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.

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.

Glycosyltransferase POMGNT1 deficiency strengthens N-cadherin-mediated cell-cell adhesion.

Defects in protein O-mannosylation lead to severe congenital muscular dystrophies collectively known as α-dystroglycanopathy. A hallmark of these diseases is the loss of the O-mannose-bound matriglycan on α-dystroglycan, which reduces cell adhesion to the extracellular matrix. Mutations in protein O-mannose ß1,2-N-acetylglucosaminyltransferase 1 (POMGNT1), which is crucial for the elongation of O-mannosyl glycans, have mainly been associated with muscle-eye-brain (MEB) disease. In addition to defects in cell-extracellular matrix adhesion, aberrant cell-cell adhesion has occasionally been observed in response to defects in POMGNT1. However, specific molecular consequences of POMGNT1 deficiency on cell-cell adhesion are largely unknown. We used POMGNT1 knockout HEK293T cells and fibroblasts from an MEB patient to gain deeper insight into the molecular changes in POMGNT1 deficiency. Biochemical and molecular biological techniques combined with proteomics, glycoproteomics, and glycomics revealed that a lack of POMGNT1 activity strengthens cell-cell adhesion. We demonstrate that the altered intrinsic adhesion properties are due to an increased abundance of N-cadherin (N-Cdh). In addition, site-specific changes in the N-glycan structures in the extracellular domain of N-Cdh were detected, which positively impact on homotypic interactions. Moreover, in POMGNT1-deficient cells, ERK1/2 and p38 signaling pathways are activated and transcriptional changes that are comparable with the epithelial-mesenchymal transition (EMT) are triggered, defining a possible molecular mechanism underlying the observed phenotype. Our study indicates that changes in cadherin-mediated cell-cell adhesion and other EMT-related processes may contribute to the complex clinical symptoms of MEB or α-dystroglycanopathy in general and suggests that the impact of changes in O-mannosylation on N-glycosylation has been underestimated.

The Metabolic Map into the Pathomechanism and Treatment of PGM1-CDG.

Phosphoglucomutase 1 (PGM1) encodes the metabolic enzyme that interconverts glucose-6-P and glucose-1-P. Mutations in PGM1 cause impairment in glycogen metabolism and glycosylation, the latter manifesting as a congenital disorder of glycosylation (CDG). This unique metabolic defect leads to abnormal N-glycan synthesis in the endoplasmic reticulum (ER) and the Golgi apparatus (GA). On the basis of the decreased galactosylation in glycan chains, galactose was administered to individuals with PGM1-CDG and was shown to markedly reverse most disease-related laboratory abnormalities. The disease and treatment mechanisms, however, have remained largely elusive. Here, we confirm the clinical benefit of galactose supplementation in PGM1-CDG-affected individuals and obtain significant insights into the functional and biochemical regulation of glycosylation. We report here that, by using tracer-based metabolomics, we found that galactose treatment of PGM1-CDG fibroblasts metabolically re-wires their sugar metabolism, and as such replenishes the depleted levels of galactose-1-P, as well as the levels of UDP-glucose and UDP-galactose, the nucleotide sugars that are required for ER- and GA-linked glycosylation, respectively. To this end, we further show that the galactose in UDP-galactose is incorporated into mature, de novo glycans. Our results also allude to the potential of monosaccharide therapy for several other CDG.

Missense variant c.1460 T > C (p.L487P) enhances protein degradation of ER mannosyltransferase ALG9 in two new ALG9-CDG patients presenting with West syndrome and review of the literature.

ALG9-CDG is a CDG-I defect within the group of Congenital Disorders of Glycosylation (CDG). We here describe the clinical symptoms of two new and unrelated ALG9-CDG patients, both carrying the novel homozygous missense variant c.1460 T > C (p.L487P) in the ALG9 gene which led to global developmental delay, psychomotor disability, facial dysmorphisms, brain and heart defects, hearing loss, hypotonia, as well as feeding problems. New clinical symptoms comprised West syndrome with hypsarrhythmia. Quantitative RT-PCR analysis revealed a significantly enhanced ALG9 mRNA transcript level, whereas the protein amount in fibroblasts was significantly reduced. This could be ascribed to a stronger degradation of the mutated ALG9 protein in patient fibroblasts. Lipid-linked oligosaccharide analysis showed an ALG9-CDG characteristic accumulation of Man6GlcNAc2-PP-dolichol and Man8GlcNAc2-PP-dolichol in patient cells. The clinical findings of our patients and of all previously published ALG9-CDG patients are brought together to further expand the knowledge about this rare N-glycosylation disorder. SYNOPSIS: Homozygosity for p.L487P in ALG9 causes protein degradation and leads to West syndrome.

Manifestation of epilepsy in a patient with EED-related overgrowth (Cohen-Gibson syndrome).

Cohen-Gibson syndrome is a rare genetic disorder, characterized by fetal or early childhood overgrowth and mild to severe intellectual disability. It is caused by heterozygous aberrations in EED, which encodes an evolutionary conserved polycomb group (PcG) protein that forms the polycomb repressive complex-2 (PRC2) together with EZH2, SUZ12, and RBBP7/4. In total, 11 affected individuals with heterozygous pathogenic variants in EED were reported, so far. All variants affect a few key residues within the EED WD40 repeat domain. By trio exome sequencing, we identified the heterozygous missense variant c.581A > G, p.(Asn194Ser) in exon 6 of the EED-gene in an individual with moderate intellectual disability, overgrowth, and epilepsy. The same pathogenic variant was detected in 2 of the 11 previously reported cases. Epilepsy, however, was only diagnosed in one other individual with Cohen-Gibson syndrome before. Our findings further confirm that the WD40 repeat domain represents a mutational hotspot; they also expand the clinical spectrum of Cohen-Gibson syndrome and highlight the clinical variability even in individuals with the same pathogenic variant. Furthermore, they indicate a possible association between Cohen-Gibson syndrome and epilepsy.

Synergistic use of glycomics and single-molecule molecular inversion probes for identification of congenital disorders of glycosylation type-1.

Congenital disorders of glycosylation type 1 (CDG-I) comprise a group of 27 genetic defects with heterogeneous multisystem phenotype, mostly presenting with nonspecific neurological symptoms. The biochemical hallmark of CDG-I is a partial absence of complete N-glycans on transferrin. However, recent findings of a diagnostic N-tetrasaccharide for ALG1-CDG and increased high-mannose N-glycans for a few other CDG suggested the potential of glycan structural analysis for CDG-I gene discovery. We analyzed the relative abundance of total plasma N-glycans by high resolution quadrupole time-of-flight mass spectrometry in a large cohort of 111 CDG-I patients with known (n = 75) or unsolved (n = 36) genetic cause. We designed single-molecule molecular inversion probes (smMIPs) for sequencing of CDG-I candidate genes on the basis of specific N-glycan signatures. Glycomics profiling in patients with known defects revealed novel features such as the N-tetrasaccharide in ALG2-CDG patients and a novel fucosylated N-pentasaccharide as specific glycomarker for ALG1-CDG. Moreover, group-specific high-mannose N-glycan signatures were found in ALG3-, ALG9-, ALG11-, ALG12-, RFT1-, SRD5A3-, DOLK-, DPM1-, DPM3-, MPDU1-, ALG13-CDG, and hereditary fructose intolerance. Further differential analysis revealed high-mannose profiles, characteristic for ALG12- and ALG9-CDG. Prediction of candidate genes by glycomics profiling in 36 patients with thus far unsolved CDG-I and subsequent smMIPs sequencing led to a yield of solved cases of 78% (28/36). Combined plasma glycomics profiling and targeted smMIPs sequencing of candidate genes is a powerful approach to identify causative mutations in CDG-I patient cohorts.

Standardized procedure prevents perioperative and early complications in totally implantable venous-access ports-a complication analysis of more than 1000 TIVAP implantations.

PURPOSE: Since their invention 40 years ago, totally implantable venous-access ports (TIVAPs) have become indispensable in cancer treatment. The aim of our study was to analyze complications under standardized operative and perioperative procedures and to identify risk factors for premature port catheter explantation. METHODS: A total of 1008 consecutive TIVAP implantations were studied for success rate, perioperative, early, and late complications. Surgical, clinical, and demographic factors were analyzed as potential risk factors for emergency port catheter explantation. RESULTS: Successful surgical TIVAP implantation was achieved in 1005/1008 (99.7%) cases. No intraoperative or perioperative complications occurred. A total of 32 early complications and 88 late complications were observed leading to explantation in 11/32 (34.4%) and 34/88 (38.6%) cases, respectively. The most common complications were infections in 4.7% followed by thrombosis in 3.6%. Parameters that correlated with unplanned TIVAP explantation were gender (port in situ: female 95% vs. male 91%, p = 0.01), underlying disease (breast cancer 97% vs. gastrointestinal 89%, p = 0.004), indication (chemotherapy 95% vs. combination of chemotherapy and parenteral nutrition 64%, p < 0.0001), and type of complication (infection 13.4% vs. TIVAP-related complication 54% and thrombosis 95%, p < 0.0001). CONCLUSION: Standardized operative and perioperative TIVAP implantation procedures provide excellent results and low explantation rate.

Feasibility of smartphone-supported, combined physical and cognitive activities in the Neighbourhood for stimulating social participation of the elderly.

BACKGROUND: Combining smartphone-assisted group activities in the neighbourhood and training in physical and cognitive skills may offer the potential to promote social participation and connectedness of older adults. This non-controlled proof-of-concept, retrospectively registered study aimed to determine the feasibility of such an intervention approach, including its evaluation. METHODS: In two consecutive six-month intervention cycles, 39 community-dwelling adults were provided with weekly smartphone, physical and cognitive training by two tutors. Using a specifically designed app, the participants were also encouraged to join and later self-organise physically and cognitively stimulating activities related to hot spots in their Bochum neighbourhood. Indicators of feasibility were documented. RESULTS: The recruitment and assessments took 3 hours per participant. Excluding smartphone support, the preparation and the implementation of the intervention amounted to nine person-hours per week. Six participants dropped out, and 13 did not complete one or more assessments. The participants attended 76 ± 15% of the weekly training sessions. The instructors deemed the programme feasible, but familiarisation with the smartphone and the app was very time-consuming. Twenty-seven of 29 participants reported high overall satisfaction, and 22 agreed that the programme helped them to establish social contacts. The smartphones attracted substantial interest and were used frequently, despite mixed satisfaction with the project-specific app. From baseline to follow-up, the six-minute walking distance, lower extremity strength and moderate to vigorous physical activity, as well as quality of life, were preserved at a high level, while balance performance was significantly improved. Of the 11 tests related to cognitive functioning, 4 tests (a memory test, the Stroop test and 2 tests of verbal fluency) indicated significant improvement. No moderate or serious adverse events occurred in relation to the assessments or the intervention. CONCLUSIONS: The multimodal approach seems safe and feasible and offers the potential to promote social connectedness, bonds in the residential neighbourhood and smartphone competency, as well as to preserve or improve physical and cognitive functions. Adaptations of the intervention and of the outcome assessments may contribute to better assessment and exploitation of the potential of this approach in a future study involving socially, physically and cognitively less active elderly persons.

A multifactorial interdisciplinary intervention to prevent functional and mobility decline for more participation in (pre-)frail community-dwelling older adults (PromeTheus): study protocol for a multicenter randomized controlled trial.

BACKGROUND: Age-related decline in physical capacity can lead to frailty, associated with an increased vulnerability to adverse health outcomes and greater healthcare utilization. In an aging population, effective strategies to prevent physical decline and frailty, and preserve independence are needed. Prevention programs for vulnerable community-dwelling older adults are, however, often not yet established and implemented in routine practice. Research on the feasibility, implementation, and (cost-)effectiveness of multifactorial, interdisciplinary intervention programs that take advantage of available services of healthcare providers is also limited. The main aim of this study is to evaluate the effectiveness of such an intervention program (PromeTheus) to prevent functional and mobility decline for more participation in community-dwelling (pre-)frail older adults. METHODS: The study is designed as a three-center, randomized controlled trial with a 12-month intervention period. Four hundred community-dwelling (pre-)frail (Clinical Frailty Scale score 4-6) older adults (≥70 years) will be randomized in a 1:1 ratio to the intervention group (IG) or the control group (CG). The IG will receive the PromeTheus program consisting of obligatory home-based physical exercises (Weight-bearing Exercise for Better Balance) accompanied by physiotherapists and facultative counseling services (person-environment-fit, coping with everyday life, nutrition, group-based activities) delivered via existing healthcare structures (e.g., social workers, nutritionists). The CG will receive usual care and a one-time counseling session on recommendations for physical activity and nutrition. Primary outcomes assessed at months 6 and 12 are the function component of the Late-Life Function and Disability Instrument and the University of Alabama at Birmingham Life-Space Assessment. Secondary outcomes are disability, physical capacity and activity, frailty, nutritional status, falls, fear of falling, health status, and psychosocial components. Process and economic evaluations are also conducted. Primary statistical analyses will be based on the intention-to-treat principle. DISCUSSION: Compared to usual care, the PromeTheus program is expected to result in higher function and mobility, greater independence and lower need for care, and more participation. As the PromeTheus program draws on existing German healthcare structures, its large-scale translation and delivery will be feasible, if evidence of (cost-)effectiveness and successful implementation can be demonstrated. TRIAL REGISTRATION: German Clinical Trials Register, . Registered on March 11, 2021.

Mutations in PIK3C2A cause syndromic short stature, skeletal abnormalities, and cataracts associated with ciliary dysfunction.

PIK3C2A is a class II member of the phosphoinositide 3-kinase (PI3K) family that catalyzes the phosphorylation of phosphatidylinositol (PI) into PI(3)P and the phosphorylation of PI(4)P into PI(3,4)P2. At the cellular level, PIK3C2A is critical for the formation of cilia and for receptor mediated endocytosis, among other biological functions. We identified homozygous loss-of-function mutations in PIK3C2A in children from three independent consanguineous families with short stature, coarse facial features, cataracts with secondary glaucoma, multiple skeletal abnormalities, neurological manifestations, among other findings. Cellular studies of patient-derived fibroblasts found that they lacked PIK3C2A protein, had impaired cilia formation and function, and demonstrated reduced proliferative capacity. Collectively, the genetic and molecular data implicate mutations in PIK3C2A in a new Mendelian disorder of PI metabolism, thereby shedding light on the critical role of a class II PI3K in growth, vision, skeletal formation and neurological development. In particular, the considerable phenotypic overlap, yet distinct features, between this syndrome and Lowe's syndrome, which is caused by mutations in the PI-5-phosphatase OCRL, highlight the key role of PI metabolizing enzymes in specific developmental processes and demonstrate the unique non-redundant functions of each enzyme. This discovery expands what is known about disorders of PI metabolism and helps unravel the role of PIK3C2A and class II PI3Ks in health and disease.

A new bipolar device for sealing and cutting: ex and in vivo studies for performance evaluation.

INTRODUCTION: A novel multipurpose bipolar radiofrequency instrument, the Erbe Dissector (EDS), which simultaneously seals and cuts tissue, was developed. Ex vivo sealing rate and time, burst pressure, jaw temperature and thermal spread were studied in porcine renal arteries. MATERIAL AND METHODS: In vivo, 13 surgical tasks were performed in two pigs: beside sealing rate and time, overall performance in sharp and blunt dissection, tissue sticking, hemostasis, precision, etc., were evaluated by four surgeons compared with ENSEAL G2 (EG2) using surveys on a Likert scale (1 = very poor; 5 = very good). RESULTS: Ex vivo, the EDS sealing rate was 91.7% (33/36 arteries) at an average sealing time of 2.1 s (range 1.7-2.8) and a burst pressure of 1040 ± 350 mmHg. The maximum jaw temperature was 87 ± 4 °C and the mean lateral thermal spread was 0.8 ± 0.2 mm. In vivo, the sealing rate for arteries and veins was 92.6% (50/54) and the median seal and cut time was 1.6 s (range: 1.3-2.9). The average EDS performance score across all tasks was 4.4 ± 0.6 Likert points. For five shared tasks, EDS was better than EG2 (4.4 ± 0.5 versus 3.4 ± 0.6 Likert points; p = 0.016). CONCLUSIONS: EDS seals and cuts arteries and veins rapidly with good safety and user-friendliness.

Mutations in the BAF-Complex Subunit DPF2 Are Associated with Coffin-Siris Syndrome.

Variants affecting the function of different subunits of the BAF chromatin-remodelling complex lead to various neurodevelopmental syndromes, including Coffin-Siris syndrome. Furthermore, variants in proteins containing PHD fingers, motifs recognizing specific histone tail modifications, have been associated with several neurological and developmental-delay disorders. Here, we report eight heterozygous de novo variants (one frameshift, two splice site, and five missense) in the gene encoding the BAF complex subunit double plant homeodomain finger 2 (DPF2). Affected individuals share common clinical features described in individuals with Coffin-Siris syndrome, including coarse facial features, global developmental delay, intellectual disability, speech impairment, and hypoplasia of fingernails and toenails. All variants occur within the highly conserved PHD1 and PHD2 motifs. Moreover, missense variants are situated close to zinc binding sites and are predicted to disrupt these sites. Pull-down assays of recombinant proteins and histone peptides revealed that a subset of the identified missense variants abolish or impaire DPF2 binding to unmodified and modified H3 histone tails. These results suggest an impairment of PHD finger structural integrity and cohesion and most likely an aberrant recognition of histone modifications. Furthermore, the overexpression of these variants in HEK293 and COS7 cell lines was associated with the formation of nuclear aggregates and the recruitment of both wild-type DPF2 and BRG1 to these aggregates. Expression analysis of truncating variants found in the affected individuals indicated that the aberrant transcripts escape nonsense-mediated decay. Altogether, we provide compelling evidence that de novo variants in DPF2 cause Coffin-Siris syndrome and propose a dominant-negative mechanism of pathogenicity.

Making sense of missense variants in TTN-related congenital myopathies.

Mutations in the sarcomeric protein titin, encoded by TTN, are emerging as a common cause of myopathies. The diagnosis of a TTN-related myopathy is, however, often not straightforward due to clinico-pathological overlap with other myopathies and the prevalence of TTN variants in control populations. Here, we present a combined clinico-pathological, genetic and biophysical approach to the diagnosis of TTN-related myopathies and the pathogenicity ascertainment of TTN missense variants. We identified 30 patients with a primary TTN-related congenital myopathy (CM) and two truncating variants, or one truncating and one missense TTN variant, or homozygous for one TTN missense variant. We found that TTN-related myopathies show considerable overlap with other myopathies but are strongly suggested by a combination of certain clinico-pathological features. Presentation was typically at birth with the clinical course characterized by variable progression of weakness, contractures, scoliosis and respiratory symptoms but sparing of extraocular muscles. Cardiac involvement depended on the variant position. Our biophysical analyses demonstrated that missense mutations associated with CMs are strongly destabilizing and exert their effect when expressed on a truncating background or in homozygosity. We hypothesise that destabilizing TTN missense mutations phenocopy truncating variants and are a key pathogenic feature of recessive titinopathies that might be amenable to therapeutic intervention.