Integrative functional genomic search for regulatory DNA sequence polymorphisms influencing DNA methylation and mRNA expression in hippocampal brain tissue

Neuropsychiatric disorders have a strong genetic predisposition, but their genetic basis remains elusive. Genome-wide association studies (GWASs) have mapped more than 2,000 susceptibility loci that were shown to increase the risk of common brain disorders. However, the majority of these susceptibility loci reside in non-coding regions and their functional consequences are unknown. The present study addresses the question whether regulatory sequence variants, affecting DNA methylation and gene expression, may be causal susceptibility alleles. I used an integrative functional genomics approach to investigate epigenetic regulation phenomena in human hippocampal brain of 115 European patients with pharmacoresistant mesial temporal lobe epilepsy. High-density SNP genotypes were correlated with genome-wide quantitative CpG methylation and mRNA expression levels using the Human Methylation450 array (HM450) and the Human HT-12 v3 array. Subsequently, a genome-wide map of methylation quantitative trait loci (meQTLs) and expression quantitative trait loci (eQTLs) was used to dissect regulatory SNPs (rSNPs) that confer susceptibility to common brain disorders at 488 known GWAS hits (P < 5.0 x 10-8). This is the first meQTL study of brain tissue applying the high-density HM450 array in specimens of fresh frozen human brain tissue obtained by epilepsy surgery at large scale. Linear regression analysis of this study implementing a correction for cell-type heterogeneity, identified 19,954 (8.5% of 362k CpGs) cis-acting meQTLs at a false-discovery rate (FDR) of 5%, which is a six-fold increase compared to previous meQTL studies that all investigated postmortem brain tissue. Specifically, cis-meQTLs were strongly enriched upstream of the gene promoter region (TSS201-1500; P = 7.7 x 10-61), highlighting the functional impact of this 5´-regulatory region that harbors binding sites of enhancers and insulators. Some of the most significant cis-meQTLs affected high-ranking candidate genes (ADARB2, HDAC4, NAPRT1, MAD1L1, PTPRN2 and RIMBP2) for neurodevelopmental disorders. To explore tissue specifity, the same approach was repeated in an additional meQTL analysis of whole blood cells originating from 496 German population controls without neuropsychiatric disorders. Results show that 65% of the meQTLs in brain tissues were also present in whole blood cells (Spearman’s Rank coefficient = 0.42). The present database of cis-meQTLs in brain and blood cells provides a key to select accessible epigenetic biomarkers for brain disorders in whole blood cells. The performed eQTL study identified 734 out of 31k mRNA probes at which expression levels were significantly influenced by cis-acting SNPs (FDR < 5%). Apart from meQTL and eQTL analyses, additionally a CpG methylation to gene expression correlation analysis was performed. This represents the first systematic delineation of methylation-driven genes in fresh frozen brain tissue. Both inverse correlations (73%) and positive correlations (27%) were observed, whereby the strongest inverse correlations were detected at NAPRT1, the gene encoding Nicotinate Phosphoribosyltransferase. Furthermore, the NAPRT1-associated meQTLs and eQTL were both genetically regulated by SNP rs9657360. The minor C allele of that very SNP was significantly associated with high methylation levels in the NAPRT1 promoter region and simultaneously associated with low gene expression of NAPRT1. Both, the tumor-specific hypermethylation of a promoter CpG island as well as loss of NAPRT1 expression have been previously proposed as predictive biomarkers for the therapy of carcinomas using NAMPT inhibitors. The additionally genetic risk constellation which has been identified by my approach – combining meQTLs and eQTLs to unravel the translational impact of epigenetic regulation of gene expression – is of high clinical relevance. It enables a diagnostically driven clinical strategy in tumorigenesis including the selection of patients which likely benefit from the administration of NAMPT inhibitors. To dissect imprinted meQTLs (imeQTLs) exhibiting differential methylation in a Parent-of-Origin (PofO) dependent manner, the CpG methylation states of blood cells in groups of 269 individuals stratified by parentally inverse heterozygous genotypes of nearby SNPs were compared. The imeQTL analysis revealed 177 CpGs at 31 genomic loci of which 22 were previously unknown. The strongest PofO effects were observed at loci harboring neurodevelopmental genes and on chromosome 3p21.1, which is a GWAS candidate region for mood disorders. Genes at genomic loci that show imprinting effects are promising candidate genes because of their potentially monoallelic gene expression which may unmask recessive susceptibility alleles. Enrichment analyses of genes associated with cis-meQTLs revealed an overrepresentation of genes implicated in GWAS hits of brain disorders (P = 5.8 x 10-4). Potential rSNPs at the GWAS candidate loci 1q31.2 (RGS1 gene locus) and 3p21.1 (PRBM1 gene locus) were identified. The allelic alteration of transcription factor binding sites by potential rSNPs is likely to result in changes of gene transcription or splicing processes which could contribute to pathogenic pathways underlying neuropsychiatric disorders. As exemplified in this thesis, the created database of autosomal meQTLs, imeQTLs and eQTLs in brain tissue provides a valuable resource to dissect rSNPs at GWAS hits and to decipher their functional effects

Genome-wide mapping of genetic determinants influencing DNA methylation and gene expression in human hippocampus

Emerging evidence emphasizes the strong impact of regulatory genomic elements in neurodevelopmental processes and the complex pathways of brain disorders. The present genome-wide quantitative trait loci analyses explore the cis-regulatory effects of single-nucleotide polymorphisms (SNPs) on DNA methylation (meQTL) and gene expression (eQTL) in 110 human hippocampal biopsies. We identify cis-meQTLs at 14,118 CpG methylation sites and cis-eQTLs for 302 3'-mRNA transcripts of 288 genes. Hippocampal cis-meQTL-CpGs are enriched in flanking regions of active promoters, CpG island shores, binding sites of the transcription factor CTCF and brain eQTLs. Cis-acting SNPs of hippocampal meQTLs and eQTLs significantly overlap schizophrenia-associated SNPs. Correlations of CpG methylation and RNA expression are found for 34 genes. Our comprehensive maps of cis-acting hippocampal meQTLs and eQTLs provide a link between disease-associated SNPs and the regulatory genome that will improve the functional interpretation of non-coding genetic variants in the molecular genetic dissection of brain disorders

Методы и механизмы геттерирования кремниевых структур в производстве интегральных микросхем

Увеличение степени интеграции элементной базы предъявляет все более жесткие требования к уменьшению концентрации загрязняющих примесей и окислительных дефектов упаковки в исходных кремниевых пластинах с ее сохранением в технологическом цикле изготовления ИМС. Это обуславливает высокую актуальность применения геттерирования в современной технологии микроэлектроники. В статье рассмотрены существующие методы геттерирования кремниевых пластин и механизмы их протекания.Збільшення ступеня інтеграції елементної бази пред'являє все більш жорсткі вимоги до зменшення концентрації забруднюючих домішок та окислювальних дефектів упаковки у вихідних кремнієвих пластинах за її збереження у технологічному циклі виготовлення ІМС. Це обумовлює високу актуальність застосування гетерування в сучасній технології мікроелектроніки. Розглянуто існуючі методи гетерування кремнієвих пластин та розглянуто механізми їх перебігу.Increasing the degree of integration of hardware components imposes more stringent requirements for the reduction of the concentration of contaminants and oxidation stacking faults in the original silicon wafers with its preservation in the IC manufacturing process cycle. This causes high relevance of the application of gettering in modern microelectronic technology. The existing methods of silicon wafers gettering and the mechanisms of their occurrence are considered

Evaluation of presumably disease causing SCN1A variants in a cohort of common epilepsy syndromes

Objective: The SCN1A gene, coding for the voltage-gated Na+ channel alpha subunit NaV1.1, is the clinically most relevant epilepsy gene. With the advent of high-throughput next-generation sequencing, clinical laboratories are generating an ever-increasing catalogue of SCN1A variants. Variants are more likely to be classified as pathogenic if they have already been identified previously in a patient with epilepsy. Here, we critically re-evaluate the pathogenicity of this class of variants in a cohort of patients with common epilepsy syndromes and subsequently ask whether a significant fraction of benign variants have been misclassified as pathogenic. Methods: We screened a discovery cohort of 448 patients with a broad range of common genetic epilepsies and 734 controls for previously reported SCN1A mutations that were assumed to be disease causing. We re-evaluated the evidence for pathogenicity of the identified variants using in silico predictions, segregation, original reports, available functional data and assessment of allele frequencies in healthy individuals as well as in a follow up cohort of 777 patients. Results and Interpretation: We identified 8 known missense mutations, previously reported as path

Genome-wide association analysis of genetic generalized epilepsies implicates susceptibility loci at 1q43, 2p16.1, 2q22.3 and 17q21.32

Genetic generalized epilepsies (GGEs) have a lifetime prevalence of 0.3% and account for 20-30% of all epilepsies. Despite their high heritability of 80%, the genetic factors predisposing to GGEs remain elusive. To identify susceptibility variants shared across common GGE syndromes, we carried out a two-stage genome-wide association study (GWAS) including 3020 patients with GGEs and 3954 controls of European ancestry. To dissect out syndrome-related variants, we also explored two distinct GGE subgroups comprising 1434 patients with genetic absence epilepsies (GAEs) and 1134 patients with juvenile myoclonic epilepsy (JME). Joint Stage-1 and 2 analyses revealed genome-wide significant associations for GGEs at 2p16.1 (rs13026414, Pmeta = 2.5 × 10−9, OR[T] = 0.81) and 17q21.32 (rs72823592, Pmeta = 9.3 × 10−9, OR[A] = 0.77). The search for syndrome-related susceptibility alleles identified significant associations for GAEs at 2q22.3 (rs10496964, Pmeta = 9.1 × 10−9, OR[T] = 0.68) and at 1q43 for JME (rs12059546, Pmeta = 4.1 × 10−8, OR[G] = 1.42). Suggestive evidence for an association with GGEs was found in the region 2q24.3 (rs11890028, Pmeta = 4.0 × 10−6) nearby the SCN1A gene, which is currently the gene with the largest number of known epilepsy-related mutations. The associated regions harbor high-ranking candidate genes: CHRM3 at 1q43, VRK2 at 2p16.1, ZEB2 at 2q22.3, SCN1A at 2q24.3 and PNPO at 17q21.32. Further replication efforts are necessary to elucidate whether these positional candidate genes contribute to the heritability of the common GGE syndrome

Alterations in the α2 δ ligand, thrombospondin-1, in a rat model of spontaneous absence epilepsy and in patients with idiopathic/genetic generalized epilepsies

Objectives Thrombospondins, which are known to interact with the α2δ subunit of voltage-sensitive calcium channels to stimulate the formation of excitatory synapses, have recently been implicated in the process of epileptogenesis. No studies have been so far performed on thrombospondins in models of absence epilepsy. We examined whether expression of the gene encoding for thrombospondin-1 was altered in the brain of WAG/Rij rats, which model absence epilepsy in humans. In addition, we examined the frequency of genetic variants of THBS1 in a large cohort of children affected by idiopathic/genetic generalized epilepsies (IGE/GGEs). Methods We measured the transcripts of thrombospondin-1 and α2δ subunit, and protein levels of α2δ, Rab3A, and the vesicular glutamate transporter, VGLUT1, in the somatosensory cortex and ventrobasal thalamus of presymptomatic and symptomatic WAG/Rij rats and in two control strains by real-time polymerase chain reaction (PCR) and immunoblotting. We examined the genetic variants of THBS1 and CACNA2D1 in two independent cohorts of patients affected by IGE/GGE recruited through the Genetic Commission of the Italian League Against Epilepsy (LICE) and the EuroEPINOMICS-CoGIE Consortium. Results Thrombospondin-1 messenger RNA (mRNA) levels were largely reduced in the ventrobasal thalamus of both presymptomatic and symptomatic WAG/Rij rats, whereas levels in the somatosensory cortex were unchanged. VGLUT1 protein levels were also reduced in the ventrobasal thalamus of WAG/Rij rats. Genetic variants of THBS1 were significantly more frequent in patients affected by IGE/GGE than in nonepileptic controls, whereas the frequency of CACNA2D1 was unchanged. Significance These findings suggest that thrombospondin-1 may have a role in the pathogenesis of IGE/GGEs

Analysis of shared heritability in common disorders of the brain

ience, this issue p. eaap8757 Structured Abstract INTRODUCTION Brain disorders may exhibit shared symptoms and substantial epidemiological comorbidity, inciting debate about their etiologic overlap. However, detailed study of phenotypes with different ages of onset, severity, and presentation poses a considerable challenge. Recently developed heritability methods allow us to accurately measure correlation of genome-wide common variant risk between two phenotypes from pools of different individuals and assess how connected they, or at least their genetic risks, are on the genomic level. We used genome-wide association data for 265,218 patients and 784,643 control participants, as well as 17 phenotypes from a total of 1,191,588 individuals, to quantify the degree of overlap for genetic risk factors of 25 common brain disorders. RATIONALE Over the past century, the classification of brain disorders has evolved to reflect the medical and scientific communities' assessments of the presumed root causes of clinical phenomena such as behavioral change, loss of motor function, or alterations of consciousness. Directly observable phenomena (such as the presence of emboli, protein tangles, or unusual electrical activity patterns) generally define and separate neurological disorders from psychiatric disorders. Understanding the genetic underpinnings and categorical distinctions for brain disorders and related phenotypes may inform the search for their biological mechanisms. RESULTS Common variant risk for psychiatric disorders was shown to correlate significantly, especially among attention deficit hyperactivity disorder (ADHD), bipolar disorder, major depressive disorder (MDD), and schizophrenia. By contrast, neurological disorders appear more distinct from one another and from the psychiatric disorders, except for migraine, which was significantly correlated to ADHD, MDD, and Tourette syndrome. We demonstrate that, in the general population, the personality trait neuroticism is significantly correlated with almost every psychiatric disorder and migraine. We also identify significant genetic sharing between disorders and early life cognitive measures (e.g., years of education and college attainment) in the general population, demonstrating positive correlation with several psychiatric disorders (e.g., anorexia nervosa and bipolar disorder) and negative correlation with several neurological phenotypes (e.g., Alzheimer's disease and ischemic stroke), even though the latter are considered to result from specific processes that occur later in life. Extensive simulations were also performed to inform how statistical power, diagnostic misclassification, and phenotypic heterogeneity influence genetic correlations. CONCLUSION The high degree of genetic correlation among many of the psychiatric disorders adds further evidence that their current clinical boundaries do not reflect distinct underlying pathogenic processes, at least on the genetic level. This suggests a deeply interconnected nature for psychiatric disorders, in contrast to neurological disorders, and underscores the need to refine psychiatric diagnostics. Genetically informed analyses may provide important "scaffolding" to support such restructuring of psychiatric nosology, which likely requires incorporating many levels of information. By contrast, we find limited evidence for widespread common genetic risk sharing among neurological disorders or across neurological and psychiatric disorders. We show that both psychiatric and neurological disorders have robust correlations with cognitive and personality measures. Further study is needed to evaluate whether overlapping genetic contributions to psychiatric pathology may influence treatment choices. Ultimately, such developments may pave the way toward reduced heterogeneity and improved diagnosis and treatment of psychiatric disorders

Deficiency of Nucleotide-binding oligomerization domain-containing proteins (NOD) 1 and 2 reduces atherosclerosis

Atherosclerosis is crucially fueled by inflammatory pathways including pattern recognition receptor (PRR)-related signaling of the innate immune system. Currently, the impact of the cytoplasmic PRRs nucleotide-binding oligomerization domain-containing protein (NOD) 1 and 2 is incompletely characterized. We, therefore, generated Nod1/Nod2 double knockout mice on a low-density lipoprotein receptor (Ldlr)-deficient background (= Ldl

A homozygous splice-site mutation in CARS2 is associated with progressive myoclonic epilepsy

Objective: We report a consanguineous family with 2 affected individuals whose clinical symptoms closely resembled MERRF (myoclonus epilepsy with ragged red fibers) syndrome including severe myoclonic epilepsy, progressive spastic tetraparesis, progressive impairment of vision and hearing, as well as progressive cognitive decline. Methods: After excluding the presence of pathogenic mitochondrial DNA mutations, whole-exome sequencing of blood DNA from the index patient was performed. Detected homozygous mutations and their cosegregation were confirmed by Sanger sequencing. CARS2 (cysteinyl-tRNA synthetase 2, mitochondrial) messenger RNA analysis was performed by reverse transcription PCR and sequencing. Results: We identified a homozygous c.655G>A mutation in the CARS2 gene cosegregating in the family. The mutation is localized at the last nucleotide of exon 6 and thus is predicted to cause aberrant splicing. Analysis of the CARS2 messenger RNA showed that the presence of the mutation resulted in removal of exon 6. This leads to an in-frame deletion of 28 amino acids in a conserved sequence motif of the protein involved in stabilization of the acceptor end hairpin of tRNA(Cys). Conclusion: CARS2 is a novel disease gene associated with a severe progressive myoclonic epilepsy most resembling MERRF syndrome