients showed homogeneous clinical presentation and systematically elevated sorbitol levels (22-fold) compared to controls, with both diagnostic and potential therapeutic implications.

Biallelic variants in SLC35B2 cause a novel chondrodysplasia with hypomyelinating leukodystrophy.

Sulphated proteoglycans are essential in skeletal and brain development. Recently, pathogenic variants in genes encoding proteins involved in the proteoglycan biosynthesis have been identified in a range of chondrodysplasia associated with intellectual disability. Nevertheless, several patients remain with unidentified molecular basis. This study aimed to contribute to the deciphering of new molecular bases in patients with chondrodysplasia and neurodevelopmental disease. Exome sequencing was performed to identify pathogenic variants in patients presenting with chondrodysplasia and intellectual disability. The pathogenic effects of the potentially causative variants were analysed by functional studies. We identified homozygous variants (c.1218_1220del and c.1224_1225del) in SLC35B2 in two patients with pre- and postnatal growth retardation, scoliosis, severe motor and intellectual disabilities and hypomyelinating leukodystrophy. By functional analyses, we showed that the variants affect SLC35B2 mRNA expression and protein subcellular localization leading to a functional impairment of the protein. Consistent with those results, we detected proteoglycan sulphation impairment in SLC35B2 patient fibroblasts and serum. Our data support that SLC35B2 functional impairment causes a novel syndromic chondrodysplasia with hypomyelinating leukodystrophy, most likely through a proteoglycan sulphation defect. This is the first time that SLC35B2 variants are associated with bone and brain development in human.

Vitamin C improves microvascular reactivity and peripheral tissue perfusion in septic shock patients.

BACKGROUND: Vitamin C has potential protective effects through antioxidant and anti-inflammatory properties. However, the effect of vitamin C supplementation on microvascular function and peripheral tissue perfusion in human sepsis remains unknown. We aimed to determine vitamin C effect on microvascular endothelial dysfunction and peripheral tissue perfusion in septic shock patients. METHODS: Patients with septic shock were prospectively included after initial resuscitation. Bedside peripheral tissue perfusion and skin microvascular reactivity in response to acetylcholine iontophoresis in the forearm area were measured before and 1 h after intravenous vitamin C supplementation (40 mg/kg). Norepinephrine dose was not modified during the studied period. RESULTS: We included 30 patients with septic shock. SOFA score was 11 [8-14], SAPS II was 66 [54-79], and in-hospital mortality was 33%. Half of these patients had vitamin C deficiency at inclusion. Vitamin C supplementation strongly improved microvascular reactivity (AUC 2263 [430-4246] vs 5362 [1744-10585] UI, p = 0.0004). In addition, vitamin C supplementation improved mottling score (p = 0.06), finger-tip (p = 0.0003) and knee capillary refill time (3.7 [2.6-5.5] vs 2.9 [1.9-4.7] s, p < 0.0001), as well as and central-to-periphery temperature gradient (6.1 [4.9-7.4] vs 4.6 [3.4-7.0] °C, p < 0.0001). The beneficial effects of vitamin C were observed both in patients with or without vitamin C deficiency. CONCLUSION: In septic shock patients being resuscitated, vitamin C supplementation improved peripheral tissue perfusion and microvascular reactivity whatever plasma levels of vitamin C. ClinicalTrials.gov Identifier: NCT04778605 registered 26 January 2021.

Expanding the phenotype of X-linked SSR4-CDG: Connective tissue implications.

Signal sequence receptor protein 4 (SSR4) is a subunit of the translocon-associated protein complex, which participates in the translocation of proteins across the endoplasmic reticulum membrane, enhancing the efficiency of N-linked glycosylation. Pathogenic variants in SSR4 cause a congenital disorder of glycosylation: SSR4-congenital disorders of glycosylation (CDG). We describe three SSR4-CDG boys and review the previously reported. All subjects presented with hypotonia, failure to thrive, developmental delay, and dysmorphic traits and showed a type 1 serum sialotransferrin profile, facilitating the diagnosis. Genetic confirmation of this X-linked CDG revealed one de novo hemizygous deletion, one maternally inherited deletion, and one de novo nonsense mutation of SSR4. The present subjects highlight the similarities with a connective tissue disorder (redundant skin, joint laxity, blue sclerae, and vascular tortuosity). The connective tissue problems are relevant, and require preventive rehabilitation measures. As an X-linked disorder, genetic counseling is essential.

Serum bikunin isoforms in congenital disorders of glycosylation and linkeropathies.

Bikunin (Bkn) isoforms are serum chondroitin sulfate (CS) proteoglycans synthesized by the liver. They include two light forms, that is, the Bkn core protein and the Bkn linked to the CS chain (urinary trypsin inhibitor [UTI]), and two heavy forms, that is, pro-α-trypsin inhibitor and inter-α-trypsin inhibitor, corresponding to UTI esterified by one or two heavy chains glycoproteins, respectively. We previously showed that the Western-blot analysis of the light forms could allow the fast and easy detection of patients with linkeropathy, deficient in enzymes involved in the synthesis of the initial common tetrasaccharide linker of glycosaminoglycans. Here, we analyzed all serum Bkn isoforms in a context of congenital disorders of glycosylation (CDG) and showed very specific abnormal patterns suggesting potential interests for their screening and diagnosis. In particular, genetic deficiencies in V-ATPase (ATP6V0A2-CDG, CCDC115-CDG, ATP6AP1-CDG), in Golgi manganese homeostasis (TMEM165-CDG) and in the N-acetyl-glucosamine Golgi transport (SLC35A3-CDG) all share specific abnormal Bkn patterns. Furthermore, for each studied linkeropathy, we show that the light abnormal Bkn could be further in-depth characterized by two-dimensional electrophoresis. Moreover, besides being interesting as a specific biomarker of both CDG and linkeropathies, Bkn isoforms' analyses can provide new insights into the pathophysiology of the aforementioned diseases.

Long term outcome of MPI-CDG patients on D-mannose therapy.

Mannose phosphate isomerase MPI-CDG (formerly CDG-1b) is a potentially fatal inherited metabolic disease which is readily treatable with oral D-mannose. We retrospectively reviewed long-term outcomes of patients with MPI-CDG, all but one of whom were treated with D-mannose. Clinical, biological, and histological data were reviewed at diagnosis and on D-mannose treatment. Nine patients were diagnosed with MPI-CDG at a median age of 3 months. The presenting symptoms were diarrhea (n = 9), hepatomegaly (n = 9), hypoglycemia (n = 8), and protein loosing enteropathy (n = 7). All patients survived except the untreated one who died at 2 years of age. Oral D-mannose was started in eight patients at a median age of 7 months (mean 38 months), with a median follow-up on treatment of 14 years 9 months (1.5-20 years). On treatment, two patients developed severe portal hypertension, two developed venous thrombosis, and 1 displayed altered kidney function. Poor compliance with D-mannose was correlated with recurrence of diarrhea, thrombosis, and abnormal biological parameters including coagulation factors and transferrin profiles. Liver fibrosis persisted despite treatment, but two patients showed improved liver architecture during follow-up. This study highlights (i) the efficacy and safety of D-mannose treatment with a median follow-up on treatment of almost 15 years (ii) the need for life-long treatment (iii) the risk of relapse with poor compliance, (iii) the importance of portal hypertension screening (iv) the need to be aware of venous and renal complications in adulthood.

Consensus guideline for the diagnosis and management of mannose phosphate isomerase-congenital disorder of glycosylation.

Mannose phosphate isomerase-congenital disorder of glycosylation (MPI-CDG) deficiency is a rare subtype of congenital disorders of protein N-glycosylation. It is characterised by deficiency of MPI caused by pathogenic variants in MPI gene. The manifestation of MPI-CDG is different from other CDGs as the patients suffer dominantly from gastrointestinal and hepatic involvement whereas they usually do not present intellectual disability or neurological impairment. It is also one of the few treatable subtypes of CDGs with proven effect of oral mannose. This article covers a complex review of the literature and recommendations for the management of MPI-CDG with an emphasis on the clinical aspect of the disease. A team of international experts elaborated summaries and recommendations for diagnostics, differential diagnosis, management, and treatment of each system/organ involvement based on evidence-based data and experts' opinions. Those guidelines also reveal more questions about MPI-CDG which need to be further studied.

Long-term follow-up in PMM2-CDG: are we ready to start treatment trials?

PURPOSE: PMM2-CDG is the most common congenital disorder of glycosylation (CDG), which presents with either a neurologic or multisystem phenotype. Little is known about the longitudinal evolution. METHODS: We performed data analysis on PMM2-CDG patients' clinical features according to the Nijmegen CDG severity score and laboratory data. Seventy-five patients (28 males) were followed up from 11.0 ± 6.91 years for an average of 7.4 ± 4.5 years. RESULTS: On a group level, there was no significant evolution in overall clinical severity. There was some improvement in mobility and communication, liver and endocrine function, and strabismus and eye movements. Educational achievement and thyroid function worsened in some patients. Overall, the current clinical function, the system-specific involvement, and the current clinical assessment remained unchanged. On follow-up there was improvement of biochemical variables with (near) normalization of activated partial thromboplastin time (aPTT), factor XI, protein C, antithrombin, thyroid stimulating hormone, and liver transaminases. CONCLUSION: PMM2-CDG patients show a spontaneous biochemical improvement and stable clinical course based on the Nijmegen CDG severity score. This information is crucial for the definition of endpoints in clinical trials.

CCDC115-CDG: A new rare and misleading inherited cause of liver disease.

Congenital disorders of glycosylation (CDG) linked to defects in Golgi apparatus homeostasis constitute an increasing part of these rare inherited diseases. Among them, COG-CDG, ATP6V0A2-CDG, TMEM199-CDG and CCDC115-CDG have been shown to disturb Golgi vesicular trafficking and/or lumen pH acidification. Here, we report 3 new unrelated cases of CCDC115-CDG with emphasis on diagnosis difficulties related to strong phenotypic similarities with mitochondriopathies, Niemann-Pick disease C and Wilson Disease. Indeed, while two individuals clinically presented with early and severe liver fibrosis and cirrhosis associated with neurological symptoms, the other one "only" showed isolated and late severe liver involvement. Biological results were similar to previously described patients, including hypercholesterolemia, elevated alkaline phosphatases and defects in copper metabolism. CDG screening and glycosylation study finally led to the molecular diagnosis of CCDC115-CDG. Besides pointing to the importance of CDG screening in patients with unexplained and severe liver disease, these reports expand the clinical and molecular phenotypes of CCDC115-CDG. The hepatic involvement is particularly addressed. Furthermore, hypothesis concerning the pathogenesis of the liver disease and of major biological abnormalities are proposed.

Complementarity of electrophoretic, mass spectrometric, and gene sequencing techniques for the diagnosis and characterization of congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are rare autosomal genetic diseases affecting the glycosylation of proteins and lipids. Since CDG-related clinical symptoms are classically extremely variable and nonspecific, a combination of electrophoretic, mass spectrometric, and gene sequencing techniques is often mandatory for obtaining a definitive CDG diagnosis, as well as identifying causative gene mutations and deciphering the underlying biochemical mechanisms. Here, we illustrate the potential of integrating data from capillary electrophoresis of transferrin, two-dimensional electrophoresis of N- and O-glycoproteins, mass spectrometry analyses of total serum N-linked glycans and mucin core1 O-glycosylated apolipoprotein C-III for the determination of various culprit CDG gene mutations. "Step-by-step" diagnosis pathways of four particular and new CDG cases, including MGAT2-CDG, ATP6V0A2-CDG, SLC35A2-CDG, and SLC35A3-CDG, are described as illustrative examples.

Clinical, laboratory and molecular findings and long-term follow-up data in 96 French patients with PMM2-CDG (phosphomannomutase 2-congenital disorder of glycosylation) and review of the literature.

BACKGROUND: Phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is a multisystem inborn error of metabolism. OBJECTIVES: To better characterise the natural history of PMM2-CDG. METHODS: Medical charts of 96 patients with PMM2-CDG (86 families, 41 males, 55 females) were retrospectively reviewed. Data on clinical, laboratory and molecular parameters at diagnosis were analysed. Follow-up data at last examination were reported for 25 patients. RESULTS: The patients were born between 1963 and 2011. Diagnosis of PMM2-CDG was made at a mean (SD) age of 6.8 (8.5) years. The presenting signs were mostly neurological (hypotonia, intellectual disability, cerebellar syndrome) and observed in almost all the patients. A total of 38 patients (14 males, 24 females) exhibited, in addition to neurological signs, visceral features including at least one of these: feeding difficulty requiring a nutritional support (n=23), cardiac features (n=20; pericarditis: 14, cardiac malformation: 9, cardiomyopathy: 2), hepato-gastrointestinal features (n=12; chronic diarrhoea: 7, protein-losing enteropathy: 1, ascites: 3, liver failure: 1, portal hypertension: 1), kidney features (n=4; nephrotic syndrome: 2, tubulopathy: 2) and hydrops fetalis (n=1). Twelve patients died at a mean age of 3.8 years (especially from pericarditis and other cardiac issues). Laboratory abnormalities mostly included elevated transaminases and abnormal coagulation parameters. High thyreostimulin levels, hypocholesterolemia, hypoalbuminemia and elevated transaminases were associated with the visceral phenotype. Besides the common Arg141His PMM2 variant harboured by half of the patients, 45 different variants were observed. CONCLUSIONS: PMM2-CDG clinical phenotype is heterogeneous in terms of clinical course, with no clear division between neurological and visceral presentations.

A Fully Automated Web-Based Program Improves Lifestyle Habits and HbA1c in Patients With Type 2 Diabetes and Abdominal Obesity: Randomized Trial of Patient E-Coaching Nutritional Support (The ANODE Study).

BACKGROUND: The prevalence of abdominal obesity and type 2 diabetes mellitus (T2DM) is a public health challenge. New solutions need to be developed to help patients implement lifestyle changes. OBJECTIVE: The objective of the study was to evaluate a fully automated Web-based intervention designed to help users improve their dietary habits and increase their physical activity. METHODS: The Accompagnement Nutritionnel de l'Obésité et du Diabète par E-coaching (ANODE) study was a 16-week, 1:1 parallel-arm, open-label randomized clinical trial. Patients with T2DM and abdominal obesity (n=120, aged 18-75 years) were recruited. Patients in the intervention arm (n=60) had access to a fully automated program (ANODE) to improve their lifestyle. Patients were asked to log on at least once per week. Human contact was limited to hotline support in cases of technical issues. The dietetic tool provided personalized menus and a shopping list for the day or the week. Stepwise physical activity was prescribed. The control arm (n=60) received general nutritional advice. The primary outcome was the change of the dietary score (International Diet Quality Index; DQI-I) between baseline and the end of the study. Secondary endpoints included changes in body weight, waist circumference, hemoglobin A1c (HbA1c) and measured maximum oxygen consumption (VO2 max). RESULTS: The mean age of the participants was 57 years (standard deviation [SD] 9), mean body mass index was 33 kg/m² (SD 4), mean HbA1c was 7.2% (SD 1.1), and 66.7% (80/120) of participants were women. Using an intention-to-treat analysis, the DQI-I score (54.0, SD 5.7 in the ANODE arm; 52.8, SD 6.2 in the control arm; P=.28) increased significantly in the ANODE arm compared to the control arm (+4.55, SD 5.91 vs -1.68, SD 5.18; between arms P<.001). Body weight, waist circumference, and HbA1c changes improved significantly in the intervention. CONCLUSIONS: Among patients with T2DM and abdominal obesity, the use of a fully automated Web-based program resulted in a significant improvement in dietary habits and favorable clinical and laboratory changes. The sustainability of these effects remains to be determined. TRIAL REGISTRATION: ClinicalTrials.gov NCT02343107; http://clinicaltrials.gov/ct2/show/NCT02343107 (Archived by WebCite at http://www.webcitation.org/6uVMKPRzs).

Reactive oxygen species, AMP-activated protein kinase, and the transcription cofactor p300 regulate α-tubulin acetyltransferase-1 (αTAT-1/MEC-17)-dependent microtubule hyperacetylation during cell stress.

Beyond its presence in stable microtubules, tubulin acetylation can be boosted after UV exposure or after nutrient deprivation, but the mechanisms of microtubule hyperacetylation are still unknown. In this study, we show that this hyperacetylation is a common response to several cellular stresses that involves the stimulation of the major tubulin acetyltransferase MEC-17. We also demonstrate that the acetyltransferase p300 negatively regulates MEC-17 expression and is sequestered on microtubules upon stress. We further show that reactive oxygen species of mitochondrial origin are required for microtubule hyperacetylation by activating the AMP kinase, which in turn mediates MEC-17 phosphorylation upon stress. Finally, we show that preventing microtubule hyperacetylation by knocking down MEC-17 affects cell survival under stress conditions and starvation-induced autophagy, thereby pointing out the importance of this rapid modification as a broad cell response to stress.

Red blood cell Thomsen-Friedenreich antigen expression and galectin-3 plasma concentrations in Streptococcus pneumoniae-associated hemolytic uremic syndrome and hemolytic anemia.

BACKGROUND: Pneumococcal hemolytic uremic syndrome (P-HUS) is a rare but severe complication of invasive pneumococcal disease (IPD) in young children. Consensual biologic diagnosis criteria are currently lacking. STUDY DESIGN AND METHODS: A prospective study was conducted on 10 children with culture-confirmed IPD. Five presented with full-blown P-HUS, three had an incomplete form with hemolytic anemia and mild or no uremia (P-HA), and two had neither HUS nor HA. Thomsen-Friedenreich (T), Th, and Tk cryptantigens and sialic acid expression were determined on red blood cells (RBCs) with peanut (PNA), Glycine soja (SBA), Bandeiraea simplicifolia II, and Maackia amurensis lectins. Plasma concentrations of the major endogenous T-antigen-binding protein, galectin-3 (Gal-3), were analyzed. RESULTS: We found that RBCs strongly reacted with PNA and SBA lectins in all P-HUS and P-HA patients. Three P-HUS and three P-HA patients showed also concomitant Tk activation. Direct antiglobulin test (DAT) was positive in three P-HUS (one with anti-C3d and two with anti-IgG) and two P-HA patients (one with anti-C3d and one with anti-IgG). RBCs derived from the two uncomplicated IPD patients reacted with PNA but not with SBA lectin. Gal-3 plasma concentrations were increased in all P-HUS patients. CONCLUSIONS: The results indicate high levels of neuraminidase activity and desialylation in both P-HUS and P-HA patients. T-antigen activation is more sensitive than DAT for P-HUS diagnosis. Combining PNA and SBA lectins is needed to improve the specificity of T-antigen activation. High concentrations of Gal-3 in P-HUS patients suggest that Gal-3 may contribute to the pathogenesis of P-HUS.

Biochemical diagnosis of congenital disorders of glycosylation.

Congenital disorders of glycosylation (CDG) are one of the fastest growing groups of inborn errors of metabolism, comprising over 160 described diseases to this day. CDG are characterized by a dysfunctional glycosylation process, with molecular defects localized in the cytosol, the endoplasmic reticulum, or the Golgi apparatus. Depending on the CDG, N-glycosylation, O-glycosylation and/or glycosaminoglycan synthesis can be affected. Various proteins, lipids, and glycosylphosphatidylinositol anchors bear glycan chains, with potential impacts on their folding, targeting, secretion, stability, and thus, functionality. Therefore, glycosylation defects can have diverse and serious clinical consequences. CDG patients often present with a non-specific, multisystemic syndrome including neurological involvement, growth delay, hepatopathy and coagulopathy. As CDG are rare diseases, and typically lack distinctive clinical signs, biochemical and genetic testing bear particularly important and complementary diagnostic roles. Here, after a brief introduction on glycosylation and CDG, we review historical and recent findings on CDG biomarkers and associated analytical techniques, with a particular emphasis on those with relevant use in the specialized clinical chemistry laboratory. We provide the reader with insights and methods which may help them properly assist the clinician in navigating the maze of glycosylation disorders.

Droplet Microfluidics with Capillary Electrophoresis for Glycan Biomarker Analysis.

Several glycoproteins are validated biomarkers of various diseases such as cancer, cardiovascular diseases, chronic alcohol abuse, or congenital disorders of glycosylation (CDG). In particular, CDG represent a group of more than 150 inherited diseases with varied symptoms affecting multiple organs. The distribution of glycans from target glycoprotein(s) can be used to extract information to help the diagnosis and possibly differentiate subtypes of CDG. Indeed, depending on the glycans and the proteins to which they are attached, glycans can play a very broad range of roles in both physical and biological properties of glycoproteins. For glycans in general, capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) has become a staple. Analysis of glycans with CE-LIF requires several sample preparation steps, including release of glycans from the target glycoprotein, fluorescent labeling of glycans, and purification of labeled glycans. Here, we describe the protocol for glycan sample treatment in a microfluidic droplet system prior to CE-LIF of labeled glycans. The microfluidic droplet approach offers full automation, sample, and reagent volume reduction and elimination of contamination from external environment.

"Hide and seek": Misleading transferrin variants in PMM2-CDG complicate diagnostics.

PURPOSE: Congenital disorders of glycosylation (CDG) are one of the fastest growing groups of inborn errors of metabolism. Despite the availability of next-generation sequencing techniques and advanced methods for evaluation of glycosylation, CDG screening mainly relies on the analysis of serum transferrin (Tf) by isoelectric focusing, HPLC or capillary electrophoresis. The main pitfall of this screening method is the presence of Tf protein variants within the general population. Although reports describe the role of Tf variants leading to falsely abnormal results, their significance in confounding diagnosis in patients with CDG has not been documented so far. Here, we describe two PMM2-CDG cases, in which Tf variants complicated the diagnostic. EXPERIMENTAL DESIGN: Glycosylation investigations included classical screening techniques (capillary electrophoresis, isoelectric focusing and HPLC of Tf) and various confirmation techniques (two-dimensional electrophoresis, western blot, N-glycome, UPLC-FLR/QTOF MS with Rapifluor). Tf variants were highlighted following neuraminidase treatment. Sequencing of PMM2 was performed. RESULTS: In both patients, Tf screening pointed to CDG-II, while second-line analyses pointed to CDG-I. Tf variants were found in both patients, explaining these discrepancies. PMM2 causative variants were identified in both patients. CONCLUSION AND CLINICAL RELEVANCE: We suggest that a neuraminidase treatment should be performed when a typical CDG Tf pattern is found upon initial screening analysis.

Efficacy of oral manganese and D-galactose therapy in a patient bearing a novel TMEM165 variant.

TMEM165-CDG has first been reported in 2012 and manganese supplementation was shown highly efficient in rescuing glycosylation in isogenic KO cells. The unreported homozygous missense c.928G>C; p.Ala310Pro variant leading to a functional but unstable protein was identified. This patient was diagnosed at 2 months and displays a predominant bone phenotype and combined defects in N-, O- and GAG glycosylation. We administered for the first time a combined D-Gal and Mn2+ therapy to the patient. This fully suppressed the N-; O- and GAG hypoglycosylation. There was also striking improvement in biochemical parameters and in gastrointestinal symptoms. This study offers exciting therapeutic perspectives for TMEM165-CDG.