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Objectives: To evaluate a virtual reality (VR) application for gestational sac fluid volume (GSFV) measurements in first-trimester pregnancies and to study the correlation between different embryonic growth parameters. Methods: This was a prospective cohort study analyzing 180 three-dimensional (3D) ultrasound scans of 42 healthy women, performed between 5 + 5 and 12 + 6 weeks' gestational age (GA). The 3D datasets were transferred to the I-Space immersive VR system. The V-Scope application was used to create a 'hologram' of the ultrasound image, allowing depth perception and interaction with the rendered objects. Volumes were measured semi-automatically using a segmentation algorithm. In addition to the GSFV, the total gestational sac volume (GSV) and its diameter (GSD) were measured. The GSV was also calculated using the ellipsoid formula. Previously obtained measurements of embryonic volume and crown-rump length (CRL) were included in the study. The outcomes were analyzed using repeated-measures analysis of variance. Results: The GSFV was measured in 78 scans, and varied from 434 to 81 491 mm(3). A positive correlation between GSFV and GA, CRL and GSD was found. Comparison of the GSD formula constructed in our study in relation to GA with a formula that is commonly used clinically showed an increasing difference with increasing GA either side of 8 + 5 weeks. The GSFV/embryonic volume ratio showed a decrease with GA. The GSV calculated using the ellipsoid formula was on average 19.8% larger compared with the GSV measured in VR. Conclusion: New charts for first-trimester GSFV were constructed using VR. These growth charts could be promising tools for studying normal and abnormal embryonic development. [ABSTRACT FROM AUTHOR]

Testicular germ cell tumours (GCTs) of adolescents and adults can be subdivided into seminomas (referred to as dysgerminomas of the ovary) and non-seminomas, all referred to as type II GCTs. They originate from carcinoma in situ (CIS), being the malignant counterparts of primordial germ cells (PGCs)/gonocytes. The invasive components mimic embryogenesis, including the stem cell component embryonal carcinoma (EC), the somatic lineage teratoma (TE), and the extra-embryonic tissues yolk sac tumour (YST) and choriocarcinoma (CH). The other type is the so-called spermatocytic seminomas (SS, type III GCT), composed of neoplastic primary spermatocytes. We reported previously that the miRNAs hsa-miR 371-373 cluster is involved in overruling cellular senescence induced by oncogenic stress, allowing cells to become malignant. Here we report the first high-throughput screen of 156 microRNAs in a series of type II and III GCTs ( n = 69, in duplicate) using a quantitative PCR-based approach. After normalization to allow inter-sample analysis, the technical replicates clustered together, and the previous hsa-miRNA 371-373 cluster finding was confirmed. Unsupervised cluster analysis demonstrated that the cell lines are different from the in vivo samples. The in vivo samples, both normal and malignant, clustered predominantly based on their maturation status. This parallels normal embryogenesis, rather than chromosomal anomalies in the tumours. miRNAs within a single cluster showed a similar expression pattern, implying common regulatory mechanisms. Normal testicular tissue expressed most discriminating miRNAs at a higher level than SE and SS. Moreover, differentiated non-seminomas showed overexpression of discriminating miRNAs. These results support the model that miRNAs are involved in regulating differentiation of stem cells, retained in GCTs. Copyright © 2007 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Refractive error (RE) and myopia are complex polygenic conditions with the majority of genome-wide associated genetic variants in non-exonic regions. Given this, and the onset during childhood, gene-regulation is expected to play an important role in its pathogenesis. This prompted us to explore beyond traditional gene finding approaches. We performed a genetic association study between variants in non-coding RNAs and enhancers, and RE and myopia. We obtained single-nucleotide polymorphisms (SNPs) in microRNA (miRNA) genes, miRNA-binding sites, long non-coding RNAs genes (lncRNAs) and enhancers from publicly available databases: miRNASNPv2, PolymiRTS, VISTA Enhancer Browser, FANTOM5 and lncRNASNP2. We investigated whether SNPs overlapping these elements were associated with RE and myopia leveraged from a large GWAS meta-analysis (N = 160,420). With genetic risk scores (GRSs) per element, we investigated the joint effect of associated variants on RE, axial length (AL)/corneal radius (CR), and AL progression in an independent child cohort, the Generation R Study (N = 3638 children). We constructed a score for biological plausibility per SNP in highly confident miRNA-binding sites and enhancers in chromatin accessible regions. We found that SNPs in two miRNA genes, 14 enhancers and 81 lncRNA genes in chromatin accessible regions and 54 highly confident miRNA-binding sites, were in RE and myopia-associated loci. GRSs from SNPs in enhancers were significantly associated with RE, AL/CR and AL progression. GRSs from lncRNAs were significantly associated with all AL/CR and AL progression. GRSs from miRNAs were not associated with any ocular biometric measurement. GRSs from miRNA-binding sites showed suggestive but inconsistent significance. We prioritized candidate miRNA binding sites and candidate enhancers for future functional validation. Pathways of target and host genes of highly ranked variants included eye development (BMP4, MPPED2), neurogenesis (DDIT4, NTM), extracellular matrix (ANTXR2, BMP3), photoreceptor metabolism (DNAJB12), photoreceptor morphogenesis (CHDR1), neural signaling (VIPR2) and TGF-beta signaling (ANAPC16). This is the first large-scale study of non-coding RNAs and enhancers for RE and myopia. Enhancers and lncRNAs could be of large importance as they are associated with childhood myopia. We provide a confident blueprint for future functional validation by prioritizing candidate miRNA binding sites and candidate enhancers., Competing Interests: Declarations. Conflict of interest: The authors declare no competing interests., (© 2025. The Author(s).)

Introduction: Acute onset of severe psychiatric symptoms or regression may occur in children with premorbid neurodevelopmental disorders, although typically developing children can also be affected. Infections or other stressors are likely triggers. The underlying causes are unclear, but a current hypothesis suggests the convergence of genes that influence neuronal and immunological function. We previously identified 11 genes in pediatric acute-onset neuropsychiatric syndrome (PANS), in which two classes of genes related to either synaptic function or the immune system were found. Among the latter, three affect the DNA damage response (DDR): PPM1D, CHK2, and RAG1. We now report an additional 17 cases with mutations in PPM1D and other DDR genes in patients with acute onset of psychiatric symptoms and/or regression that their clinicians classified as PANS or another inflammatory brain condition., Methods: We analyzed genetic findings obtained from parents and carried out whole-exome sequencing on a total of 17 cases, which included 3 sibling pairs and a family with 4 affected children., Results: The DDR genes include clusters affecting p53 DNA repair (PPM1D, ATM, ATR, 53BP1, and RMRP), and the Fanconi Anemia Complex (FANCE, SLX4/FANCP, FANCA, FANCI, and FANCC). We hypothesize that defects in DNA repair genes, in the context of infection or other stressors, could contribute to decompensated states through an increase in genomic instability with a concomitant accumulation of cytosolic DNA in immune cells triggering DNA sensors, such as cGAS-STING and AIM2 inflammasomes, as well as central deficits on neuroplasticity. In addition, increased senescence and defective apoptosis affecting immunological responses could be playing a role., Conclusion: These compelling preliminary findings motivate further genetic and functional characterization as the downstream impact of DDR deficits may point to novel treatment strategies., (© 2024 The Author(s). Published by S. Karger AG, Basel.)

Immunological aetiology is supported for a subgroup with obsessive compulsive disorder (OCD) and conceptualized as autoimmune OCD. The longitudinal clinical course is detailed for three severely ill cases with OCD and indications of immunological involvement with off-label rituximab treatment every six months. All cases showed clear and sustained gains regarding symptom burden and function for over 2.5 years. Brief Psychiatric Rating Scale and Yale-Brown Obsessive-Compulsive Inventory Scale scores decreased 67-100% and 44-92%, respectively. These complex cases, prior to rituximab, had very low functioning and disease duration has been eight, nine and 16 years respectively. All three patients had been unsuccessfully treated with at least two antidepressants or anxiolytics, one neuroleptic and cognitive behavioural therapy. Clinical phenotypes and findings were suggestive of possible autoimmune OCD. Indirect immunohistochemistry detected cerebral spinal fluid (CSF) antibodies in all three cases including a novel anti-neuronal staining pattern against mouse thalamic cells. Exploratory analyses of CSF markers and proteomics identified elevated levels of sCD27 and markers indicative of complement pathway activation when compared to CSF from healthy controls. Multidisciplinary collaboration, advanced clinical investigations and rituximab treatment are feasible in a psychiatric setting. The case histories provide a proof of principle for the newly proposed criteria for autoimmune OCD. The findings suggest that clinical red flags and biological measures may predict rituximab response in chronic treatment-resistant OCD. The report provides orientation that may inform the hypotheses and design of future treatment trials., (© 2024. The Author(s).)

STAT3 gain-of-function (GOF) variants results in a heterogeneous clinical syndrome characterized by early onset immunodeficiency, multi-organ autoimmunity, and lymphoproliferation. While 191 documented cases with STAT3 GOF variants have been reported, the impact of individual variants on immune regulation and the broad clinical spectrum remains unclear. We developed a Stat3 p.L387R mouse model, mirroring a variant identified in a family exhibiting common STAT3 GOF symptoms, and rare phenotypes including pulmonary hypertension and retinal vasculitis. In vitro experiments revealed increased STAT3 phosphorylation, nuclear migration, and DNA binding of the variant. Our Stat3 p.L387R model displayed similar traits from previous Stat3 GOF strains, such as splenomegaly and lymphadenopathy. Notably, Stat3 p.L387R/+ mice exhibited heightened embryonic lethality compared to prior Stat3 GOF/+ models and ocular abnormalities were observed. This research underscores the variant-specific pathology in Stat3 p.L387R/+ mice, highlighting the ability to recapitulate human STAT3 GOF syndrome in patient-specific transgenic murine models. Additionally, such models could facilitate tailored treatment development., Competing Interests: Declaration of competing interest There is no conflict of interest to declare., (Copyright © 2024 Erasmus Medical Center. Published by Elsevier Inc. All rights reserved.)

Objective: Heart failure (HF) pathophysiology in patients with obesity may be distinct. To study these features, we identified obesity-related biomarkers from 4210 circulating proteins in patients with HF with reduced ejection fraction (HFrEF) and examined associations of these proteins with HF prognosis and biological mechanisms., Methods: In 373 patients with trimonthly blood sampling during a median follow-up of 2.1 (25th-75th percentile: 1.1-2.6) years, we applied an aptamer-based multiplex approach measuring 4210 proteins in baseline samples and the last two samples before study end. Associations between obesity (BMI > 30 kg/m 2 ) and baseline protein levels were analyzed. Subsequently, associations of serially measured obesity-related proteins with biological mechanisms and the primary endpoint (PEP; composite of cardiovascular mortality, HF hospitalization, left ventricular assist device implantation, and heart transplantation) were examined., Results: Obesity was identified in 26% (96/373) of patients. A total of 30% (112/373) experienced a PEP (with obesity: 26% [25/96] vs. without obesity: 31% [87/277]). A total of 141/4210 proteins were linked to obesity, reflecting mechanisms of neuron projection development, cell adhesion, and muscle cell migration. A total of 50/141 proteins were associated with the PEP, of which 12 proteins related to atherosclerosis or hypertrophy provided prognostic information beyond clinical characteristics, N-terminal pro-B-type natriuretic peptide, and high-sensitivity troponin T., Conclusions: Patients with HFrEF and obesity show distinct proteomic profiles compared to patients with HFrEF without obesity. Obesity-related proteins are independently associated with HF outcome. These proteins carry potential to improve management of obesity-related HF and could be leads for future research., (© 2024 The Author(s). Obesity published by Wiley Periodicals LLC on behalf of The Obesity Society.)

Introduction: Sarcoidosis is a multi-system inflammatory disease of unknown origin with heterogeneous clinical manifestations varying from a single organ non-caseating granuloma site to chronic systemic inflammation and fibrosis. Gene expression studies have suggested several genes and pathways implicated in the pathogenesis of sarcoidosis, however, due to differences in study design and variable statistical approaches, results were frequently not reproducible or concordant. Therefore, meta-analysis of sarcoidosis gene-expression datasets is of great importance to robustly establish differentially expressed genes and signalling pathways., Methods: We performed meta-analysis on 22 published gene-expression studies on sarcoidosis. Datasets were analysed systematically using same statistical cut-offs. Differentially expressed genes were identified by pooling of p -values using Edgington's method and analysed for pathways using Ingenuity Pathway Analysis software., Results: A consistent and significant signature of novel and well-known genes was identified, those collectively implicated both type I and type II interferon mediated signalling pathways in sarcoidosis. In silico functional analysis showed consistent downregulation of eukaryotic initiation factor 2 signalling, whereas cytokines like interferons and transcription factor STAT1 were upregulated. Furthermore, we analysed affected tissues to detect differentially expressed genes likely to be involved in granuloma biology. This revealed that matrix metallopeptidase 12 was exclusively upregulated in affected tissues, suggesting a crucial role in disease pathogenesis., Discussion: Our analysis provides a concise gene signature in sarcoidosis and expands our knowledge about the pathogenesis. Our results are of importance to improve current diagnostic approaches and monitoring strategies as well as in the development of targeted therapeutics., 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 © 2024 van Wijck, Sharma, Swagemakers, Dik, IJspeert, Dalm, van Daele, van Hagen and van der Spek.)

Despite its uniform appearance, the cerebellar cortex is highly heterogeneous in terms of structure, genetics and physiology. Purkinje cells (PCs), the principal and sole output neurons of the cerebellar cortex, can be categorized into multiple populations that differentially express molecular markers and display distinctive physiological features. Such features include action potential rate, but also their propensity for synaptic and intrinsic plasticity. However, the precise molecular and genetic factors that correlate with the differential physiological properties of PCs remain elusive. In this article, we provide a detailed overview of the cellular mechanisms that regulate PC activity and plasticity. We further perform a pathway analysis to highlight how molecular characteristics of specific PC populations may influence their physiology and plasticity mechanisms., (© 2024 The Authors. BioEssays published by Wiley Periodicals LLC.)

Purpose: Herpes simplex virus (HSV) and varicella-zoster virus (VZV) are neurotropic human alphaherpesviruses endemic worldwide. Upon primary infection, both viruses establish lifelong latency in neurons and reactivate intermittently to cause a variety of mild to severe diseases. Acute retinal necrosis (ARN) is a rare, sight-threatening eye disease induced by ocular VZV or HSV infection. The virus and host factors involved in ARN pathogenesis remain incompletely described. We hypothesize an underlying genetic defect in at least part of ARN cases., Methods: We collected blood from 17 patients with HSV-or VZV-induced ARN, isolated DNA and performed Whole Exome Sequencing by Illumina followed by analysis in Varseq with criteria of CADD score > 15 and frequency in GnomAD < 0.1% combined with biological filters. Gene modifications relative to healthy control genomes were filtered according to high quality and read-depth, low frequency, high deleteriousness predictions and biological relevance., Results: We identified a total of 50 potentially disease-causing genetic variants, including missense, frameshift and splice site variants and on in-frame deletion in 16 of the 17 patients. The vast majority of these genes are involved in innate immunity, followed by adaptive immunity, autophagy, and apoptosis; in several instances variants within a given gene or pathway was identified in several patients., Discussion: We propose that the identified variants may contribute to insufficient viral control and increased necrosis ocular disease presentation in the patients and serve as a knowledge base and starting point for the development of improved diagnostic, prophylactic, and therapeutic applications., 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 Heinz, Swagemakers, von Hofsten, Helleberg, Thomsen, De Keukeleere, de Boer, Ilginis, Verjans, van Hagen, van der Spek and Mogensen.)

Aims: Risk assessment tools are needed for timely identification of patients with heart failure (HF) with reduced ejection fraction (HFrEF) who are at high risk of adverse events. In this study, we aim to derive a small set out of 4210 repeatedly measured proteins, which, along with clinical characteristics and established biomarkers, carry optimal prognostic capacity for adverse events, in patients with HFrEF., Methods and Results: In 382 patients, we performed repeated blood sampling (median follow-up: 2.1 years) and applied an aptamer-based multiplex proteomic approach. We used machine learning to select the optimal set of predictors for the primary endpoint (PEP: composite of cardiovascular death, heart transplantation, left ventricular assist device implantation, and HF hospitalization). The association between repeated measures of selected proteins and PEP was investigated by multivariable joint models. Internal validation (cross-validated c -index) and external validation (Henry Ford HF PharmacoGenomic Registry cohort) were performed. Nine proteins were selected in addition to the MAGGIC risk score, N-terminal pro-hormone B-type natriuretic peptide, and troponin T: suppression of tumourigenicity 2, tryptophanyl-tRNA synthetase cytoplasmic, histone H2A Type 3, angiotensinogen, deltex-1, thrombospondin-4, ADAMTS-like protein 2, anthrax toxin receptor 1, and cathepsin D. N-terminal pro-hormone B-type natriuretic peptide and angiotensinogen showed the strongest associations [hazard ratio (95% confidence interval): 1.96 (1.17-3.40) and 0.66 (0.49-0.88), respectively]. The multivariable model yielded a c -index of 0.85 upon internal validation and c -indices up to 0.80 upon external validation. The c -index was higher than that of a model containing established risk factors ( P = 0.021)., Conclusion: Nine serially measured proteins captured the most essential prognostic information for the occurrence of adverse events in patients with HFrEF, and provided incremental value for HF prognostication beyond established risk factors. These proteins could be used for dynamic, individual risk assessment in a prospective setting. These findings also illustrate the potential value of relatively 'novel' biomarkers for prognostication., Clinical Trial Registration: https://clinicaltrials.gov/ct2/show/NCT01851538?term=nCT01851538&draw=2&rank=1 24., Competing Interests: Conflict of interest: R.O. is employed by Somalogic Inc. D.L. reports non-financial support from Somalogic Inc., during the conduct of the study; grants and personal fees from Janssen, personal fees from Ortho Diagnostics, personal fees from DCRI (Novartis), grants from Bayer, grants from Astra Zeneca, grants from Critical Diagnostics, non-financial support from Somalogic, grants from Lilly, personal fees from ACI (Abbott Laboratories), personal fees from Martin Pharmaceuticals, personal fees from Illumina, personal fees from Vicardia, other from Hridaya, grants and personal fees from Amgen, personal fees from Cytokinetics, outside the submitted work. In addition, D.L. has a patent genomic predictors of BB response issued. The other authors have no disclosures to report., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Colour agnosia is a disorder that impairs colour knowledge (naming, recognition) despite intact colour perception. Previously, we have identified the first and only-known family with hereditary developmental colour agnosia. The aim of the current study was to explore genomic regions and candidate genes that potentially cause this trait in this family. For three family members with developmental colour agnosia and three unaffected family members CGH-array analysis and exome sequencing was performed, and linkage analysis was carried out using DominantMapper, resulting in the identification of 19 cosegregating chromosomal regions. Whole exome sequencing resulted in 11 rare coding variants present in all affected family members with developmental colour agnosia and absent in unaffected members. These variants affected genes that have been implicated in neural processes and functions (CACNA2D4, DDX25, GRINA, MYO15A) or that have an indirect link to brain function, development or disease (MAML2, STAU1, TMED3, RABEPK), and a remaining group lacking brain expression or involved in non-neural traits (DEPDC7, OR1J1, OR8D4). Although this is an explorative study, the small set of candidate genes that could serve as a starting point for unravelling mechanisms of higher level cognitive functions and cortical specialization, and disorders therein such as developmental colour agnosia., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2023 Nijboer et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Background: HFrEF is a heterogenous condition with high mortality. We used serial assessments of 4210 circulating proteins to identify distinct novel protein-based HFrEF subphenotypes and to investigate underlying dynamic biological mechanisms. Herewith we aimed to gain pathophysiological insights and fuel opportunities for personalised treatment., Methods: In 382 patients, we performed trimonthly blood sampling during a median follow-up of 2.1 [IQR:1.1-2.6] years. We selected all baseline samples and two samples closest to the primary endpoint (PEP; composite of cardiovascular mortality, HF hospitalization, LVAD implantation, and heart transplantation) or censoring, and applied an aptamer-based multiplex proteomic approach. Using unsupervised machine learning methods, we derived clusters from 4210 repeatedly measured proteomic biomarkers. Sets of proteins that drove cluster allocation were analysed via an enrichment analysis. Differences in clinical characteristics and PEP occurrence were evaluated., Findings: We identified four subphenotypes with different protein profiles, prognosis and clinical characteristics, including age (median [IQR] for subphenotypes 1-4, respectively:70 [64, 76], 68 [60, 79], 57 [47, 65], 59 [56, 66]years), EF (30 [26, 36], 26 [20, 38], 26 [22, 32], 33 [28, 37]%), and chronic renal failure (45%, 65%, 36%, 37%). Subphenotype allocation was driven by subsets of proteins associated with various biological functions, such as oxidative stress, inflammation and extracellular matrix organisation. Clinical characteristics of the subphenotypes were aligned with these associations. Subphenotypes 2 and 3 had the worst prognosis compared to subphenotype 1 (adjHR (95%CI):3.43 (1.76-6.69), and 2.88 (1.37-6.03), respectively)., Interpretation: Four circulating-protein based subphenotypes are present in HFrEF, which are driven by varying combinations of protein subsets, and have different clinical characteristics and prognosis., Clinical Trial Registration: ClinicalTrials.gov Identifier: NCT01851538https://clinicaltrials.gov/ct2/show/NCT01851538., Funding: EU/EFPIA IMI2JU BigData@Heart grant n°116074, Jaap Schouten Foundation and Noordwest Academie., Competing Interests: Declaration of interests Rachel Ostroff is an employee of Somalogic Inc. with stock options. Jasper J. Brugts has received speaker fees or honoraria for advisory boards from Abbott, Novartis, Astra Zeneca, Vifor and Bayer in the past three years. The other authors have no conflicts of interest to report., (Copyright © 2023 The Author(s). Published by Elsevier B.V. All rights reserved.)

Background: Studies focusing on sex differences in circulating proteins in patients with heart failure with reduced ejection fraction (HFrEF) are scarce. Insight into sex-specific cardiovascular protein profiles and their associations with the risk of adverse outcomes may contribute to a better understanding of the pathophysiological processes involved in HFrEF. Moreover, it could provide a basis for the use of circulating protein measurements for prognostication in women and men, wherein the most relevant protein measurements are applied in each of the sexes., Methods: In 382 patients with HFrEF, we performed tri-monthly blood sampling (median follow-up: 25 [13-31] months). We selected all baseline samples and two samples closest to the primary endpoint (PEP: composite of cardiovascular death, heart transplantation, left ventricular assist device implantation, and HF hospitalization) or censoring. We then applied an aptamer-based multiplex proteomic assay identifying 1105 proteins previously associated with cardiovascular disease. We used linear regression models and gene-enrichment analysis to study sex-based differences in baseline levels. We used time-dependent Cox models to study differences in the prognostic value of serially measured proteins. All models were adjusted for the MAGGIC HF mortality risk score and p-values for multiple testing., Results: In 104 women and 278 men (mean age 62 and 64 years, respectively) cumulative PEP incidence at 30 months was 25% and 35%, respectively. At baseline, 55 (5%) out of the 1105 proteins were significantly different between women and men. The female protein profile was most strongly associated with extracellular matrix organization, while the male profile was dominated by regulation of cell death. The association of endothelin-1 (P interaction  < 0.001) and somatostatin (P interaction  = 0.040) with the PEP was modified by sex, independent of clinical characteristics. Endothelin-1 was more strongly associated with the PEP in men (HR 2.62 [95%CI, 1.98, 3.46], p < 0.001) compared to women (1.14 [1.01, 1.29], p = 0.036). Somatostatin was positively associated with the PEP in men (1.23 [1.10, 1.38], p < 0.001), but inversely associated in women (0.33 [0.12, 0.93], p = 0.036)., Conclusion: Baseline cardiovascular protein levels differ between women and men. However, the predictive value of repeatedly measured circulating proteins does not seem to differ except for endothelin-1 and somatostatin., (© 2023. The Author(s).)

CLEC16A is a membrane-associated C-type lectin protein that functions as a E3-ubiquitin ligase. CLEC16A regulates autophagy and mitophagy, and reportedly localizes to late endosomes. GWAS studies have associated CLEC16A SNPs to various auto-immune and neurological disorders, including multiple sclerosis and Parkinson disease. Studies in mouse models imply a role for CLEC16A in neurodegeneration. We identified bi-allelic CLEC16A truncating variants in siblings from unrelated families presenting with a severe neurodevelopmental disorder including microcephaly, brain atrophy, corpus callosum dysgenesis, and growth retardation. To understand the function of CLEC16A in neurodevelopment we used in vitro models and zebrafish embryos. We observed CLEC16A localization to early endosomes in HEK293T cells. Mass spectrometry of human CLEC16A showed interaction with endosomal retromer complex subunits and the endosomal ubiquitin ligase TRIM27. Expression of the human variant leading to C-terminal truncated CLEC16A, abolishes both its endosomal localization and interaction with TRIM27, suggesting a loss-of-function effect. CLEC16A knockdown increased TRIM27 adhesion to early endosomes and abnormal accumulation of endosomal F-actin, a sign of disrupted vesicle sorting. Mutagenesis of clec16a by CRISPR-Cas9 in zebrafish embryos resulted in accumulated acidic/phagolysosome compartments, in neurons and microglia, and dysregulated mitophagy. The autophagocytic phenotype was rescued by wild-type human CLEC16A but not the C-terminal truncated CLEC16A. Our results demonstrate that CLEC16A closely interacts with retromer components and regulates endosomal fate by fine-tuning levels of TRIM27 and polymerized F-actin on the endosome surface. Dysregulation of CLEC16A-mediated endosomal sorting is associated with neurodegeneration, but it also causes accumulation of autophagosomes and unhealthy mitochondria during brain development., (© 2022. The Author(s).)

Background: The signal transducer and activator of transcription 6 (STAT6) signaling pathway plays a central role in allergic inflammation. To date, however, there have been no descriptions of STAT6 gain-of-function variants leading to allergies in humans., Objective: We report a STAT6 gain-of-function variant associated with early-onset multiorgan allergies in a family with 3 affected members., Methods: Exome sequencing and immunophenotyping of T-helper cell subsets were conducted. The function of the STAT6 protein was analyzed by Western blot, immunofluorescence, electrophoretic mobility shift assays, and luciferase assays. Gastric organoids obtained from the index patient were used to study downstream effector cytokines., Results: We identified a heterozygous missense variant (c.1129G>A;p.Glu377Lys) in the DNA binding domain of STAT6 that was de novo in the index patient's father and was inherited by 2 of his 3 children. Severe atopic dermatitis and food allergy were key presentations. Clinical heterogeneity was observed among the affected individuals. Higher levels of peripheral blood T H 2 lymphocytes were detected. The mutant STAT6 displayed a strong preference for nuclear localization, increased DNA binding affinity, and spontaneous transcriptional activity. Moreover, gastric organoids showed constitutive activation of STAT6 downstream signaling molecules., Conclusions: A germline STAT6 gain-of-function variant results in spontaneous activation of the STAT6 signaling pathway and is associated with an early-onset and severe allergic phenotype in humans. These observations enhance our knowledge of the molecular mechanisms underlying allergic diseases and will potentially contribute to novel therapeutic interventions., (Copyright © 2022 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.)

Objectives: To test the hypothesis that ROSAH (retinal dystrophy, optic nerve oedema, splenomegaly, anhidrosis and headache) syndrome, caused by dominant mutation in ALPK1 , is an autoinflammatory disease., Methods: This cohort study systematically evaluated 27 patients with ROSAH syndrome for inflammatory features and investigated the effect of ALPK1 mutations on immune signalling. Clinical, immunologic and radiographical examinations were performed, and 10 patients were empirically initiated on anticytokine therapy and monitored. Exome sequencing was used to identify a new pathogenic variant. Cytokine profiling, transcriptomics, immunoblotting and knock-in mice were used to assess the impact of ALPK1 mutations on protein function and immune signalling., Results: The majority of the cohort carried the p.Thr237Met mutation but we also identified a new ROSAH-associated mutation, p.Tyr254Cys.Nearly all patients exhibited at least one feature consistent with inflammation including recurrent fever, headaches with meningeal enhancement and premature basal ganglia/brainstem mineralisation on MRI, deforming arthritis and AA amyloidosis. However, there was significant phenotypic variation, even within families and some adults lacked functional visual deficits. While anti-TNF and anti-IL-1 therapies suppressed systemic inflammation and improved quality of life, anti-IL-6 (tocilizumab) was the only anticytokine therapy that improved intraocular inflammation (two of two patients).Patients' primary samples and in vitro assays with mutated ALPK1 constructs showed immune activation with increased NF-κB signalling, STAT1 phosphorylation and interferon gene expression signature. Knock-in mice with the Alpk1 T237M mutation exhibited subclinical inflammation.Clinical features not conventionally attributed to inflammation were also common in the cohort and included short dental roots, enamel defects and decreased salivary flow., Conclusion: ROSAH syndrome is an autoinflammatory disease caused by gain-of-function mutations in ALPK1 and some features of disease are amenable to immunomodulatory therapy., Competing Interests: Competing interests: FS is a cofounder and stockholder of Pyrotech therapeutics, a company that aims to develop agonist/inhibitor drugs for ALPK1. RM has received honorary fees for lectures from SHIRE-TAKEDA- SANOFI- NOVARTIS-SOBI and Nida Sen is employed by Janssen., (© Author(s) (or their employer(s)) 2022. Re-use permitted under CC BY. Published by BMJ.)

Purpose: Although haploinsufficiency of ANKRD11 is among the most common genetic causes of neurodevelopmental disorders, the role of rare ANKRD11 missense variation remains unclear. We characterized clinical, molecular, and functional spectra of ANKRD11 missense variants., Methods: We collected clinical information of individuals with ANKRD11 missense variants and evaluated phenotypic fit to KBG syndrome. We assessed pathogenicity of variants through in silico analyses and cell-based experiments., Results: We identified 20 unique, mostly de novo, ANKRD11 missense variants in 29 individuals, presenting with syndromic neurodevelopmental disorders similar to KBG syndrome caused by ANKRD11 protein truncating variants or 16q24.3 microdeletions. Missense variants significantly clustered in repression domain 2 at the ANKRD11 C-terminus. Of the 10 functionally studied missense variants, 6 reduced ANKRD11 stability. One variant caused decreased proteasome degradation and loss of ANKRD11 transcriptional activity., Conclusion: Our study indicates that pathogenic heterozygous ANKRD11 missense variants cause the clinically recognizable KBG syndrome. Disrupted transrepression capacity and reduced protein stability each independently lead to ANKRD11 loss-of-function, consistent with haploinsufficiency. This highlights the diagnostic relevance of ANKRD11 missense variants, but also poses diagnostic challenges because the KBG-associated phenotype may be mild and inherited pathogenic ANKRD11 (missense) variants are increasingly observed, warranting stringent variant classification and careful phenotyping., Competing Interests: Conflict of Interest H.Z.E. and K.G.M. are employees of GeneDx, Inc. All other authors declare no conflicts of interest., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Asthma, COPD, COVID-19, EGPA, Lung cancer, and Pneumonia are major chronic respiratory diseases (or CRDs) affecting millions worldwide and account for substantial morbidity and mortality. These CRDs are irreversible diseases that affect different parts of the respiratory system, imposing a considerable burden on different socio-economic classes. All these CRDs have been linked to increased eosinophils in the lungs. Eosinophils are essential immune mediators that contribute to tissue homeostasis and the pathophysiology of various diseases. Interestingly, elevated eosinophil level is associated with cellular processes that regulate airway hyperresponsiveness, airway remodeling, mucus hypersecretion, and inflammation in the lung. Therefore, eosinophil is considered the therapeutic target in eosinophil-mediated lung diseases. Although, conventional medicines like antibiotics, anti-inflammatory drugs, and bronchodilators are available to prevent CRDs. But the development of resistance to these therapeutic agents after long-term usage remains a challenge. However, progressive development in nanotechnology has unveiled the targeted nanocarrier approach that can significantly improve the pharmacokinetics of a therapeutic drug. The potential of the nanocarrier system can be specifically targeted on eosinophils and their associated components to obtain promising results in the pharmacotherapy of CRDs. This review intends to provide knowledge about eosinophils and their role in CRDs. Moreover, it also discusses nanocarrier drug delivery systems for the targeted treatment of CRDs., 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 © 2022. Published by Elsevier B.V.)

Background: Indolent systemic mastocytosis (ISM) is characterized by pathologic accumulation of mast cells. The mechanism behind its phenotypic heterogeneity is not well understood. Interaction of mast cells with other immune cells might cause systemic inflammation and thereby associated symptoms., Objective: We investigated peripheral leukocyte compartments and serum immune proteome in ISM., Methods: Peripheral blood leukocyte phenotyping using flow cytometry in a cohort of 18 adults with ISM and 12 healthy controls. Targeted proteomics was performed to measure 169 proteins associated with inflammation on serum of another 20 ISM patients and 20 healthy controls., Results: Proportions of plasmacytoid dendritic cells and monocytes were significantly decreased while T H 2 cells were increased in peripheral blood of ISM patients. Furthermore, a shift from naive to memory T cells was observed. Hierarchical clustering of the serum proteome revealed 2 distinct subgroups within ISM patients. In subgroup A (n = 8), 62 proteins were significantly overexpressed, whereas those of subgroup B (n = 12) were comparable to healthy controls. Patients in subgroup A displayed upregulated signaling pathways downstream of Toll-like receptor 4, TNF-α, and IFN-γ. Fatigue was more often present in subgroup A compared to B (75% vs 33% respectively, P = .06)., Conclusions: Altered distribution of leukocyte subsets and a proinflammatory proteome were observed in subsequent 2 cohorts of ISM patients. We hypothesize that neoplastic mast cells recruit and activate plasmacytoid dendritic cells, monocytes, and T cells, leading to a vicious cycle of inflammation., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Abrupt onset of severe neuropsychiatric symptoms including obsessive-compulsive disorder, tics, anxiety, mood swings, irritability, and restricted eating is described in children with Pediatric Acute-Onset Neuropsychiatric Syndrome (PANS). Symptom onset is often temporally associated with infections, suggesting an underlying autoimmune/autoinflammatory etiology, although direct evidence is often lacking. The pathological mechanisms are likely heterogeneous, but we hypothesize convergence on one or more biological pathways. Consequently, we conducted whole exome sequencing (WES) on a U.S. cohort of 386 cases, and whole genome sequencing (WGS) on ten cases from the European Union who were selected because of severe PANS. We focused on identifying potentially deleterious genetic variants that were de novo or ultra-rare (MAF) < 0.001. Candidate mutations were found in 11 genes (PPM1D, SGCE, PLCG2, NLRC4, CACNA1B, SHANK3, CHK2, GRIN2A, RAG1, GABRG2, and SYNGAP1) in 21 cases, which included two or more unrelated subjects with ultra-rare variants in four genes. These genes converge into two broad functional categories. One regulates peripheral immune responses and microglia (PPM1D, CHK2, NLRC4, RAG1, PLCG2). The other is expressed primarily at neuronal synapses (SHANK3, SYNGAP1, GRIN2A, GABRG2, CACNA1B, SGCE). Mutations in these neuronal genes are also described in autism spectrum disorder and myoclonus-dystonia. In fact, 12/21 cases developed PANS superimposed on a preexisting neurodevelopmental disorder. Genes in both categories are also highly expressed in the enteric nervous system and the choroid plexus. Thus, genetic variation in PANS candidate genes may function by disrupting peripheral and central immune functions, neurotransmission, and/or the blood-CSF/brain barriers following stressors such as infection., (© 2022. The Author(s).)

Purpose: The vitreous proteome might provide an attractive gateway to discriminate between various uveitis aetiologies and gain novel insights into the underlying pathophysiological processes. Here, we investigated 180 vitreous proteins to discover novel biomarkers and broaden disease insights by comparing (1). primary vitreoretinal lymphoma ((P)VRL) versus other aetiologies, (2). sarcoid uveitis versus tuberculosis (TB)-associated uveitis and (3). granulomatous (sarcoid and TB) uveitis versus other aetiologies., Methods: Vitreous protein levels were determined by proximity extension assay in 47 patients with intraocular inflammation and a prestudy diagnosis (cohort 1; training) and 22 patients with a blinded diagnosis (cohort 2; validation). Differentially expressed proteins identified by t-tests on cohort 1 were used to calculate Youden's indices. Pathway and network analysis was performed by ingenuity pathway analysis. A random forest classifier was trained to predict the diagnosis of blinded patients., Results: For (P)VRL stratification, the previously reported combined diagnostic value of IL-10 and IL-6 was confirmed. Additionally, CD70 was identified as potential novel marker for (P)VRL. However, the classifier trained on the entire cohort (cohort 1 and 2) relied primarily on the interleukin score for intraocular lymphoma diagnosis (ISOLD) or IL-10/IL-6 ratio and only showed a supportive role for CD70. Furthermore, sarcoid uveitis displayed increased levels of vitreous CCL17 as compared to TB-associated uveitis., Conclusion: We underline the previously reported value of the ISOLD and the IL-10/IL-6 ratio for (P)VRL identification and present CD70 as a potentially valuable target for (P)VRL stratification. Finally, we also show that increased CCL17 levels might help to distinguish sarcoid uveitis from TB-associated uveitis., (© 2021 The Authors. Acta Ophthalmologica published by John Wiley & Sons Ltd on behalf of Acta Ophthalmologica Scandinavica Foundation.)

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) have been identified in bacteria, archaea and mitochondria of plants, but not in eukaryotes. Here, we report the discovery of 12,572 putative CRISPRs randomly distributed across the human chromosomes, which we termed hCRISPRs. By using available transcriptome datasets, we demonstrate that hCRISPRs are distinctively expressed as small non-coding RNAs (sncRNAs) in cell lines and human tissues. Moreover, expression patterns thereof enabled us to distinguish normal from malignant tissues. In prostate cancer, we confirmed the differential hCRISPR expression between normal adjacent and malignant primary prostate tissue by RT-qPCR and demonstrate that the SHERLOCK and DETECTR dipstick tools are suitable to detect these sncRNAs. We anticipate that the discovery of CRISPRs in the human genome can be further exploited for diagnostic purposes in cancer and other medical conditions, which certainly will lead to the development of point-of-care tests based on the differential expression of the hCRISPRs., (© 2022. The Author(s).)

Economic evaluation is an integral component of informed public health decision-making in personalized medicine. However, performing economic evaluation assessments often requires specialized knowledge, expertise, and significant resources. To this end, developing generic models can significantly assist towards providing the necessary evidence for the cost-effectiveness of genome-guided therapeutic interventions, compared to the traditional drug treatment modalities. Here, we report a generic cost-utility analysis model, developed in R, which encompasses essential economic evaluation steps. Specifically, critical steps towards a comprehensive deterministic and probabilistic sensitivity analysis were incorporated in our model, while also providing an easy-to-use graphical user interface, which allows even non-experts in the field to produce a fully comprehensive cost-utility analysis report. To further demonstrate the model's reproducibility, two sets of data were assessed, one stemming from in-house clinical data and one based on previously published data. By implementing the generic model presented herein, we show that the model produces results in complete concordance with the traditionally performed cost-utility analysis for both datasets. Overall, this work demonstrates the potential of generic models to provide useful economic evidence for personalized medicine interventions., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Hyperinflammation plays an important role in severe and critical COVID-19. Using inconsistent criteria, many researchers define hyperinflammation as a form of very severe inflammation with cytokine storm. Therefore, COVID-19 patients are treated with anti-inflammatory drugs. These drugs appear to be less efficacious than expected and are sometimes accompanied by serious adverse effects. SARS-CoV-2 promotes cellular ATP release. Increased levels of extracellular ATP activate the purinergic receptors of the immune cells initiating the physiologic pro-inflammatory immune response. Persisting viral infection drives the ATP release even further leading to the activation of the P2X7 purinergic receptors (P2X7Rs) and a severe yet physiologic inflammation. Disease progression promotes prolonged vigorous activation of the P2X7R causing cell death and uncontrolled ATP release leading to cytokine storm and desensitisation of all other purinergic receptors of the immune cells. This results in immune paralysis with co-infections or secondary infections. We refer to this pathologic condition as hyperinflammation. The readily available and affordable P2X7R antagonist lidocaine can abrogate hyperinflammation and restore the normal immune function. The issue is that the half-maximal effective concentration for P2X7R inhibition of lidocaine is much higher than the maximal tolerable plasma concentration where adverse effects start to develop. To overcome this, we selectively inhibit the P2X7Rs of the immune cells of the lymphatic system inducing clonal expansion of Tregs in local lymph nodes. Subsequently, these Tregs migrate throughout the body exerting anti-inflammatory activities suppressing systemic and (distant) local hyperinflammation. We illustrate this with six critically ill COVID-19 patients treated with lidocaine., (© 2021. The Author(s).)

Craniosynostosis is a birth defect occurring in approximately one in 2000 live births, where premature fusion of the cranial bones inhibits growth of the skull during critical periods of brain development. The resulting changes in skull shape can lead to compression of the brain, causing severe complications. While we have some understanding of the molecular pathology of craniosynostosis, a large proportion of cases are of unknown genetic aetiology. Based on studies in mouse, we previously proposed that the ciliopathy gene Fuz should be considered a candidate craniosynostosis gene. Here, we report a novel variant of FUZ (c.851 G > C, p.(Arg284Pro)) found in monozygotic twins presenting with craniosynostosis. To investigate whether Fuz has a direct role in regulating osteogenic fate and mineralisation, we cultured primary osteoblasts and mouse embryonic fibroblasts (MEFs) from Fuz mutant mice. Loss of Fuz resulted in increased osteoblastic mineralisation. This suggests that FUZ protein normally acts as a negative regulator of osteogenesis. We then used Fuz mutant MEFs, which lose functional primary cilia, to test whether the FUZ p.(Arg284Pro) variant could restore FUZ function during ciliogenesis. We found that expression of the FUZ p.(Arg284Pro) variant was sufficient to partially restore cilia numbers, but did not mediate a comparable response to Hedgehog pathway activation. Together, this suggests the osteogenic effects of FUZ p.(Arg284Pro) do not depend upon initiation of ciliogenesis., (© 2021. The Author(s).)

Childhood obesity has increased worldwide, and many clinical and public interventions have attempted to reduce morbidity. We aimed to determine the metabolomic signatures associated with weight control interventions in children with obesity. Forty children from the "Intervention for Children and Adolescent Obesity via Activity and Nutrition (ICAAN)" cohort were selected according to intervention responses. Based on changes in body mass index z-scores, 20 were responders and the remaining non-responders. Their serum metabolites were quantitatively analyzed using capillary electrophoresis time-of-flight mass spectrometry at baseline and after 6 and 18 months of intervention. After 18 months of intervention, the metabolite cluster changes in the responders and non-responders showed a difference on the heatmap, but significant metabolites were not clear. However, regardless of the responses, 13 and 49 metabolites were significant in the group of children with obesity intervention at 6 months and 18 months post-intervention compared to baseline. In addition, the top five metabolic pathways (D-glutamine and D-glutamate metabolism; arginine biosynthesis; alanine, aspartate, and glutamate metabolism; TCA cycle (tricarboxylic acid cycle); valine, leucine, and isoleucine biosynthesis) including several amino acids in the metabolites of obese children after 18 months were significantly changed. Our study showed significantly different metabolomic profiles based on time post obesity-related intervention. Through this study, we can better understand and predict childhood obesity through metabolite analysis and monitoring.

Background: Fusion genes are typically identified by RNA sequencing (RNA-seq) without elucidating the causal genomic breakpoints. However, non-poly(A)-enriched RNA-seq contains large proportions of intronic reads that also span genomic breakpoints., Results: We have developed an algorithm, Dr. Disco, that searches for fusion transcripts by taking an entire reference genome into account as search space. This includes exons but also introns, intergenic regions, and sequences that do not meet splice junction motifs. Using 1,275 RNA-seq samples, we investigated to what extent genomic breakpoints can be extracted from RNA-seq data and their implications regarding poly(A)-enriched and ribosomal RNA-minus RNA-seq data. Comparison with whole-genome sequencing data revealed that most genomic breakpoints are not, or minimally, transcribed while, in contrast, the genomic breakpoints of all 32 TMPRSS2-ERG-positive tumours were present at RNA level. We also revealed tumours in which the ERG breakpoint was located before ERG, which co-existed with additional deletions and messenger RNA that incorporated intergenic cryptic exons. In breast cancer we identified rearrangement hot spots near CCND1 and in glioma near CDK4 and MDM2 and could directly associate this with increased expression. Furthermore, in all datasets we find fusions to intergenic regions, often spanning multiple cryptic exons that potentially encode neo-antigens. Thus, fusion transcripts other than classical gene-to-gene fusions are prominently present and can be identified using RNA-seq., Conclusion: By using the full potential of non-poly(A)-enriched RNA-seq data, sophisticated analysis can reliably identify expressed genomic breakpoints and their transcriptional effects., (© The Author(s) 2021. Published by Oxford University Press GigaScience.)

Glucose-6-phosphate dehydrogenase (G6PD) deficiency caused by genetic variants in the G6PD gene, constitutes the most common enzymopathy worldwide, affecting approximately 5% of the global population. While carriers are mostly asymptomatic, they are at substantial risk of acute hemolytic anemia upon certain infections or exposure to various medications. As such, information about G6PD activity status in a given patient can constitute an important parameter to guide clinical decision-making. Here, we leveraged whole genome sequencing data from 142,069 unrelated individuals across seven human populations to provide a global comprehensive map of G6PD variability. By integrating established functional classifications with stringent computational predictions using 13 partly orthogonal algorithms for uncharacterized and novel variants, we reveal the large extent of ethnogeographic variability in G6PD deficiency and highlight its population-specific genetic composition. Overall, estimated disease prevalence in males ranged between 12.2% in Africans, 2.7-3.5% across Asia and 2.1% in Middle Easterners to < 0.3% in Europeans, Finnish and Amish. In Africans, the major deficient alleles were A- 202A/376 G (minor allele frequency 11.6%) and A- 968 C/376 G (0.5%). In contrast, G6PD deficiency in Middle Easterners was primarily due to the Mediterranean allele (1.3%) and the population-specific Cairo variant (0.4%). In South Asia, the most prevalent deficient alleles were Mediterranean (1.7%), Kerala (1.1%), Gond (0.9%) and Orissa (0.2%), whereas in East Asian populations the Canton (1.1%), Kaiping (0.7%) and Viangchan (0.3%) variants were predominant. Combined, our analyses provide a large dataset of G6PD variability across major ethnogeographic groups and can instruct population-specific genotyping strategies to optimize genetically guided therapeutic interventions., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Aims: Lifestyle changes, such as increasing physical activity (PA), are a cornerstone of treatment of patients with chronic heart failure (HF). However, improving PA in HF patients is challenging, and low participation rates for cardiac rehabilitation (CR) as well as relapse to low PA levels after CR are major issues. We designed a randomized controlled trial to investigate if PA monitoring with motivational feedback before and after centre-based CR in HF patients with reduced ejection fraction (HFrEF) will lead to a clinically meaningful increase in physical fitness., Methods and Results: A randomized controlled trial will be conducted in a sample of 180 HFrEF patients (New York Heart Association Class II/III) who are referred to 12-week standard CR. Patients will be randomized (2:1) to (1) standard of care (SoC) plus wearing a PA monitoring device (Fitbit Charge 3) with personalized step goals, feedback and motivation or (2) SoC only. The intervention lasts ±7 months: 4-5 weeks before CR, 12 weeks during CR and 12 weeks after CR. Measurements will take place at three time points. The primary endpoint is the change in the distance in 6-min walking test (6MWT) over the entire study period. Other endpoints include step count, grip strength, quality of life and all-cause mortality or hospitalization., Conclusions: HF-aPProACH will provide novel information on the effectiveness of remote PA stimulation and feedback before, during and after standard CR using a commercially available device to improve physical fitness in HFrEF patients., (© 2021 The Authors. ESC Heart Failure published by John Wiley & Sons Ltd on behalf of European Society of Cardiology.)

Trichothiodystrophy (TTD) is a rare hereditary neurodevelopmental disorder defined by sulfur-deficient brittle hair and nails and scaly skin, but with otherwise remarkably variable clinical features. The photosensitive TTD (PS-TTD) forms exhibits in addition to progressive neuropathy and other features of segmental accelerated aging and is associated with impaired genome maintenance and transcription. New factors involved in various steps of gene expression have been identified for the different non-photosensitive forms of TTD (NPS-TTD), which do not appear to show features of premature aging. Here, we identify alanyl-tRNA synthetase 1 and methionyl-tRNA synthetase 1 variants as new gene defects that cause NPS-TTD. These variants result in the instability of the respective gene products alanyl- and methionyl-tRNA synthetase. These findings extend our previous observations that TTD mutations affect the stability of the corresponding proteins and emphasize this phenomenon as a common feature of TTD. Functional studies in skin fibroblasts from affected individuals demonstrate that these new variants also impact on the rate of tRNA charging, which is the first step in protein translation. The extension of reduced abundance of TTD factors to translation as well as transcription redefines TTD as a syndrome in which proteins involved in gene expression are unstable., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Background: The field of pharmacogenomics focuses on the way a person's genome affects his or her response to a certain dose of a specified medication. The main aim is to utilize this information to guide and personalize the treatment in a way that maximizes the clinical benefits and minimizes the risks for the patients, thus fulfilling the promises of personalized medicine. Technological advances in genome sequencing, combined with the development of improved computational methods for the efficient analysis of the huge amount of generated data, have allowed the fast and inexpensive sequencing of a patient's genome, hence rendering its incorporation into clinical routine practice a realistic possibility., Methods: This study exploited thoroughly characterized in functional level SNVs within genes involved in drug metabolism and transport, to train a classifier that would categorize novel variants according to their expected effect on protein functionality. This categorization is based on the available in silico prediction and/or conservation scores, which are selected with the use of recursive feature elimination process. Toward this end, information regarding 190 pharmacovariants was leveraged, alongside with 4 machine learning algorithms, namely AdaBoost, XGBoost, multinomial logistic regression, and random forest, of which the performance was assessed through 5-fold cross validation., Results: All models achieved similar performance toward making informed conclusions, with RF model achieving the highest accuracy (85%, 95% CI: 0.79, 0.90), as well as improved overall performance (precision 85%, sensitivity 84%, specificity 94%) and being used for subsequent analyses. When applied on real world WGS data, the selected RF model identified 2 missense variants, expected to lead to decreased function proteins and 1 to increased. As expected, a greater number of variants were highlighted when the approach was used on NGS data derived from targeted resequencing of coding regions. Specifically, 71 variants (out of 156 with sufficient annotation information) were classified as to "Decreased function," 41 variants as "No" function proteins, and 1 variant in "Increased function.", Conclusion: Overall, the proposed RF-based classification model holds promise to lead to an extremely useful variant prioritization and act as a scoring tool with interesting clinical applications in the fields of pharmacogenomics and personalized medicine., (© 2021. The Author(s).)

DNA methylation is the most widely studied mechanism of epigenetic modification, which can influence gene expression without alterations in DNA sequences. Aberrations in DNA methylation are known to play a role in carcinogenesis, and methylation profiling has enabled the identification of biomarkers of potential clinical interest for several cancers. For vulvar squamous cell carcinoma (VSCC), however, methylation profiling remains an under-studied area. We sought to identify differentially methylated genes (DMGs) in VSCC, by performing Infinium MethylationEPIC BeadChip (Illumina) array sequencing, on a set of primary VSCC ( n = 18), and normal vulvar tissue from women with no history of vulvar (pre)malignancies ( n = 6). Using a false-discovery rate of 0.05, beta-difference (Δβ) of ±0.5, and CpG-island probes as cut-offs, 199 DMGs (195 hyper-methylated, 4 hypo-methylated) were identified for VSCC. Most of the hyper-methylated genes were found to be involved in transcription regulator activity, indicating that disruption of this process plays a vital role in VSCC development. The majority of VSCCs harbored amplifications of chromosomes 3, 8, and 9. We identified a set of DMGs in this exploratory, hypothesis-generating study, which we hope will facilitate epigenetic profiling of VSCCs. Prognostic relevance of these DMGs deserves further exploration in larger cohorts of VSCC and its precursor lesions.

Vulvar squamous cell carcinoma (VSCC) comprises two distinct etiopathological subtypes: i) Human papilloma virus (HPV)-related VSCC, which arises via the precursor high grade squamous intraepithelial lesion (HSIL); and ii) HPV-independent VSCC, which arises via precursor, differentiated vulvar intraepithelial neoplasia (dVIN), driven by TP53 mutations. However, the mechanism of carcinogenesis of VSCC is poorly understood. The current study aimed to gain insight into VSCC carcinogenesis by identifying differentially expressed genes (DEGs) for each VSCC subtype. The expression of certain DEGs was then further assessed by performing immunohistochemistry (IHC) on whole tissue sections of VSCC and its precursors. Statistical analysis of microarrays was performed on two independent gene expression datasets (GSE38228 and a study from Erasmus MC) on VSCC and normal vulva. DEGs were identified that were similarly (up/down) regulated with statistical significance in both datasets. For HPV-related VSCCs, this constituted 88 DEGs, and for HPV-independent VSCCs, this comprised 46 DEGs. IHC was performed on VSCC (n=11), dVIN (n=6), HSIL (n=6) and normal vulvar tissue (n=7) with i) signal transducer and activator of transcription 1 (STAT1; an upregulated DEGs); ii) nuclear factor IB (NFIB; a downregulated DEG); iii) p16 (to determine the HPV status of tissues); and iv) p53 (to confirm the histological diagnoses). Strong and diffuse NFIB expression was observed in the basal and para-basal layers of normal vulvar tissue, whereas NFIB expression was minimal or completely negative in dVIN and in both subtypes of VSCC. In contrast, no discernable difference was observed in STAT1 expression among normal vulvar tissue, dVIN, HSIL or VSCC. By leveraging bioinformatics, the current study identified DEGs that can facilitate research into VSCC carcinogenesis. The results suggested that NFIB is downregulated in VSCC and its relevance as a diagnostic/prognostic biomarker deserves further exploration., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Dasgupta et al.)

Histological diagnosis of differentiated vulvar intraepithelial neoplasia (dVIN), the precursor of human papillomavirus (HPV)-independent vulvar squamous cell carcinoma (VSCC), can be challenging, as features of dVIN may mimic those of non-dysplastic dermatoses. To aid the diagnosis, p53-immunohistochemistry (IHC) is commonly used, and mutant expression patterns are used to support a histological diagnosis of dVIN. However, a proportion of dVIN can show wild-type p53-expression, which is characteristic of non-dysplastic dermatoses. Furthermore, recent research has identified a novel precursor of HPV-independent VSCC-the p53-wild-type differentiated exophytic vulvar intraepithelial lesion (de-VIL). Currently, there are no established diagnostic IHC-markers for p53-wild-type dVIN or de-VIL. We evaluated IHC-markers, cytokeratin 17 (CK17), and SRY-box 2 (SOX2), as diagnostic adjuncts for dVIN. For this, IHC-expression of CK17, SOX2, and p53 was studied in dVIN ( n = 56), de-VIL ( n = 8), and non-dysplastic vulvar tissues ( n = 46). For CK17 and SOX2, the percentage of cells showing expression, and the intensity and distribution of expression were recorded. We also performed next generation targeted sequencing (NGTS) on a subset of dVIN ( n = 8) and de-VIL ( n = 8). With p53-IHC, 74% of dVIN showed mutant patterns and 26% showed wild-type expression. Median percentage of cells expressing CK17 or SOX2 was significantly higher in dVIN (p53-mutant or p53-wild-type) and de-VIL than in non-dysplastic tissues ( p < 0.01). Diffuse, moderate-to-strong, full epithelial expression of CK17 or SOX2 was highly specific for dVIN and de-VIL. With NGTS, TP53 mutations were detected in both dVIN and de-VIL. We infer that immunohistochemical markers CK17 and SOX2, when used along with p53, may help support the histological diagnosis of dVIN.

Background: Graves' ophthalmopathy (GO) is a frequent extrathyroidal complication of Graves' hyperthyroidism. Orbital fibroblasts contribute to both orbital tissue inflammation and remodeling in GO, and as such are crucial cellular elements in active GO and inactive GO. However, so far it is largely unknown whether GO disease progression is associated with functional reprogramming of the orbital fibroblast effector function. Therefore, the aim of this study was to compare both the proteome and global DNA methylation patterns between orbital fibroblasts isolated from active GO, inactive GO and healthy controls., Methods: Orbital fibroblasts from inactive GO (n=5), active GO (n=4) and controls (n=5) were cultured and total protein and DNA was isolated. Labelled and fractionated proteins were analyzed with a liquid chromatography tandem-mass spectrometer (LC-MS/MS). Data are available via ProteomeXchange with identifier PXD022257. Furthermore, bisulphite-treated DNA was analyzed for methylation pattern with the Illumina Infinium Human Methylation 450K beadchip. In addition, RNA was isolated from the orbital fibroblasts for real-time quantitative (RQ)-PCR. Network and pathway analyses were performed., Results: Orbital fibroblasts from active GO displayed overexpression of proteins that are typically involved in inflammation, cellular proliferation, hyaluronan synthesis and adipogenesis, while various proteins associated with extracellular matrix (ECM) biology and fibrotic disease, were typically overexpressed in orbital fibroblasts from inactive GO. Moreover, orbital fibroblasts from active GO displayed hypermethylation of genes that linked to inflammation and hypomethylated genes that linked to adipogenesis and autoimmunity. Further analysis revealed networks that contained molecules to which both hypermethylated and hypomethylated genes were linked, including NF-κB, ERK1/2, Alp, RNA polymerase II, Akt and IFNα. In addition, NF-κB, Akt and IFNα were also identified in networks that were derived from the differentially expressed proteins. Generally, poor correlation between protein expression, DNA methylation and mRNA expression was observed., Conclusions: Both the proteomics and DNA methylation data support that orbital fibroblasts from active GO are involved in inflammation, adipogenesis, and glycosaminoglycan production, while orbital fibroblasts from inactive disease are more skewed towards an active role in extracellular matrix remodeling. This switch in orbital fibroblast effector function may have therapeutic implications and further studies into the underlying mechanism are thus warranted., 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 © 2021 Virakul, Somparn, Pisitkun, van der Spek, Dalm, Paridaens, van Hagen, Hirankarn, Palaga and Dik.)

Epigenetic programming is essential for lineage differentiation, embryogenesis and placentation in early pregnancy. In epigenetic association studies, DNA methylation is often examined in DNA derived from white blood cells, although its validity to other tissues of interest remains questionable. Therefore, we investigated the tissue specificity of epigenome-wide DNA methylation in newborn and placental tissues. Umbilical cord white blood cells (UC-WBC, n = 25), umbilical cord blood mononuclear cells (UC-MNC, n = 10), human umbilical vein endothelial cells (HUVEC, n = 25) and placental tissue (n = 25) were obtained from 36 uncomplicated pregnancies. Genome-wide DNA methylation was measured by the Illumina HumanMethylation450K BeadChip. Using UC-WBC as a reference tissue, we identified 3595 HUVEC tissue-specific differentially methylated regions (tDMRs) and 11,938 placental tDMRs. Functional enrichment analysis showed that HUVEC and placental tDMRs were involved in embryogenesis, vascular development and regulation of gene expression. No tDMRs were identified in UC-MNC. In conclusion, the extensive amount of genome-wide HUVEC and placental tDMRs underlines the relevance of tissue-specific approaches in future epigenetic association studies, or the use of validated representative tissues for a certain disease of interest, if available. To this purpose, we herewith provide a relevant dataset of paired, tissue-specific, genome-wide methylation measurements in newborn tissues.

Text mining in biomedical literature is an emerging field which has already been shown to have a variety of implementations in many research areas, including genetics, personalized medicine, and pharmacogenomics. In this study, we describe a novel text-mining approach for the extraction of pharmacogenomics associations. The code that was used toward this end was implemented using R programming language, either through custom scripts, where needed, or through utilizing functions from existing libraries. Articles (abstracts or full texts) that correspond to a specified query were extracted from PubMed, while concept annotations were derived by PubTator Central. Terms that denote a Mutation or a Gene as well as Chemical compound terms corresponding to drug compounds were normalized and the sentences containing the aforementioned terms were filtered and preprocessed to create appropriate training sets. Finally, after training and adequate hyperparameter tuning, four text classifiers were created and evaluated (FastText, Linear kernel SVMs, XGBoost, Lasso, and Elastic-Net Regularized Generalized Linear Models) with regard to their performance in identifying pharmacogenomics associations. Although further improvements are essential toward proper implementation of this text-mining approach in the clinical practice, our study stands as a comprehensive, simplified, and up-to-date approach for the identification and assessment of research articles enriched in clinically relevant pharmacogenomics relationships. Furthermore, this work highlights a series of challenges concerning the effective application of text mining in biomedical literature, whose resolution could substantially contribute to the further development of this field., (Copyright © 2020 Patrinos, Pandi, Van Der Spek and Koromina.)

Originating in the city of Wuhan in China in December 2019, COVID-19 has emerged now as a global health emergency with a high number of deaths worldwide. COVID-19 is caused by a novel coronavirus, referred to as severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), resulting in pandemic conditions around the globe. We are in the battleground to fight against the virus by rapidly developing therapeutic strategies in tackling SARS-CoV-2 and saving human lives from COVID-19. Scientists are evaluating several known drugs either for the pathogen or the host; however, many of them are reported to be associated with side effects. In the present study, we report the molecular binding mechanisms of the natural alkaloid, noscapine, for repurposing against the main protease of SARS-CoV-2, a key enzyme involved in its reproduction. We performed the molecular dynamics (MD) simulation in an explicit solvent to investigate the molecular mechanisms of noscapine for stable binding and conformational changes to the main protease (Mpro) of SARS-CoV-2. The drug repurposing study revealed the high potential of noscapine and proximal binding to the Mpro enzyme in a comparative binding pattern analyzed with chloroquine, ribavirin, and favipiravir. Noscapine binds closely to binding pocket-3 of the Mpro enzyme and depicted stable binding with RMSD 0.1-1.9 Å and RMSF profile peak conformational fluctuations at 202-306 residues, and a Rg score ranging from 21.9 to 22.4 Å. The MM/PB (GB) SA calculation landscape revealed the most significant contribution in terms of binding energy with ΔPB -19.08 and ΔGB -27.17 kcal/mol. The electrostatic energy distribution in MM energy was obtained to be -71.16 kcal/mol and depicted high free energy decomposition (electrostatic energy) at 155-306 residues (binding pocket-3) of Mpro by a MM force field. Moreover, the dynamical residue cross-correlation map also stated that the high pairwise correlation occurred at binding residues 200-306 of the Mpro enzyme (binding pocket-3) with noscapine. Principal component analysis depicted the enhanced movement of protein atoms with a high number of static hydrogen bonds. The obtained binding results of noscapine were also well correlated with the pharmacokinetic parameters of antiviral drugs.