Guideline-based and bioinformatic reassessment of lesion-associated gene and variant pathogenicity in focal human epilepsies

in Epilepsia (2018)

Objective: Increasing availability of surgically resected brain tissue from patients with focal epilepsy and Focal Cortical Dysplasia (FCD) or low-grade glio-neuronal tumors has fostered large-scale ... [more ▼]

Objective: Increasing availability of surgically resected brain tissue from patients with focal epilepsy and Focal Cortical Dysplasia (FCD) or low-grade glio-neuronal tumors has fostered large-scale genetic examination. However, assessment of pathogenicity of germline and somatic variants remains difficult. Here, we present a state of the art evaluation of reported genes and variants associated with epileptic brain lesions. Methods: We critically re-evaluated the pathogenicity for all neuropathology-associated variants reported to date in PubMed and ClinVar databases including 101 neuropathology-associated missense variants encompassing 11 disease-related genes. We assessed gene variant tolerance and classified all identified missense variants according to guidelines from the American College of Medical Genetics and Genomics (ACMG). We further extended the bioinformatic variant prediction by introducing a novel gene-specific deleteriousness ranking for prediction scores. Results: Application of ACMG guidelines and in silico gene variant tolerance analysis classified only seven out of 11 genes to be likely disease-associated according to the reported a disease mechanism, while 61 (60.4%) of 101 variants of those genes were classified as of uncertain significance (VUS), 37 (36.6%) as being likely pathogenic (LP) and 3 (3%) as being pathogenic (P). Significance: We concluded that the majority of neuropathology-associated variants reported to date do not have enough evidence to be classified as pathogenic. Interpretation of lesion-associated variants remains challenging and application of current ACMG guidelines is recommended for interpretation and prediction. [less ▲]

Major changes of cell function and toxicant sensitivity in cultured cells undergoing mild, quasi-natural genetic drift

in Archives of Toxicology (2018)

Genomic drift affects the functional properties of cell lines, and the reproducibility of data from in vitro studies. While chromosomal aberrations and mutations in single pivotal genes are well explored ... [more ▼]

Genomic drift affects the functional properties of cell lines, and the reproducibility of data from in vitro studies. While chromosomal aberrations and mutations in single pivotal genes are well explored, little is known about effects of minor, possibly pleiotropic, genome changes. We addressed this question for the human dopaminergic neuronal precursor cell line LUHMES by comparing two subpopulations (SP) maintained either at the American-Type-Culture-Collection (ATCC) or by the original provider (UKN). Drastic differences in susceptibility towards the specific dopaminergic toxicant 1-methyl-4-phenylpyridinium (MPP+) were observed. Whole-genome sequencing was performed to identify underlying genetic differences. While both SP had normal chromosome structures, they displayed about 70 differences on the level of amino acid changing events. Some of these differences were confirmed biochemically, but none offered a direct explanation for the altered toxicant sensitivity pattern. As second approach, markers known to be relevant for the intended use of the cells were specifically tested. The “ATCC” cells rapidly down-regulated the dopamine-transporter and tyrosine-hydroxylase after differentiation, while “UKN” cells maintained functional levels. As the respective genes were not altered themselves, we conclude that polygenic complex upstream changes can have drastic effects on biochemical features and toxicological responses of relatively similar SP of cells. [less ▲]

Rare gene deletions in genetic generalized and Rolandic epilepsies

in PLoS ONE (2018)

Genetic Generalized Epilepsy (GGE) and benign epilepsy with centro-temporal spikes or Rolandic Epilepsy (RE) are common forms of genetic epilepsies. Rare copy number variants have been recognized as ... [more ▼]

Genetic Generalized Epilepsy (GGE) and benign epilepsy with centro-temporal spikes or Rolandic Epilepsy (RE) are common forms of genetic epilepsies. Rare copy number variants have been recognized as important risk factors in brain disorders. We performed a systematic survey of rare deletions affecting protein-coding genes derived from exome data of patients with common forms of genetic epilepsies. We analysed exomes from 390 European patients (196 GGE and 194 RE) and 572 population controls to identify low-frequency genic deletions. We found that 75 (32 GGE and 43 RE) patients out of 390, i.e. ~19%, carried rare genic deletions. In particular, large deletions (>400 kb) represent a higher burden in both GGE and RE syndromes as compared to controls. The detected low-frequency deletions (1) share genes with brain-expressed exons that are under negative selection, (2) overlap with known autism and epilepsy-associated candidate genes, (3) are enriched for CNV intolerant genes recorded by the Exome Aggregation Consortium (ExAC) and (4) coincide with likely disruptive de novo mutations from the NPdenovo database. Employing several knowledge databases, we discuss the most prominent epilepsy candidate genes and their protein-protein networks for GGE and RE. [less ▲]

Microbiome-derived Multi-omicBiomarkers for Early-stage Detection and Stratification of Parkinson's Disease

Poster (2018, August)

The RNA Complement of Outer Membrane Vesicles From Salmonella enterica Serovar Typhimurium Under Distinct Culture Conditions

in Frontiers in Microbiology (2018), 9

Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host–pathogen interactions. The presence of larger RNA transcripts in OMVs has been ... [more ▼]

Bacterial outer membrane vesicles (OMVs), as well as OMV-associated small RNAs, have been demonstrated to play a role in host–pathogen interactions. The presence of larger RNA transcripts in OMVs has been less studied and their potential role in host–pathogen interactions remains largely unknown. Here we analyze RNA from OMVs secreted by Salmonella enterica serovar Typhimurium (S. Typhimurium) cultured under different conditions, which mimic host–pathogen interactions. S. Typhimurium was grown to exponential and stationary growth phases in minimal growth control medium (phosphate-carbon-nitrogen, PCN), as well as in acidic and phosphate-depleted PCN, comparable to the macrophage environment and inducing therefore the expression of Salmonella pathogenicity island 2 (SPI-2) genes. Moreover, Salmonella pathogenicity island 1 (SPI-1), which is required for virulence during the intestinal phase of infection, was induced by culturing S. Typhimurium to the stationary phase in Lysogeny Broth (LB). For each condition, we identified OMV-associated RNAs that are enriched in the extracellular environment relative to the intracellular space. All RNA classes could be observed, but a vast majority of rRNA was exported in all conditions in variable proportions with a notable decrease in LB SPI-1 inducing media. Several mRNAs and ncRNAs were specifically enriched in/on OMVs dependent on the growth conditions. Important to note is that some RNAs showed identical read coverage profiles intracellularly and extracellularly, whereas distinct coverage patterns were observed for other transcripts, suggesting a specific processing or degradation. Moreover, PCR experiments confirmed that distinct RNAs were present in or on OMVs as full-length transcripts (IsrB-1/2; IsrA; ffs; SsrS; CsrC; pSLT035; 10Sa; rnpB; STM0277; sseB; STM0972; STM2606), whereas others seemed to be rather present in a processed or degraded form. Finally, we show by a digestion protection assay that OMVs are able to prevent enzymatic degradation of given full-length transcripts (SsrS, CsrC, 10Sa, and rnpB). In summary, we show that OMV-associated RNA is clearly different in distinct culture conditions and that at least a fraction of the extracellular RNA is associated as a full-length transcripts with OMVs, indicating that some RNAs are protected by OMVs and thereby leaving open the possibility that those might be functionally active. [less ▲]

Small RNA profiling of low biomass samples: identification and removal of contaminants

in BMC Biology (2018), 16(52),

Background: Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and ... [more ▼]

Background: Sequencing-based analyses of low-biomass samples are known to be prone to misinterpretation due to the potential presence of contaminating molecules derived from laboratory reagents and environments. DNA contamination has been previously reported, however contamination with RNA is usually considered to be unlikely due to its inherent instability. Small RNAs (sRNAs) identified in tissues and bodily fluids such as blood plasma, have implications for physiology and pathology, and therefore the potential to act as disease biomarkers. Thus, the possibility for RNA contaminants demands a careful evaluation. Results: Here we report the presence of small RNA contaminants in widely used microRNA extraction kits and propose an approach for their depletion. We sequenced sRNAs extracted from human plasma samples and detected important levels of non-human (exogenous) sequences whose source could be traced to the microRNA extraction columns through a careful qPCR-based analysis of several laboratory reagents. Furthermore, we also detected the presence of artefactual sequences related to these contaminants in a range of published datasets, arguing for a re-evaluation of reports suggesting the presence of exogenous RNAs of microbial and dietary origins in blood plasma. To avoid artefacts in future experiments, we also devise several protocols of contaminant RNAs, define minimal amounts of starting material for artefact-free analyses, and confirm the reduction of contaminant levels for identification of bona fide sequences using ‘ultra-clean’ extraction kits. Conclusion: This is the first report of the presence of RNA molecules as contaminants in RNA extraction kits. The described protocols should be applied in the future to avoid confounding sRNA studies. [less ▲]

Natural variation of chronological aging in the Saccharomyces cerevisiae species reveals diet-dependent mechanisms of life span control

in npj Aging and Mechanisms of Disease (2018), 4(3),

Aging is a complex trait of broad scientific interest, especially because of its intrinsic link with common human diseases. Pioneering work on aging-related mechanisms has been made in Saccharomyces ... [more ▼]

Aging is a complex trait of broad scientific interest, especially because of its intrinsic link with common human diseases. Pioneering work on aging-related mechanisms has been made in Saccharomyces cerevisiae, mainly through the use of deletion collections isogenic to the S288c reference strain. In this study, using a recently published high-throughput approach, we quantified chronological life span (CLS) within a collection of 58 natural strains across seven different conditions. We observed a broad aging variability suggesting the implication of diverse genetic and environmental factors in chronological aging control. Two major Quantitative Trait Loci (QTLs) were identified within a biparental population obtained by crossing two natural isolates with contrasting aging behavior. Detection of these QTLs was dependent upon the nature and concentration of the carbon sources available for growth. In the first QTL, the RIM15 gene was identified as major regulator of aging under low glucose condition, lending further support to the importance of nutrient-sensing pathways in longevity control under calorie restriction. In the second QTL, we could show that the SER1 gene, encoding a conserved aminotransferase of the serine synthesis pathway not previously linked to aging, is causally associated with CLS regulation, especially under high glucose condition. These findings hint toward a new mechanism of life span control involving a trade-off between serine synthesis and aging, most likely through modulation of acetate and trehalose metabolism. More generally it shows that genetic linkage studies across natural strains represent a promising strategy to further unravel the molecular basis of aging. [less ▲]

Identification and Characterization of Variant Intolerant Sites across Human Protein 3-Dimensional Structures

in Biophysical Journal (2018, February 02), 114(3, Suppl. 1), 664

The functional interpretation of genetic variation in disease-associated genes is far outpaced by data generation. Existing algorithms for prediction of variant consequences do not adequately distinguish ... [more ▼]

The functional interpretation of genetic variation in disease-associated genes is far outpaced by data generation. Existing algorithms for prediction of variant consequences do not adequately distinguish pathogenic variants from benign rare variants. This lack of statistical and bioinformatics analyses, accompanied by an ever-increasing number of identified variants in biomedical research and clinical applications, has become a major challenge. Established methods to predict the functional effect of genetic variation use the degree of amino acid conservation across species in linear protein sequence alignment. More recent methods include the spatial distribution pattern of known patient and control variants. Here, we propose to combine the linear conservation and spatial constrained based scores to devise a novel score that incorporates 3-dimensional structural properties of amino acid residues, such as the solvent-accessible surface area, degree of flexibility, secondary structure propensity and binding tendency, to quantify the effect of amino acid substitutions. For this study, we develop a framework for large-scale mapping of established linear sequence-based paralog and ortholog conservation scores onto the tertiary structures of human proteins. This framework can be utilized to map the spatial distribution of mutations on solved protein structures as well as homology models. As a proof of concept, using a homology model of the human Nav1.2 voltage-gated sodium channel structure, we observe spatial clustering in distinct domains of mutations, associated with Autism Spectrum Disorder (>20 variants) and Epilepsy (>100 variants), that exert opposing effects on channel function. We are currently characterizing all variants (>300k individuals) found in ClinVar, the largest disease variant database, as well as variants identified in >140k individuals from general population. The variant mapping framework and our score, informed with structural information, will be useful in identifying structural motifs of proteins associated with disease risk. [less ▲]

Exome-wide analysis of mutational burden in patients with typical and atypical Rolandic Epilepsy

in European Journal of Human Genetics (2018)

Rolandic Epilepsy (RE) is the most common focal epilepsy in childhood. To date no hypothesis-free exome-wide mutational screen has been conducted for RE and Atypical RE (ARE). Here we report on whole ... [more ▼]

Rolandic Epilepsy (RE) is the most common focal epilepsy in childhood. To date no hypothesis-free exome-wide mutational screen has been conducted for RE and Atypical RE (ARE). Here we report on whole-exome sequencing of 194 unrelated patients with RE/ARE and 567 ethnically matched population controls. We identified an exome-wide significantly enriched burden for deleterious and loss-of-function variants only for the established RE/ARE gene GRIN2A. The statistical significance of the enrichment disappeared after removing ARE patients. For several disease-related gene-sets, an odds ratio > 1 was detected for loss-of-function variants. [less ▲]

Birth mode is associated with earliest strain-conferred gut microbiome functions and immunostimulatory potential

in Nature Communications (2018)