Active site variants in STT3A cause a dominant type I congenital disorder of glycosylation with neuromusculoskeletal findings.

Congenital disorders of glycosylation (CDGs) form a group of rare diseases characterized by hypoglycosylation. We here report the identification of 16 individuals from nine families who have either inherited or de novo heterozygous missense variants in STT3A, leading to an autosomal-dominant CDG. STT3A encodes the catalytic subunit of the STT3A-containing oligosaccharyltransferase (OST) complex, essential for protein N-glycosylation. Affected individuals presented with variable skeletal anomalies, short stature, macrocephaly, and dysmorphic features; half had intellectual disability. Additional features included increased muscle tone and muscle cramps. Modeling of the variants in the 3D structure of the OST complex indicated that all variants are located in the catalytic site of STT3A, suggesting a direct mechanistic link to the transfer of oligosaccharides onto nascent glycoproteins. Indeed, expression of STT3A at mRNA and steady-state protein level in fibroblasts was normal, while glycosylation was abnormal. In S. cerevisiae, expression of STT3 containing variants homologous to those in affected individuals induced defective glycosylation of carboxypeptidase Y in a wild-type yeast strain and expression of the same mutants in the STT3 hypomorphic stt3-7 yeast strain worsened the already observed glycosylation defect. These data support a dominant pathomechanism underlying the glycosylation defect. Recessive mutations in STT3A have previously been described to lead to a CDG. We present here a dominant form of STT3A-CDG that, because of the presence of abnormal transferrin glycoforms, is unusual among dominant type I CDGs.

A patient-based medaka alg2 mutant as a model for hypo-N-glycosylation.

Defects in the evolutionarily conserved protein-glycosylation machinery during embryonic development are often fatal. Consequently, congenital disorders of glycosylation (CDG) in human are rare. We modelled a putative hypomorphic mutation described in an alpha-1,3/1,6-mannosyltransferase (ALG2) index patient (ALG2-CDG) to address the developmental consequences in the teleost medaka (Oryzias latipes). We observed specific, multisystemic, late-onset phenotypes, closely resembling the patient's syndrome, prominently in the facial skeleton and in neuronal tissue. Molecularly, we detected reduced levels of N-glycans in medaka and in the patient's fibroblasts. This hypo-N-glycosylation prominently affected protein abundance. Proteins of the basic glycosylation and glycoprotein-processing machinery were over-represented in a compensatory response, highlighting the regulatory topology of the network. Proteins of the retinal phototransduction machinery, conversely, were massively under-represented in the alg2 model. These deficiencies relate to a specific failure to maintain rod photoreceptors, resulting in retinitis pigmentosa characterized by the progressive loss of these photoreceptors. Our work has explored only the tip of the iceberg of N-glycosylation-sensitive proteins, the function of which specifically impacts on cells, tissues and organs. Taking advantage of the well-described human mutation has allowed the complex interplay of N-glycosylated proteins and their contribution to development and disease to be addressed.

Assessing carnosinase 1 activity for diagnosing congenital disorders of glycosylation.

Diagnosing Congenital Disorders of Glycosylation (CDG) is challenging due to clinical heterogeneity and the limited sensitivity of the classic serum transferrin isoelectric focusing (IEF) or capillary zone electrophoresis test. This study investigates the potential of using the glycoprotein carnosinase 1 (CN1) activity as a diagnostic marker for CDG patients. CN1 activity was measured photometrically in serum from 81 genetically confirmed CDG patients and healthy individuals. While the IEF transferrin method detected 77 patients, four remained undetected. In healthy individuals, serum CN1 activity ranged from 0.1 to 6.4 µmol/ml/h depending on age, with mean CN1 activities up to four-fold higher than in CDG patients. CDG patients´ CN1 activities never exceeded 2,04 µmol/ml/h. Using the 25th percentile to differentiate between groups, the test performance varied by age. For children over 10 years old, the sensitivity and specificity were 96 % and 83 %, respectively. For those under 10, sensitivity and specificity dropped to 71 % and to 64 %. However, CN1 activity successfully identified three of four patients with normal IEF patterns. Although mean CN1 activity in CDG patients is significantly lower than in healthy controls, the test's reliability for classic CDG diagnosis is limited, as the diagnosis is usually made at a young age. Nevertheless, it is a simple, cost-effective assay that can complement classic tests, especially in settings with limited access to complex methods or for patients with normal transferrin patterns but suspicious for CDG.

[DNVF Memorandum: Objectives and Methods of Physical Activity-Related Health Services Research]. / DNVF Memorandum ­ Ziele und Methoden bewegungsbezogener Versorgungsforschung.

The DNVF Memorandum: Objectives and Methods of Physical Activity-Related Health Services Research summarizes, for the first time, the highly interdisciplinary and interprofessional field of physical activity-based health care in the German healthcare system. In addition to providing a conceptual framework and definition of key measures and concepts in physical activity-related health care research, existing research gaps and needs are identified, and methods for advancing this relatively young field of research are described. A particular focus of this study is the relevant outcome parameters and their standardized assessment using established and valid measurement tools. The memorandum aims to establish a general understanding of the complex subject of promoting physical activity and sports therapy in the context of healthcare, to give an impulse to new research initiatives, and to integrate the currently available strong evidence on the effectiveness of physical activity and exercise into healthcare.

Targeted Proteomics Reveals Quantitative Differences in Low-Abundance Glycosyltransferases of Patients with Congenital Disorders of Glycosylation.

Protein glycosylation is an essential post-translational modification in all domains of life. Its impairment in humans can result in severe diseases named congenital disorders of glycosylation (CDGs). Most of the glycosyltransferases (GTs) responsible for proper glycosylation are polytopic membrane proteins that represent challenging targets in proteomics. We established a multiple reaction monitoring (MRM) assay to comprehensively quantify GTs involved in the processes of N-glycosylation and O- and C-mannosylation in the endoplasmic reticulum. High robustness was achieved by using an enriched membrane protein fraction of isotopically labeled HEK 293T cells as an internal protein standard. The analysis of primary skin fibroblasts from eight CDG type I patients with impaired ALG1, ALG2, and ALG11 genes, respectively, revealed a substantial reduction in the corresponding protein levels. The abundance of the other GTs, however, remained unchanged at the transcript and protein levels, indicating that there is no fail-safe mechanism for the early steps of glycosylation in the endoplasmic reticulum. The established MRM assay was shared with the scientific community via the commonly used open source Skyline software environment, including Skyline Batch for automated data analysis. We demonstrate that another research group could easily reproduce all analysis steps, even while using different LC-MS hardware.

Myeloperoxidase Modulates Inflammation in Generalized Pustular Psoriasis and Additional Rare Pustular Skin Diseases.

Generalized pustular psoriasis (GPP) is a severe multi-systemic inflammatory disease characterized by neutrophilic pustulosis and triggered by pro-inflammatory IL-36 cytokines in skin. While 19%-41% of affected individuals harbor bi-allelic mutations in IL36RN, the genetic cause is not known in most cases. To identify and characterize new pathways involved in the pathogenesis of GPP, we performed whole-exome sequencing in 31 individuals with GPP and demonstrated effects of mutations in MPO encoding the neutrophilic enzyme myeloperoxidase (MPO). We discovered eight MPO mutations resulting in MPO -deficiency in neutrophils and monocytes. MPO mutations, primarily those resulting in complete MPO deficiency, cumulatively associated with GPP (p = 1.85E-08; OR = 6.47). The number of mutant MPO alleles significantly differed between 82 affected individuals and >4,900 control subjects (p = 1.04E-09); this effect was stronger when including IL36RN mutations (1.48E-13) and correlated with a younger age of onset (p = 0.0018). The activity of four proteases, previously implicated as activating enzymes of IL-36 precursors, correlated with MPO deficiency. Phorbol-myristate-acetate-induced formation of neutrophil extracellular traps (NETs) was reduced in affected cells (p = 0.015), and phagocytosis assays in MPO-deficient mice and human cells revealed altered neutrophil function and impaired clearance of neutrophils by monocytes (efferocytosis) allowing prolonged neutrophil persistence in inflammatory skin. MPO mutations contribute significantly to GPP's pathogenesis. We implicate MPO as an inflammatory modulator in humans that regulates protease activity and NET formation and modifies efferocytosis. Our findings indicate possible implications for the application of MPO inhibitors in cardiovascular diseases. MPO and affected pathways represent attractive targets for inducing resolution of inflammation in neutrophil-mediated skin diseases.

Quantification of Dolichyl Phosphates Using Phosphate Methylation and Reverse-Phase Liquid Chromatography-High Resolution Mass Spectrometry.

Dolichyl monophosphates (DolPs) are essential lipids in glycosylation pathways that are highly conserved across almost all domains of life. The availability of DolP is critical for all glycosylation processes, as these lipids serve as membrane-anchored building blocks used by various types of glycosyltransferases to generate complex post-translational modifications of proteins and lipids. The analysis of DolP species by reverse-phase liquid chromatography-mass spectrometry (RPLC-MS) remains a challenge due to their very low abundance and wide range of lipophilicities. Until now, a method for the simultaneous qualitative and quantitative assessment of DolP species from biological membranes has been lacking. Here, we describe a novel approach based on simple sample preparation, rapid and efficient trimethylsilyl diazomethane-dependent phosphate methylation, and RPLC-MS analysis for quantification of DolP species with different isoprene chain lengths. We used this workflow to selectively quantify DolP species from lipid extracts derived of Saccharomyces cerevisiae, HeLa, and human skin fibroblasts from steroid 5-α-reductase 3- congenital disorders of glycosylation (SRD5A3-CDG) patients and healthy controls. Integration of this workflow with global lipidomics analyses will be a powerful tool to expand our understanding of the role of DolPs in pathophysiological alterations of metabolic pathways downstream of HMG-CoA reductase, associated with CDGs, hypercholesterolemia, neurodegeneration, and cancer.

Complex metabolic disharmony in PMM2-CDG paves the way to new therapeutic approaches.

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.

Additional effects of pain neuroscience education combined with physiotherapy on the headache frequency of adult patients with migraine: A randomized controlled trial.

Aim To assess the efficacy of pain neuroscience education combined with physiotherapy for the management of migraine.Background Physiotherapy can significantly reduce the frequency of migraine, but the evidence is based only on a few studies. Pain neuroscience education might pose a promising treatment, as it addresses migraine as a chronic pain disease.Methods In this non-blinded randomized controlled trial, migraine patients received physiotherapy + pain neuroscience education or physiotherapy alone, preceded by a three-month waiting period. Primary outcomes were frequency of headache (with and without migraine features), frequency of migraine and associated disability.Results Eighty-two participants were randomized and analyzed. Both groups showed a decrease of headache frequency (p = 0.02, d = 0.46) at post-treatment (physiotherapy: 0.77 days, 95%CI: -0.75 to 2.29 and physiotherapy + pain neuroscience education: 1.25 days, 95%CI: -0.05 to 2.55) and at follow-up (physiotherapy: 1.93, 95%CI: 0.07 to 3.78 and physiotherapy + pain neuroscience education: 3.48 days, 95%CI: 1.89 to 5.06), with no difference between groups (p = 0.26, d = 0.26). Migraine frequency was reduced significantly in the physiotherapy + pain neuroscience education group, and not in the physiotherapy group, at post-treatment (1.28 days, 95%CI: 0.34 to 2.22, p = 0.004) and follow-up (3.05 days, 95%CI: 1.98 to 5.06, p < 0.0001), with a difference between groups at follow-up (2.06 days, p = 0.003). Migraine-related disability decreased significantly in both groups (physiotherapy: 19.8, physiotherapy + pain neuroscience education: 24.0 points, p < 0.001, d = 1.15) at follow-up, with no difference between groups (p = 0.583). Secondary outcomes demonstrated a significant effect of time with no interaction between time and group. No harm or adverse events were observed during the study.Conclusion In comparison to physiotherapy alone, pain neuroscience education combined with physiotherapy can further reduce the frequency of migraine, but had no additional effect on general headache frequency or migraine-related disability.Trial Registration The study was pre-registered at the German Clinical Trials Register (DRKS00020804).

Smartphone-assisted training with education for patients with hip and/or knee osteoarthritis (SmArt-E): study protocol for a multicentre pragmatic randomized controlled trial.

INTRODUCTION: Hip and knee osteoarthritis are associated with functional limitations, pain and restrictions in quality of life and the ability to work. Furthermore, with growing prevalence, osteoarthritis is increasingly causing (in)direct costs. Guidelines recommend exercise therapy and education as primary treatment strategies. Available options for treatment based on physical activity promotion and lifestyle change are often insufficiently provided and used. In addition, the quality of current exercise programmes often does not meet the changing care needs of older people with comorbidities and exercise adherence is a challenge beyond personal physiotherapy. The main objective of this study is to investigate the short- and long-term (cost-)effectiveness of the SmArt-E programme in people with hip and/or knee osteoarthritis in terms of pain and physical functioning compared to usual care. METHODS: This study is designed as a multicentre randomized controlled trial with a target sample size of 330 patients. The intervention is based on the e-Exercise intervention from the Netherlands, consists of a training and education programme and is conducted as a blended care intervention over 12 months. We use an app to support independent training and the development of self-management skills. The primary and secondary hypotheses are that participants in the SmArt-E intervention will have less pain (numerical rating scale) and better physical functioning (Hip Disability and Osteoarthritis Outcome Score, Knee Injury and Osteoarthritis Outcome Score) compared to participants in the usual care group after 12 and 3 months. Other secondary outcomes are based on domains of the Osteoarthritis Research Society International (OARSI). The study will be accompanied by a process evaluation. DISCUSSION: After a positive evaluation, SmArt-E can be offered in usual care, flexibly addressing different care situations. The desired sustainability and the support of the participants' behavioural change are initiated via the app through audio-visual contact with their physiotherapists. Furthermore, the app supports the repetition and consolidation of learned training and educational content. For people with osteoarthritis, the new form of care with proven effectiveness can lead to a reduction in underuse and misuse of care as well as contribute to a reduction in (in)direct costs. TRIAL REGISTRATION: German Clinical Trials Register, DRKS00028477. Registered on August 10, 2022.