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INTRODUCTION: Several different methods for emergent airway management are feasible in the tactical environment. Current studies fail to identify which method minimizes the exposure of the tactical medic or which is most rapid with the greatest chance of first-attempt success. METHODS: We evaluated three commonly used airway management techniques, including standard direct laryngoscopy with endotracheal intubation, digital endotracheal intubation, and use of the King LT laryngotracheal airway device. Study participants were volunteer emergency medicine (EM) residents and medical flight crew members with difficult airway management skills. We compared the times to successful ventilation, numbers of attempts to successful ventilation, and heights of presentation of the participants above a barricade used to simulate concealment. RESULTS: Thirty-one subjects completed the study, of whom 12 (39%) were medical flight crew members and 19 (61%) were EM residents. All subjects were able to successfully ventilate manikins using each of the three methods. The mean number of attempts to intubate and ventilate the manikin was 1.03 for direct laryngoscopy, 1.26 for the King LT, and 1.67 for digital endotracheal intubation. Mean time to ventilation was 59.7 seconds for the King LT, 63.3 seconds for laryngoscopy, and 125.4 seconds for digital intubation. The maximum height the medic reached above the barricade during airway management was 17.7 inches for the King LT, 19.7 inches for laryngoscopy, and 23.5 inches for digital intubation. Comparison of all three factors across groups showed significance, with the exception of time to ventilation between laryngoscopy and use of the King LT. CONCLUSION: In a simulated tactical airway management scenario, use of the King LT provided less exposure than digital or standard endotracheal intubation techniques. Digital intubation behind the simulated barricade was the least successful by all measures. Although direct laryngoscopy was the most successful on the first attempt, use of the King LT in our scenario provided the least exposure of the medic and was as effective as direct laryngoscopy with regard to time to ventilation. Key words: TEMS; airway management; simulation; tactical environment; combat medicine. [ABSTRACT FROM AUTHOR]

After a topical fluoride application two resulting features can be observed and measured: the formation of calcium fluoride and a decrease in caries incidence. For the formation of solid calcium fluoride the source of calcium may be either the enamel itself or remaining saliva or--in plaque--plaque fluid and calculus. The aim of the present study was to examine whether saliva through its calcium content has any influence on the amount of calcium fluoride formed by a topical fluoride treatment of enamel. The roots and the fissure systems of 64 freshly extracted intact third molars were covered with nail varnish and allocated to eight groups of 8. Half of the groups were put in fresh paraffin-stimulated saliva for 5 min after which surplus of saliva was shaken off whilst the other groups remained in distilled water. The teeth were given a 2-min topical treatment with either distilled water (control), neutral 0.2% NaF, neutral 2% NaF or 2% NaF acidified with 0.1 mol/l H3PO4. After the topical treatment the teeth were rinsed in running tap water for 5 min, dried and revarnished before determination of calcium fluoride. It was found that the amount of calcium fluoride formed depended on the concentration of fluoride (the higher the fluoride concentration, the higher was the amount of calcium fluoride), the acidity of the solution (presumably due to the increase in available calcium through enamel dissolution) and the presence of saliva (presumably due to its calcium content and its mucinous nature). As there was always more calcium fluoride formed in the presence of saliva, thorough drying of the teeth prior to topical treatments may be superfluous. [ABSTRACT FROM AUTHOR]

Introduction: Long-read whole genome sequencing like Oxford Nanopore Technology, is increasingly being introduced in clinical settings. With its ability to simultaneously call sequence variation and DNA modifications including 5-methylcytosine, nanopore is a promising technology to improve diagnostics of imprinting disorders., Methods: Currently, no tools to analyze DNA methylation patterns at known clinically relevant imprinted regions are available. Here we present NanoImprint, which generates an easily interpretable report, based on long-read nanopore sequencing, to use for identifying clinical relevant abnormalities in methylation levels at 14 imprinted regions and diagnosis of common imprinting disorders., Results and Conclusion: NanoImprint outputs a summarizing table and visualization plots displays methylation frequency (%) and chromosomal positions for all regions, with phased data color-coded for the two alleles. We demonstrate the utility of NanoImprint using three imprinting disorder samples from patients with Beckwith-Wiedemann syndrome (BWS), Angelman syndrome (AS) and Prader-Willi syndrome (PWS). NanoImprint script is available from https://github.com/carolinehey/NanoImprint., (© 2024 The Authors. Annals of Human Genetics published by University College London (UCL) and John Wiley & Sons Ltd.)

Background: Current clinical markers overestimate the recurrence risk in many lymph node negative (LNN) breast cancer (BC) patients such that a majority of these low-risk patients unnecessarily receive systemic treatments. We tested if differential microRNA expression in primary tumors allows reliable identification of indolent LNN BC patients to provide an improved classification tool for overtreatment reduction in this patient group., Methods: We collected freshly frozen primary tumors of 80 LNN BC patients with recurrence and 80 recurrence-free patients (mean follow-up: 20.9 years). The study comprises solely systemically untreated patients to exclude that administered treatments confound the metastasis status. Samples were pairwise matched for clinical-pathological characteristics to minimize dependence of current markers. Patients were classified into risk-subgroups according to the differential microRNA expression of their tumors via classification model building with cross-validation using seven classification methods and a voting scheme. The methodology was validated using available data of two independent cohorts (n = 123, n = 339)., Results: Of the 80 indolent patients (who would all likely receive systemic treatments today) our ultralow-risk classifier correctly identified 37 while keeping a sensitivity of 100% in the recurrence group. Multivariable logistic regression analysis confirmed independence of voting results from current clinical markers. Application of the method in two validation cohorts confirmed successful classification of ultralow-risk BC patients with significantly prolonged recurrence-free survival., Conclusion: Profiles of differential microRNAs expression can identify LNN BC patients who could spare systemic treatments demanded by currently applied classifications. However, further validation studies are required for clinical implementation of the applied methodology., (© 2024 The Authors. Cancer Medicine published by John Wiley & Sons Ltd.)

Background: Reports of dual carriers of pathogenic BRCA1 variants in trans are extremely rare, and so far, most individuals have been associated with a Fanconi Anemia-like phenotype., Methods: We identified two families with a BRCA1 in-frame exon 20 duplication (Ex20dup). In one male individual, the variant was in trans with the BRCA1 frameshift variant c.2475delC p.(Asp825Glufs*21). We performed splicing analysis and used a transcription activation domain (TAD) assay to assess the functional impact of Ex20dup. We collected pedigrees and mapped the breakpoints of the duplication by long- and short-read genome sequencing. In addition, we performed a mitomycin C (MMC) assay from the dual carrier using cultured lymphoblastoid cells., Results: Genome sequencing and RNA analysis revealed the BRCA1 exon 20 duplication to be in tandem. The duplication was expressed without skipping any one of the two exon 20 copies, resulting in a lack of wild-type transcripts from this allele. TAD assay indicated that the Ex20dup variant has a functional level similar to the well-known moderate penetrant pathogenic BRCA1 variant c.5096G > A p.(Arg1699Gln). MMC assay of the dual carrier indicated a slightly impaired chromosomal repair ability., Conclusions: This is the first reported case where two BRCA1 variants with demonstrated functional impact are identified in trans in a male patient with an apparently normal clinical phenotype and no BRCA1-associated cancer. The results pinpoint a minimum necessary BRCA1 protein activity to avoid a Fanconi Anemia-like phenotype in compound heterozygous status and yet still predispose carriers to hormone-related cancers. These findings urge caution when counseling families regarding potential Fanconi Anemia risk. Furthermore, prudence should be taken when classifying individual variants as benign based on co-occurrence in trans with well-established pathogenic variants., (© 2024. The Author(s).)

WDR44 prevents ciliogenesis initiation by regulating RAB11-dependent vesicle trafficking. Here, we describe male patients with missense and nonsense variants within the WD40 repeats (WDR) of WDR44, an X-linked gene product, who display ciliopathy-related developmental phenotypes that we can model in zebrafish. The patient phenotypic spectrum includes developmental delay/intellectual disability, hypotonia, distinct craniofacial features and variable presence of brain, renal, cardiac and musculoskeletal abnormalities. We demonstrate that WDR44 variants associated with more severe disease impair ciliogenesis initiation and ciliary signaling. Because WDR44 negatively regulates ciliogenesis, it was surprising that pathogenic missense variants showed reduced abundance, which we link to misfolding of WDR autonomous repeats and degradation by the proteasome. We discover that disease severity correlates with increased RAB11 binding, which we propose drives ciliogenesis initiation dysregulation. Finally, we discover interdomain interactions between the WDR and NH 2 -terminal region that contains the RAB11 binding domain (RBD) and show patient variants disrupt this association. This study provides new insights into WDR44 WDR structure and characterizes a new syndrome that could result from impaired ciliogenesis., (© 2024. This is a U.S. Government work and not under copyright protection in the US; foreign copyright protection may apply.)

We have developed a recovery, regeneration, and reapplication process for Nafion, a perfluorinated sulfonic acid (PFSA) ionomer, from end-of-life (EoL) low-temperature proton-exchange membrane (PEM) fuel cells (FCs). Samples of PFSA PEM recovered from EoL membrane-electrode assemblies (MEAs) with a history of close to 19,000 h of operation were recycled by dissolving the polymeric material in ethanol and applying the dissolved PFSA ionomer for producing the ionomer phase of the catalyst layer of new PEMFC cathodes. Structural characterizations show a marginally lower abundance of sulfonic groups for the EoL PEM compared to a fresh sample. Sulfonation of the former was employed to regenerate sulfonic groups to compensate for the lost ones. New gas-diffusion electrodes (GDEs) were prepared with the recycled PFSA ionomer both with and without sulfonation, and MEAs with these GDEs as cathodes were assembled through a state-of-the-art procedure. Electrochemical characterizations of the GDEs and single-cell studies of the MEAs showed that the electrochemical performances of catalyst layers containing recycled PFSA ionomer were at least similar to those containing fresh. Durability studies of the GDEs and MEAs, performed through a three-electrode liquid cell and a single cell, respectively, show the highest durability for the GDE/MEA with PFSA ionomer recycled without applying the sulfonation step. However, the GDE with PFSA ionomer obtained from recycling a re-sulfonated PEM shows a durability comparable to that of the GDE with fresh PFSA ionomer. Hence, PFSA material aged during PEMFC operation may be employed to produce highly functional and durable regenerated PFSA ionomer for PEMFC catalyst layers. The studied process of PFSA ionomer recycling is highly attractive for industrial adoption.

Objective: This study aimed to assess the diagnostic yield of prenatal genetic testing using trio whole exome sequencing (WES) and trio whole genome sequencing (WGS) in pregnancies with fetal anomalies by comparing the results with conventional chromosomal microarray (CMA) analysis., Methods: A total of 40 pregnancies with fetal anomalies or increased nuchal translucency (NT ≥ 5 mm) were included between the 12th and 21st week of gestation. Trio WES/WGS and CMA were performed in all cases., Results: The trio WES/WGS analysis increased the diagnostic yield by 25% in cases with negative CMA results. Furthermore, all six chromosomal aberrations identified by CMA were independently detected by WES/WGS analysis. In total, 16 out of 40 cases obtained a genetic sequence variant, copy number variant, or aneuploidy explaining the phenotype, resulting in an overall WES/WGS diagnostic yield of 40%. WES analysis provided a more reliable identification of mosaic sequence variants than WGS because of its higher sequencing depth., Conclusions: Prenatal WES/WGS proved to be powerful diagnostic tools for fetal anomalies, surpassing the diagnostic yield of CMA. They have the potential to serve as standalone methods for prenatal diagnosis. The study highlighted the limitations of WGS in accurately detecting mosaic variants, which is particularly relevant when analyzing chorionic villus samples., (© 2023 The Authors. Prenatal Diagnosis published by John Wiley & Sons Ltd.)

Background: Familial breast cancer is in most cases unexplained due to the lack of identifiable pathogenic variants in the BRCA1 and BRCA2 genes. The somatic mutational landscape and in particular the extent of BRCA-like tumour features (BRCAness) in these familial breast cancers where germline BRCA1 or BRCA2 mutations have not been identified is to a large extent unknown., Methods: We performed whole-genome sequencing on matched tumour and normal samples from high-risk non-BRCA1/BRCA2 breast cancer families to understand the germline and somatic mutational landscape and mutational signatures. We measured BRCAness using HRDetect. As a comparator, we also analysed samples from BRCA1 and BRCA2 germline mutation carriers., Results: We noted for non-BRCA1/BRCA2 tumours, only a small proportion displayed high HRDetect scores and were characterized by concomitant promoter hypermethylation or in one case a RAD51D splice variant previously reported as having unknown significance to potentially explain their BRCAness. Another small proportion showed no features of BRCAness but had mutationally active tumours. The remaining tumours lacked features of BRCAness and were mutationally quiescent., Conclusions: A limited fraction of high-risk familial non-BRCA1/BRCA2 breast cancer patients is expected to benefit from treatment strategies against homologue repair deficient cancer cells., (© 2023. The Author(s).)

Neural differentiation, synaptic transmission, and action potential propagation depend on membrane sphingolipids, whose metabolism is tightly regulated. Mutations in the ceramide transporter CERT (CERT1), which is involved in sphingolipid biosynthesis, are associated with intellectual disability, but the pathogenic mechanism remains obscure. Here, we characterize 31 individuals with de novo missense variants in CERT1. Several variants fall into a previously uncharacterized dimeric helical domain that enables CERT homeostatic inactivation, without which sphingolipid production goes unchecked. The clinical severity reflects the degree to which CERT autoregulation is disrupted, and inhibiting CERT pharmacologically corrects morphological and motor abnormalities in a Drosophila model of the disease, which we call ceramide transporter (CerTra) syndrome. These findings uncover a central role for CERT autoregulation in the control of sphingolipid biosynthetic flux, provide unexpected insight into the structural organization of CERT, and suggest a possible therapeutic approach for patients with CerTra syndrome.

Disease causing variants in the Ryanodine receptor 1 (RYR1) gene are a common cause for congenital myopathy and for malignant hyperthermia susceptibility. We report a 17 year old boy with congenital muscle weakness progressing to a myasthenia like myopathy with muscle weakness, fatigability, ptosis, and ophthalmoplegia. Muscle biopsy showed predominance and atrophy of type 1 fibers. Whole-exome trio sequencing revealed three variants in the RYR1-gene in the patient: c.6721C > T,p.(Arg2241*) and c.2122G > A,p.(Asp708Asn) in cis position, and the c.325C > T,p.(Arg109Trp) variant in trans. Treatment with pyridostigmine improved symptoms. This case supports that a myasthenia like phenotype is part of the phenotypic spectrum of RYR1 related disorders, and that treatment with pyridostigmine can be beneficial for patients with this phenotype., Competing Interests: Declaration of competing interest There exist no conflicts of interest., (Copyright © 2023. Published by Elsevier Masson SAS.)

The application of next-generation sequencing (NGS) to clinical practice is still hampered by the ability to interpret the clinical relevance of novel variants and the difficulty of evaluating their effect in specific tissues. Here, we applied integrated genomic approaches for interrogating blood samples of two unrelated individuals with neurodevelopmental disorders and identified a novel neuro-pathogenic role for the Mitogen-Activated Protein Kinase 4 gene (MAP4K4). In particular, we identified two novel frameshift variants in coding exons expressed in the blood and neuronal isoforms. Both variants were predicted to generate non-sense-mediated decay. By transcriptome analysis, we simultaneously demonstrated the deleterious effect of the identified variants on the splicing activity and stability of MAP4K4 mRNA. Therefore, we propose MAP4K4 as a novel causative gene for non-syndromic and syndromic neurodevelopmental disorders. Altogether, we prove the efficacy of an integrated approach of exome and transcriptome sequencing in the resolution of undiagnosed cases by leveraging the analysis of variants in genes expressed in peripheral blood., (© 2022. The Author(s).)

Developmental dysplasia of the hip (DDH) is a common condition involving instability of the hip with multifactorial etiology. Early diagnosis and treatment are critical as undetected DDH is an important cause of long-term hip complications. Better diagnostics may be achieved through genetic methods, especially for patients with positive family history. Several candidate genes have been reported but the exact molecular etiology of the disease is yet unknown. In the present study, we performed whole exome sequencing of DDH patients from 28 families with at least two affected first-degree relatives. Four genes previously not associated with DDH (METTL21B, DIS3L2, PPP6R2, and TM4SF19) were identified with the same variants shared among affected family members, in more than two families. Among known association genes, we found damaging variants in DACH1, MYH10, NOTCH2, TBX4, EVC2, OTOG, and SHC3. Mutational burden analysis across the families identified 322 candidate genes, and enriched pathways include the extracellular matrix, cytoskeleton, ion-binding, and detection of mechanical stimulus. Taken altogether, our data suggest a polygenic mode of inheritance for DDH, and we propose that an impaired transduction of the mechanical stimulus is involved in the etiopathological mechanism. Our findings refine our current understanding of candidate causal genes in DDH, and provide a foundation for downstream functional studies., (© 2022. The Author(s).)

Background: Autosomal recessive polycystic kidney disease is a cystic kidney disease with early onset and clinically characterized by enlarged echogenic kidneys, hypertension, varying degrees of kidney dysfunction, and liver fibrosis. It is most frequently caused by sequence variants in the PKHD1 gene, encoding fibrocystin. In more rare cases, sequence variants in DZIP1L are seen, encoding the basal body protein DAZ interacting protein 1-like protein (DZIP1L). So far, only four different DZIP1L variants have been reported., Methods: Four children from three consanguineous families presenting with polycystic kidney disease were selected for targeted or untargeted exome sequencing., Results: We identified two different, previously not reported homozygous DZIP1L sequence variants: c.193 T > C; p.(Cys65Arg), and c.216C > G; p.(Cys72Trp). Functional analyses of the c.216C > G; p.(Cys72Trp) variant indicated mislocalization of mutant DZIP1L., Conclusions: In line with published data, our results suggest a critical role of the N-terminal domain for proper protein function. Although patients with PKHD1-associated autosomal recessive polycystic kidney disease often have liver abnormalities, none of the present four patients showed any clinically relevant liver involvement. Our data demonstrate the power and efficiency of next-generation sequencing-based approaches. While DZIP1L-related polycystic kidney disease certainly represents a rare form of the disease, our results emphasize the importance of including DZIP1L in multigene panels and in the data analysis of whole-exome sequencing for cystic kidney diseases. A higher resolution version of the Graphical abstract is available as Supplementary information., (© 2022. The Author(s).)

Here, we report a study on the structural characteristics of membrane electrode assembly (MEA) samples obtained from a low-temperature (LT) polymer electrolyte membrane (PEM) fuel cell (FC) stack subjected to long-term durability testing for ∼18,500 h of nominal operation along with ∼900 on/off cycles accumulated over the operation time, with the total power production being 3.39 kW h/cm 2 of MEA and the overall degradation being 87% based on performance loss. The chemical and physical states of the degraded MEAs were investigated through structural characterizations aiming to probe their different components, namely the cathode and anode electrocatalysts, the Nafion ionomer in the catalyst layers (CLs), the gas diffusion layers (GDLs), and the PEM. Surprisingly, X-ray diffraction and electron microscopy studies suggested no significant degradation of the electrocatalysts. Similarly, the cathode and anode GDLs exhibited no significant change in porosity and structure as indicated by BET analysis and helium ion microscopy. Nevertheless, X-ray fluorescence spectroscopy, elemental analysis through a CHNS analyzer, and comprehensive investigations by X-ray photoelectron spectroscopy suggested significant degradation of the Nafion, especially in terms of sulfur content, that is, the abundance of the -SO 3 - groups responsible for H + conduction. Hence, the degradation of the Nafion, in both of the CLs and in the PEM, was found to be the principal mechanism for performance degradation, while the Pt/C catalyst degradation in terms of particle size enlargement or mass loss was minimal. The study suggests that under real-life operating conditions, ionomer degradation plays a more significant role than electrocatalyst degradation in LT-PEMFCs, in contrast to many scientific studies under artificial stress conditions. Mitigation of the ionomer degradation must be emphasized as a strategy to improve the PEMFC's durability.

Shank proteins are major scaffolds of the postsynaptic density of excitatory synapses. Mutations in SHANK genes are associated with autism and intellectual disability. The effects of missense mutations on Shank3 function, and therefore the pathomechanisms are unclear. Several missense mutations in SHANK3 affect the N-terminal region, consisting of the Shank/ProSAP N-terminal (SPN) domain and a set of Ankyrin (Ank) repeats. Here we identify a novel SHANK3 missense mutation (p.L270M) in the Ankyrin repeats in patients with an ADHD-like phenotype. We functionally analysed this and a series of other mutations, using biochemical and biophysical techniques. We observe two major effects: (1) a loss of binding to δ-catenin (e.g. in the p.L270M variant), and (2) interference with the intramolecular interaction between N-terminal SPN domain and the Ank repeats. This also interferes with binding to the α-subunit of the calcium-/calmodulin dependent kinase II (αCaMKII), and appears to be associated with a more severe neurodevelopmental pathology., (© 2022. The Author(s).)

Early infantile epileptic encephalopathy 38 (EIEE38, MIM #617020) is caused by biallelic variants in ARV1, encoding a transmembrane protein of the endoplasmic reticulum with a pivotal role in glycosylphosphatidylinositol (GPI) biosynthesis. We ascertained seven new patients from six unrelated families harboring biallelic variants in ARV1, including five novel variants. Affected individuals showed psychomotor delay, hypotonia, early onset refractory seizures followed by regression and specific neuroimaging features. Flow cytometric analysis on patient fibroblasts showed a decrease in GPI-anchored proteins on the cell surface, supporting a lower residual activity of the mutant ARV1 as compared to the wildtype. A rescue assay through the transduction of lentivirus expressing wild type ARV1 cDNA effectively rescued these alterations. This study expands the clinical and molecular spectrum of the ARV1-related encephalopathy, confirming the essential role of ARV1 in GPI biosynthesis and brain function., (© 2021 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Several gene expression signatures based on mRNAs and a few based on long non-coding RNAs (lncRNAs) have been developed to provide prognostic information beyond clinical evaluation in breast cancer (BC). However, the comparison of such signatures for predicting recurrence is very scarce. Therefore, we compared the prognostic utility of mRNAs and lncRNAs in low-risk BC patients using two different classification strategies. Frozen primary tumor samples from 160 lymph node negative and systemically untreated BC patients were included; 80 developed recurrence-i.e., regional or distant metastasis while 80 remained recurrence-free (mean follow-up of 20.9 years). Patients were pairwise matched for clinicopathological characteristics. Classification based on differential mRNA or lncRNA expression using seven individual machine learning methods and a voting scheme classified patients into risk-subgroups. Classification by the seven methods with a fixed sensitivity of ≥90% resulted in specificities ranging from 16-40% for mRNA and 38-58% for lncRNA, and after voting, specificities of 38% and 60% respectively. Classifier performance based on an alternative classification approach of balanced accuracy optimization also provided higher specificities for lncRNA than mRNA at comparable sensitivities. Thus, our results suggested that classification followed by voting improved prognostic power using lncRNAs compared to mRNAs regardless of classification strategy.

Cognitive impairment is the most prominent symptom in neurodegenerative disorders affecting quality of life and mortality. However, despite years of research, the molecular mechanism underlying the regulation of cognitive function and its impairment is poorly understood. This study aims to elucidate the role of long non-coding RNAs (lncRNAs) expression and lncRNA-mRNA interaction networks, by analyzing lncRNA expression in whole blood samples of 400 middle and old aged monozygotic twins in association with cognitive function using both linear models and a generalized correlation coefficient (GCC) to capture the diverse patterns of correlation. We detected 13 probes (p < 1e-03) displaying nonlinear and 7 probes (p < 1e-03) showing linear correlations. After combining the results, we identified 20 lncRNA probes with p < 1e-03. The top lncRNA probes were annotated to genes, along with the non-coding MALAT1, that play roles in neurodegenerative diseases. The top lncRNAs were linked to functional clusters including peptidyl-glycine modification, vascular smooth muscle cells, mitotic spindle organization and protein tyrosine phosphatase. In addition, mapping of the top significant lncRNAs to the lncRNA-mRNA interaction network detected significantly enriched biological pathways involving neuroactive ligand-receptor interaction, proteasome and chemokines. We show that GCC served as a complementary approach in detecting lncRNAs missed by the conventional linear models. A combination of GCC and linear models identified lncRNAs of diverse patterns of association enriched for GO biological and molecular functions meaningful in cognitive performance and cognitive decline. The novel lncRNA regulatory network further contributed to detect significant pathways implicated in cognition., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Cognitive aging is one of the major problems worldwide, especially as people get older. This study aimed to perform global gene expression profiling of cognitive function to identify associated genes and pathways and a novel transcriptional regulatory network analysis to identify important regulons. We performed single transcript analysis on 400 monozygotic twins using an assumption-free generalized correlation coefficient (GCC), linear mixed-effect model (LME) and kinship model and identified six probes (one significant at the standard FDR < 0.05 while the other results were suggestive with 0.18 ≤ FDR ≤ 0.28). We combined the GCC and linear model results to cover diverse patterns of relationships, and meaningful and novel genes like APOBEC3G, H6PD, SLC45A1, GRIN3B , and PDE4D were detected. Our exploratory study showed the downregulation of all these genes with increasing cognitive function or vice versa except the SLC45A1 gene, which was upregulated with increasing cognitive function. Linear models found only H6PD and SLC45A1 , the other genes were captured by GCC. Significant functional pathways (FDR < 3.95e-10) such as focal adhesion, ribosome, cysteine and methionine metabolism, Huntington's disease, eukaryotic translation elongation, nervous system development, influenza infection, metabolism of RNA, and cell cycle were identified. A total of five regulons (FDR< 1.3e-4) were enriched in a transcriptional regulatory analysis in which CTCF and REST were activated and SP3, SRF , and XBP1 were repressed regulons. The genome-wide transcription analysis using both assumption-free GCC and linear models identified important genes and biological pathways implicated in cognitive performance, cognitive aging, and neurological diseases. Also, the regulatory network analysis revealed significant activated and repressed regulons on cognitive function., 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 Mohammadnejad, Li, Lund, Li, Larsen, Mengel-From, Michel, Christiansen, Christensen, Hjelmborg, Baumbach and Tan.)

Purpose: We describe a novel neurobehavioral phenotype of autism spectrum disorder (ASD), intellectual disability, and/or attention-deficit/hyperactivity disorder (ADHD) associated with de novo or inherited deleterious variants in members of the RFX family of genes. RFX genes are evolutionarily conserved transcription factors that act as master regulators of central nervous system development and ciliogenesis., Methods: We assembled a cohort of 38 individuals (from 33 unrelated families) with de novo variants in RFX3, RFX4, and RFX7. We describe their common clinical phenotypes and present bioinformatic analyses of expression patterns and downstream targets of these genes as they relate to other neurodevelopmental risk genes., Results: These individuals share neurobehavioral features including ASD, intellectual disability, and/or ADHD; other frequent features include hypersensitivity to sensory stimuli and sleep problems. RFX3, RFX4, and RFX7 are strongly expressed in developing and adult human brain, and X-box binding motifs as well as RFX ChIP-seq peaks are enriched in the cis-regulatory regions of known ASD risk genes., Conclusion: These results establish a likely role of deleterious variation in RFX3, RFX4, and RFX7 in cases of monogenic intellectual disability, ADHD and ASD, and position these genes as potentially critical transcriptional regulators of neurobiological pathways associated with neurodevelopmental disease pathogenesis.

Background: Sufficient fetal fraction (FF) is crucial for quality control of NIPT (Non-Invasive Prenatal Test) results. Different factors influencing bioinformatic estimation of FF should be considered when implementing NIPT. To what extent the total number of sequencing reads influences FF estimate has been unexplored. In this study, to test the robustness of SeqFF FF estimation and provide additional recommendations for NIPT analysis quality control, we compared the SeqFF FF estimates with two other methods and investigated how the number of sequencing reads and FF level affects the accuracy and precision of FF estimates., Methods: WGS data of 516 NIPT samples from a prenatal screening program was obtained. Sample data were randomly downsampled by the read count, and FF was calculated by SeqFF software. Then, the outcome was compared with FF estimates from SNP- and chrY-based methods. FF estimated with different read counts and FF levels were compared with FF at 30 M reads as a reference., Results: SeqFF FF highly correlates with SNP- and chrY-based FF estimates. Raising read count from 2 M to 10 M drastically increased the accuracy of FF estimates. After adding more reads, we saw a further improvement in FF accuracy, reaching a plateau at 20 M reads. Precision of SeqFF FF estimate is independent of FF level in the sample., Conclusion: SeqFF is a robust method for FF estimation for both genders and for any FF level in range 2-13%. Accuracy of FF estimates highly depends on the read count. We recommend using no less than 10 M reads to achieve accurate FF estimates for NIPT analysis in clinical settings., (© 2021 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Although somatic mutations in Histone 3.3 (H3.3) are well-studied drivers of oncogenesis, the role of germline mutations remains unreported. We analyze 46 patients bearing de novo germline mutations in histone 3 family 3A ( H3F3A ) or H3F3B with progressive neurologic dysfunction and congenital anomalies without malignancies. Molecular modeling of all 37 variants demonstrated clear disruptions in interactions with DNA, other histones, and histone chaperone proteins. Patient histone posttranslational modifications (PTMs) analysis revealed notably aberrant local PTM patterns distinct from the somatic lysine mutations that cause global PTM dysregulation. RNA sequencing on patient cells demonstrated up-regulated gene expression related to mitosis and cell division, and cellular assays confirmed an increased proliferative capacity. A zebrafish model showed craniofacial anomalies and a defect in Foxd3-derived glia. These data suggest that the mechanism of germline mutations are distinct from cancer-associated somatic histone mutations but may converge on control of cell proliferation., (Copyright © 2020 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works. Distributed under a Creative Commons Attribution NonCommercial License 4.0 (CC BY-NC).)

Background: Patients with germline variants in SMAD4 can present symptoms of both juvenile polyposis syndrome (JPS) and Hereditary Hemorrhagic Telangiectasia (HHT): JP-HHT syndrome. Next-Generation Sequencing (NGS) techniques disclose causative sequence variants in around 90% of HHT patients fulfilling the Curaçao criteria. Here we report a translocation event involving SMAD4 resulting in JP-HHT., Methods: A patient fulfilling the Curaçao criteria was analyzed for variants in ENG, ACVRL1, and SMAD4 using standard techniques. Whole-genome sequencing (WGS) using both short-read NGS technology and long-read Oxford Nanopore technology was performed to define the structural variant and exact breakpoints., Results: No pathogenic variant was detected in ENG, ACVRL1, or SMAD4 in DNA extracted from blood. Due to abortus habitualis, the proband´s daughter was submitted for chromosomal analysis, and a cytogenetically balanced chromosomal reciprocal translocation t(1;18)(p36.1;q21.1) was detected in the daughter and the patient. The balanced translocation segregated with both gastrointestinal cancer and HHT in the family. WGS provided the exact breakpoints of the reciprocal translocation proving disruption of the SMAD4 gene., Discussion: A disease-causing reciprocal translocation between chromosome 1 and 18 with a breakpoint in the SMAD4 locus co-segregated with JP-HHT in an extended family. This observation warrants further analysis for chromosomal rearrangements in individuals with clinical HHT or JP-HHT of unknown cause., (© 2020 The Authors. Molecular Genetics & Genomic Medicine published by Wiley Periodicals LLC.)

Aim: Many efforts have been deployed to identify genetic variants associated with BMI. Alternatively, we explore epigenetic contribution to BMI variation by focusing on long noncoding RNAs (lncRNAs) which represents a key layer of epigenetic control. Materials & methods: We analyzed lncRNA expression in whole blood of 229 monozygotic twin pairs in association with BMI using generalized estimating equations. Results & conclusion: Six lncRNA probes were identified as significant (false discovery rate <0.05), with BMI showing causal effects on the expression of the significant lncRNAs. Functional annotation of differential profiles identified Gene ontology biological processes including kidney development, regulations of lipid biosynthetic process, circadian rhythm, notch signaling, etc. Whole blood lncRNAs are significantly expressed in response to BMI variation.

Nucleoporins (NUPs) are an essential component of the nuclear-pore complex, which regulates nucleocytoplasmic transport of macromolecules. Pathogenic variants in NUP genes have been linked to several inherited human diseases, including a number with progressive neurological degeneration. We present six affected individuals with bi-allelic truncating variants in NUP188 and strikingly similar phenotypes and clinical courses, representing a recognizable genetic syndrome; the individuals are from four unrelated families. Key clinical features include congenital cataracts, hypotonia, prenatal-onset ventriculomegaly, white-matter abnormalities, hypoplastic corpus callosum, congenital heart defects, and central hypoventilation. Characteristic dysmorphic features include small palpebral fissures, a wide nasal bridge and nose, micrognathia, and digital anomalies. All affected individuals died as a result of respiratory failure, and five of them died within the first year of life. Nuclear import of proteins was decreased in affected individuals' fibroblasts, supporting a possible disease mechanism. CRISPR-mediated knockout of NUP188 in Drosophila revealed motor deficits and seizure susceptibility, partially recapitulating the neurological phenotype seen in affected individuals. Removal of NUP188 also resulted in aberrant dendrite tiling, suggesting a potential role of NUP188 in dendritic development. Two of the NUP188 pathogenic variants are enriched in the Ashkenazi Jewish population in gnomAD, a finding we confirmed with a separate targeted population screen of an international sampling of 3,225 healthy Ashkenazi Jewish individuals. Taken together, our results implicate bi-allelic loss-of-function NUP188 variants in a recessive syndrome characterized by a distinct neurologic, ophthalmologic, and facial phenotype., (Copyright © 2020 American Society of Human Genetics. Published by Elsevier Inc. All rights reserved.)

Monozygotic twins are genetically identical but rarely phenotypically identical. Epigenetic and transcriptional variation could influence this phenotypic discordance. Investigation of intra-pair differences in molecular markers and a given phenotype in monozygotic twins controls most of the genetic contribution, enabling studies of the molecular features of the phenotype. This study aimed to identify genes associated with cognition in later life using integrated enrichment analyses of the results of blood-derived intra-pair epigenome-wide and transcriptome-wide association analyses of cognition in 452 middle-aged and old-aged monozygotic twins (56-80 years). Integrated analyses were performed with an unsupervised approach using KeyPathwayMiner, and a supervised approach using the KEGG and Reactome databases. The supervised approach identified several enriched gene sets, including "neuroactive ligand receptor interaction" ( p -value = 1.62 ∗ 10-2), "Neurotrophin signaling" ( p -value = 2.52 ∗ 10-3), "Alzheimer's disease" ( p -value = 1.20 ∗ 10-2), and "long-term depression" ( p -value = 1.62 ∗ 10-2). The unsupervised approach resulted in a 238 gene network, including the Alzheimer's disease gene APP (Amyloid Beta Precursor Protein) as an exception node, and several novel candidate genes. The strength of the unsupervised method is that it can reveal previously uncharacterized sub-pathways and detect interplay between biological processes, which remain undetected by the current supervised methods. In conclusion, this study identified several previously reported cognition genes and pathways and, additionally, puts forward novel candidates for further verification and validation., (Copyright © 2020 Soerensen, Hozakowska-Roszkowska, Nygaard, Larsen, Schwämmle, Christensen, Christiansen and Tan.)

Heterologous sensitization of adenylyl cyclase (AC) is revealed as enhanced or exaggerated AC/cAMP signaling that occurs following persistent activation of G α i/o -coupled receptors. This paradoxical phenomenon was discovered more than 40 years ago and was proposed as a cellular mechanism to explain the adaptive changes that occur following chronic exposure to drugs of abuse. However, the underlying molecular mechanisms of heterologous sensitization of AC remain largely unknown. In the present study, we performed a genome-wide cell-based RNA interference screen as an unbiased approach to identify genes associated with heterologous sensitization of AC. Following a series of validation and confirmation assays, three genes that form an E3 ligase complex, cullin3 ( CUL3 ), neural precursor-cell-expressed and developmentally downregulated 8 ( NEDD8 ), and really interesting new gene (RING)-box protein 1 ( RBX1 ), were identified as specific modulators of heterologous sensitization of AC. Furthermore, based on the downstream actions of these genes, we evaluated the activity of proteasome inhibitors as well as the specific NEDD8-activating enzyme inhibitor, MLN4924 (Pevonedistat), in AC sensitization. We demonstrate that MG-132 and bortezomib treatments could mimic the inhibitory effects observed with gene knockdown, and MLN4924 was potent and efficacious in blocking the development of heterologous sensitization of endogenous and recombinant AC isoforms, including AC1, AC2, AC5, and AC6. Together, by using genetic and pharmacological approaches, we identified, for the first time, cullin3-RING ligases and the protein degradation pathway as essential modulators for heterologous sensitization of AC. SIGNIFICANCE STATEMENT: Through a genome-wide cell-based RNA interference screening, we identified three genes that form an E3 ligase complex, cullin3, neural precursor-cell-expressed and developmentally downregulated 8 ( NEDD8 ), and really interesting new gene-box protein 1, as specific modulators of heterologous sensitization of AC. The effect of cullin3 , NEDD8, or really interesting new gene-box protein 1 small interfering RNAs on heterologous sensitization was recapitulated by proteasome inhibitors, MG132 and bortezomib, and the specific NEDD8-activating enzyme inhibitor, MLN4924. These results suggest a novel hypothesis in which protein degradation is involved in the sensitization of AC signaling that occurs following chronic activation of Gα i/o -coupled receptors., (Copyright © 2020 by The American Society for Pharmacology and Experimental Therapeutics.)

Unexplained or idiopathic ketotic hypoglycemia (KH) is the most common type of hypoglycemia in children. The diagnosis is based on the exclusion of routine hormonal and metabolic causes of hypoglycemia. We aimed to identify novel genes that cause KH, as this may lead to a more targeted treatment. Deep phenotyping of ten preschool age at onset KH patients (boys, n = 5; girls, n = 5) was performed followed by trio exome sequencing and comprehensive bioinformatics analysis. Data analysis revealed four novel candidate genes: (1) NCOR1 in a patient with KH, iron deficiency and loose stools; (2) IGF2BP1 in a proband with KH, short stature and delayed bone age; (3) SLC5A2 in a proband with KH, intermittent glucosuria and extremely elevated p-GLP-1; and (4) NEK11 in a proband with ketotic hypoglycemia and liver affliction. These genes are associated with different metabolic processes, such as gluconeogenesis, translational regulation, and glucose transport. In conclusion, WES identified DNA variants in four different genes as potential novel causes of IKH, suggesting that IKH is a heterogeneous disorder that can be split into several novel diseases: NCOR1-KH, IGF2BP1-KH, SGLT2-KH or familial renal glucosuria KH, and NEK11-KH. Precision medicine treatment based on exome sequencing may lead to advances in the management of IKH.

Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance., (© The Author(s) (2019). Published by Oxford University Press on behalf of the Guarantors of Brain. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Only few studies have investigated the genomewide transcriptome of normative cognitive aging. We therefore aimed at investigating blood gene expression patterns associated with cognitive aging using a population-based sample of 235 middle-aged monozygotic twin pairs with longitudinal data on cognitive function. This unique setup enabled examination of gene expression differences associated with individual and intrapair differences in cognitive level and change while controlling for underlying genetic variation and shared early environment. Overall, increased expression of several gene sets was found to strongly correlate with a lower cognitive level and cognitive decline. The most significantly correlated gene sets were related to protein metabolism, translation, RNA metabolism, infectious disease, and the immune system, which are all processes previously linked to transcription signatures of pathological and normal brain aging, and aging in blood. The results of our study thus suggest that gene expression patterns of cognitive level and decline in our sample mirror those seen in cognitively impaired individuals, which could point toward a more generic response to cognitive aging and aging in general., (Copyright © 2019 Elsevier Inc. All rights reserved.)

The tumor content of a biopsy and the average depth of coverage are two essential aspects when performing DNA sequencing using next-generation sequencing technologies. The heterogeneous nature of cancer necessitates the identification of distinct clonal cell populations to better understand and treat cancer. Deep sequencing enables researchers to identify these populations, but no consensus on an optimal depth exists for identifying clonal populations. Data from eight deep-sequenced oral squamous cell carcinoma biopsies obtained from three stage IV patients, with various degrees of tumor content, were used to randomly down sample the depth before being subjected to cluster analysis. An increase in coverage resulted in an increase in resolution for clusters of mutations, enabling the identification of distinct clonal cell populations and clonal events. From a depth of 800×, limited gain in resolution can be achieved; and from a depth of 1200×, the resolution stabilizes. Overall, researchers should aim for an average depth of 1000× to 1200× when performing deep sequencing. The tumor content will, however, dictate the resolution and fidelity of the analysis, as an increase in tumor complexity increases the need for higher tumor content., (Copyright © 2019 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

In this review, we discuss the management of genomic medicine, which is based on very large data sets with up to billions of data points when analysing the whole genome. By using artificial intelligence (AI) it is possible to find patterns in such data sets and thereby identify subgroups of patients differing clinically, or to extract informative data points and construct models, which will predict disease risk, prognosis or treatment response most often in a process including training and testing (machine learning). Future clinical decision making will increasingly be based on patterns and models obtained by AI analysis of many parameters.

Rett syndrome is rarely suspected in males because of the X-linked dominant inheritance. In the literature, only six male patients have been reported with methyl-CpG-binding protein 2 (MECP2) mosaicism. Next-generation sequencing (NGS) methods have enabled better detection of somatic mosaicism compared to conventional Sanger sequencing; however, mosaics can still be difficult to detect. We present clinical and molecular findings in two males mosaic for a pathogenic MECP2 variant. Both have been reexamined using deep sequencing of DNA isolated from four different cell tissues (blood, muscle, fibroblasts and oral mucosa). Deep sequencing of the different tissues revealed that the variants were present in all tissues. In one patient, the molecular diagnosis could only be established by reexamination after a normal whole exome sequencing, and the other case is an example of reverse genetic diagnostics. Rett syndrome should be considered in males with neurodevelopmental delay and stereotypical hand movements. Subsequent to clinical diagnosis males should be investigated with NGS-based technologies of MECP2 with high read depth and a low threshold for variant calls. If the initial analysis on full blood derived DNA fails to confirm the suspicion, we recommend repeating the analysis on another tissue, preferentially fibroblasts to increase the diagnostic yield., (© 2018 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Patients with a BRCA1 or BRCA2 mutation (BRCA-mutated breast cancer) are frequently diagnosed with low differentiated and highly proliferating breast cancer characterized by high amounts of tumor-infiltrating lymphocytes (Tils). Stromal Tils (sTils) are highly prognostic in sporadic triple-negative and HER2 positive breast cancer however, their prognostic importance in BRCA-mutated breast cancers is unknown., Material and Methods: Formalin-fixed paraffin-embedded primary tumor tissue from 411 patients with a germline BRCA1 or BRCA2 mutation and diagnosed with early breast cancer was included. The percentage of sTils was quantified on full HE sections according to guidelines proposed by the Immuno-Oncology Biomarker in Breast Cancer Working Group. Distribution of sTils and associates with patient and tumor characteristics were assessed according to categorical sTils groups defined as low (<10%), intermediate (10-59%) and high (≥60%). Prognostic associations of sTils were evaluated as a continuous variable in univariate and multivariate models. Only follow-up time beyond date of BRCA mutation test was included., Results: A large proportion had high sTils (27% in the full cohort, 36% in BRCA1-mutated, and 44% in ER negative breast cancers). Higher sTils were associated with BRCA1, ER negative breast cancer, high histological grade and medullary histology. In combined analysis for BRCA1 and BRCA2-mutated breast cancers, increasing sTils in 10% intervals were significantly associated with OS (HR 0.92, 95% CI 0.84-1.00, p = .05). For each 10% increment of sTils in BRCA1 breast cancers, a 10% reduction of mortality (adjusted HR 0.90 95% CI 0.81-0.99, p = .03) and a 13% reduction in risk of DFS-event (HR 0.87 95% CI 0.76-1.00, p = .05) was observed even after adjustment for ER status. No significant association with survival was of observed in the BRCA2 subgroup. Test for interaction of sTils and BRCA status was not statistically significant (p = .3)., Conclusions: Breast cancer patients with a germline BRCA mutation had higher sTils than previously reported in sporadic breast cancers, and sTils were associated with favorable survival among BRCA carriers.

Introduction: MED13L-related intellectual disability is characterized by moderate intellectual disability (ID), speech impairment, and dysmorphic facial features. We present 8 patients with MED13L-related intellectual disability and review the literature for phenotypical and genetic aspects of previously described patients., Materials and Methods: In the search for genetic aberrations in individuals with ID, two of the patients were identified by chromosomal microarray analysis, and five by exome sequencing. One of the individuals, suspected of MED13L-related intellectual disability, based on clinical features, was identified by Sanger sequencing., Results: All 8 individuals had de novo MED13L aberrations, including two intragenic microdeletions, two frameshift, three nonsense variants, and one missense variant. Phenotypically, they all had intellectual disability, speech and motor delay, and features of the mouth (open mouth appearance, macroglossia, and/or macrostomia). Two individuals were diagnosed with autism, and one had autistic features. One had complex congenital heart defect, and one had persistent foramen ovale. The literature was reviewed with respect to clinical and dysmorphic features, and genetic aberrations., Conclusions: Even if most clinical features of MED13L-related intellectual disability are rather non-specific, the syndrome may be suspected in some individuals based on the association of developmental delay, speech impairment, bulbous nasal tip, and macroglossia, macrostomia, or open mouth appearance., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Mutations in ALDH18A1 can cause autosomal recessive and dominant hereditary spastic paraplegia and autosomal recessive and dominant cutis laxa. ALDH18A1 encodes delta-1-pyrroline-5-carboxylate synthetase (P5CS), which consists of two domains, the glutamate 5-kinase (G5K) and the gamma-glutamyl phosphate reductase (GR5P) domain. The location of the mutations in the gene has influence on whether the amino acid levels are affected. Mutations affecting the G5K domain have previously been found to cause reduced plasma levels of proline, citrulline and arginine, whereas such effect is not seen with mutations affecting the GR5P domain. We present a 19-year old male patient with autosomal recessive spastic paraplegia and compound heterozygosity for two ALDH18A1 mutations, one in each of the P5CS domains. This young man has spastic paraplegia with onset in childhood and temporal lobe epilepsy, but normal levels of proline, ornithine and arginine. To our knowledge, this is the first case with compound heterozygous mutations affecting both P5CS domains, where levels of plasma amino acids have been reported.