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Abstract Background We sought to analyze, in well-defined clinical setting, the first 100 patients treated at the intraoperative MRI (iMRI) hybrid surgical theatre at our facility in a population-based setting to evaluate which pathologies are best approached with iMRI assisted surgeries, as this is not yet clearly defined. Methods Patients undergoing surgery in the 3T iMRI hybrid surgical theatre at our neurosurgical department between December 2017 to May 2021 were included after informed consent. Demographic, clinical, surgical, histological, radiological and outcome parameters, as well as variables related to iMRI, were retrospectively collected and analyzed. Patients were subdivided into adult and pediatric cohorts. Results Various neurosurgical procedures were performed; resection of tumors and epileptic foci, endoscopic skull base procedures including pituitary lesions, deep brain stimulation (DBS) and laser interstitial thermal therapy (LITT). In total, 41 patients were pediatric. An iMRI scan was carried out in 96% of cases and led to continuation of surgery in 50% of cases, mainly due to visualized remaining pathological tissue (95.2%). Median time to iMRI from intubation was 280 min and median total duration of surgery was 445 min. The majority of patients experienced no postoperative complications (70%), 13 patients suffered permanent postoperative deficits, predominantly visual. Conclusion Herein, we demonstrate the first 100 patients undergoing neurosurgery aided by iMRI at our facility since introduction. Indications for surgery differed between pediatric and adult patients. The iMRI was utilized for tumor surgeries, particularly adult low-grade gliomas and pediatric tumors, as well as for epilepsy surgery and DBS. In this heterogenous population, iMRI led to continuation of surgery in 50%. To establish the benefit in maximizing the extent of resection in these brain pathologies future studies are recommended. Clinical trial number Not applicable.

Introduction: The surgical procedure for severe, drug-resistant, unilateral hemispheric epilepsy is challenging. Over the last decades the surgical landscape for hemispheric disconnection procedures changed from anatomical hemispherectomy to functional hemispherotomy with a reduction of complications and stable good seizure outcome. Here, a task force of European epilepsy surgeons prepared, on behalf of the EANS Section for Functional Neurosurgery, a consensus statement on different aspects of the hemispheric disconnection procedure. Research question: To determine history, indication, timing, techniques, complications and current practice in Europe for hemispheric disconnection procedures in drug-resistant epilepsy. Material and methods: Relevant literature on the topic was collected by a literature search based on the PRISMA 2020 guidelines. Results: A comprehensive overview on the historical development of hemispheric disconnection procedures for epilepsy is presented, while discussing indications, timing, surgical techniques and complications. Current practice for this procedure in European epilepsy surgery centers is provided. At present, our knowledge of long-term seizure outcomes primarily stems from open surgical disconnection procedures. Although minimal invasive surgical techniques in epilepsy are rapidly developing and reported in case reports or small case series, long-term seizure outcome remain uncertain and needs to be reported. Discussion and conclusion: This is the first paper presenting a European consensus statement regarding history, indications, techniques and complications of hemispheric disconnection procedures for different causes of chronic, drug-resistant epilepsy. Furthermore, it serves as the pioneering document to report a comprehensive overview of the current surgical practices regarding this type of surgery employed in renowned epilepsy surgery centers across Europe.

Measurement(s) highway vehicle routing Technology Type(s) GPS location tracking Sample Characteristic - Location Sonoma County, California, USA

FOXC1 is a ubiquitously expressed forkhead transcription factor that plays a critical role during early development. Germline pathogenic variants in FOXC1 are associated with anterior segment dysgenesis and Axenfeld-Rieger syndrome (ARS, #602482), an autosomal dominant condition with ophthalmologic anterior segment abnormalities, high risk for glaucoma and extraocular findings including distinctive facial features, as well as dental, skeletal, audiologic, and cardiac anomalies. De Hauwere syndrome is an ultrarare condition previously associated with 6p microdeletions and characterized by anterior segment dysgenesis, joint instability, short stature, hydrocephalus, and skeletal abnormalities. Here, we report clinical findings of two unrelated adult females with FOXC1 haploinsufficiency who have ARS and skeletal abnormalities. Final molecular diagnoses of both patients were achieved using genome sequencing. Patient 1 had a complex rearrangement involving a 4.9 kB deletion including FOXC1 coding region (Hg19; chr6:1,609,721-1,614,709), as well as a 7 MB inversion (Hg19; chr6:1,614,710-8,676,899) and a second deletion of 7.1 kb (Hg19; chr6:8,676,900-8,684,071). Patient 2 had a heterozygous single nucleotide deletion, resulting in a frameshift and a premature stop codon in FOXC1 (NM_001453.3): c.467del, p.(Pro156Argfs*25). Both individuals had moderate short stature, skeletal abnormalities, anterior segment dysgenesis, glaucoma, joint laxity, pes planovalgus, dental anomalies, hydrocephalus, distinctive facial features, and normal intelligence. Skeletal surveys revealed dolichospondyly, epiphyseal hypoplasia of femoral and humeral heads, dolichocephaly with frontal bossin gand gracile long bones. We conclude that haploinsufficiency of FOXC1 causes ARS and a broad spectrum of symptoms with variable expressivity that at its most severe end also includes a phenotype overlapping with De Hauwere syndrome.

Abstract DNA methylation is increasingly used for tumour classification and has expanded upon the > 100 currently known brain tumour entities. A correct diagnosis is the basis for suitable treatment for patients with brain tumours, which is the leading cause of cancer-related death in children. DNA methylation profiling is required for diagnosis of certain tumours, and used clinically for paediatric brain tumours in several countries. We therefore evaluated if the methylation-based classification is robust in different locations of the same tumour, and determined how the methylation pattern changed over time to relapse. We sampled 3–7 spatially separated biopsies per patient, and collected samples from paired primary and relapse brain tumours from children. Altogether, 121 samples from 46 paediatric patients with brain tumours were profiled with EPIC methylation arrays. The methylation-based classification was mainly homogeneous for all included tumour types that were successfully classified, which is promising for clinical diagnostics. There were indications of multiple subclasses within tumours and switches in the relapse setting, but not confirmed as the classification scores were below the threshold. Site-specific methylation alterations did occur within the tumours and varied significantly between tumour types for the temporal samples, and as a trend in spatial samples. More alterations were present in high-grade tumours compared to low-grade, and significantly more alterations with longer relapse times. The alterations in the spatial and temporal samples were significantly depleted in CpG islands, exons and transcription start sites, while enriched in OpenSea and regions not affiliated with a gene, suggesting a random location of the alterations in less conserved regions. In conclusion, more DNA methylation changes accumulated over time and more alterations occurred in high-grade tumours. The alterations mainly occurred in regions without gene affiliation, and did not affect the methylation-based classification, which largely remained homogeneous in paediatric brain tumours.

IntroductionNeuromuscular disorders (NMDs) have a heterogeneous etiology. A genetic diagnosis is key to personalized healthcare and access to targeted treatment for the affected individuals.MethodsIn this study, 861 patients with NMDs were analyzed with genome sequencing and comprehensive variant calling including single nucleotide variants, small insertions/deletions (SNVs/INDELs), and structural variants (SVs) in a panel of 895 NMD genes, as well as short tandem repeat expansions (STRs) at 28 loci. In addition, for unsolved cases with an unspecific clinical presentation, the analysis of a panel with OMIM disease genes was added.ResultsIn the cohort, 27% (232/861) of the patients harbored pathogenic variants, of which STRs and SVs accounted for one-third of the patients (71/232). The variants were found in 107 different NMD genes. Furthermore, 18 pediatric patients harbored pathogenic variants in non-NMD genes.DiscussionOur results highlight that for children with unspecific hypotonia, a genome-wide analysis rather than a disease-based gene panel should be considered as a diagnostic approach. More importantly, our results clearly show that it is crucial to include STR- and SV-analyses in the diagnostics of patients with neuromuscular disorders.

Abstract Background We report the findings from 4437 individuals (3219 patients and 1218 relatives) who have been analyzed by whole genome sequencing (WGS) at the Genomic Medicine Center Karolinska-Rare Diseases (GMCK-RD) since mid-2015. GMCK-RD represents a long-term collaborative initiative between Karolinska University Hospital and Science for Life Laboratory to establish advanced, genomics-based diagnostics in the Stockholm healthcare setting. Methods Our analysis covers detection and interpretation of SNVs, INDELs, uniparental disomy, CNVs, balanced structural variants, and short tandem repeat expansions. Visualization of results for clinical interpretation is carried out in Scout—a custom-developed decision support system. Results from both singleton (84%) and trio/family (16%) analyses are reported. Variant interpretation is done by 15 expert teams at the hospital involving staff from three clinics. For patients with complex phenotypes, data is shared between the teams. Results Overall, 40% of the patients received a molecular diagnosis ranging from 19 to 54% for specific disease groups. There was heterogeneity regarding causative genes (n = 754) with some of the most common ones being COL2A1 (n = 12; skeletal dysplasia), SCN1A (n = 8; epilepsy), and TNFRSF13B (n = 4; inborn errors of immunity). Some causative variants were recurrent, including previously known founder mutations, some novel mutations, and recurrent de novo mutations. Overall, GMCK-RD has resulted in a large number of patients receiving specific molecular diagnoses. Furthermore, negative cases have been included in research studies that have resulted in the discovery of 17 published, novel disease-causing genes. To facilitate the discovery of new disease genes, GMCK-RD has joined international data sharing initiatives, including ClinVar, UDNI, Beacon, and MatchMaker Exchange. Conclusions Clinical WGS at GMCK-RD has provided molecular diagnoses to over 1200 individuals with a broad range of rare diseases. Consolidation and spread of this clinical-academic partnership will enable large-scale national collaboration.

Abstract Background Exome and genome sequencing is becoming the method of choice for rare disease diagnostics. One of the key challenges remaining is distinguishing the disease causing variants from the benign background variation. After analysis and annotation of the sequencing data there are typically thousands of candidate variants requiring further investigation. One of the most effective and least biased ways to reduce this number is to assess the rarity of a variant in any population. Currently, there are a number of reliable sources of information for major population frequencies when considering single nucleotide variants (SNVs) and small insertion and deletions (INDELs), with gnomAD as the most prominent public resource available. However, local variation or frequencies in sub-populations may be underrepresented in these public resources. In contrast, for structural variation (SV), the background frequency in the general population is more or less unknown mostly due to challenges in calling SVs in a consistent way. Keeping track of local variation is one way to overcome these problems and significantly reduce the number of potential disease causing variants retained for manual inspection, both for SNVs and SVs. Results Here, we present loqusdb, a tool to solve the challenge of keeping track of any type of variant observations from genome sequencing data. Loqusdb was designed to handle a large flow of samples and unlike other solutions, samples can be added continuously to the database without rebuilding it, facilitating improvements and additions. We assessed the added value of a local observations database using 98 samples annotated with information from a background of 888 unrelated individuals. Conclusions We show both how powerful SV analysis can be when filtering for population frequencies and how the number of apparently rare SNVs/INDELs can be reduced by adding local population information even after annotating the data with other large frequency databases, such as gnomAD. In conclusion, we show that a local frequency database is an attractive, and a necessary addition to the publicly available databases that facilitate the analysis of exome and genome data in a clinical setting.

Abstract Background DNA damage accumulates over the course of cancer development. The often-substantial amount of somatic mutations in cancer poses a challenge to traditional methods to characterize tumors based on driver mutations. However, advances in machine learning technology can take advantage of this substantial amount of data. Results We developed a command line interface python package, pyCancerSig, to perform sample profiling by integrating single nucleotide variation (SNV), structural variation (SV) and microsatellite instability (MSI) profiles into a unified profile. It also provides a command to decipher underlying cancer processes, employing an unsupervised learning technique, Non-negative Matrix Factorization, and a command to visualize the results. The package accepts common standard file formats (vcf, bam). The program was evaluated using a cohort of breast- and colorectal cancer from The Cancer Genome Atlas project (TCGA). The result showed that by integrating multiple mutations modes, the tool can correctly identify cases with known clear mutational signatures and can strengthen signatures in cases with unclear signal from an SNV-only profile. The software package is available at https://github.com/jessada/pyCancerSig. Conclusions pyCancerSig has demonstrated its capability in identifying known and unknown cancer processes, and at the same time, illuminates the association within and between the mutation modes.

Induced pluripotent stem cells (iPSCs) from patients are an attractive disease model to study tissues with poor accessibility such as the brain. Using this approach, we and others have shown that trisomy 21 results in genome-wide transcriptional dysregulations. The effects of loss of genes on chromosome 21 is much less characterized. Here, we use patient-derived neural cells from an individual with neurodevelopmental delay and a ring chromosome 21 with two deletions spanning 3.8 Mb at the terminal end of 21q22.3, containing 60 protein-coding genes. To investigate the molecular perturbations of the partial monosomy on neural cells, we established patient-derived iPSCs from fibroblasts retaining the ring chromosome 21, and we then induced iPSCs into neuroepithelial stem cells. RNA-Seq analysis of NESCs with the ring chromosome revealed downregulation of 18 genes within the deleted region together with global transcriptomic dysregulations when compared to euploid NESCs. Since the deletions on chromosome 21 represent a genetic “contrary” to trisomy of the corresponding region, we further compared the dysregulated transcriptomic profile in with that of two NESC lines with trisomy 21. The analysis revealed opposed expression changes for 23 genes on chromosome 21 as well as 149 non-chromosome 21 genes. Taken together, our results bring insights into the effects on the global and chromosome 21 specific gene expression from a partial monosomy of chromosome 21qter during early neuronal differentiation.

Background: The influence of daily life exposure on the gastrointestinal tract is not fully understood. This study aimed to examine associations between functional gastrointestinal symptoms and sociodemographic status and lifestyle habits in the general population.Methods: The Malmö Offspring Study (MOS) included 2,648 participants from the general population who had answered a questionnaire about sociodemographic status, lifestyle habits, medical health, and self-reported irritable bowel syndrome (IBS). The visual analog scale for IBS (VAS-IBS) was completed to assess gastrointestinal symptoms the past 2 weeks. Subjects with organic gastrointestinal diseases were excluded. Presence of self-reported IBS and gastrointestinal symptoms the past 2 weeks were used as dependent variables to study the associations with age, sex, body mass index, education, occupation, marital status, smoking, snuff using, alcohol drinking frequency, alcohol amount per drinking occasion, physical activity at work, and physical activity during leisure time, using logistic regression and generalized linear model.Results: Self-reported IBS was associated with gastrointestinal symptoms the past 2 weeks (p < 0.001). There was an association between IBS and female sex (p < 0.001), former smoking (p < 0.001), present smoking (p < 0.001), and an inverse association with drinking 3–4 standard glasses per occasion (p = 0.038). Gastrointestinal symptoms were associated with age 50–59 years (p = 0.009), ≥60 years (p = 0.004), female sex (p < 0.001), studying (p = 0.036), unemployment (p = 0.009), former smoking (p = 0.001), and present smoking (p = 0.012). In men, IBS was associated with middle-age and both IBS and gastrointestinal symptoms were associated with unemployment (p < 0.001 and p = 0.001, respectively). In women, IBS was associated with present smoking (p = 0.022), and gastrointestinal symptoms were associated with former smoking and inversely associated with higher age (p = 0.006) and intermediate physical activity at work (p = 0.008). No associations were found with BMI, education, marital status, or snuff using.Conclusion: Self-reported IBS in the general population shows strongest association with female sex and smoking, whereas gastrointestinal symptoms also are associated with unemployment and inversely associated with higher age. In men, both IBS and gastrointestinal symptoms are associated with unemployment. In women, both IBS and gastrointestinal symptoms are associated with smoking, whereas symptoms are inversely associated with higher age and intermediate physical activity.

Abstract Background Since different types of genetic variants, from single nucleotide variants (SNVs) to large chromosomal rearrangements, underlie intellectual disability, we evaluated the use of whole-genome sequencing (WGS) rather than chromosomal microarray analysis (CMA) as a first-line genetic diagnostic test. Methods We analyzed three cohorts with short-read WGS: (i) a retrospective cohort with validated copy number variants (CNVs) (cohort 1, n = 68), (ii) individuals referred for monogenic multi-gene panels (cohort 2, n = 156), and (iii) 100 prospective, consecutive cases referred to our center for CMA (cohort 3). Bioinformatic tools developed include FindSV, SVDB, Rhocall, Rhoviz, and vcf2cytosure. Results First, we validated our structural variant (SV)-calling pipeline on cohort 1, consisting of three trisomies and 79 deletions and duplications with a median size of 850 kb (min 500 bp, max 155 Mb). All variants were detected. Second, we utilized the same pipeline in cohort 2 and analyzed with monogenic WGS panels, increasing the diagnostic yield to 8%. Next, cohort 3 was analyzed by both CMA and WGS. The WGS data was processed for large (> 10 kb) SVs genome-wide and for exonic SVs and SNVs in a panel of 887 genes linked to intellectual disability as well as genes matched to patient-specific Human Phenotype Ontology (HPO) phenotypes. This yielded a total of 25 pathogenic variants (SNVs or SVs), of which 12 were detected by CMA as well. We also applied short tandem repeat (STR) expansion detection and discovered one pathologic expansion in ATXN7. Finally, a case of Prader-Willi syndrome with uniparental disomy (UPD) was validated in the WGS data. Important positional information was obtained in all cohorts. Remarkably, 7% of the analyzed cases harbored complex structural variants, as exemplified by a ring chromosome and two duplications found to be an insertional translocation and part of a cryptic unbalanced translocation, respectively. Conclusion The overall diagnostic rate of 27% was more than doubled compared to clinical microarray (12%). Using WGS, we detected a wide range of SVs with high accuracy. Since the WGS data also allowed for analysis of SNVs, UPD, and STRs, it represents a powerful comprehensive genetic test in a clinical diagnostic laboratory setting.

Arachnoid cysts are benign, congenital, non-neoplastic, extra-axial-intra cerebrospinal fluid (CF) collection lesions. They are often diagnosed incidentally during imaging examinations and are usually asymptomatic.We present a case of a subdural hygroma, secondary to an arachnoid cyst which had ruptured following minor head injury. This is a rarely seen complication although it has previously been described in the literature.

BackgroundAnalysis of cell-free tumour DNA, a liquid biopsy, is a promising biomarker for cancer. We have performed a proof-of principle study to test the applicability in the clinical setting, analysing copy number alterations (CNAs) in plasma and tumour tissue from 44 patients with gastro-oesophageal cancer.MethodsDNA was isolated from blood plasma and a tissue sample from each patient. Array-CGH was applied to the tissue DNA. The cell-free plasma DNA was sequenced by low-coverage whole-genome sequencing using a clinical pipeline for non-invasive prenatal testing. WISECONDOR and ichorCNA, two bioinformatic tools, were used to process the output data and were compared to each other.ResultsCancer-associated CNAs could be seen in 59% (26/44) of the tissue biopsies. In the plasma samples, a targeted approach analysing 61 regions of special interest in gastro-oesophageal cancer detected cancer-associated CNAs with a z-score >5 in 11 patients. Broadening the analysis to a whole-genome view, 17/44 patients (39%) had cancer-associated CNAs using WISECONDOR and 13 (30%) using ichorCNA. Of the 26 patients with tissue-verified cancer-associated CNAs, 14 (54%) had corresponding CNAs in plasma. Potentially clinically actionable amplifications overlapping the genes VEGFA, EGFR and FGFR2 were detected in the plasma from three patients.ConclusionsWe conclude that low-coverage whole-genome sequencing without prior knowledge of the tumour alterations could become a useful tool for cell-free tumour DNA analysis of total CNAs in plasma from patients with gastro-oesophageal cancer.

Advanced electrochemical impedance spectroscopy (EIS) was applied to characterize industrial Ni-Cd batteries and to investigate the electrochemical redox processes. A two-term calibration workflow was used for accurate complex impedance measurements across a broad frequency range of 10 mHz to 2 kHz, resulting in calibrated resistance and reactance values. The EIS calibration significantly improved the measurements, particularly at high frequencies above 200 Hz, with differences of 6–8% to the uncalibrated impedance. With an electromagnetic finite element method (FEM) model, we showed that the impedance is strongly influenced by the cable fixturing and the self-inductance of the wire conductors due to alternating currents, which are efficiently removed by the proposed calibration workflow. For single cells, we measured the resistance and the reactance with respect to the state-of-charge (SoC) at different frequencies and a given rest period. For Ni-Cd blocks that include two cells in series, we found good agreement of EIS curves with single cells. As such, EIS can be used as a fast and reliable method to estimate the cell or block capacity status. For electrochemical interpretation, we used an equivalent electric circuit (EEC) model to fit the impedance spectra and to extract the main electrochemical parameters based on calibrated EIS, including charge-transfer kinetics, mass transport, and ohmic resistances. From the charge-transfer resistance, we computed the exchange current density, resulting in 0.23 A/cm2, reflecting high intrinsic rates of the redox electron transfer processes in Ni-Cd cells.

Wind power coupled to hydrogen (H2) production is an interesting strategy to reduce power curtailment and to provide clean fuel for decarbonizing agricultural activities. However, such implementation is challenging for several reasons, including uncertainties in wind power availability, seasonalities in agricultural fuel demand, capital-intensive gas storage systems, and high specific investment costs of small-scale electrolysers. To investigate whether on-site H2 production could be a feasible alternative to conventional diesel farming, a model was built for dynamic simulations of H2 production from wind power driven by the fuel demand of a cereal farm located on the island of Gotland, Sweden. Different cases and technological scenarios were considered to assess the effects of future developments, H2 end-use, as well as production scale on the levelised- and farmers’ equivalent annual costs. In a single-farm application, H2 production costs varied between 21.20–14.82 €/kg. By sharing a power-to-H2 facility among four different farms of 300-ha each, the specific investment costs could be significantly decreased, resulting in 28% lower H2 production costs than when facilities are not shared. By including delivery vans as additional H2 consumers in each farm, costs of H2 production decreased by 35% due to the higher production scale and more distributed demand. However, in all cases and technological scenarios assessed, projected diesel price in retailers was cheaper than H2. Nevertheless, revenues from leasing the land to wind power developers could make H2 a more attractive option even in single-farm applications as early as 2020. Without such revenues, H2 is more competitive than diesel where power-to-H2 plants are shared by at least two farms, if technological developments predicted for 2030 come true. Also, out of 20 different cases assessed, nine of them showed a carbon abatement cost lower than the current carbon tax in Sweden of 110 €/tCO2, which demonstrate the potential of power-to-H2 as an effective strategy to decarbonize agricultural systems.

Objective: Peroxisome proliferator-activated receptors (PPARs) are key transcription factors that regulate adipose development and function, and the conversion of white into brown-like adipocytes. Here we investigated whether PPARα and PPARγ activation synergize to induce the browning of white fat. Methods: A selection of PPAR activators was tested for their ability to induce the browning of both mouse and human white adipocytes in vitro, and in vivo in lean and obese mice. Results: All dual PPARα/γ activators tested robustly increased uncoupling protein 1 (Ucp1) expression in both mouse and human adipocytes in vitro, with tesaglitazar leading to the largest Ucp1 induction. Importantly, dual PPARα/γ activator tesaglitazar strongly induced browning of white fat in vivo in both lean and obese male mice at thermoneutrality, greatly exceeding the increase in Ucp1 observed with the selective PPARγ activator rosiglitazone. While selective PPARγ activation was sufficient for the conversion of white into brown-like adipocytes in vitro, dual PPARα/γ activation was superior to selective PPARγ activation at inducing white fat browning in vivo. Mechanistically, the superiority of dual PPARα/γ activators is mediated at least in part via a PPARα-driven increase in fibroblast growth factor 21 (FGF21). Combined treatment with rosiglitazone and FGF21 resulted in a synergistic increase in Ucp1 mRNA levels both in vitro and in vivo. Tesaglitazar-induced browning was associated with increased energy expenditure, enhanced insulin sensitivity, reduced liver steatosis, and an overall improved metabolic profile compared to rosiglitazone and vehicle control groups. Conclusions: PPARγ and PPARα synergize to induce robust browning of white fat in vivo, via PPARγ activation in adipose, and PPARα-mediated increase in FGF21.

The etiology of hereditary ataxia syndromes is heterogeneous, and the mechanisms underlying these disorders are often unknown. Here, we utilized exome sequencing in two siblings with progressive ataxia and muscular weakness and identified a novel homozygous splice mutation (c.3020-1G > A) in neurofascin (NFASC). In RNA extracted from fibroblasts, we showed that the mutation resulted in inframe skipping of exon 26, with a deprived expression of the full-length transcript that corresponds to NFASC isoform NF186. To further investigate the disease mechanisms, we reprogrammed fibroblasts from one affected sibling to induced pluripotent stem cells, directed them to neuroepithelial stem cells and finally differentiated to neurons. In early neurogenesis, differentiating cells with selective depletion of the NF186 isoform showed significantly reduced neurite outgrowth as well as fewer emerging neurites. Furthermore, whole-cell patch-clamp recordings of patient-derived neuronal cells revealed a lower threshold for openings, indicating altered Na+ channel kinetics, suggesting a lower threshold for openings as compared to neuronal cells without the NFASC mutation. Taken together, our results suggest that loss of the full-length NFASC isoform NF186 causes perturbed neurogenesis and impaired neuronal biophysical properties resulting in a novel early-onset autosomal recessive ataxia syndrome.

Abstract Background The bladder exstrophy‐epispadias complex (BEEC) is a congenital malformation of the bladder and urethra. The underlying causes of this malformation are still largely unknown; however, aside from environment, genetics is thought to play an essential role. The recurrent 22q11.2 microduplication is the most persistently detected genetic aberration found in BEEC cases. Methods We performed array comparative genomic hybridization (array‐CGH) analysis of 76 Swedish BEEC patients. Statistical analysis was performed on current dataset pooled with previously published data on the 22q11.2 microduplication in BEEC patients. We performed massive parallel sequencing (MPS) of the 22q11.2 region in 20 BEEC patients without the 22q11.2 microduplication followed by functional studies. Results We identified three additional cases with the 22q11.2 microduplication. Pooling data from this study with previously published reports showed a statistically significant enrichment of the 22q11.2 microduplication in BEEC patients (2.61% in cases vs. 0.08% in controls; OR = 32.6; p = 8.7 × 10−4). MPS of the 22q11.2 region in 20 BEEC patients without the 22q11.2 microduplication identified a novel variant in LZTR1 (p.Ser698Phe) in one patient. Functional evaluation of the LZTR1 p.Ser698Phe variant in live NIH 3T3 cells showed that the concentration and cytoplasmic mobility differ between the Lztr1wt and Lztr1mut, indicating a potential functional effect of the LZTR1mut. Conclusion Our study further emphasizes the involvement of the 22q11.2 region in BEEC development and highlights LZTR1 as a candidate gene underlying the urogenital malformation.

Complex chromosomal rearrangements (CCRs) are rearrangements involving more than two chromosomes or more than two breakpoints. Whole genome sequencing (WGS) allows for outstanding high resolution characterization on the nucleotide level in unique sequences of such rearrangements, but problems remain for mapping breakpoints in repetitive regions of the genome, which are known to be prone to rearrangements. Hence, multiple complementary WGS experiments are sometimes needed to solve the structures of CCRs. We have studied three individuals with CCRs: Case 1 and Case 2 presented with de novo karyotypically balanced, complex interchromosomal rearrangements (46,XX,t(2;8;15)(q35;q24.1;q22) and 46,XY,t(1;10;5)(q32;p12;q31)), and Case 3 presented with a de novo, extremely complex intrachromosomal rearrangement on chromosome 1. Molecular cytogenetic investigation revealed cryptic deletions in the breakpoints of chromosome 2 and 8 in Case 1, and on chromosome 10 in Case 2, explaining their clinical symptoms. In Case 3, 26 breakpoints were identified using WGS, disrupting five known disease genes. All rearrangements were subsequently analyzed using optical maps, linked-read WGS, and short-read WGS. In conclusion, we present a case series of three unique de novo CCRs where we by combining the results from the different technologies fully solved the structure of each rearrangement. The power in combining short-read WGS with long-molecule sequencing or optical mapping in these unique de novo CCRs in a clinical setting is demonstrated.

The incidence of stillbirth in Sweden has essentially remained constant since the 1980's, and despite thorough investigation, many cases remain unexplained. It has been suggested that a proportion of stillbirth cases is caused by heart disease, mainly channelopathies. The aim of this study was to analyze DNA from 290 stillbirth cases without chromosomal abnormalities for pathogenic single nucleotide variants (SNVs) in 70 genes associated with cardiac channelopathies and cardiomyopathies. The HaloPlex Target Enrichment System (Agilent Technologies) was utilized to prepare sequencing libraries which were sequenced on the Illumina NextSeq platform. We found that 12.1% of the 290 investigated stillbirth cases had one (n = 31) or two (n = 4) variants with evidence supporting pathogenicity, i.e. loss-of-function variants (nonsense, frameshift, splice site substitutions), evidence from functional studies, or previous identification of the variants in affected individuals. Regarding identified putative pathogenic variants in genes associated with channelopathies, the prevalence was significantly higher in the stillbirth cohort (n = 23, 7.93%) than the corresponding prevalence of the same variants in the non-Finnish European population of the Exome Aggregation Consortium (2.70%, p

Clustered copy number variants (CNVs) as detected by chromosomal microarray analysis (CMA) are often reported as germline chromothripsis. However, such cases might need further investigations by massive parallel whole genome sequencing (WGS) in order to accurately define the underlying complex rearrangement, predict the occurrence mechanisms and identify additional complexities. Here, we utilized WGS to delineate the rearrangement structure of 21 clustered CNV carriers first investigated by CMA and identified a total of 83 breakpoint junctions (BPJs). The rearrangements were further sub-classified depending on the patterns observed: I) Cases with only deletions (n = 8) often had additional structural rearrangements, such as insertions and inversions typical to chromothripsis; II) cases with only duplications (n = 7) or III) combinations of deletions and duplications (n = 6) demonstrated mostly interspersed duplications and BPJs enriched with microhomology. In two cases the rearrangement mutational signatures indicated both a breakage-fusion-bridge cycle process and haltered formation of a ring chromosome. Finally, we observed two cases with Alu- and LINE-mediated rearrangements as well as two unrelated individuals with seemingly identical clustered CNVs on 2p25.3, possibly a rare European founder rearrangement. In conclusion, through detailed characterization of the derivative chromosomes we show that multiple mechanisms are likely involved in the formation of clustered CNVs and add further evidence for chromoanagenesis mechanisms in both "simple" and highly complex chromosomal rearrangements. Finally, WGS characterization adds positional information, important for a correct clinical interpretation and deciphering mechanisms involved in the formation of these rearrangements.

The detection of recurrent somatic chromosomal rearrangements is standard of care for most leukemia types. Even though karyotype analysis-a low-resolution genome-wide chromosome analysis-is still the gold standard, it often needs to be complemented with other methods to increase resolution. To evaluate the feasibility and applicability of mate pair whole genome sequencing (MP-WGS) to detect structural chromosomal rearrangements in the diagnostic setting, we sequenced ten bone marrow samples from leukemia patients with recurrent rearrangements. Samples were selected based on cytogenetic and FISH results at leukemia diagnosis to include common rearrangements of prognostic relevance. Using MP-WGS and in-house bioinformatic analysis all sought rearrangements were successfully detected. In addition, unexpected complexity or additional, previously undetected rearrangements was unraveled in three samples. Finally, the MP-WGS analysis pinpointed the location of chromosome junctions at high resolution and we were able to identify the exact exons involved in the resulting fusion genes in all samples and the specific junction at the nucleotide level in half of the samples. The results show that our approach combines the screening character from karyotype analysis with the specificity and resolution of cytogenetic and molecular methods. As a result of the straightforward analysis and high-resolution detection of clinically relevant rearrangements, we conclude that MP-WGS is a feasible method for routine leukemia diagnostics of structural chromosomal rearrangements.

Copy number variations (CNVs) within the human genome have been linked to a diversity of inherited diseases and phenotypic traits. The currently used methodology to measure copy numbers has limited resolution and/or precision, especially for regions with more than 4 copies. Whole genome sequencing (WGS) offers an alternative data source to allow for the detection and characterization of the copy number across different genomic regions in a single experiment. A plethora of tools have been developed to utilize WGS data for CNV detection. None of these tools are designed specifically to accurately estimate copy numbers of complex regions in a small cohort or clinical setting. Herein, we present AMYCNE (automatic modeling functionality for copy number estimation), a CNV analysis tool using WGS data. AMYCNE is multifunctional and performs copy number estimation of complex regions, annotation of VCF files, and CNV detection on individual samples. The performance of AMYCNE was evaluated using AMY1A ddPCR measurements from 86 unrelated individuals. In addition, we validated the accuracy of AMYCNE copy number predictions on two additional genes (FCGR3A and FCGR3B) using datasets available through the 1000 genomes consortium. Finally, we simulated levels of mosaic loss and gain of chromosome X and used this dataset for benchmarking AMYCNE. The results show a high concordance between AMYCNE and ddPCR, validating the use of AMYCNE to measure tandem AMY1 repeats with high accuracy. This opens up new possibilities for the use of WGS for accurate copy number determination of other complex regions in the genome in small cohorts or single individuals.

It is well known that a large proportion of available logging residues intended for extraction will not reach the energy-conversion industry, because some are lost during transportation or left on the clear-felled area. However, there is little understanding of where logging residue losses occur in the supply chain. In this study, the distribution of logging residues for two methods (dried- and fresh-stacked method) to extract logging residues were studied in one clear-felled area. In addition, residue fractions were examined in a detailed comparison. Even though the fresh-stacked method left somewhat more logging residues at the clear-felled area, the differences are small between the methods. Approximately 30% of the total amount of logging residues was left behind between the harvester heaps, with an additional 10%–15% under these heaps and approximately 2%–3% beneath the windrows. The final product that was delivered to the energy-conversion industry was very similar, regardless of the extraction method used. The delivered chipped logging residues had moisture contents of 37% and 36% following fresh- and dried-stacked methods respectively, and in both cases the needle content in the processed logging residues was approximately 10%. However, the total amount of fine fractions (needles and fines) was slightly higher following dried-stacking.

Reliable detection of large structural variation ( > 1000 bp) is important in both rare and common genetic disorders. Whole genome sequencing (WGS) is a technology that may be used to identify a large proportion of the genomic structural variants (SVs) in an individual in a single experiment. Even though SV callers have been extensively used in research to detect mutations, the potential usage of SV callers within routine clinical diagnostics is still limited. One well known, but not well-addressed problem is the large number of benign variants and reference errors present in the human genome that further complicates analysis. Even though there is a wide range of SV-callers available, the number of callers that allow detection of the entire spectra of SV at a low computational cost is still relatively limited.

BACKGROUND:Cardiomyopathies represent a rare group of disorders often of genetic origin. While approximately 50% of genetic causes are known for other types of cardiomyopathies, the genetic spectrum of restrictive cardiomyopathy (RCM) is largely unknown. The aim of the present study was to identify the genetic background of idiopathic RCM and to compile the obtained genetic variants to the novel signalling pathways using in silico protein network analysis. PATIENTS AND METHODS:We used Illumina MiSeq setup to screen for 108 cardiomyopathy and arrhythmia-associated genes in 24 patients with idiopathic RCM. Pathogenicity of genetic variants was classified according to American College of Medical Genetics and Genomics classification. RESULTS:Pathogenic and likely-pathogenic variants were detected in 13 of 24 patients resulting in an overall genotype-positive rate of 54%. Half of the genotype-positive patients carried a combination of pathogenic, likely-pathogenic variants and variants of unknown significance. The most frequent combination included mutations in sarcomeric and cytoskeletal genes (38%). A bioinformatics approach underlined the mechanotransducing protein networks important for RCM pathogenesis. CONCLUSIONS:Multiple gene mutations were detected in half of the RCM cases, with a combination of sarcomeric and cytoskeletal gene mutations being the most common. Mutations of genes encoding sarcomeric, cytoskeletal, and Z-line-associated proteins appear to have a predominant role in the development of RCM.

Replication of reported associations is crucial to the investigation of complex disease. More than 100 SNPs have previously been reported as associated with allergic rhinitis (AR), but few of these have been replicated successfully. To investigate the general reproducibility of reported AR-associations in candidate gene studies, one Swedish (352 AR-cases, 709 controls) and one Singapore Chinese population (948 AR-cases, 580 controls) were analyzed using 49 AR-associated SNPs. The overall pattern of P-values indicated that very few of the investigated SNPs were associated with AR. Given published odds ratios (ORs) most SNPs showed high power to detect an association, but no correlations were found between the ORs of the two study populations or with published ORs. None of the association signals were in common to the two genome-wide association studies published in AR, indicating that the associations represent false positives or have much lower effect-sizes than reported.

The pathway for RNA interference is widespread in metazoans and participates in numerous cellular tasks, from gene silencing to chromatin remodeling and protection against retrotransposition. The unicellular eukaryote Trypanosoma cruzi is missing the canonical RNAi pathway and is unable to induce RNAi-related processes. To further understand alternative RNA pathways operating in this organism, we have performed deep sequencing and genome-wide analyses of a size-fractioned cDNA library (16-61 nt) from the epimastigote life stage. Deep sequencing generated 582,243 short sequences of which 91% could be aligned with the genome sequence. About 95-98% of the aligned data (depending on the haplotype) corresponded to small RNAs derived from tRNAs, rRNAs, snRNAs and snoRNAs. The largest class consisted of tRNA-derived small RNAs which primarily originated from the 3' end of tRNAs, followed by small RNAs derived from rRNA. The remaining sequences revealed the presence of 92 novel transcribed loci, of which 79 did not show homology to known RNA classes.

Among the many strategies employed by parasites for immune evasion and host manipulation, one of the most fascinating is molecular mimicry. With genome sequences available for host and parasite, mimicry of linear amino acid epitopes can be investigated by comparative genomics. Here we developed an in silico pipeline for genome-wide identification of molecular mimicry candidate proteins or epitopes. The predicted proteome of a given parasite was broken down into overlapping fragments, each of which was screened for close hits in the human proteome. Control searches were carried out against unrelated, free-living eukaryotes to eliminate the generally conserved proteins, and with randomized versions of the parasite proteins to get an estimate of statistical significance. This simple but computation-intensive approach yielded interesting candidates from human-pathogenic parasites. From Plasmodium falciparum, it returned a 14 amino acid motif in several of the PfEMP1 variants identical to part of the heparin-binding domain in the immunosuppressive serum protein vitronectin. And in Brugia malayi, fragments were detected that matched to periphilin-1, a protein of cell-cell junctions involved in barrier formation. All the results are publicly available by means of mimicDB, a searchable online database for molecular mimicry candidates from pathogens. To our knowledge, this is the first genome-wide survey for molecular mimicry proteins in parasites. The strategy can be adopted to any pair of host and pathogen, once appropriate negative control organisms are chosen. MimicDB provides a host of new starting points to gain insights into the molecular nature of host-pathogen interactions.

Trans-splicing of leader sequences onto the 5'ends of mRNAs is a widespread phenomenon in protozoa, nematodes and some chordates. Using parallel sequencing we have developed a method to simultaneously map 5'splice sites and analyze the corresponding gene expression profile, that we term spliced leader trapping (SLT). The method can be applied to any organism with a sequenced genome and trans-splicing of a conserved leader sequence. We analyzed the expression profiles and splicing patterns of bloodstream and insect forms of the parasite Trypanosoma brucei. We detected the 5' splice sites of 85% of the annotated protein-coding genes and, contrary to previous reports, found up to 40% of transcripts to be differentially expressed. Furthermore, we discovered more than 2500 alternative splicing events, many of which appear to be stage-regulated. Based on our findings we hypothesize that alternatively spliced transcripts present a new means of regulating gene expression and could potentially contribute to protein diversity in the parasite. The entire dataset can be accessed online at TriTrypDB or through: http://splicer.unibe.ch/.

The recently launched veterinary anthelmintic drench for sheep (Novartis Animal Health Inc., Switzerland) containing the nematocide monepantel represents a new class of anthelmintics: the amino-acetonitrile derivatives (AADs), much needed in view of widespread resistance to the classical drugs. Recently, it was shown that the ACR-23 protein in Caenorhabditis elegans and a homologous protein, MPTL-1 in Haemonchus contortus, are potential targets for AAD action. Both proteins belong to the DEG-3 subfamily of acetylcholine receptors, which are thought to be nematode-specific, and different from those targeted by the imidazothiazoles (e.g. levamisole). Here we provide further evidence that Cel-ACR-23 and Hco-MPTL-1-like subunits are involved in the monepantel-sensitive phenotype. We performed comparative genomics of ligand-gated ion channel genes from several nematodes and subsequently assessed their sensitivity to anthelmintics. The nematode species in the Caenorhabditis genus, equipped with ACR-23/MPTL-1-like receptor subunits, are sensitive to monepantel (EC(50)43 µM). Genome sequence information has long been used to identify putative targets for therapeutic intervention. We show how comparative genomics can be applied to predict drug sensitivity when molecular targets of a compound are known or suspected.

Multiple primary cancers, defined as three or more primary tumours, are rare, and there are few genetic studies concerning them. There is a need for increased knowledge on the heritability of multiple primary cancers and genotype-phenotype correlations. We have performed whole-genome/exome sequencing (WGS/WES) in ten individuals with three or more primary tumours, with no previous findings on standard clinical genetic investigations. In one individual with a clinical diagnosis of MEN1, a likely pathogenic cryptic splice site variant was detected in the MEN1 gene. The variant (c.654C > A) is synonymous but we showed in a cDNA analysis that it affects splicing and leads to a frameshift, with the theoretical new amino acid sequence p.(Gly219Glufs*13). In one individual with metachronous colorectal cancers, ovarian cancer, endometrial cancer and chronic lymphocytic leukaemia, we found a likely pathogenic variant in the MLH1 gene (c.27G > A), and two risk factor variants in the genes CHEK2 and HOXB13. The MLH1 variant is synonymous but has previously been shown to be associated to constitutional low-grade hypermethylation of the MLH1 promoter, and segregates with disease in families with colorectal and endometrial cancer. No pathogenic single nucleotide or structural variants were detected in the remaining eight individuals in the study. The pathogenic variants found by WGS/WES were in genes already sequenced by Sanger sequencing and WES in the clinic, without any findings. We conclude that, in individuals with an unequivocal clinical diagnosis of a specific hereditary cancer syndrome, where standard clinical testing failed to detect a causative variant, re-analysis may lead to a diagnosis. Keywords: Hereditary cancer, Re-analysis, WGS, WES, Multiple primary, MEN1, MLH1, Author(s): Wallander Karin[sup.1,2], Thonberg Håkan[sup.1,2], Nilsson Daniel[sup.1,2] and Tham Emma[sup.1,2] Introduction Globally, cancer is the second most common killer and accounts for 16% of all deaths [1]. In Sweden, the [...]

Reliable detection of large structural variation ( > 1000 bp) is important in both rare and common genetic disorders. Whole genome sequencing (WGS) is a technology that may be used to identify a large proportion of the genomic structural variants (SVs) in an individual in a single experiment. Even though SV callers have been extensively used in research to detect mutations, the potential usage of SV callers within routine clinical diagnostics is hindered by high computational costs, usage of non-standard output format, and limited support for the various sequencing platforms and libraries. Another well known, but not well-addressed problem is the large number of benign variants and reference errors present in the human genome that further complicates analysis. Here we present TIDDIT, a time efficient variant caller, that uses discordant read pairs as well as the depth of coverage and split reads to detect and classify a large spectrum of SVs. As part of the software suite, TIDDIT also includes a database functionality that enables filtering for rare variants and reduces the number of false positive calls and background noise. Benchmarked against five state-of-the-art SV callers, TIDDIT performs at an equal/superior level while using only 2 CPU hours per sample. Thanks to its speed, sensitivity, flexibility and ability to easily detect variants on a wide range of WGS library types, TIDDIT solves many of the problems that are currently hindering the utilization of WGS for SV calling in clinical settings.