Mette Nyegaard

Genome-wide study of association and interaction with maternal cytomegalovirus infection suggests new schizophrenia loci

Genetic and environmental components as well as their interaction contribute to the risk of schizophrenia, making it highly relevant to include environmental factors in genetic studies of schizophrenia. This study comprises genome-wide association (GWA) and follow-up analyses of all individuals born in Denmark since 1981 and diagnosed with schizophrenia as well as controls from the same birth cohort. Furthermore, we present the first genome-wide interaction survey of single nucleotide polymorphisms (SNPs) and maternal cytomegalovirus (CMV) infection. The GWA analysis included 888 cases and 882 controls, and the follow-up investigation of the top GWA results was performed in independent Danish (1396 cases and 1803 controls) and German-Dutch (1169 cases, 3714 controls) samples. The SNPs most strongly associated in the single-marker analysis of the combined Danish samples were rs4757144 in ARNTL (P=3.78 × 10−6) and rs8057927 in CDH13 (P=1.39 × 10−5). Both genes have previously been linked to schizophrenia or other psychiatric disorders. The strongest associated SNP in the combined analysis, including Danish and German-Dutch samples, was rs12922317 in RUNDC2A (P=9.04 × 10−7). A region-based analysis summarizing independent signals in segments of 100u2009kb identified a new region-based genome-wide significant locus overlapping the gene ZEB1 (P=7.0 × 10−7). This signal was replicated in the follow-up analysis (P=2.3 × 10−2). Significant interaction with maternal CMV infection was found for rs7902091 (PSNP × CMV=7.3 × 10−7) in CTNNA3, a gene not previously implicated in schizophrenia, stressing the importance of including environmental factors in genetic studies.

Blood DNA methylation age is not associated with cognitive functioning in middle-aged monozygotic twins.

The epigenetic clock, also known as DNA methylation age (DNAmAge), represents age-related changes of DNA methylation at multiple sites of the genome and is suggested to be a biomarker for biological age. Elevated blood DNAmAge is associated with all-cause mortality, with the strongest effects reported in a recent intrapair twin study where epigenetically older twins had increased mortality risk in comparison to their co-twins. In the study presented here, we hypothesize that DNAmAge in blood is associated with cross-sectional and longitudinal cognitive abilities in middle-aged individuals. In 486 monozygotic twins, we investigated the association of DNAmAge, difference between DNAmAge and chronological age and age acceleration with cognition. Despite using a powerful paired twin design, we found no evidence for association of blood DNAmAge with cognitive abilities. This observation was confirmed in unpaired analyses, where DNAmAge initially correlated with cognitive abilities, until adjusting for chronological age. Overall, our study shows that for middle-aged individuals DNAmAge calculated in blood does not correlate with cognitive abilities.

Identification of the BRD1 interaction network and its impact on mental disorder risk

BackgroundThe bromodomain containing 1 (BRD1) gene has been implicated with transcriptional regulation, brain development, and susceptibility to schizophrenia and bipolar disorder. To advance the understanding of BRD1 and its role in mental disorders, we characterized the protein and chromatin interactions of the BRD1 isoforms, BRD1-S and BRD1-L.MethodsStable human cell lines expressing epitope tagged BRD1-S and BRD1-L were generated and used as discovery systems for identifying protein and chromatin interactions. Protein-protein interactions were identified using co-immunoprecipitation followed by mass spectrometry and chromatin interactions were identified using chromatin immunoprecipitation followed by next generation sequencing. Gene expression profiles and differentially expressed genes were identified after upregulating and downregulating BRD1 expression using microarrays. The presented functional molecular data were integrated with human genomic and transcriptomic data using available GWAS, exome-sequencing datasets as well as spatiotemporal transcriptomic datasets from the human brain.ResultsWe present several novel protein interactions of BRD1, including isoform-specific interactions as well as proteins previously implicated with mental disorders. By BRD1-S and BRD1-L chromatin immunoprecipitation followed by next generation sequencing we identified binding to promoter regions of 1540 and 823 genes, respectively, and showed correlation between BRD1-S and BRD1-L binding and regulation of gene expression. The identified BRD1 interaction network was found to be predominantly co-expressed with BRD1 mRNA in the human brain and enriched for pathways involved in gene expression and brain function. By interrogation of large datasets from genome-wide association studies, we further demonstrate that the BRD1 interaction network is enriched for schizophrenia risk.ConclusionOur results show that BRD1 interacts with chromatin remodeling proteins, e.g. PBRM1, as well as histone modifiers, e.g. MYST2 and SUV420H1. We find that BRD1 primarily binds in close proximity to transcription start sites and regulates expression of numerous genes, many of which are involved with brain development and susceptibility to mental disorders. Our findings indicate that BRD1 acts as a regulatory hub in a comprehensive schizophrenia risk network which plays a role in many brain regions throughout life, implicating e.g. striatum, hippocampus, and amygdala at mid-fetal stages.

Genome-wide DNA methylation profiling with MeDIP-seq using archived dried blood spots

BackgroundIn utero and early-life experienced environmental exposures are suggested to play an important role in many multifactorial diseases potentially mediated through lasting effects on the epigenome. As the epigenome in addition remains modifiable throughout life, identifying specific disease-relevant biomarkers may prove challenging. This has led to an increased interest in epigenome-wide association studies using dried blood spots (DBS) routinely collected in perinatal screening programs. Such programs are in place in numerous countries around the world producing large and unique biobanks. However, availability of this biological material is highly limited as each DBS is made only from a few droplets of blood and storage conditions may be suboptimal for epigenetic studies. Furthermore, as relevant markers may reside outside gene bodies, epigenome-wide interrogation is needed.ResultsHere we demonstrate, as a proof of principle, that genome-wide interrogation of the methylome based on methylated DNA immunoprecipitation coupled with next-generation sequencing (MeDIP-seq) is feasible using a single 3.2 mm DBS punch (60xa0ng DNA) from filter cards archived for up to 16xa0years. The enrichment profile, sequence quality and distribution of reads across genetic regions were comparable between samples archived 16xa0years, 4xa0years and a freshly prepared control sample.ConclusionsIn summary, we show that high-quality MeDIP-seq data is achievable from neonatal screening filter cards stored at room temperature, thereby providing information on annotated as well as on non-RefSeq genes and repetitive elements. Moreover, the quantity of DNA from one DBS punch proved sufficient allowing for multiple epigenome studies using one single DBS.

Differential DNA methylation at birth associated with mental disorder in individuals with 22q11.2 deletion syndrome

Individuals with 22q11.2 deletion syndrome (DS) have an increased risk of comorbid mental disorders including schizophrenia, attention deficit hyperactivity disorder, depression, as well as intellectual disability. Although most 22q11.2 deletion carriers have the long 3-Mb form of the hemizygous deletion, there remains a large variation in the development and progression of psychiatric disorders, which suggests that alternative factors contribute to the pathogenesis. In this study we investigated whether neonatal DNA methylation signatures in individuals with the 22q11.2 deletion associate with mental disorder later in life. DNA methylation was measured genome-wide from neonatal dried blood spots in a cohort of 164 individuals with 22q11.2DS, including 48 individuals diagnosed with a psychiatric disorder. Among several CpG sites with P-value<10−6, we identified cg23546855 (P-value=2.15 × 10−7) mapping to STK32C to be associated with a later psychiatric diagnosis. Pathway analysis of the top findings resulted in the identification of several Gene Ontology pathways to be significantly enriched (P-value<0.05 after Benjamini–Hochberg correction); among them are the following: neurogenesis, neuron development, neuron projection development, astrocyte development, axonogenesis and axon guidance. In addition, we identified differentially methylated CpG sites in LRP2BP (P-value=5.37 × 10−8) to be associated with intellectual disability (F70–79), in TOP1 (P-value=1.86 × 10−7) with behavioral disorders (F90–98), in NOSIP (P-value=5.12 × 10−8) with disorders of psychological development (F80–89) and in SEMA4B (P-value=4.02 × 10−7) with schizophrenia spectrum disorders (F20–29). In conclusion, our study suggests an association of DNA methylation differences at birth with development of mental disorder later in life in 22q11.2DS individuals.

Analysis of potential protein-modifying variants in 9000 endometriosis patients and 150000 controls of European ancestry

Genome-wide association (GWA) studies have identified 19 independent common risk loci for endometriosis. Most of the GWA variants are non-coding and the genes responsible for the association signals have not been identified. Herein, we aimed to assess the potential role of protein-modifying variants in endometriosis using exome-array genotyping in 7164 cases and 21005 controls, and a replication set of 1840 cases and 129016 controls of European ancestry. Results in the discovery sample identified significant evidence for association with coding variants in single-variant (rs1801232-CUBN) and gene-level (CIITA and PARP4) meta-analyses, but these did not survive replication. In the combined analysis, there was genome-wide significant evidence for rs13394619 (Pu2009=u20092.3u2009×u200910−9) in GREB1 at 2p25.1 — a locus previously identified in a GWA meta-analysis of European and Japanese samples. Despite sufficient power, our results did not identify any protein-modifying variants (MAF > 0.01) with moderate or large effect sizes in endometriosis, although these variants may exist in non-European populations or in high-risk families. The results suggest continued discovery efforts should focus on genotyping large numbers of surgically-confirmed endometriosis cases and controls, and/or sequencing high-risk families to identify novel rare variants to provide greater insights into the molecular pathogenesis of the disease.

The schizophrenia-associated BRD1 gene regulates behavior, neurotransmission, and expression of schizophrenia risk enriched gene sets in mice

BACKGROUNDnThe schizophrenia-associated BRD1 gene encodes a transcriptional regulator whose comprehensive chromatin interactome is enriched with schizophrenia risk genes. However, the biology underlying the disease association of BRD1 remains speculative.nnnMETHODSnThis study assessed the transcriptional drive of a schizophrenia-associated BRD1 risk variant in vitro. Accordingly, to examine the effects of reduced Brd1 expression, we generated a genetically modified Brd1+/- mouse and subjected it to behavioral, electrophysiological, molecular, and integrative genomic analyses with focus on schizophrenia-relevant parameters.nnnRESULTSnBrd1+/- mice displayed cerebral histone H3K14 hypoacetylation and a broad range of behavioral changes with translational relevance to schizophrenia. These behaviors were accompanied by striatal dopamine/serotonin abnormalities and cortical excitation-inhibition imbalances involving loss of parvalbumin immunoreactive interneurons. RNA-sequencing analyses of cortical and striatal micropunches from Brd1+/- and wild-type mice revealed differential expression of genes enriched for schizophrenia risk, including several schizophrenia genome-wide association study risk genes (e.g., calcium channel subunits [Cacna1c and Cacnb2], cholinergic muscarinic receptor 4 [Chrm4)], dopamine receptor D2 [Drd2], and transcription factor 4 [Tcf4]). Integrative analyses further found differentially expressed genes to cluster in functional networks and canonical pathways associated with mental illness and molecular signaling processes (e.g., glutamatergic, monoaminergic, calcium, cyclic adenosine monophosphate [cAMP], dopamine- and cAMP-regulated neuronal phosphoprotein 32 kDa [DARPP-32], and cAMP responsive element binding protein signaling [CREB]).nnnCONCLUSIONSnOur study bridges the gap between genetic association and pathogenic effects and yields novel insights into the unfolding molecular changes in the brain of a new schizophrenia model that incorporates genetic risk at three levels: allelic, chromatin interactomic, and brain transcriptomic.

Investigating interactions between early life stress and two single nucleotide polymorphisms in HSD11B2 on the risk of schizophrenia

BACKGROUNDnTo examine the risk of schizophrenia in a Danish population after exposure to early life stress, and whether this risk is modified by DNA sequence variation, specifically two single nucleotide polymorphisms (SNPs) (rs5479 and rs56303414) from the gene HSD11B2. This gene encodes the enzyme 11-β hydroxysteroid dehydrogenase type 2 which converts active cortisol into inactive cortisone.nnnMETHODSnA two-stage analysis involving (1) a population-based cohort study, and (2) a nested case-control study using genotype information. Stage 1 included 1,141,447 people; here, we calculated incidence rate ratios (IRR) for the risk of schizophrenia among children of mothers who experienced loss or serious illness of close relatives before, during, and after pregnancy. In stage 2, we genotyped rs5479 and rs56303414 among 1275 schizophrenia cases and 1367 controls, and investigated interactions between genotypes and early life stress on the risk of schizophrenia.nnnRESULTSnIn stage 1, no increased risk of schizophrenia was found in offspring after exposure during pregnancy, but offspring exposed to early life stress at age 0-2 years had a significantly increased risk of schizophrenia (adjusted IRR 1.18, 95% confidence interval 1.07-1.31). For rs5479, the minor allele was nucleotide A, and the major allele was nucleotide C. No interaction was found between rs5479 and exposure during pregnancy. Individuals with the minor A allele of rs5479, however, had a significantly increased risk of schizophrenia after exposure to early life stress at age 3-9 years (adjusted IRR 2.06, 1.04-4.06). No interaction was found between rs56303414 and exposure in any of the time periods.nnnCONCLUSIONnNo association was found between exposure to early life stress during pregnancy and schizophrenia in the offspring investigated, whereas individuals exposed to early life stress within the first two years of life had an increased risk. No interaction was found between HSD11B2 and exposure during pregnancy, but individuals with the A allele of rs5479 had an increased risk of schizophrenia after exposure at age 3-9 years.

Evaluating the Feasibility of DNA Methylation Analyses Using Long-Term Archived Brain Formalin-Fixed Paraffin-Embedded Samples

We here characterize the usability of archival formalin-fixed paraffin-embedded (FFPE) brain tissue as a resource for genetic and DNA methylation analyses with potential relevance for brain-manifested diseases. We analyzed FFPE samples from The Brain Collection, Aarhus University Hospital Risskov, Denmark (AUBC), constituting 9479 formalin-fixated brains making it one of the largest collections worldwide. DNA extracted from brain FFPE tissue blocks was interrogated for quality and usability in genetic and DNA methylation analyses by different molecular techniques. Overall, we found that DNA quality was inversely correlated with storage time and DNA quality was insufficient for Illumina methylation arrays; data from methylated DNA immunoprecipitation, clonal bisulfite sequencing, and pyrosequencing of BDNF and ST6GALNAC1 suggested that the original methylation pattern is indeed preserved. Proof-of-principle experiments predicting sex based on the methylation status of the X-inactivated SLC9A7 gene, or genotype differences of the Y and X chromosomes, showed consistency between predicted and actual sex for a subset of FFPE samples. In conclusion, even though DNA from FFPE samples is of low quality and technically challenging, it is likely that a subset of samples can provide reliable data given that the methodology used is designed for small DNA fragments. We propose that simple PCR-based quality control experiments at the genetic and DNA methylation level, carried out at the beginning of any given project, can be used to enrich for the best-performing FFPE samples. The apparent preservation of genetic and DNA methylation patterns in archival FFPE samples may bring along new perspectives for the identification of genetic and epigenetic changes associated with brain-manifested diseases.

Epigenome-wide association study of cognitive functioning in middle-aged monozygotic twins

As the worlds population ages, the age-related cognitive decline presents a great challenge to worlds healthcare systems. One of the molecular mechanisms implicated in cognitive ageing is DNA methylation, an epigenetic modification known to be a key player in memory formation, maintenance, and synaptic plasticity. Using the twin design we performed an epigenome-wide association study (EWAS) in a population of 486 middle-aged monozygotic twins (mean age at follow-up 65.9, SD = 6.1) and correlated their blood DNA methylation to their level (cross-sectional analysis) and change in cognitive abilities over 10 years (longitudinal analysis). We identified several CpG sites where cross-sectional cognitive functioning was associated with DNA methylation levels. The top identified loci were located in ZBTB46 (p = 5.84 × 10−7), and TAF12 (p = 4.91 × 10−7). KEGGs enrichment analyses of the most associated findings identified “Neuroactive ligand-receptor interaction” as the most enriched pathway (p = 0.0098). Change in cognitive functioning over 10 years was associated with DNA methylation levels in AGBL4 (p = 9.01 × 10−7) and SORBS1 (p = 5.28 × 10−6), with the first gene playing an important role in neuronal survival and the latter gene implicated before in Alzheimers disease and ischemic stroke. Our findings point to an association between changes in DNA methylation of genes related to neuronal survival and change of cognitive functioning in aging individuals.