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Transferrin isoform analysis is an established laboratory test for congenital disorders of glycosylation (CDG). Despite its long history of clinical use, little has been published about its empirical sensitivity for specific conditions. We conducted a retrospective analysis of ten years of testing data and report our experience with transferrin testing for type I profiles and its sensitivity for the most common congenital disorder of glycosylation, PMM2-CDG. The data demonstrate 94% overall test sensitivity for PMM2-CDG and importantly demonstrate two known, recurrent variants enriched in false positive cases highlighting an important limitation of the test. The data confirm the clinical validity of transferrin isotype analysis as a screening test for disorders of protein N-linked glycosylation and as functional test for PMM2 genotypes of uncertain significance., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Inc.)

Phosphomannomutase 2 deficiency (PMM2-CDG), the most frequent congenital disorder of glycosylation, is an autosomal recessive disease caused by biallelic pathogenic variants in the PMM2 gene. There is no cure for this multisystemic syndrome. Some of the therapeutic approaches that are currently in development include mannose-1-phosphate replacement therapy, drug repurposing, and the use of small chemical molecules to correct folding defects. Preclinical models are needed to evaluate the efficacy of treatments to overcome the high lethality of the available animal model. In addition, the number of variants with unknown significance is increasing in clinical settings. This study presents the generation of a cellular disease model by knocking out the PMM2 gene in the hepatoma HepG2 cell line using CRISPR-Cas9 gene editing. The HepG2 knockout model accurately replicates the PMM2-CDG phenotype, exhibiting a complete absence of PMM2 protein and mRNA, a 90% decrease in PMM enzymatic activity, and altered ICAM-1, LAMP1 and A1AT glycoprotein patterns. The evaluation of PMM2 disease-causing variants validates the model's utility for studying new PMM2 clinical variants, providing insights for diagnosis and potentially for evaluating therapies. A CRISPR-Cas9-generated HepG2 knockout model accurately recapitulates the PMM2-CDG phenotype, providing a valuable tool for assessing disease-causing variants and advancing therapeutic strategies., Competing Interests: Declaration of competing interest The authors declare that they do not have any known personal or financial interests that could have affected the work that is described in this manuscript., (Copyright © 2024 Universidad Autónoma de Madrid. Published by Elsevier Inc. All rights reserved.)

PMM2-CDG is the most common congenital disorder of glycosylation (CDG). Patients with this disease often carry compound heterozygous mutations of the gene encoding the phosphomannomutase 2 (PMM2) enzyme. PMM2 converts mannose-6-phosphate (M6P) to mannose-1-phosphate (M1P), which is a critical upstream metabolite for proper protein N-glycosylation. Therapeutic options for PMM2-CDG patients are limited to management of the disease symptoms, as no drug is currently approved to treat this disease. GLM101 is a M1P-loaded liposomal formulation being developed as a candidate drug to treat PMM2-CDG. This report describes the effect of GLM101 treatment on protein N-glycosylation of PMM2-CDG patient-derived fibroblasts. This treatment normalized intracellular GDP-mannose, increased the relative glycoprotein mannosylation content and TNFα-induced ICAM-1 expression. Moreover, glycomics profiling revealed that GLM101 treatment of PMM2-CDG fibroblasts resulted in normalization of most high mannose glycans and partial correction of multiple complex and hybrid glycans. In vivo characterization of GLM101 revealed its favorable pharmacokinetics, liver-targeted biodistribution, and tolerability profile with achieved systemic concentrations significantly greater than its effective in vitro potency. Taken as a whole, the results described in this report support further exploration of GLM101's safety, tolerability, and efficacy in PMM2-CDG patients., Competing Interests: Declaration of competing interest None., (Copyright © 2024. Published by Elsevier Inc.)

PMM2-CDG, a disease caused by mutations in phosphomannomutase-2, is the most common congenital disorder of glycosylation. Yet, it still lacks a cure. Targeting phosphomannomutase-2 with pharmacological chaperones or inhibiting the phosphatase activity of phosphomannomutase-1 to enhance intracellular glucose-1,6-bisphosphate have been proposed as therapeutical approaches. We used Recombinant Bacterial Thermal Shift Assay to assess the binding of a substrate analog to phosphomannomutase-2 and the specific binding to phosphomannomutase-1 of an FDA-approved drug - clodronate. We also deepened the clodronate binding by enzyme activity assays and in silico docking. Our results confirmed the selective binding of clodronate to phosphomannomutase-1 and shed light on such binding., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Congenital disorder of glycosylation (CDG) is inherited metabolicdiseasecaused by defects in the genes important for the process of protein and lipidglycosylation. We established an induced pluripotent stem cell (iPSC) line from peripheral blood mononuclear cells of a 6-month-old boy with congenital disorder of glycosylation carrying heterozygous mutations c.1193 T > C (p.I398T) and c.376_384dup CCGCAGCAC (p.P126_H128 dupPQH) in MPI gene. This iPSC line was free of exogenous gene, expressed pluripotency markers, has normal karyotype, exhibited differentiation potential and harbored the same mutations found in the patient. This cell line will provide a reliable cell model for further studies on the potential therapeutic targets of CDG., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

PMM2-CDG (MIM # 212065), the most common congenital disorder of glycosylation, is caused by the deficiency of phosphomannomutase 2 (PMM2). It is a multisystemic disease of variable severity that particularly affects the nervous system; however, its molecular pathophysiology remains poorly understood. Currently, there is no effective treatment. We performed an RNA-seq based transcriptomic study using patient-derived fibroblasts to gain insight into the mechanisms underlying the clinical symptomatology and to identify druggable targets. Systems biology methods were used to identify cellular pathways potentially affected by PMM2 deficiency, including Senescence, Bone regulation, Cell adhesion and Extracellular Matrix (ECM) and Response to cytokines. Functional validation assays using patients' fibroblasts revealed defects related to cell proliferation, cell cycle, the composition of the ECM and cell migration, and showed a potential role of the inflammatory response in the pathophysiology of the disease. Furthermore, treatment with a previously described pharmacological chaperone reverted the differential expression of some of the dysregulated genes. The results presented from transcriptomic data might serve as a platform for identifying therapeutic targets for PMM2-CDG, as well as for monitoring the effectiveness of therapeutic strategies, including pharmacological candidates and mannose-1-P, drug repurposing., Competing Interests: Declaration of competing interest The authors declare no conflict of interest., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Phosphomannomutase 2 (PMM2) converts mannose-6-phospahate to mannose-1-phosphate; the substrate for GDP-mannose, a building block of the glycosylation biosynthetic pathway. Pathogenic variants in the PMM2 gene have been shown to be associated with protein hypoglycosylation causing PMM2-congenital disorder of glycosylation (PMM2-CDG). While mannose supplementation improves glycosylation in vitro, but not in vivo, we hypothesized that liposomal delivery of mannose-1-phosphate could increase the stability and delivery of the activated sugar to enter the targeted compartments of cells. Thus, we studied the effect of liposome-encapsulated mannose-1-P (GLM101) on global protein glycosylation and on the cellular proteome in skin fibroblasts from individuals with PMM2-CDG, as well as in individuals with two N-glycosylation defects early in the pathway, namely ALG2-CDG and ALG11-CDG. We leveraged multiplexed proteomics and N-glycoproteomics in fibroblasts derived from different individuals with various pathogenic variants in PMM2, ALG2 and ALG11 genes. Proteomics data revealed a moderate but significant change in the abundance of some of the proteins in all CDG fibroblasts upon GLM101 treatment. On the other hand, N-glycoproteomics revealed the GLM101 treatment enhanced the expression levels of several high-mannose and complex/hybrid glycopeptides from numerous cellular proteins in individuals with defects in PMM2 and ALG2 genes. Both PMM2-CDG and ALG2-CDG exhibited several-fold increase in glycopeptides bearing Man 6 and higher glycans and a decrease in Man 5 and smaller glycan moieties, suggesting that GLM101 helps in the formation of mature glycoforms. These changes in protein glycosylation were observed in all individuals irrespective of their genetic variants. ALG11-CDG fibroblasts also showed increase in high mannose glycopeptides upon treatment; however, the improvement was not as dramatic as the other two CDG. Overall, our findings suggest that treatment with GLM101 overcomes the genetic block in the glycosylation pathway and can be used as a potential therapy for CDG with enzymatic defects in early steps in protein N-glycosylation., Competing Interests: Declaration of competing interest E.M. and M.H.A.I. are principal investigators in a clinical trial sponsored by Glycomine. Other authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Background: Congenital disorders of glycosylation (CDG) are genetic diseases caused by impaired synthesis of glycan moieties linked to glycoconjugates. Phosphomannomutase 2 deficiency (PMM2-CDG), the most frequent CDG, is characterized by prominent neurological involvement. Gait disturbance is a major cause of functional disability in patients with PMM2-CDG. However, no specific gait assessment for PMM2-CDG is available. This study analyses gait-related parameters in PMM2-CDG patients using a standardized clinical assessment and instrumented gait analysis (IGA)., Results: Seven adult patients with a molecular diagnosis of PMM2-CDG were followed-up from February 2021 to December 2022 and compared to a group of healthy control (HC) subjects, matched for age and sex. Standardized assessment of disease severity including ataxia and peripheral neuropathy along with isometric muscle strength and echo-biometry measurements at lower limbs were performed. IGA spatiotemporal parameters were obtained by means of a wearable sensor in basal conditions. PMM2-CDG patients displayed lower gait speed, stride length, cadence and symmetry index, compared to HC. Significant correlations were found among the used clinical scales and between disease severity (NCRS) scores and the gait speed measured by IGA. Variable reduction of knee extension strength and a significant decrease of lower limb muscle thickness with conserved echo intensity were found in PMM2-CDG compared to HC., Conclusions: The study elucidates different components of gait disturbance in PMM2-CDG patients and shows advantages of using wearable sensor-based IGA in this frame. IGA parameters may potentially serve as quantitative measures for follow-up or outcome quantification in PMM2-CDG., (© 2024. The Author(s).)

PMM2-CDG is the most prevalent type of congenital disorders of glycosylation (CDG). It is caused by pathogenic variants in the gene encoding phosphomannomutase 2 (PMM2), which converts mannose-6-phosphate to mannose-1-phosphate and thus activates this saccharide for further glycosylation processes. Defective glycosylation can lead to an abnormal accumulation of unfolded proteins in endoplasmic reticulum (ER) and cause its stress. The ER is a key compartment for glycosylation, and its connection and communication with mitochondria has been described extensively in literature. Their crosstalk is important for cell proliferation, calcium homeostasis, apoptosis, mitochondrial fission regulation, bioenergetics, autophagy, lipid metabolism, inflammasome formation and unfolded protein response. Therefore, in the present study we posed a question, whether defective glycosylation leads to bioenergetic disruption. Our data reveal possible chronic stress in ER and activated unfolded protein response via PERK pathway in PMM2-CDG fibroblasts. Presumably, it leads to bioenergetic reorganization and increased assembly of respiratory chain complexes into supercomplexes together with suppressed glycolysis in PMM2-CDG patient cells. These changes cause alterations in Krebs cycle, which is tightly connected to electron transport system in mitochondria. In summary, we present data showing metabolic adaptation of cells to glycosylation defect caused by various pathogenic variants in PMM2., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 The Authors. Published by Elsevier Inc. All rights reserved.)

PMM2-CDG is the most common defect among the congenital disorders of glycosylation. In order to investigate the effect of hypoglycosylation on important cellular pathways, we performed extensive biochemical studies on skin fibroblasts of PMM2-CDG patients. Among others, acylcarnitines, amino acids, lysosomal proteins, organic acids and lipids were measured, which all revealed significant abnormalities. There was an increased expression of acylcarnitines and amino acids associated with increased amounts of calnexin, calreticulin and protein-disulfid-isomerase in combination with intensified amounts of ubiquitinylated proteins. Lysosomal enzyme activities were widely decreased as well as citrate and pyruvate levels indicating mitochondrial dysfunction. Main lipid classes such as phosphatidylethanolamine, cholesterol or alkyl-phosphatidylcholine, as well as minor lipid species like hexosylceramide, lysophosphatidylcholines or phosphatidylglycerol, were abnormal. Biotinidase and catalase activities were severely reduced. In this study we discuss the impact of metabolite abnormalities on the phenotype of PMM2-CDG. In addition, based on our data we propose new and easy-to-implement therapeutic approaches for PMM2-CDG patients., Competing Interests: Declaration of Competing Interest The authors declare no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Background: Given the lack of reliable data on the prevalence of bleeding abnormalities and thrombotic episodes in PMM2-CDG patients, and whether coagulation abnormalities change over time, we prospectively collected and reviewed natural history data. Patients with PMM2-CDG often have abnormal coagulation studies due to glycosylation abnormalities but the frequency of complications resulting from these has not been prospectively studied., Methods: We studied fifty individuals enrolled in the Frontiers in Congenital Disorders of Glycosylation Consortium (FCDGC) natural history study with molecularly confirmed diagnosis of PMM2-CDG. We collected data on prothrombin time (PT), international normalized ratio (INR), activated partial thromboplastin time (aPTT), platelets, factor IX activity (FIX), factor XI activity (FXI), protein C activity (PC), protein S activity (PS) and antithrombin activity (AT)., Results: Prothrombotic and antithrombotic factor activities were frequently abnormal in PMM2-CDG patients, including AT, PC, PT, INR, and FXI. AT deficiency was the most common abnormality in 83.3% of patients. AT activity was below 50% in 62.5% of all patients (normal range 80-130%). Interestingly, 16% of the cohort experienced symptoms of spontaneous bleeding and 10% had thrombosis. Stroke-like episodes (SLE) were reported in 18% of patients in our cohort. Based on the linear growth models, on average, patients did not show significant change in AT (n = 48; t(23.8) = 1.75, p = 0.09), FIX (n = 36; t(61) = 1.60, p = 0.12), FXI (n = 39; t(22.8) = 1.88, p = 0.07), PS (n = 25; t(28.8) = 1.08, p = 0.29), PC (n = 38; t(68) = 1.61, p = 0.11), INR (n = 44; t(184) = -1.06, p = 0.29), or PT (n = 43; t(192) = -0.69, p = 0.49) over time. AT activity positively correlated with FIX activity. PS activity was significantly lower in males., Conclusion: Based on our natural history data and previous literature, we conclude that caution should be exercised when the AT levels are lower than 65%, as most thrombotic events occur in patients with AT below this level. All five, male PMM2-CDG patients in our cohort who developed thrombosis had abnormal AT levels, ranging between 19% and 63%. Thrombosis was associated with infection in all cases. We did not find significant change in AT levels over time. Several PMM2-CDG patients had an increased bleeding tendency. More long-term follow-up is necessary on coagulation abnormalities and the associated clinical symptoms to provide guidelines for therapy, patient management, and appropriate counseling., Synopsis: Most PMM2-CDG patients display chronic coagulation abnormalities without significant improvement, associated with a frequency of 16% clinical bleeding abnormalities, and 10% thrombotic episodes in patients with severe antithrombin deficiency., Competing Interests: Declaration of Competing Interest Diederik De Graef, Anna N. Ligezka, Joseph Rezents, Gina L. Mazza, Graeme Preston, Kaitlin Schwartz, Wirginia Krzysciak, Christina Lam, Andrew C. Edmondson, Christin Johnsen, Tamas Kozicz, and Eva Morava report no conflict of interest., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Phosphomannomutase-2-congenital disorder of glycosylation (PMM2-CDG) is the most common CDG and presents with highly variable features ranging from isolated neurologic involvement to severe multi-organ dysfunction. Liver abnormalities occur in in almost all patients and frequently include hepatomegaly and elevated aminotransferases, although only a minority of patients develop progressive hepatic fibrosis and liver failure. No curative therapies are currently available for PMM2-CDG, although investigation into several novel therapies is ongoing. We report the first successful liver transplantation in a 4-year-old patient with PMM2-CDG. Over a 3-year follow-up period, she demonstrated improved growth and neurocognitive development and complete normalization of liver enzymes, coagulation parameters, and carbohydrate-deficient transferrin profile, but persistently abnormal IgG glycosylation and recurrent upper airway infections that did not require hospitalization. Liver transplant should be considered as a treatment option for PMM2-CDG patients with end-stage liver disease, however these patients may be at increased risk for recurrent bacterial infections post-transplant., Competing Interests: Declaration of Competing Interest The authors Shawn Tahata, MD, Jody Weckwerth, PA-C, Anna Ligezka, Miao He, PhD, Hee Eun Lee, MD, PhD6 Julie Heimbach, MD, Samar H Ibrahim, M.B.Ch.B, Tamas Kozicz, MD PhD, Katryn Furuya, MD, Eva Morava, MD, PhD have no conflicts of interest to declare., (Copyright © 2023 Elsevier Inc. All rights reserved.)

Congenital disorders of glycosylation (CDG) are severe metabolic disorders caused by an imbalance in the glycosylation pathway. Phosphomannomutase2 (PMM2-CDG), the most prevalent CDG, is mainly due to the disorder of PMM2. Pathogenic variants in cysteine have been found in various diseases, and cysteine residues have a potential as therapeutic targets. PMM2 harbor six cysteines; the variants Cys9Tyr (C9Y) and Cys241Ser (C241S) of PMM2 have been identified to associate with CDG, but the underlying molecular mechanisms remain uncharacterized. Here, we purified PMM2 wild type (WT), C9Y, and C241S to investigate their structural characteristics and biophysical properties by spectroscopic experiments under physiological temperature and environmental stress. Notably, the variants led to drastic changes in the protein properties and were prone to aggregate at physiological temperature. Meanwhile, PMM2 was sensitive to oxidative stress, and the cysteine pathogenic variants led to obvious aggregate formation and a higher cellular apoptosis ratio under oxidative stress. Molecular dynamic simulations indicated that the pathogenic variants changed the core domain of homomeric PMM2 and subunit binding free energy. Moreover, we tested the potential drug targeting PMM2-celastrol in cell level and explained the result by molecular docking simulation. In this study, we delineated the pathological mechanism of the cysteine substitution in PMM2, which addressed the vital role of cysteine in PMM2 and provided novel insights into prevention and treatment strategies for PMM2-CDG., Competing Interests: The authors declare no competing interests., (Copyright © 2023 Fan Yu et al.)

Congenital hyperinsulinemia (CHI), is a clinically heterogeneous disorder that presents as a major cause of persistent and recurrent hypoglycemia during infancy and childhood. There are 16 subtypes of CHI-related genes. Phosphomannomutase 2 hyperinsulinemia (PMM2-HI) is an extremely rare subtype which is first reported in 2017, with only 18 families reported so far. This review provides a structured description of the genetic pathogenesis, and current diagnostic and therapeutic advances of PMM2-HI to increase clinicians' awareness of PMM2-HI., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Chen and Sang.)

Background: Congenital disorders of glycosylation (CDG) are a growing group of rare genetic disorders. The most common CDG is phosphomannomutase 2 (PMM2)-CDG which often has a severe clinical presentation and life-limiting consequences. There are no approved therapies for this condition. Also, there are no validated disease-specific quality of life (QoL) scales to assess the heterogeneous clinical burden of PMM2-CDG which presents a challenge for the assessment of the disease severity and the impact of a certain treatment on the course of the disease., Aim and Methods: This study aimed to identify the most impactful clinical signs and symptoms of PMM2-CDG, and specific patient and observer reported outcome measures (PROMs and ObsROMs, respectively) that can adequately measure such impact on patients' QoL. The most burdensome signs and symptoms were identified through input from the CDG community using a survey targeting PMM2-CDG families and experts, followed by family interviews to understand the real burden of these symptoms in daily life. The list of signs and symptoms was then verified and refined by patient representatives and medical experts in the field. Finally, a literature search for PROMs and ObsROMs used in other rare or common diseases with similar signs and symptoms to those of PMM2-CDG was performed., Results: Twenty-four signs/symptoms were identified as the most impactful throughout PMM2-CDG patients' lifetime. We found 239 articles that included tools to measure those community-selected PMM2-CDG symptoms. Among them, we identified 80 QoL scales that address those signs and symptoms and, subsequently, their psychometric quality was analysed. These scales could be applied directly to the PMM2-CDG population or adapted to create the first PMM2-CDG-specific QoL questionnaire., Conclusion: Identifying the impactful clinical manifestations of PMM2-CDG, along with the collection of PROMs/ObsROMs assessing QoL using a creative and community-centric methodology are the first step towards the development of a new, tailored, and specific PMM2-CDG QoL questionnaire. These findings can be used to fill a gap in PMM2-CDG clinical development. Importantly, this methodology is transferable to other CDG and rare diseases with multiple signs and symptoms., (© 2022. The Author(s).)

In this study, we explored a novel approach to enhancing the production and bioactivities of Ganoderma exopolysaccharides. The homologous phosphomannomutase gene (PMM1) was cloned and overexpressed in Ganoderma for the first time. As a result, the maximum production of exopolysaccharides by the PMM1 transformant was 1.53 g/L, which was 1.41-fold higher than of a wild-type (WT) strain in a 5-L bioreactor. The transcription levels of PMM1 and PMM2 increased 40.5- and 2.4-fold, respectively, whereas the value of the GDP-D-mannose pyrophosphorylase gene did not change significantly in this transgenic strain. Furthermore, the major exopolysaccharide fractions from PMM1 transformants contained higher amounts of mannose (56.5 % and 21.1 %) than those from a WT strain (26.7 % and 9.3 %). Moreover, the major fractions from PMM1 transformants exhibited stronger regulation effects on macrophage. In conclusion, this study is helpful for the efficient production and application of Ganoderma exopolysaccharides and facilitates an understanding of polysaccharide biosynthesis regulation., (Copyright © 2022. Published by Elsevier Ltd.)

Different strategies are being investigated for treating PMM2-CDG, the most common congenital disorder of glycosylation. The use of pharmacochaperones (PCs) is one of the most promising. The present work characterizes the expression, stability, and enzymatic properties of 15 previously described clinical variants of the PMM2 protein, four novel variants, the Pmm2 mouse variant p.Phe115Leu, and its p.Phe119Leu human counterpart, with the aim of extending the potential use of pharmacochaperoning treatment. PMM2 variants were purified as stable homodimers, except for p.Asp65Gly, p.Ile120Thr, and p.Thr237Lys (no expression detected), p.Thr226Ser and p.Val231Met (aggregates), and p.Glu93Ala, p.Phe119Leu, and p.Phe115Leu (partial dissociated). Enzyme activity analyses identified severe variants and milder ones. Pure dimeric mutant proteins showed a reduction in thermal stability except for p.Asn216Asp. The thermal stability of all the unstable mutants was recovered in the presence of the PC compound VIII. This study adds to the list of destabilizing human variants amenable to rescue by small chemical compounds that increase the stability/activity of PMM2. The proposed platform can be reliably used for assessing the disease-causing effects of PMM2 missense variants, for assessing the correlation between genotype and phenotype, for confirming new clinical defects, and for identifying destabilizing mutations amenable to rescue by PCs., (© 2022 The Authors. Human Mutation published by Wiley Periodicals LLC.)

Patient-reported outcomes (PROs) measure important aspects of disease burden, however they have received limited attention in the care of patients with Congenital Disorders of Glycosylation (CDG). We evaluated the PROs and correlation between clinical disease severity scoring and reported quality of life (QoL) in a PMM2-CDG patient cohort. Twenty-five patients with diagnosis of PMM2-CDG were enrolled as part of the Frontiers in Congenital Disorders of Glycosylation Consortium (FCDGC) natural history study. Patient- Reported Outcomes Measurement Information System (PROMIS) was completed by caregivers to assess health-related QoL. Clinical disease severity was scored by medical providers using the Nijmegen Progression CDG Rating Scale (NPCRS). The domains such as physical activity, strength impact, upper extremity, physical mobility, and a satisfaction in social roles (peer relationships) were found to be the most affected in the PMM2-CDG population compared to US general population. We found a strong correlation between NPCRS 1 (current functional ability) and three out of ten PROMIS subscales. NPCRS 2 (laboratory and organ function) and NPCRS 3 (neurological involvement) did not correlate with PROMIS. Mental health domains, such as anxiety, were positively correlated with depressive symptoms (r = 0.76, p = 0.004), fatigue (r = 0.67, p = 0.04). Surprisingly, patients with severely affected physical mobility showed low anxiety scores according to PROMIS (inverse correlation, r = -0.74, p = 0.005). Additionally, there was a positive correlation between upper extremity and physical mobility (r = 0.75, p = 002). Here, we found that PROMIS is an informative additional tool to measure CDG disease burden, which could be used as clinical trial outcome measures. The addition of PROMIS to clinical follow-up could help improve the quality of care for PMM2-CDG by facilitating a holistic approach for clinical decision-making. SYNOPSIS: We recommend PROMIS as an informative tool to measure disease burden in PMM2-CDG in addition to traditional CDG disease severity scores., Competing Interests: Declaration of Competing Interest Authors have no conflicts of interest to declare., (Copyright © 2022 Elsevier Inc. All rights reserved.)

The malaria-causing parasite Plasmodium falciparum is responsible for over 200 million infections and 400,000 deaths per year. At multiple stages during its complex life cycle, P. falciparum expresses several essential proteins tethered to its surface by glycosylphosphatidylinositol (GPI) anchors, which are critical for biological processes such as parasite egress and reinvasion of host red blood cells. Targeting this pathway therapeutically has the potential to broadly impact parasite development across several life stages. Here, we characterize an upstream component of parasite GPI anchor biosynthesis, the putative phosphomannomutase (PMM) (EC 5.4.2.8), HAD5 (PF3D7_1017400). We confirmed the PMM and phosphoglucomutase activities of purified recombinant HAD5 by developing novel linked enzyme biochemical assays. By regulating the expression of HAD5 in transgenic parasites with a TetR-DOZI-inducible knockdown system, we demonstrated that HAD5 is required for malaria parasite egress and erythrocyte reinvasion, and we assessed the role of HAD5 in GPI anchor synthesis by autoradiography of radiolabeled glucosamine and thin layer chromatography. Finally, we determined the three-dimensional X-ray crystal structure of HAD5 and identified a substrate analog that specifically inhibits HAD5 compared to orthologous human PMMs in a time-dependent manner. These findings demonstrate that the GPI anchor biosynthesis pathway is exceptionally sensitive to inhibition in parasites and that HAD5 has potential as a specific, multistage antimalarial target., Competing Interests: Conflict of interest A. R. O. J. reports financial support was provided by National Institutes of Health and Burroughs Wellcome Fund. D. L. J. reports financial support was provided by Natural Sciences and Engineering Research Council of Canada and Canadian Institutes of Health Research. A. R. O. J. reports a relationship with Pluton Biosciences that includes board membership and with American Society for Microbiology that includes consulting or advisory. A. R. O. J. has patent issued and pending patents on antimalarials pending to none., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Background: Polycystic kidney disease with hyperinsulinaemic hypoglycaemia (HIPKD) is a recently described disease caused by a single nucleotide variant, c.-167G>T, in the promoter region of PMM2 (encoding phosphomannomutase 2), either in homozygosity or compound heterozygosity with a pathogenic coding variant in trans. All patients identified so far are of European descent, suggesting a possible founder effect., Methods: We generated high density genotyping data from 11 patients from seven unrelated families, and used this information to identify a common haplotype that included the promoter variant. We estimated the age of the promoter mutation with DMLE+ software, using demographic parameters corresponding to the European population., Results: All patients shared a 0.312 Mb haplotype which was absent in 503 European controls available in the 1000 Genomes Project. The age of this mutation was estimated as 105-110 generations, indicating its occurrence around 600 BC, a time of intense migration, which might explain the presence of the same mutations in Europeans around the globe., Conclusion: The shared unique haplotype among seemingly unrelated patients is consistent with a founder effect in Europeans., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Phosphomannomutase 2 deficiency, PMM2-CDG, is the most frequent disorder of protein N-glycosylation. It is an autosomal recessive disease with a broad clinical and biochemical phenotype. Trying to predict the impact of novel variants is often a challenge due to the high number of variants and the difficulty to establish solid genotype-phenotype correlations. A potential useful strategy is to use computational chemistry calculations as a tool from which relevant information on the structural impact of novel variants may be deduced. Here we present our analyses based on four well-known PMM2 deleterious variants (p.(Leu32Arg), p.(Asp65Tyr), p.(Phe119Leu), p.(Arg141His)) and the polymorphic p.(Glu197Ala) for which we have predicted the effect on protein stability. Our work predicts the effect of different amino acid residues on the conformation and stability of PMM2. These computational simulations are, therefore, an extremely useful methodology which, in combination with routinely used in silico methods of pathogenicity prediction, may help to reveal the structural impact of novel variants at the protein level, potentially leading to a better understanding of target biological molecules., Competing Interests: Declaration of Competing Interest All authors hereby declare that they have no financial or personal relationships with other people or organizations that could inappropriately influence (bias) this work., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Nephrotic syndrome appears as a group of symptoms like proteinuria, edema and hyperlipidemia. Identification of monogenic forms revealed the physiology and pathogenesis of the SRNS., Methods and Results: We performed Illumina panel sequencing of seven genes in 90 Indian patients to determine the role of these genetic mutations in nephrotic syndrome prognosis. Samtool was used for variants calling, and SnpEff and Snpsift did variants annotation. Clinical significance and variant classification were performed by the ClinVar database. In SSNS and SRNS patients, we found 0.78% pathogenic and 3.41% likely pathogenic mutations. Pathogenic mutations were found in LAMB2, LMX1B and WT1 genes, while likely pathogenic mutations were found in (6/13) LAMB2, (2/13) LMX1B, (2/13) TRPC6, (2/13) PTPRO and (1/13) PMM2 genes. Approximately 46% likely pathogenic mutations were contributed to the LAMB2 gene in SSNS and SRNS patients. We also detect 30 VUS (variants of uncertain significance), which were found (17/30) pathogenic and (13/30) likely pathogenic by different prediction tools., Conclusions: Multigene panels were used for genetic screening of heterogeneous disorders like nephrotic syndrome in the Indian population. We found pathogenic, likely pathogenic and certain VUS, which were responsible for the pathogenesis of the disease. Therefore, mutational analysis of SSNS and SRNS is necessary to avoid adverse effects of corticosteroids, modify the intensity of immunosuppressing agents, and prevent the disease's progression., (© 2021. The Author(s), under exclusive licence to Springer Nature B.V.)

PMM2-CDG is a rare disease, causing hypoglycosylation of multiple proteins, hence preventing full functionality. So far, no direct genotype-phenotype correlations have been identified. We carried out a retrospective cohort study on 26 PMM2-CDG patients. We collected the identified genotype, as well as continuous variables indicating the disease severity (based on Nijmegen Pediatric CDG Rating Score or NPCRS) and dichotomous variables reflecting the patients' phenotype. The phenotypic effects of patients' genotype were studied using non-parametric and Chi-Square tests. Seventeen different pathogenic variants have been studied. Variants with zero enzyme activity had no significant impact on the Nijmegen score. Pathogenic variants involving the stabilization/folding domain have a significantly lower total NPCRS ( p = 0.017): presence of the p.Cys241Ser mutation had a significantly lower subscore 1,3 and NPCRS ( p = 0.04) and thus result in a less severe phenotype. On the other hand, variants involving the dimerization domain, p.Pro113Leu and p.Phe119Leu, resulted in a significantly higher NPCRS score ( p = 0.002), which indicates a worse clinical course. These concepts give a better insight in the phenotypic prognosis of PMM2-CDG, according to their molecular base.

We aimed to identify clinical, molecular and radiological correlates of activities of daily living (ADL) in patients with cerebellar atrophy caused by PMM2 mutations (PMM2-CDG), the most frequent congenital disorder of glycosylation. Twenty-six PMM2-CDG patients (12 males; mean age 13 ± 11.1 years) underwent a standardized assessment to measure ADL, ataxia (brief ataxia rating scale, BARS) and phenotype severity (Nijmegen CDG rating scale, NCRS). MRI biometry of the cerebellum and the brainstem were performed in 23 patients (11 males; aged 5 months-18 years) and 19 control subjects with equal gender and age distributions. The average total ADL score was 15.3 ± 8.5 (range 3-32 out of 36 indicating severe functional disability), representing variable functional outcome in PMM2-CDG patients. Total ADL scores were significantly correlated with NCRS (r 2 = 0.55, p < 0.001) and BARS scores (r 2 = 0.764; p < 0.001). Severe intellectual disability, peripheral neuropathy, and severe PMM2 variants were all significantly associated with worse functional outcome. Higher ADL scores were significantly associated with decreased diameters of cerebellar vermis (r 2 = 0.347; p = 0.004), hemispheres (r 2 = 0.436; p = 0.005), and brainstem, particularly the mid-pons (r 2 = 0.64; p < 0.001) representing the major radiological predictor of functional disability score in multivariate regression analysis. We show that cerebellar syndrome severity, cognitive level, peripheral neuropathy, and genotype correlate with ADL used to quantify disease-related deficits in PMM2-CDG. Brainstem involvement should be regarded among functional outcome predictors in patients with cerebellar atrophy caused by PMM2-CDG., (© 2021. The Author(s).)

PMM2-CDG is the most common congenital disorder of glycosylation (CDG) accounting for almost 65% of known CDG cases affecting N-glycosylation. Abnormalities in N-glycosylation could have a negative impact on many endocrine axes. There is very little known on the effect of impaired N-glycosylation on the hypothalamic-pituitary-adrenal axis function and whether CDG patients are at risk of secondary adrenal insufficiency and decreased adrenal cortisol production. Cortisol and ACTH concentrations were simultaneously measured between 7:44 am to 1 pm in forty-three subjects (20 female, median age 12.8 years, range 0.1 to 48.6 years) participating in an ongoing international, multi-center Natural History study for PMM2-CDG (ClinicalTrials.gov Identifier: NCT03173300). Of the 43 subjects, 11 (25.6%) had cortisol below 5 μg/dl and low to normal ACTH levels, suggestive of secondary adrenal insufficiency. Two of the 11 subjects have confirmed central adrenal insufficiency and are on hydrocortisone replacement and/or stress dosing during illness; 3 had normal and 1 had subnormal cortisol response to ACTH low-dose stimulation test but has not yet been started on therapy; the remaining 5 have upcoming stimulation testing planned. Our findings suggest that patients with PMM2-CDG may be at risk for adrenal insufficiency. Monitoring of morning cortisol and ACTH levels should be part of the standard care in patients with PMM2-CDG., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Aspergillus fumigatus is an opportunistic mold responsible for severe life-threatening fungal infections in immunocompromised patients. The cell wall, an essential structure composed of glucan, chitin, and galactomannan, is considered to be a target for the development of antifungal drugs. The nucleotide sugar donor GDP-mannose (GDP-Man) is required for the biosynthesis of galactomannan, glycosylphosphatidylinositol (GPI) anchors, glycolipid, and protein glycosylation. Starting from fructose-6-phosphate, GDP-Man is produced by the sequential action of the enzymes phosphomannose isomerase, phosphomannomutase (Pmm), and GDP-mannose pyrophosphorylase. Here, using heterokaryon rescue and gene knockdown approaches we demonstrate that the phosphomannomutase encoding gene in A. fumigatus (pmmA) is essential for survival. Reduced expression of pmmA is associated with significant morphological defects including retarded germination, growth, reduced conidiation, and abnormal polarity. Moreover, the knockdown strain exhibited an altered cell wall organization and sensitivity toward cell wall perturbing agents. By solving the first crystal structure of A. fumigatus phosphomannomutase (AfPmmA) we identified non-conservative substitutions near the active site when compared to the human orthologues. Taken together, this work provides a genetic and structural foundation for the exploitation of AfPmmA as a potential antifungal target., (© 2021 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Primary ovarian insufficiency (POI) is a highly heterogeneous condition, and its underlying causes remain to be clarified in a large fraction of patients. Congenital disorders of glycosylation (CDG) are multisystem diseases caused by mutations of a number of genes involved in N-glycosylation or O-glycosylation, and the most frequent form is PMM2-CDG (alias, CDG-Ia) resulting from biallelic mutations in PMM2 encoding phosphomannomutase-2 involved in N-glycosylation. Here, we examined a 46,XX Japanese female with syndromic POI accompanied by an undetectable level of serum anti-Müllerian hormone (AMH). Whole exome sequencing identified biallelic pathogenic mutations of PMM2 (a novel c.34G>C:p.(Asp12His) of maternal origin and a recurrent c.310C>G:p.(Leu104Val) of paternal origin) (NM&#95;000303.3), and N-glycosylation studies detected asialotransferrin and disialotransferrin characteristic of PMM2-CDG, in addition to normally glycosylated tetrasialotransferrin. Clinical assessment showed cerebellar hypotrophy, which is a fairly characteristic and highly prevalent feature in PMM2-CDG, together with multiple non-specific features reported in PMM2-CDG such as characteristic face, intellectual disability, skeletal abnormalities, and low blood antithrombin III value. These results including the undetectable level of serum AMH, in conjunction with previously reported findings suggestive of the critical role of glycosylation in oocyte development and function, imply that PMM2-CDG almost invariably leads to POI primarily because of the defective oogenesis and/or oocyte-dependent early folliculogenesis rather than the compromised bioactivity of FSH/LH with defective glycosylation. Thus, it is recommended to examine PMM2 in patients with syndromic POI, especially in those with cerebellar ataxia/hypotrophy.

The autosomal recessive phosphomannomutase 2-congenital disorder of glycosylation (PMM2-CDG) is characterized by defective functioning of the PMM2 enzyme, which is necessary for the conversion of mannose-6-phosphate into mannose-1-phosphate. Here, a computational pipeline was drawn to identify the most significant mutations, and further, we used a virtual screening approach to identify a new lead compound to treat the identified significant mutations. We searched for missense mutation data related to PMM2-CDG in HGMD®, UniProt, and ClinVar. Our search yielded a total of 103 mutations, of which 91 are missense mutations. The D65Y, I132N, I132T, and F183S mutations were classified as deleterious, destabilizing, and altering the biophysical properties using the PredictSNP, iStable, and Align GVGD in silico prediction tools. Additionally, we applied a multistep protocol to screen for an alternative lead compound to the existing CID2876053 (1-(3-chlorophenyl)-3,3-bis(pyridine-2-yl)urea) with affinity to these identified significant mutants. Two compounds, CHEMBL1491007 (6-chloro-4-phenyl-3-(4-pyridin-2-ylpiperazin-1-yl)-1H-quinolin-2-one) and CHEMBL3653029 (5-chloro-4-[6-[(3-fluorophenyl)methylamino]pyridin-2-yl]-N-(piperidin-4-ylmethyl)pyridin-2-amine), exhibited the highest binding affinity with the selected mutants and were chosen for further analysis. Through molecular docking, molecular dynamics simulation, and MMPBSA analysis, we found that the known compound, i.e. CID2876053, has stronger interaction with the D65Y mutant. The newly identified lead compound CHEMBL1491007 showed stronger interaction with the I132N and I132T mutants, whereas the most deleterious mutant, F183S, showed stronger interaction with CHEMBL3653029. This study is expected to aid in the field of precision medicine, and further to in vivo and in vitro analysis of these lead compounds might shed light on the treatment of PMM2-CDG. Communicated by Ramaswamy H. Sarma.

Enzyme regulation is vital for metabolic adaptability in living systems. Fine control of enzyme activity is often delivered through post-translational mechanisms, such as allostery or allokairy. β-phosphoglucomutase (βPGM) from Lactococcus lactis is a phosphoryl transfer enzyme required for complete catabolism of trehalose and maltose, through the isomerisation of β-glucose 1-phosphate to glucose 6-phosphate via β-glucose 1,6-bisphosphate. Surprisingly for a gatekeeper of glycolysis, no fine control mechanism of βPGM has yet been reported. Herein, we describe allomorphy, a post-translational control mechanism of enzyme activity. In βPGM, isomerisation of the K145-P146 peptide bond results in the population of two conformers that have different activities owing to repositioning of the K145 sidechain. In vivo phosphorylating agents, such as fructose 1,6-bisphosphate, generate phosphorylated forms of both conformers, leading to a lag phase in activity until the more active phosphorylated conformer dominates. In contrast, the reaction intermediate β-glucose 1,6-bisphosphate, whose concentration depends on the β-glucose 1-phosphate concentration, couples the conformational switch and the phosphorylation step, resulting in the rapid generation of the more active phosphorylated conformer. In enabling different behaviours for different allomorphic activators, allomorphy allows an organism to maximise its responsiveness to environmental changes while minimising the diversion of valuable metabolites.

Background: Mutations in the PMM2 gene cause phosphomannomutase 2 deficiency (PMM2; MIM# 212065), which manifests as a congenital disorder of glycosylation (PMM2-CDG). Mutant PMM2 leads to the reduced conversion of Man-6-P to Man-1-P, which results in low concentrations of guanosine 5'-diphospho-D-mannose, a nucleotide-activated sugar essential for the construction of protein oligosaccharide chains. To date the only therapeutic options are preventive and symptomatic., Scope of Review: This review covers the latest advances in the search for a treatment for PMM2-CDG., Major Conclusions: Treatments based on increasing Man-1-P levels have been proposed, along with the administration of different mannose derivates, employing enzyme inhibitors or repurposed drugs to increase the synthesis of GDP-Man. A single repurposed drug that might alleviate a severe neurological symptom associated with the disorder is now in clinical use. Proof of concept also exists regarding the use of pharmacological chaperones and/or proteostatic regulators to increase the concentration of hypomorphic PMM2 mutant proteins., General Significance: The ongoing challenges facing the discovery of drugs to treat this orphan disease are discussed., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Background: Congenital disorders of glycosylation (CDG) are rare diseases with impaired glycosylation and multiorgan disfunction, including hemostatic and inflammatory disorders. Factor XII (FXII), the first element of the contact phase, has an emerging role in hemostasia and inflammation. FXII deficiency protects against thrombosis and the p.Thr309Lys variant is involved in hereditary angioedema through the hyperreactivity caused by the associated defective O-glycosylation. We studied FXII in CDG aiming to supply further information of the glycosylation of this molecule, and its functional and clinical effects. Plasma FXII from 46 PMM2-CDG patients was evaluated by coagulometric and by Western Blot in basal conditions, treated with N-glycosydase F or activated by silica or dextran sulfate. A recombinant FXII expression model was used to validate the secretion and glycosylation of wild-type and variants targeting the two described FXII N-glycosylation sites (p.Asn230Lys; p.Asn414Lys) as well as the p.Thr309Lys variant., Results: PMM2-CDG patients had normal FXII levels (117%) but high proportions of a form lacking N-glycosylation at Asn414. Recombinant FXII p.Asn230Lys, and p.Asn230Lys&p.Asn414Lys had impaired secretion and increased intracellular retention compared to wild-type, p.Thr309Lys and p.Asn414Lys variants. The hypoglycosylated form of PMM2-CDG activated similarly than FXII fully glycosylated. Accordingly, no PMM2-CDG had angioedema. FXII levels did not associate to vascular events, but hypoglycosylated FXII, like hypoglycosylated transferrin, antithrombin and FXI levels did it., Conclusions: N-glycosylation at Asn230 is essential for FXII secretion. PMM2-CDG have high levels of FXII lacking N-glycosylation at Asn414, but this glycoform displays similar activation than fully glycosylated, explaining the absence of angioedema in CDG.

Background: PMM2-CDG (CDG-Ia) is the most frequent N-glycosylation disorder. While supplying mannose to PMM2-deficient fibroblasts corrects the altered N-glycosylation in vitro, short term therapeutic approaches with mannose supplementation in PMM2-CDG patients have been unsuccessful. Mannose found no further mention in the design of a potential therapy for PMM2-CDG in the past years, as it applies to be ineffective. This retrospective study analyzes the first long term mannose supplementation in 20 PMM2-CDG patients. Mannose was given at a total of 1-2 g mannose/kg b.w./d divided into 5 single doses over a mean time of 57,75 ± 25,85 months. Protein glycosylation, blood mannose concentration and clinical presentation were monitored in everyday clinical practice., Results: After a mean time period of more than 1 year the majority of patients showed significant improvements in protein glycosylation., Conclusion: Dietary mannose supplementation shows biological effects in PMM2-CDG patients improving glycosylation in the majority of patients. A double-blind randomized study is needed to examine the role of mannose in the design of a therapy for children with PMM2-CDG in more detail.

Objectives Hyperinsulinemic hypoglucemia (HH) is characterized by a dysregulation of insulin secretion from pancreatic β cells. Congenital hyperinsulinism has been associated with specific genes in monogenic forms and also with other diseases with a yet unknown genetic cause. In 2017, Rubio Cabezas et al. described the association of HH and autosomal recessive polycystic kidney disease (ARPKD) with a promoter mutation in the PMM2 gene. They found that all the patients carried a promoter mutation (c-167G>T) in PMM2, either homozygous or in trans with a second PMM2 coding mutation. Methods We performed the study of the PMM2 gene in six patients from four unrelated families, previously diagnosed with ARPKD and HH. Results All these patients had in common the heterozygous variant c-167G>T in the promoter region for PMM2. Additionally, each patient carried a compound heterozygote for a second missense mutation in this gene (p.Arg141His, p.Asp148Asn or p.Phe157Ser), previously reported as pathogenic for congenital disorder of glycosylation type Ia, with an autosomal recessive inheritance pattern. Unlike the previous published article, two of our patients showed altered type 1 pattern and one of them with rectal bleeding that could be a sign of PMM2-congenital disorders of glycosylation. Conclusion We propose the study of this gene when carrying out the diagnosis of patients with HH, especially in the neonatal period and when a recessive polycystic kidney disease without alterations in PKDH1 is diagnosed.

Phosphomannomutase 2 deficiency (PMM2-CDG) is the most common N-glycosylation disorder. To date there is no treatment. Following the identification of a number of destabilizing pathogenic variants, our group suggested PMM2-CDG to be a conformational disease. The aim of the present study was to evaluate the possible use of proteostasis network regulators to increase the stability, and subsequently the enzymatic activity, of misfolded PMM2 mutant proteins. Patient-derived fibroblasts transduced with their own PMM2 folding or oligomerization variants were treated with different concentrations of the proteostasis regulators celastrol or MG132. Celastrol treatment led to a significant increase in mutant PMM2 protein concentration and activity, while MG132 had a small effect on protein concentration only. The increase in enzymatic activity with celastrol correlated with an increase in the transcriptional and proteome levels of the heat shock proteins Hsp90 and Hsp70. The use of specific Hsp70 or Hsp90 inhibitors showed the positive effect of celastrol on PMM2 stability and activity to occur through Hsp90-driven modulation of the proteostasis network. The synergistic effect of celastrol and a previously described pharmacological chaperone was also examined, and a mutation-dependent synergistic effect on PMM2 activity was noted. These results provide proof-of-concept regarding the potential treatment of PMM2-CDG by proteostasis regulators, either alone or in combination with pharmacological chaperones., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020. Published by Elsevier B.V.)

Phosphomannomutase 2 deficiency (PMM2-CDG) is an autosomal recessive congenital disorder of glycosylation, characterized by multisystem phenotypes, mostly including neurological involvement. In Turkey, due to high rates of consanguinity, many patients with autosomal recessive disorders have homozygous variants and these diseases are more common, compared to Europe. However, published reports of PMM2-CDG from Turkey are scarce. Here, we describe clinical and molecular characteristics of PMM2-CDG patients diagnosed in three centers in Turkey, using data obtained retrospectively from hospital records. We also analyzed an in-house exome database of 1,313 individuals for PMM2 variants and estimated allele, carrier and disease frequencies, using the Hardy-Weinberg law. Eleven patients were identified from 10 families, displaying similar characteristics to previous publications, with the exception of the first report of epilepsia partialis continua and increased prevalence of sensorineural hearing loss. p.Val231Met was the most common variant, and was homozygous in four patients. This novel genotype results in a neurological phenotype with subclinical visceral involvement. Exome database analysis showed an estimated prevalence of 1:286,726 for PMM2-CDG, which is much lower than expected (1:20,000 in Europe) because of the lack of predominance of the common European p.Asp141His allele, associated with a severe phenotype (allele frequency of 1:2,622 compared to 1:252 in gnomAD). These data suggest that prevalence, phenotypes and genotypes of PMM2-CDG in Turkey differ significantly from those in Europe: Milder phenotypes may be more common, but the disease itself rarer, requiring a higher clinical suspicion for diagnosis. The association of sensorineural hearing loss with PMM2-CDG warrants further study., (© 2020 Wiley Periodicals, Inc.)

Human phosphoglucomutase 1 (PGM1) is an evolutionary conserved enzyme that belongs to the ubiquitous and ancient α-D-phosphohexomutases, a large enzyme superfamily with members in all three domains of life. PGM1 catalyzes the bi-directional interconversion between α-D-glucose 1-phosphate (G1P) and α-D-glucose 6-phosphate (G6P), a reaction that is essential for normal carbohydrate metabolism and also important in the cytoplasmic biosynthesis of nucleotide sugars needed for glycan biosynthesis. Clinical studies have shown that mutations in the PGM1 gene may cause PGM1 deficiency, an inborn error of metabolism previously classified as a glycogen storage disease, and PGM1 deficiency was recently also shown to be a congenital disorder of glycosylation. Here we present three crystal structures of the isoform 2 variant of PGM1, both as a free enzyme and in complex with its substrate and product. The structures show the longer N-terminal of this PGM1 variant, and the ligand complex structures reveal for the first time the detailed structural basis for both G1P substrate and G6P product recognition by human PGM1. We also show that PGM1 and the paralogous gene PGM5 are the results of a gene duplication event in a common ancestor of jawed vertebrates, and, importantly, that both PGM1 isoforms are conserved and of functional significance in all vertebrates. Our finding that PGM1 encodes two equally conserved and functionally important isoforms in the human organism should be taken into account in the evaluation of disease-related missense mutations in patients in the future.

Numerous etiologies may lead to nonimmune hydrops fetalis (NIHF) including congenital disorders of glycosylation (CDG). Recognition of CDG in NIHF is challenging. This study reviews prenatal and neonatal characteristics of CDG presenting with NIHF. A systematic literature search was performed. Thirteen articles met the inclusion criteria. Twenty-one cases with NIHF associated with a CDG were reported. There were 17 live births, three pregnancy terminations, and one fetal demise. Timing of CDG diagnosis was reported mostly postnatally (90%; 10/11). Postnatal genetic testing was reported in 18 patients; three patients were diagnosed by isoelectric focusing of serum transferrin that showed a type 1 pattern. The genes reported for CDG with NIHF for 15 distinct families include: PMM2 in 47% (7/15), ALG9 in 20% (3/15), ALG8 in 13% (2/15), ALG1 in 7% (1/15), MGAT2 in 7% (1/15), and COG6 7% (1/15). In our review, 81% (17/21) reported facial dysmorphism, 52% (11/21) reported CNS abnormalities, most commonly cerebellar atrophy (64%; 7/11), and 38% (8/21) reported cardiovascular abnormalities, most commonly hypertrophic cardiomyopathy (63%; 5/8). Among live births, 71% (12/17) infants died at a median age of 34 days (range 1-185). Thrombocytopenia was reported in 53% (9/17) patients. Of those who survived past the neonatal period, 80% (4/5) had significant reported developmental delays. CDG should be on the differential diagnosis of NIHF in the presence of cerebellar atrophy, hypertrophic cardiomyopathy, or thrombocytopenia. Our review highlights the poor prognosis in infants with NIHF due to CDG and demonstrates the importance of identifying these disorders prenatally to guide providers in their counseling with families regarding pregnancy management. SYNOPSIS: Poor prognosis in fetuses and infants with nonimmune hydrops fetalis due to congenital disorders of glycosylation highlights the importance of prenatal diagnosis of this disorder., (© 2019 SSIEM.)

Purpose: This study sought to identify a disease-related gene in a consanguineous Chinese family in which there were two premature ovarian insufficiency (POI) sisters., Method: We used whole-exome sequencing and Sanger sequencing to identify the disease-causing gene. Results were verified using an assay of mutant protein and in silico analyses., Result: We identified a novel missense mutation (NM&#95;000303: c.556G>A, p.Gly186Arg) in the PMM2 gene. The two sisters suffer from premature ovarian insufficiency (POI) only and have no other symptoms of congenital disorder of glycosylation type-1a (CDG-Ia). We found that the enzymic activity of the mutant PMM2 protein was reduced by 55.21% (p < 0.05) when compared with wild type, and many in silico tools suggested the mutation is disease-related., Conclusion: This particular gene modification results in changes in activity of phosphomannomutase modification, which could lead to PMM2-CDG-Ia with an uncommon phenotype.

Background: Proprotein convertase subtilisin/kexin type 9 (PCSK9) inhibitors are novel therapeutics for reducing low-density lipoprotein cholesterol (LDLc). While serious side-effects have not been observed in short-term clinical trials, there remain concerns that long-term PCSK9 inhibition may cause neurocognitive side-effects., Methods and Results: An adult male with childhood-onset global developmental delay, cerebellar atrophy and severe hypolipidaemia underwent extensive biochemical and genetic investigations. Initial testing revealed low circulating PCSK9 levels and a common loss-of-function PCSK9 polymorphism, but these findings did not fully account for severe hypolipidaemia. Whole-exome sequencing was subsequently performed and identified two pathogenic phosphomannose mutase 2 (PMM2) variants (p.Arg141His and p.Pro69Ser) known to cause PMM2-associated congenital disorder of glycosylation (PMM2-CDG). A diagnosis of PMM2-CDG was consistent with the proband's neurological symptoms and severe hypolipidaemia. Given that PMM2-CDG is characterised by defective protein N-glycosylation and that PCSK9 is a negative regulator of LDLc, we postulated that loss of PCSK9 N-glycosylation mediates hypolipidaemia among patients with PMM2-CDG. First, in an independent cohort of patients with PMM2-CDG (N=8), we verified that circulating PCSK9 levels were significantly lower in patients than controls (p=0.0006). Second, we conducted in vitro experiments in hepatocyte-derived cells to evaluate the effects of PCSK9 N-glycosylation loss on LDL receptor (LDLR) activity. Experimental results suggest that defective PCSK9 N-glycosylation reduces the ability of circulating PCSK9 to degrade LDLR., Conclusion: Life-long exposure to genetically lower PCSK9 per se is unlikely to cause neurocognitive impairment. Both observational and experimental findings suggest that hypolipidaemia in PMM2-CDG may be partially mediated by loss of PCSK9 N-glycosylation and/or its regulators., Competing Interests: Competing interests: GP has received consulting fees from Sanofi, Bristol-Myers Squibb, Lexicomp and Amgen and has received support for research through his institution from Sanofi. MC, GY, DSR, AC, DC and NS declare no competing interests., (© Author(s) (or their employer(s)) 2020. No commercial re-use. See rights and permissions. Published by BMJ.)

Background: PMM2-CDG, is the most common N-linked glycosylation disorder and subtype among all CDG syndromes, which are a series of genetic disorders involving the synthesis and attachment of glycoproteins and glycolipid glycans. The mutations of PMM2-CDG might lead to the loss of PMM2, which is responsible for the conversion of mannose 6- phosphate into mannose 1-phosphate. Most patients with PMM2-CDG have central nervous system involvement, abnormal coagulation, and hepatopathy. The neurological symptoms of PMM2-CDG are intellectual disability (ID), cerebellar ataxia, and peripheral neuropathy. Now, over 100 new CDG cases have been reported. However, each type of CDG is very rare, and CDGs are problematic to diagnose. In addition, few CDGs have been reported in the Chinese population., Case Presentation: Here we present a Hani ethnic minority family including two siblings with congenital glycosylation disorders. Whole-exome sequencing revealed compound heterozygous for one novel mutation (c.241-242 del variant) and previously reported mutation (c.395 T > C) in gene of PMM2. Two mutations were found in proband and her sibling by whole-exome sequencing. The mutations were identified in this family by Sanger sequencing and no mutations were detected in the normal control., Conclusions: This is the first report to describe mutations in two siblings of Hani ethnic minority which is one of five ethnic groups found only in Yunnan with a population of more than 1 million.

Phosphomannomutase 2 deficiency, or PMM2-CDG, is the most common congenital disorder of glycosylation and affects over 1000 patients globally. There are no approved drugs that treat the symptoms or root cause of PMM2-CDG. To identify clinically actionable compounds that boost human PMM2 enzyme function, we performed a multispecies drug repurposing screen using a novel worm model of PMM2-CDG, followed by PMM2 enzyme functional studies in PMM2-CDG patient fibroblasts. Drug repurposing candidates from this study, and drug repurposing candidates from a previously published study using yeast models of PMM2-CDG, were tested for their effect on human PMM2 enzyme activity in PMM2-CDG fibroblasts. Of the 20 repurposing candidates discovered in the worm-based phenotypic screen, 12 were plant-based polyphenols. Insights from structure-activity relationships revealed epalrestat, the only antidiabetic aldose reductase inhibitor approved for use in humans, as a first-in-class PMM2 enzyme activator. Epalrestat increased PMM2 enzymatic activity in four PMM2-CDG patient fibroblast lines with genotypes R141H/F119L, R141H/E139K, R141H/N216I and R141H/F183S. PMM2 enzyme activity gains ranged from 30% to 400% over baseline, depending on genotype. Pharmacological inhibition of aldose reductase by epalrestat may shunt glucose from the polyol pathway to glucose-1,6-bisphosphate, which is an endogenous stabilizer and coactivator of PMM2 homodimerization. Epalrestat is a safe, oral and brain penetrant drug that was approved 27 years ago in Japan to treat diabetic neuropathy in geriatric populations. We demonstrate that epalrestat is the first small molecule activator of PMM2 enzyme activity with the potential to treat peripheral neuropathy and correct the underlying enzyme deficiency in a majority of pediatric and adult PMM2-CDG patients., Competing Interests: Competing interestsThe following authors are shareholders of Perlara PBC: S.I., N.D., F.S.S., Z.P., K.M., H.T. and E.O.P., (© 2019. Published by The Company of Biologists Ltd.)

Lysobacter enzymogenes, a member of Xanthomonadaceae, is a promising tool to control crop-destroying fungal pathogens. One of its key antifungal virulence factors is the type IV pili that are required for twitching motility. Transposon mutagenesis of L. enzymogenes revealed that the production of type IV pili required the presence of the Le2152 gene, which encodes an AlgC-type phosphomannomutase/phosphoglucomutase (PMM). However, in addition to the cytoplasmic PMM domain, the Le2152 gene product contains a ~200-aa N-terminal periplasmic domain that is anchored in the membrane by two transmembrane segments and belongs to the dCache superfamily of periplasmic sensor domains. Sequence analysis identified similar membrane-anchored PMMs, encoded in conserved coaBC-dut-algC gene clusters, in a variety of gammaproteobacteria, either as the sole PMM gene in the entire genome or in addition to the gene encoding the stand-alone enzymatic domain. Previously overlooked N-terminal periplasmic sensor domains were detected in the well-characterized PMMs of Pseudomonas aeruginosa and Xanthomonas campestris, albeit not in the enzymes from Pseudomonas fluorescens, Pseudomonas putida or Azotobacter vinelandii. It appears that after the initial cloning of the enzymatically active soluble part of P. aeruginosa AlgC in 1991, all subsequent studies utilized N-terminally truncated open reading frames. The N-terminal dCache sensor domain of AlgC is predicted to modulate the PMM activity of the cytoplasmic domain in response to as yet unidentified environmental signal(s). AlgC-like membrane-bound PMMs appear to comprise yet another environmental signalling system that regulates the production of type IV pili and potentially other systems in certain gammaproteobacteria., (© 2019 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Chagas' disease, which is caused by the Trypanosoma cruzi parasite, has become a global health problem that is currently treated with poorly tolerated drugs that require prolonged dosing. Therefore, there is a clinical need for new therapeutic agents that can mitigate these issues. The phosphomannomutase (PMM) and GDP-mannose pyrophosphorylase (GDP-MP) enzymes form part of the de novo biosynthetic pathway to the nucleotide sugar GDP-mannose. This nucleotide sugar is used either directly, or indirectly via the formation of dolichol-phosphomannose, for the assembly of all mannose-containing glycoconjugates. In T. cruzi , mannose-containing glycoconjugates include the cell-surface glycoinositol-phospholipids and the glycosylphosphatidylinositol-anchored mucin-like glycoproteins that dominate the cell surface architectures of all life cycle stages. This makes PMM and GDP-MP potentially attractive targets for a drug discovery program against Chagas' disease. To assess the ligandability of these enzymes in T. cruzi , we have screened 18,117 structurally diverse compounds exploring drug-like chemical space and 16,845 small polar fragment compounds using an assay interrogating the activities of both PMM and GDP-MP enzymes simultaneously. This resulted in 48 small fragment hits, and on retesting 20 were found to be active against the enzymes. Deconvolution revealed that these were all inhibitors of T. cruzi GDP-MP, with compounds 2 and 3 acting as uncompetitive and competitive inhibitors, respectively. Based on these findings, the T. cruzi PMM and GDP-MP enzymes were deemed not ligandable and poorly ligandable, respectively, using small molecules from conventional drug discovery chemical space. This presents a significant hurdle to exploiting these enzymes as therapeutic targets for Chagas' disease., (Copyright © 2019 Zmuda et al.)

A large number of mutations causing PMM2-CDG, which is the most frequent disorder of glycosylation, destabilize phosphomannomutase2. We looked for a pharmacological chaperone to cure PMM2-CDG, starting from the structure of a natural ligand of phosphomannomutase2, α-glucose-1,6-bisphosphate. The compound, β-glucose-1,6-bisphosphate, was synthesized and characterized via 31 P-NMR. β-glucose-1,6-bisphosphate binds its target enzyme in silico. The binding induces a large conformational change that was predicted by the program PELE and validated in vitro by limited proteolysis. The ability of the compound to stabilize wild type phosphomannomutase2, as well as frequently encountered pathogenic mutants, was measured using thermal shift assay. β-glucose-1,6-bisphosphate is relatively resistant to the enzyme that specifically hydrolyses natural esose-bisphosphates.