Differential glycomics of epithelial membrane glycoproteins from urinary exovesicles reveals shifts towards complex-type N-glycosylation in classical galactosemia

Classical galactosemia is caused by deficiency of the enzyme galactose-1-phosphate uridyltransferase (GALT). The reason for the deficiency is a specific gene mutation causing an amino acid exchange near the active site of the enzyme. The turnover rate of the enzyme is therefore more than thousand-fold decreased. Dietary galactose uptake, for instance drinking milk, is followed by an intracellular stress response. The co-substrate of the GALT galactose-1-phosphate accumulates intracellularly and disturbs carbohydrate-dependent metabolic pathways and presumably the Leloir pathway. The Leloir pathway supplies UDP-galactose and UDP-glucose for glycosylation of proteins and lipids. UDP-galactose incorporation is necessary, for example, to build up complex-type antennae of N-glycans on glycoproteins. It was supposed that, if the Leloir pathway is affected, the N-glycosylation, especially galactosylation in complex-type chains, will be affected. For examination of changes in glycosylation patterns mass spectrometric glycomics methods were chosen as most suitable. The N-linked carbohydrate branches were detached by PNGaseF digestion and analyzed applying matrix-assisted laser ionization mass spectrometry after glycan methylation. For examination of glycan pattern changes we isolated vesicles, mainly exosomes from urine of galactosemia patients as easily accessible source of epithelial membranes, and compared the N-glycome with the corresponding samples of healthy controls. The comparison of the mass spectra reveals a dramatic glycosylation shift from prevalent high-mannose-type N-glycans found in healthy controls towards complex-type glycosylation in patients. The estimated ratio of the amounts of high-mannose-type versus complex-type was about 3:1 respecting the healthy control samples. Especially the N-glycans that either carry five or six mannoses were detected as most abundant. In contrast the ratio for patients shifted to nearly 1:1 towards complex-type glycosylation. The most abundant complex-type glycans from exovesicular membranes of GALT-deficient patients were of bi-antennary structure, terminal disialylated and without core fucosylation. Glycans with higher antennary branching were found in marginal amounts, only. The observed glycosylation shift can be regarded as a new finding and it may be assumed that not only secreted proteins like transferrin become dysglycosylated in galactosemia. Furthermore, cell surface located glycoproteins, especially receptors, were affected. Changes in glycosylation patterns were suspected to be responsible for missorting of glycoproteins especially for glyco-receptors like the EGFR (epidermal growth factor receptor) which is regularly expressed at apical membranes of epithelial cells. Quantitative and qualitative alterations of membranous exosomal glycoproteins indirectly provide information of alterations at the cell membrane. To study alterations of glycoproteoms, especially glyco-receptors, in exosomal lipid rafts, an iTRAQ (isobaric tags for relative and absolute quantitation) labeling was chosen as suitable for a quantitative in vitro experiment based on urinary exosomes. Four different individual samples were selected: two galactosemic samples (from patients showing drastic shifts from high-mannose to complex-type N-glycosylation) and two healthy controls. The samples were labeled with the isobaric tags, mixed and analyzed by offline nano-LC MALDI-MS/MS (liquid chromatography-matrix-assisted laser desorption-ionization-mass spectrometry). Although EGFR was not detected in the experiment we found strongly increased amounts of N-glycoproteins and some receptors associated with galactosemic exosomes: tyrosine-protein kinase receptor UFO, zinc-alpha-2-glycoprotein (ZA2G, MHC I -family) and aminopeptidase N (CD13), to name some. The cubilin receptor and megalin were also found. They dimerize to form the megalin-cubilin-complex and are responsible for protein reabsorption at the proximal tubulus of the nephritic kidney. This cubilin receptor was detected in a further experiment to be one of the major differences between samples of galactosemia patients and control persons. Finally, the iTRAQ experiment revealed another unexpected result. The samples of the galactosemia patients contained increased amounts of serum proteins, in particular N-glycoproteins. A large variety of immunoglobulins were found increased; further albumin, fetuin, transferrin and many more. All together these results strongly suggest renal failure for these examined galactosemic patients. Biopsy is usually the most common method to detect renal diseases. The examination of urinary exosomal samples therefore possibly represents a new diagnostic method to detect renal failure without surgical intervention

Evaluating the use of ABBA-BABA statistics to locate introgressed loci

Several methods have been proposed to test for introgression across genomes. One method tests for a genome-wide excess of shared derived alleles between taxa using Patterson’s D statistic, but does not establish which loci show such an excess or whether the excess is due to introgression or ancestral population structure. Several recent studies have extended the use of D by applying the statistic to small genomic regions, rather than genome-wide. Here, we use simulations and whole-genome data from Heliconius butterflies to investigate the behavior of D in small genomic regions. We find that D is unreliable in this situation as it gives inflated values when effective population size is low, causing D outliers to cluster in genomic regions of reduced diversity. As an alternative, we propose a related statistic f ̂ d, a modified version of a statistic originally developed to estimate the genome-wide fraction of admixture. f ̂ d is not subject to the same biases as D, and is better at identifying introgressed loci. Finally, we show that both D and f ̂ d outliers tend to cluster in regions of low absolute divergence (dXY), which can confound a recently proposed test for differentiating introgression from shared ancestral variation at individual loci

The international EAACI/GA²LEN/EuroGuiDerm/APAAACI guideline for the definition, classification, diagnosis, and management of urticaria

This update and revision of the international guideline for urticaria was developed following the methods recommended by Cochrane and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) working group. It is a joint initiative of the Dermatology Section of the European Academy of Allergology and Clinical Immunology (EAACI), the Global Allergy and Asthma European Network (GA(2)LEN) and its Urticaria and Angioedema Centers of Reference and Excellence (UCAREs and ACAREs), the European Dermatology Forum (EDF; EuroGuiDerm), and the Asia Pacific Association of Allergy, Asthma and Clinical Immunology with the participation of 64 delegates of 50 national and international societies and from 31 countries. The consensus conference was held on 3 December 2020. This guideline was acknowledged and accepted by the European Union of Medical Specialists (UEMS). Urticaria is a frequent, mast cell-driven disease that presents with wheals, angioedema, or both. The lifetime prevalence for acute urticaria is approximately 20%. Chronic spontaneous or inducible urticaria is disabling, impairs quality of life, and affects performance at work and school. This updated version of the international guideline for urticaria covers the definition and classification of urticaria and outlines expert-guided and evidence-based diagnostic and therapeutic approaches for the different subtypes of urticaria

The international EAACI/GA(2)LEN/EuroGuiDerm/APAAACI guideline for the definition, classification, diagnosis, and management of urticaria

Publisher Copyright: © 2021 GA²LEN. Allergy published by European Academy of Allergy and Clinical Immunology and John Wiley & Sons Ltd.This update and revision of the international guideline for urticaria was developed following the methods recommended by Cochrane and the Grading of Recommendations Assessment, Development and Evaluation (GRADE) working group. It is a joint initiative of the Dermatology Section of the European Academy of Allergology and Clinical Immunology (EAACI), the Global Allergy and Asthma European Network (GA(2)LEN) and its Urticaria and Angioedema Centers of Reference and Excellence (UCAREs and ACAREs), the European Dermatology Forum (EDF; EuroGuiDerm), and the Asia Pacific Association of Allergy, Asthma and Clinical Immunology with the participation of 64 delegates of 50 national and international societies and from 31 countries. The consensus conference was held on 3 December 2020. This guideline was acknowledged and accepted by the European Union of Medical Specialists (UEMS). Urticaria is a frequent, mast cell-driven disease that presents with wheals, angioedema, or both. The lifetime prevalence for acute urticaria is approximately 20%. Chronic spontaneous or inducible urticaria is disabling, impairs quality of life, and affects performance at work and school. This updated version of the international guideline for urticaria covers the definition and classification of urticaria and outlines expert-guided and evidence-based diagnostic and therapeutic approaches for the different subtypes of urticaria.Peer reviewe

Genomic copy number variation in Mus musculus.

BACKGROUND: Copy number variation is an important dimension of genetic diversity and has implications in development and disease. As an important model organism, the mouse is a prime candidate for copy number variant (CNV) characterization, but this has yet to be completed for a large sample size. Here we report CNV analysis of publicly available, high-density microarray data files for 351 mouse tail samples, including 290 mice that had not been characterized for CNVs previously. RESULTS: We found 9634 putative autosomal CNVs across the samples affecting 6.87% of the mouse reference genome. We find significant differences in the degree of CNV uniqueness (single sample occurrence) and the nature of CNV-gene overlap between wild-caught mice and classical laboratory strains. CNV-gene overlap was associated with lipid metabolism, pheromone response and olfaction compared to immunity, carbohydrate metabolism and amino-acid metabolism for wild-caught mice and classical laboratory strains, respectively. Using two subspecies of wild-caught Mus musculus, we identified putative CNVs unique to those subspecies and show this diversity is better captured by wild-derived laboratory strains than by the classical laboratory strains. A total of 9 genic copy number variable regions (CNVRs) were selected for experimental confirmation by droplet digital PCR (ddPCR). CONCLUSION: The analysis we present is a comprehensive, genome-wide analysis of CNVs in Mus musculus, which increases the number of known variants in the species and will accelerate the identification of novel variants in future studies

Phase 3 trials of ixekizumab in moderate-to-severe plaque psoriasis

BACKGROUND Two phase 3 trials (UNCOVER-2 and UNCOVER-3) showed that at 12 weeks of treatment, ixekizumab, a monoclonal antibody against interleukin-17A, was superior to placebo and etanercept in the treatment of moderate-to-severe psoriasis. We report the 60-week data from the UNCOVER-2 and UNCOVER-3 trials, as well as 12-week and 60-week data from a third phase 3 trial, UNCOVER-1. METHODS We randomly assigned 1296 patients in the UNCOVER-1 trial, 1224 patients in the UNCOVER-2 trial, and 1346 patients in the UNCOVER-3 trial to receive subcutaneous injections of placebo (placebo group), 80 mg of ixekizumab every 2 weeks after a starting dose of 160 mg (2-wk dosing group), or 80 mg of ixekizumab every 4 weeks after a starting dose of 160 mg (4-wk dosing group). Additional cohorts in the UNCOVER-2 and UNCOVER-3 trials were randomly assigned to receive 50 mg of etanercept twice weekly. At week 12 in the UNCOVER-3 trial, the patients entered a long-term extension period during which they received 80 mg of ixekizumab every 4 weeks through week 60; at week 12 in the UNCOVER-1 and UNCOVER-2 trials, the patients who had a response to ixekizumab (defined as a static Physicians Global Assessment [sPGA] score of 0 [clear] or 1 [minimal psoriasis]) were randomly reassigned to receive placebo, 80 mg of ixekizumab every 4 weeks, or 80 mg of ixekizumab every 12 weeks through week 60. Coprimary end points were the percentage of patients who had a score on the sPGA of 0 or 1 and a 75% or greater reduction from baseline in Psoriasis Area and Severity Index (PASI 75) at week 12. RESULTS In the UNCOVER-1 trial, at week 12, the patients had better responses to ixekizumab than to placebo; in the 2-wk dosing group, 81.8% had an sPGA score of 0 or 1 and 89.1% had a PASI 75 response; in the 4-wk dosing group, the respective rates were 76.4% and 82.6%; and in the placebo group, the rates were 3.2% and 3.9% (P<0.001 for all comparisons of ixekizumab with placebo). In the UNCOVER-1 and UNCOVER-2 trials, among the patients who were randomly reassigned at week 12 to receive 80 mg of ixekizumab every 4 weeks, 80 mg of ixekizumab every 12 weeks, or placebo, an sPGA score of 0 or 1 was maintained by 73.8%, 39.0%, and 7.0% of the patients, respectively. Patients in the UNCOVER-3 trial received continuous treatment of ixekizumab from weeks 0 through 60, and at week 60, at least 73% had an sPGA score of 0 or 1 and at least 80% had a PASI 75 response. Adverse events reported during ixekizumab use included neutropenia, candidal infections, and inflammatory bowel disease. CONCLUSIONS In three phase 3 trials involving patients with psoriasis, ixekizumab was effective through 60 weeks of treatment. As with any treatment, the benefits need to be weighed against the risks of adverse events. The efficacy and safety of ixekizumab beyond 60 weeks of treatment are not yet known