Exploring the mediation of DNA methylation across the epigenome between childhood adversity and First Episode of Psychosis-findings from the EU-GEI study.

ABTRACT: Studies conducted in psychotic disorders have shown that DNA-methylation (DNAm) is sensitive to the impact of Childhood Adversity (CA). However, whether it mediates the association between CA and psychosis is yet to be explored. Epigenome wide association studies (EWAS) using the Illumina Infinium-Methylation EPIC array in peripheral blood tissue from 366 First-episode of psychosis and 517 healthy controls was performed. Adversity scores were created for abuse, neglect and composite adversity with the Childhood Trauma Questionnaire (CTQ). Regressions examining (I) CTQ scores with psychosis; (II) with DNAm EWAS level and (III) between DNAm and caseness, adjusted for a variety of confounders were conducted. Divide-Aggregate Composite-null Test for the composite null-hypothesis of no mediation effect was conducted. Enrichment analyses were conducted with missMethyl package and the KEGG database. Our results show that CA was associated with psychosis (Composite: OR = 1.68; p = <0.001; abuse: OR = 2.16; p < 0.001; neglect: OR = 2.27; p = <0.001). None of the CpG sites significantly mediated the adversity-psychosis association after Bonferroni correction (p < 8.1 × 10-8). However, 28, 34 and 29 differentially methylated probes associated with 21, 27, 20 genes passed a less stringent discovery threshold (p < 5 × 10-5) for composite, abuse and neglect respectively, with a lack of overlap between abuse and neglect. These included genes previously associated to psychosis in EWAS studies, such as PANK1, SPEG TBKBP1, TSNARE1 or H2R. Downstream gene ontology analyses did not reveal any biological pathways that survived false discovery rate correction. Although at a non-significant level, DNAm changes in genes previously associated with schizophrenia in EWAS studies may mediate the CA-psychosis association. These results and associated involved processes such as mitochondrial or histaminergic disfunction, immunity or neural signalling requires replication in well powered samples. The lack of overlap between mediating genes associated with abuse and neglect suggests differential biological trajectories linking CA subtypes and psychosis.

Predicting Alcohol Use From Genome-Wide Polygenic Scores, Environmental Factors, and Their Interactions in Young Adulthood.

OBJECTIVE: Alcohol use during emerging adulthood is associated with adverse life outcomes, but its risk factors are not well known. Here, we predicted alcohol use in 3153 young adults aged 22 years from a) genome-wide polygenic scores (GPS) based on genome-wide association studies for the target phenotypes number of drinks per week and Alcohol Use Disorders Identification Test scores, b) 30 environmental factors, and c) their interactions (i.e., G × E effects). METHODS: Data were collected from 1994 to 2018 as a part of the UK Twins Early Development Study. RESULTS: GPS accounted for up to 1.9% of the variance in alcohol use (i.e., Alcohol Use Disorders Identification Test score), whereas the 30 measures of environmental factors together accounted for 21.1%. The 30 GPS by environment interactions did not explain any additional variance, and none of the interaction terms exceeded the significance threshold after correcting for multiple testing. CONCLUSIONS: GPS and some environmental factors significantly predicted alcohol use in young adulthood, but we observed no GPS by environment interactions in our study.

Predictive validity of genome-wide polygenic scores for alcohol use from adolescence to young adulthood.

BACKGROUND: Adolescence is a critical period for experimenting with alcohol, and these early experiences have long-term influences on alcohol-related behaviours throughout adulthood. This study examined the utility of genome-wide polygenic scores (GPS) for predicting alcohol use during adolescence and young adulthood. METHODS: We used GPS based on the Genome-wide association study and Sequencing Consortium of Alcohol and Nicotine use (GSCAN) study on drinks per week to predict alcohol use in a longitudinal, UK-representative sample of unrelated adolescents aged 16 through to 22 years (Nmax = 3390). RESULTS: At age 16, the GSCAN GPS predicted variance in alcohol consumption on a typical day (0.58 %), intake frequency (0.89 %), and hazardous drinking (i.e. ≥6 units at one occasion) (1.07 %). At age 22, the predictive power of the GPS had increased, explaining variance in alcohol consumption (0.61 %), intake frequency (1.69 %), and hazardous drinking (1.19 %). CONCLUSIONS: The predictive validity of GPS for phenotypic alcohol use was evident in adolescence and increased in young adulthood. The findings suggest that GPS, which are available from birth, may be potentially useful for identifying individuals at risk for harmful and hazardous alcohol use. However, because the overall effect sizes were small, the utility of the GPS that are currently available is limited for the prediction of individual-level alcohol use.

DNA methylation signatures of adolescent victimization: analysis of a longitudinal monozygotic twin sample.

Accumulating evidence suggests that individuals exposed to victimization at key developmental stages may have different epigenetic fingerprints compared to those exposed to no/minimal stressful events, however results are inconclusive. This study aimed to strengthen causal inference regarding the impact of adolescent victimization on the epigenome by controlling for genetic variation, age, gender, and shared environmental exposures. We conducted longitudinal epigenome-wide association analyses (EWAS) on DNA methylation (DNAm) profiles of 118 monozygotic (MZ) twin pairs from the Environmental Risk study with and without severe adolescent victimization generated using buccal DNA collected at ages 5, 10 and 18, and the Illumina EPIC array. Additionally, we performed cross-sectional EWAS on age-18 blood and buccal DNA from the same individuals to elucidate tissue-specific signatures of severe adolescent victimization. Our analyses identified 20 suggestive differentially methylated positions (DMPs) (P < 5e-05), with altered DNAm trajectories between ages 10-18 associated with severe adolescent victimization (∆Beta range = -5.5%-5.3%). Age-18 cross-sectional analyses revealed 72 blood (∆Beta range = -2.2%-3.4%) and 42 buccal (∆Beta range = -3.6%-4.6%) suggestive severe adolescent victimization-associated DMPs, with some evidence of convergent signals between these two tissue types. Downstream regional analysis identified significant differentially methylated regions (DMRs) in LGR6 and ANK3 (Sidák P = 5e-09 and 4.07e-06), and one upstream of CCL27 (Sidák P = 2.80e-06) in age-18 blood and buccal EWAS, respectively. Our study represents the first longitudinal MZ twin analysis of DNAm and severe adolescent victimization, providing initial evidence for altered DNA methylomic signatures in individuals exposed to adolescent victimization.

Patterns of Reliability: Assessing the Reproducibility and Integrity of DNA Methylation Measurement.

DNA methylation plays an important role in both normal human development and risk of disease. The most utilized method of assessing DNA methylation uses BeadChips, generating an epigenome-wide "snapshot" of >450,000 observations (probe measurements) per assay. However, the reliability of each of these measurements is not equal, and little consideration is paid to consequences for research. We correlated repeat measurements of the same DNA samples using the Illumina HumanMethylation450K and the Infinium MethylationEPIC BeadChips in 350 blood DNA samples. Probes that were reliably measured were more heritable and showed consistent associations with environmental exposures, gene expression, and greater cross-tissue concordance. Unreliable probes were less replicable and generated an unknown volume of false negatives. This serves as a lesson for working with DNA methylation data, but the lessons are equally applicable to working with other data: as we advance toward generating increasingly greater volumes of data, failure to document reliability risks harming reproducibility.

Identification of rare nonsynonymous variants in SYNE1/CPG2 in bipolar affective disorder.

BACKGROUND: Bipolar affective disorder (BPD) is a severe mood disorder with a prevalence of ∼1.5% in the population. The pathogenesis of BPD is poorly understood; however, a strong heritable component has been identified. Previous genome-wide association studies have indicated a region on 6q25, coding for the SYNE1 gene, which increases disease susceptibility. SYNE1 encodes the synaptic nuclear envelope protein-1, nesprin-1. A brain-specific splice variant of SYNE1, CPG2 encoding candidate plasticity gene 2, has been identified. The intronic single-nucleotide polymorphism with the strongest genome-wide significant association in BPD, rs9371601, is present in both SYNE1 and CPG2. METHODS: We screened 937 BPD samples for genetic variation in SYNE1 exons 14-33, which covers the CPG2 region, using high-resolution melt analysis. In addition, we screened two regions of increased transcriptional activity, one of them proposed to be the CPG2 promoter region. RESULTS AND CONCLUSION: We identified six nonsynonymous and six synonymous variants. We genotyped three rare nonsynonymous variants, rs374866393, rs148346599 and rs200629713, in a total of 1099 BPD samples and 1056 controls. Burden analysis of these rare variants did not show a significant association with BPD. However, nine patients are compound heterozygotes for variants in SYNE1/CPG2, suggesting that rare coding variants may contribute significantly towards the complex genetic architecture underlying BPD. Imputation analysis in our own whole-genome sequencing sample of 99 BPD individuals identified an additional eight risk variants in the CPG2 region of SYNE1.

Genetic Predisposition to Chronic Lymphocytic Leukemia Is Mediated by a BMF Super-Enhancer Polymorphism.

Chronic lymphocytic leukemia (CLL) is an adult B cell malignancy. Genome-wide association studies show that variation at 15q15.1 influences CLL risk. We deciphered the causal variant at 15q15.1 and the mechanism by which it influences tumorigenesis. We imputed all possible genotypes across the locus and then mapped highly associated SNPs to areas of chromatin accessibility, evolutionary conservation, and transcription factor binding. SNP rs539846 C>A, the most highly associated variant (p = 1.42 × 10(-13), odds ratio = 1.35), localizes to a super-enhancer defined by extensive histone H3 lysine 27 acetylation in intron 3 of B cell lymphoma 2 (BCL2)-modifying factor (BMF). The rs539846-A risk allele alters a conserved RELA-binding motif, disrupts RELA binding, and is associated with decreased BMF expression in CLL. These findings are consistent with rs539846 influencing CLL susceptibility through differential RELA binding, with direct modulation of BMF expression impacting on anti-apoptotic BCL2, a hallmark of oncogenic dependency in CLL.

Capture Hi-C identifies the chromatin interactome of colorectal cancer risk loci.

Multiple regulatory elements distant from their targets on the linear genome can influence the expression of a single gene through chromatin looping. Chromosome conformation capture implemented in Hi-C allows for genome-wide agnostic characterization of chromatin contacts. However, detection of functional enhancer-promoter interactions is precluded by its effective resolution that is determined by both restriction fragmentation and sensitivity of the experiment. Here we develop a capture Hi-C (cHi-C) approach to allow an agnostic characterization of these physical interactions on a genome-wide scale. Single-nucleotide polymorphisms associated with complex diseases often reside within regulatory elements and exert effects through long-range regulation of gene expression. Applying this cHi-C approach to 14 colorectal cancer risk loci allows us to identify key long-range chromatin interactions in cis and trans involving these loci.

Genetic variants in or near ADH1B and ADH1C affect susceptibility to alcohol dependence in a British and Irish population.

Certain single nucleotide polymorphisms (SNPs) in genes encoding alcohol dehydrogenase (ADH) enzymes confer a significant protective effect against alcohol dependence syndrome (ADS) in East Asian populations. Recently, attention has focused on the role of these SNPs in determining ADS risk in European populations. To further elucidate these associations, SNPs of interest in ADH1B, ADH1C and the ADH1B/1C intergenic region were genotyped in a British and Irish population (ADS cases n = 1076: controls n = 1027) to assess their relative contribution to ADS risk. A highly significant, protective association was observed between the minor allele of rs1229984 in ADH1B and ADS risk [allelic P = 8.4 × 10(-6) , odds ratio (OR) = 0.26, 95 percent confidence interval, 0.14, 0.49]. Significant associations were also observed between ADS risk and the ADH1B/1C intergenic variant, rs1789891 [allelic P = 7.2 × 10(-5) , OR = 1.4 (1.2, 1.6)] and three non-synonymous SNPs rs698, rs1693482 and rs283413 in ADH1C. However, these associations were not completely independent; thus, while the ADH1B rs1229984 minor allele association was independent of those of the intergenic variant rs1789891 and the three ADH1C variants, the three ADH1C variants were not individually independent. In conclusion, the rare ADH1B rs1229984 mutation provides significant protection against ADS in this British and Irish population; other variants in the ADH gene cluster also alter ADS risk, although the strong linkage disequilibrium between SNPs at this location precluded clear identification of the variant(s) driving the associations.

Allelic association, DNA resequencing and copy number variation at the metabotropic glutamate receptor GRM7 gene locus in bipolar disorder.

Genetic markers at the GRM7 gene have shown allelic association with bipolar disorder (BP) in several case-control samples including our own sample. In this report, we present results of resequencing the GRM7 gene in 32 bipolar samples and 32 random controls selected from 553 bipolar cases and 547 control samples (UCL1). Novel and potential etiological base pair changes discovered by resequencing were genotyped in the entire UCL case-control sample. We also report on the association between GRM7 and BP in a second sample of 593 patients and 642 controls (UCL2). The three most significantly associated SNPs in the original UCL1 BP GWAS sample were genotyped in the UCL2 sample, of which none were associated. After combining the genotype data for the two samples only two (rs1508724 and rs6769814) of the original three SNP markers remained significantly associated with BP. DNA sequencing revealed mutations in three cases which were absent in control subjects. A 3'-UTR SNP rs56173829 was found to be significantly associated with BP in the whole UCL sample (P = 0.035; OR = 0.482), the rare allele being less common in cases compared to controls. Bioinformatic analyses predicted a change in the centroid secondary structure of RNA and alterations in the miRNA binding sites for the mutated base of rs56173829. We also validated two deletions and a duplication within GRM7 using quantitative-PCR which provides further support for the pre-existing evidence that copy number variants at GRM7 may have a role in the etiology of BP.

Analysis of ANK3 and CACNA1C variants identified in bipolar disorder whole genome sequence data.

OBJECTIVES: Genetic markers in the genes encoding ankyrin 3 (ANK3) and the α-calcium channel subunit (CACNA1C) are associated with bipolar disorder (BP). The associated variants in the CACNA1C gene are mainly within intron 3 of the gene. ANK3 BP-associated variants are in two distinct clusters at the ends of the gene, indicating disease allele heterogeneity. METHODS: In order to screen both coding and non-coding regions to identify potential aetiological variants, we used whole-genome sequencing in 99 BP cases. Variants with markedly different allele frequencies in the BP samples and the 1,000 genomes project European data were genotyped in 1,510 BP cases and 1,095 controls. RESULTS: We found that the CACNA1C intron 3 variant, rs79398153, potentially affecting an ENCyclopedia of DNA Elements (ENCODE)-defined region, showed an association with BP (p = 0.015). We also found the ANK3 BP-associated variant rs139972937, responsible for an asparagine to serine change (p = 0.042). However, a previous study had not found support for an association between rs139972937 and BP. The variants at ANK3 and CACNA1C previously known to be associated with BP were not in linkage disequilibrium with either of the two variants that we identified and these are therefore independent of the previous haplotypes implicated by genome-wide association. CONCLUSIONS: Sequencing in additional BP samples is needed to find the molecular pathology that explains the previous association findings. If changes similar to those we have found can be shown to have an effect on the expression and function of ANK3 and CACNA1C, they might help to explain the so-called 'missing heritability' of BP.

Genetic association, mutation screening, and functional analysis of a Kozak sequence variant in the metabotropic glutamate receptor 3 gene in bipolar disorder.

IMPORTANCE: Genetic markers at the gene encoding the metabotropic glutamate receptor 3 (GRM3) showed allelic association with bipolar disorder. OBJECTIVE: To screen the GRM3 gene and adjacent control regions of genomic DNA in volunteers with bipolar affective disorder for mutations increasing susceptibility to bipolar disorder. DESIGN: Sequencing and high-resolution melting curve analysis of DNA followed by genotyping was carried out in 1099 patients with bipolar affective disorder and 1152 healthy comparator individuals. SETTING: Participants with bipolar disorder were recruited from National Health Service psychiatric services and from patient organizations. PARTICIPANTS: Individuals were included if they had Research Diagnostic Criteria diagnoses of bipolar I and bipolar II disorder and were of British or Irish ancestry. MAIN OUTCOMES AND MEASURES: Identification of base pair changes in the GRM3 gene that affected expression or function of the GRM3 receptor that also showed an allelic association with bipolar disorder. RESULTS: A base pair variant (rs148754219) was found in the Kozak sequence of exon 1 of the GRM3 gene, 2 bases before the translation start codon of one of the receptor isoforms, in 23 of 2251 people who were screened and genotyped. Nineteen of the 1099 bipolar cases (1.7%) were mutation carriers compared with 4 of 1152 healthy comparators (0.3%). The variant was associated with bipolar disorder (P = .005; odds ratio, 4.20). Bioinformatic, electrophoretic mobility shift assay, and gene expression analysis found that the variant created a new transcription factor protein binding site and had a strong effect on gene transcription and translation. CONCLUSIONS AND RELEVANCE: Confirmation of these findings is needed before the Kozak sequence variant can be accepted as a potential marker for personalized treatment of affective disorders with drugs targeting the metabotropic glutamate receptor 3.

Sequencing of the ANKYRIN 3 gene (ANK3) encoding ankyrin G in bipolar disorder reveals a non-conservative amino acid change in a short isoform of ankyrin G.

Significant association between polymorphisms at the ANK3 gene with bipolar disorder has previously been reported and confirmed in several samples. Here we report on association between ANK3 and bipolar disorder in a new sample of 593 patients and 642 controls (UCL2) as well as the results of sequencing of the exons and flanking regions of ANK3 from bipolar patients. Single nucleotide polymorphisms (SNPs) associated with bipolar disorder in our original GWA study (UCL1) were genotyped and tested for association in the new sample. Novel SNPs found by sequencing were genotyped in both samples to test for association with bipolar disorder. None of the SNPs previously associated with bipolar disorder were associated in the UCL2 sample. One of the four SNPs associated in the UCL1 sample, rs1938526, was still significantly associated with bipolar disorder when the UCL1 and UCL2 samples were combined (P = 0.0095). The results demonstrate the impact of heterogeneity on replication of allelic associations even within well-defined ancestral populations. DNA sequencing revealed a novel low frequency (0.007) ANK3 SNP (ss469104599) which causes a non-conservative amino acid change at position 794 in the shorter isoforms of the ankyrin G protein. Protein-function analysis software predicted the amino acid change to be "probably damaging" and it could therefore be detrimental to the function of this isoform. Given that there was only a modest increase in the allele frequency of ss469104599 in cases compared to controls further association studies are needed in additional samples to establish a possible etiological role for this amino acid change.

Tests of linkage and allelic association between markers in the 1p36 PRKCZ (protein kinase C zeta) gene region and bipolar affective disorder.

Three linkage studies of families with multiple cases of bipolar disorder and/or unipolar affective disorder have confirmed the involvement of the chromosome 1p36 region in the etiology of affective disorders with LOD scores of 2.7, 3.6, and 3.97. We investigated the protein kinase C zeta gene (PRKCZ) as a susceptibility locus for bipolar disorder because it is highly brain expressed and is localized close to the marker D1S243 which was linked to affective disorder in a single large UCL bipolar disorder family with a LOD of 3.1. PRKCZ encodes an unusual type of protein kinase which affects axonal differentiation through Wnt-signaling. We genotyped four microsatellite markers and nine single nucleotide polymorphism (SNP) markers within or near the PRKCZ gene in the UCL case-control sample of 600 bipolar disorder patients and up to 605 supernormal controls. Markers D1S243 and rs3128396 were significantly associated with bipolar disorder (empirical P = 0.037 and P = 0.040, respectively). We also included data from eight SNPs which were genotyped as part of our GWA study on bipolar disorder for association analysis. Tests of haplotypic association found that a haplotype block comprising markers rs3128296, rs2503706, and rs3128309 was associated with bipolar disorder (empirical P = 0.004). A previous linkage study had shown greater evidence for linkage within female cases compared to males. Therefore, to assess if the association was sex-specific, we performed a female-only allelic-association analysis, which resulted in SNPs rs3128296 and rs3128309 becoming associated with bipolar disorder (P = 0.004 and P = 0.016, respectively). PRKCZ may play a role in susceptibility to bipolar affective disorder.

Confirmation of prior evidence of genetic susceptibility to alcoholism in a genome-wide association study of comorbid alcoholism and bipolar disorder.

OBJECTIVES: Alcoholism and affective disorders are both strongly comorbid and heritable. We have investigated the genetic comorbidity between bipolar affective disorder and alcoholism. METHODS: A genome-wide allelic association study of 506 patients from the University College London bipolar disorder case-control sample and 510 ancestrally matched supernormal controls. One hundred forty-three of the bipolar patients fulfilled the Research Diagnostic Criteria diagnosis of alcoholism. A total of 372 193 single nucleotide polymorphisms (SNPs) were genotyped. Genes previously shown to be associated with alcoholism and addiction phenotypes were then tested for association in the bipolar alcoholic sample using gene-wise permutation tests of all SNPs genotyped within a 50-kb region flanking each gene. RESULTS: Several central nervous system genes showed significant (P<0.05) gene-wise evidence of association with bipolar alcoholism. The genes implicated, which replicated genes previously shown to be associated with alcoholism were: cadherin 11, collagen type 11 α2, neuromedin U receptor 2, exportin7, and semaphorin-associated protein 5A. The SNPs most strongly implicated in bipolar alcoholism, but, which did not meet conventional genome-wide significance criteria were the insulin-like growth factor-binding protein 7, carboxypeptidase O, cerebellin 2, and the cadherin 12 genes. CONCLUSION: We have confirmed the role of some genes previously shown to be associated with alcoholism in the comorbid bipolar alcoholism subgroup. In this subgroup, bipolar disorder may lower the threshold for the phenotypic expression of these alcoholism susceptibility genes. We also show that some genes may independently increase susceptibility to affective disorder and alcoholism.

Genetic association and sequencing of the insulin-like growth factor 1 gene in bipolar affective disorder.

Insulin-like growth factor 1 (IGF1) has been shown to have an important role in brain development and function. Studies of IGF1 administration in rodents have shown that it has an anxiolytic and antidepressant effect. A genome-wide association study (GWAS) of the first University College London (UCL) cohort of 506 bipolar affective disorder subjects and 510 controls was carried out. The exons and flanking regions of IGF1 were resequenced, any new polymorphisms found were genotyped in an enlarged UCL sample of 937 cases and 941 controls. GWAS data gave good evidence of allelic and haplotypic association between multiple IGF1 SNP's and bipolar disorder (BD). New polymorphisms were found by resequencing IGF1 region. Data from GWAS and the new markers showed that twelve out of 43 SNPs showed association with BD with the four most significant SNPs having values of 3.7 × 10(-5) , 8.4 × 10(-4) , 2.6 × 10(-4) , and 2.5 × 10(-4) . A 5' promoter microsatellite polymorphism previously correlated with plasma lipoprotein concentration was also associated with BD (P = 0.013). Haplotypic association confirmed association with BD with significance values similar to the single marker SNP values. The marker rs12426318 has also been found to be associated with BD in a second sample. A test of gene wide significance with permutation testing for all markers genotyped at IGF1 was also significant. These data implicate IGF1 as a candidate gene to cause genetic susceptibility to BD.