Missense variants in ANO4 cause sporadic encephalopathic or familial epilepsy with evidence for a dominant-negative effect

Anoctamins are a family of Ca$^{2+}$-activated proteins that may act as ion channels and/or phospholipid scramblases with limited understanding of function and disease association. Here, we identified five de novo and two inherited missense variants in ANO4 (alias TMEM16D) as a cause of fever-sensitive developmental and epileptic or epileptic encephalopathy (DEE/EE) and generalized epilepsy with febrile seizures plus (GEFS+) or temporal lobe epilepsy. In silico modeling of the ANO4 structure predicted that all identified variants lead to destabilization of the ANO4 structure. Four variants are localized close to the Ca$^{2+}$ binding sites of ANO4, suggesting impaired protein function. Variant mapping to the protein topology suggests a preliminary genotype-phenotype correlation. Moreover, the observation of a heterozygous ANO4 deletion in a healthy individual suggests a dysfunctional protein as disease mechanism rather than haploinsufficiency. To test this hypothesis, we examined mutant ANO4 functional properties in a heterologous expression system by patchclamp recordings, immunocytochemistry, and surface expression of annexin A5 as a measure of phosphatidylserine scramblase activity. All ANO4 variants showed severe loss of ion channel function and DEE/EE associated variants presented mild loss of surface expression due to impaired plasma membrane trafficking. Increased levels of Ca$^{2+}$-independent annexin A5 at the cell surface suggested an increased apoptosis rate in DEE-mutant expressing cells, but no changes in Ca$^{2+}$-dependent scramblase activity were observed. Co-transfection with ANO4 wild-type suggested a dominant-negative effect. In summary, we expand the genetic base for both encephalopathic sporadic and inherited fever-sensitive epilepsies and link germline variants in ANO4 to a hereditary disease

Twist exome capture allows for lower average sequence coverage in clinical exome sequencing

Background Exome and genome sequencing are the predominant techniques in the diagnosis and research of genetic disorders. Sufficient, uniform and reproducible/consistent sequence coverage is a main determinant for the sensitivity to detect single-nucleotide (SNVs) and copy number variants (CNVs). Here we compared the ability to obtain comprehensive exome coverage for recent exome capture kits and genome sequencing techniques. Results We compared three different widely used enrichment kits (Agilent SureSelect Human All Exon V5, Agilent SureSelect Human All Exon V7 and Twist Bioscience) as well as short-read and long-read WGS. We show that the Twist exome capture significantly improves complete coverage and coverage uniformity across coding regions compared to other exome capture kits. Twist performance is comparable to that of both short- and long-read whole genome sequencing. Additionally, we show that even at a reduced average coverage of 70× there is only minimal loss in sensitivity for SNV and CNV detection. Conclusion We conclude that exome sequencing with Twist represents a significant improvement and could be performed at lower sequence coverage compared to other exome capture techniques

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Sponge EEG is equivalent regarding signal quality, but faster than routine EEG

Objective: Emergency diagnostics, such as acquisition of an electroencephalogram (EEG), are of great diagnostic importance, but there is often a lack of experienced personnel. Wet active electrode sponge-based electroencephalogram (sp-EEG) systems can be applied rapidly and by inexperienced personnel. This makes them an attractive alternative to routine EEG (r-EEG) systems in these settings. Here, we examined the feasibility and signal quality of sp-EEG compared to r-EEG. Methods: In this case-control, single-blind, non-randomized study, EEG recordings using a sp- and a r-EEG system were performed in 18 individuals with a variety of epileptiform discharges and 11 healthy control subjects. The time was stopped until all electrodes in both systems displayed adequate skin-electrode impedances. The resulting 58 EEGs were visually inspected by 7 experienced, blinded neurologists. Raters were asked to score physiological and pathological graphoelements, and to distinguish between the different systems by visual inspection of the EEGs. Results: Time to signal acquisition for sp-EEG was significantly faster (4.8 min (SD 2.01) vs. r-EEG 13.3 min (SD 2.72), p < 0.001). All physiological and pathological graphoelements of all 58 EEGs could be identified. Raters were unable to distinguish between sp-EEG or r-EEG based on visual inspection of the EEGs alone. Conclusions: Sp-EEG represents a feasible alternative to r-EEG in emergency diagnostics or resource-limited settings. Significance: Given shortage of trained personnel or resources, the easy implementation and comparable quality of a novel sp-EEG system may increase general availability of EEG and thus improve patient care

CSF and Serum Levels of Inflammatory Markers in PD: Sparse Correlation, Sex Differences and Association With Neurodegenerative Biomarkers

BACKGROUND: An involvement of the central-nervous and peripheral, innate and adaptive immune system in the pathogenesis of Parkinson's disease (PD) is nowadays well established. OBJECTIVES: We face several open questions in preparation of clinical trials aiming at disease-modification by targeting the immune system: Do peripheral (blood) inflammatory profiles reflect central (CSF) inflammatory processes? Are blood/CSF inflammatory markers associated with CSF levels of neurodegenerative/PD-specific biomarkers? METHODS: Using a multiplex assay we assessed 41 inflammatory markers in CSF/serum pairs in 453 sporadic PD patients. We analyzed CSF/serum correlation as well as associations of inflammatory markers with clinical outcome measures (UPDRS-III, H&Y, MoCA) and with CSF levels of α-synuclein, Aβ(1−42), t-Tau, p181-Tau and NFL. All analyses were stratified by sex as the immune system shows relevant sex-specific differences. RESULTS: Correlations between CSF and serum were sparse and detected in only 25% (9 out of 36) of the analysable inflammatory markers in male PD patients and in only 38% (12 out of 32) of female PD patients. The most important pro-inflammatory mediators associated with motor and cognitive decline as well as with neurodegenerative/PD-specific biomarkers were FABP, ICAM-1, IL-8, MCP-1, MIP-1-beta, and SCF. Results were more robust for CSF than for serum. INTERPRETATION: Levels of central-nervous and peripheral inflammatory markers might be regulated independently of each other with CSF inflammatory markers reflecting CNS pathology more accurately than peripheral markers. These findings along with sex-specific characteristics have to be considered when designing clinical trials aiming at disease-modification by targeting the immune system