A large-scale genome-wide association study meta-analysis of cannabis use disorder

Summary Background Variation in liability to cannabis use disorder has a strong genetic component (estimated twin and family heritability about 50–70%) and is associated with negative outcomes, including increased risk of psychopathology. The aim of the study was to conduct a large genome-wide association study (GWAS) to identify novel genetic variants associated with cannabis use disorder. Methods To conduct this GWAS meta-analysis of cannabis use disorder and identify associations with genetic loci, we used samples from the Psychiatric Genomics Consortium Substance Use Disorders working group, iPSYCH, and deCODE (20 916 case samples, 363 116 control samples in total), contrasting cannabis use disorder cases with controls. To examine the genetic overlap between cannabis use disorder and 22 traits of interest (chosen because of previously published phenotypic correlations [eg, psychiatric disorders] or hypothesised associations [eg, chronotype] with cannabis use disorder), we used linkage disequilibrium score regression to calculate genetic correlations. Findings We identified two genome-wide significant loci: a novel chromosome 7 locus (FOXP2, lead single-nucleotide polymorphism [SNP] rs7783012; odds ratio [OR] 1·11, 95% CI 1·07–1·15, p=1·84 × 10−9) and the previously identified chromosome 8 locus (near CHRNA2 and EPHX2, lead SNP rs4732724; OR 0·89, 95% CI 0·86–0·93, p=6·46 × 10−9). Cannabis use disorder and cannabis use were genetically correlated (rg 0·50, p=1·50 × 10−21), but they showed significantly different genetic correlations with 12 of the 22 traits we tested, suggesting at least partially different genetic underpinnings of cannabis use and cannabis use disorder. Cannabis use disorder was positively genetically correlated with other psychopathology, including ADHD, major depression, and schizophrenia. Interpretation These findings support the theory that cannabis use disorder has shared genetic liability with other psychopathology, and there is a distinction between genetic liability to cannabis use and cannabis use disorder. Funding National Institute of Mental Health; National Institute on Alcohol Abuse and Alcoholism; National Institute on Drug Abuse; Center for Genomics and Personalized Medicine and the Centre for Integrative Sequencing; The European Commission, Horizon 2020; National Institute of Child Health and Human Development; Health Research Council of New Zealand; National Institute on Aging; Wellcome Trust Case Control Consortium; UK Research and Innovation Medical Research Council (UKRI MRC); The Brain & Behavior Research Foundation; National Institute on Deafness and Other Communication Disorders; Substance Abuse and Mental Health Services Administration (SAMHSA); National Institute of Biomedical Imaging and Bioengineering; National Health and Medical Research Council (NHMRC) Australia; Tobacco-Related Disease Research Program of the University of California; Families for Borderline Personality Disorder Research (Beth and Rob Elliott) 2018 NARSAD Young Investigator Grant; The National Child Health Research Foundation (Cure Kids); The Canterbury Medical Research Foundation; The New Zealand Lottery Grants Board; The University of Otago; The Carney Centre for Pharmacogenomics; The James Hume Bequest Fund; National Institutes of Health: Genes, Environment and Health Initiative; National Institutes of Health; National Cancer Institute; The William T Grant Foundation; Australian Research Council; The Virginia Tobacco Settlement Foundation; The VISN 1 and VISN 4 Mental Illness Research, Education, and Clinical Centers of the US Department of Veterans Affairs; The 5th Framework Programme (FP-5) GenomEUtwin Project; The Lundbeck Foundation; NIH-funded Shared Instrumentation Grant S10RR025141; Clinical Translational Sciences Award grants; National Institute of Neurological Disorders and Stroke; National Heart, Lung, and Blood Institute; National Institute of General Medical Sciences.Peer reviewe

Shared genetic risk between eating disorder- and substance-use-related phenotypes:Evidence from genome-wide association studies

First published: 16 February 202

Transancestral GWAS of alcohol dependence reveals common genetic underpinnings with psychiatric disorders

Liability to alcohol dependence (AD) is heritable, but little is known about its complex polygenic architecture or its genetic relationship with other disorders. To discover loci associated with AD and characterize the relationship between AD and other psychiatric and behavioral outcomes, we carried out the largest genome-wide association study to date of DSM-IV-diagnosed AD. Genome-wide data on 14,904 individuals with AD and 37,944 controls from 28 case-control and family-based studies were meta-analyzed, stratified by genetic ancestry (European, n = 46,568; African, n = 6,280). Independent, genome-wide significant effects of different ADH1B variants were identified in European (rs1229984; P = 9.8 x 10(-13)) and African ancestries (rs2066702; P = 2.2 x 10(-9)). Significant genetic correlations were observed with 17 phenotypes, including schizophrenia, attention deficit-hyperactivity disorder, depression, and use of cigarettes and cannabis. The genetic underpinnings of AD only partially overlap with those for alcohol consumption, underscoring the genetic distinction between pathological and nonpathological drinking behaviors.Peer reviewe

Circular non-coding RNA ANRIL modulates ribosomal RNA maturation and atherosclerosis in humans

Circular RNAs (circRNAs) are broadly expressed in eukaryotic cells, but their molecular mechanism in human disease remains obscure. Here we show that circular antisense non-coding RNA in the INK4 locus (circANRIL), which is transcribed at a locus of atherosclerotic cardiovascular disease on chromosome 9p21, confers atheroprotection by controlling ribosomal RNA (rRNA) maturation and modulating pathways of atherogenesis. CircANRIL binds to pescadillo homologue 1 (PES1), an essential 60S-preribosomal assembly factor, thereby impairing exonuclease-mediated pre-rRNA processing and ribosome biogenesis in vascular smooth muscle cells and macrophages. As a consequence, circANRIL induces nucleolar stress and p53 activation, resulting in the induction of apoptosis and inhibition of proliferation, which are key cell functions in atherosclerosis. Collectively, these findings identify circANRIL as a prototype of a circRNA regulating ribosome biogenesis and conferring atheroprotection, thereby showing that circularization of long non-coding RNAs may alter RNA function and protect from human disease

Alu Elements in <i>ANRIL</i> Non-Coding RNA at Chromosome 9p21 Modulate Atherogenic Cell Functions through <i>Trans</i>-Regulation of Gene Networks

<div><p>The chromosome 9p21 (Chr9p21) locus of coronary artery disease has been identified in the first surge of genome-wide association and is the strongest genetic factor of atherosclerosis known today. Chr9p21 encodes the long non-coding RNA (ncRNA) <i>antisense non-coding RNA in the INK4 locus</i> (<i>ANRIL</i>). <i>ANRIL</i> expression is associated with the Chr9p21 genotype and correlated with atherosclerosis severity. Here, we report on the molecular mechanisms through which <i>ANRIL</i> regulates target-genes <i>in trans</i>, leading to increased cell proliferation, increased cell adhesion and decreased apoptosis, which are all essential mechanisms of atherogenesis. Importantly, <i>trans</i>-regulation was dependent on Alu motifs, which marked the promoters of <i>ANRIL</i> target genes and were mirrored in <i>ANRIL</i> RNA transcripts. <i>ANRIL</i> bound Polycomb group proteins that were highly enriched in the proximity of Alu motifs across the genome and were recruited to promoters of target genes upon <i>ANRIL</i> over-expression. The functional relevance of Alu motifs in <i>ANRIL</i> was confirmed by deletion and mutagenesis, reversing <i>trans</i>-regulation and atherogenic cell functions. <i>ANRIL</i>-regulated networks were confirmed in 2280 individuals with and without coronary artery disease and functionally validated in primary cells from patients carrying the Chr9p21 risk allele. Our study provides a molecular mechanism for pro-atherogenic effects of <i>ANRIL</i> at Chr9p21 and suggests a novel role for Alu elements in epigenetic gene regulation by long ncRNAs.</p></div

<i>ANRIL</i> binds to PRC1 and 2 proteins and recruits CBX7 and SUZ12 to promoters of target genes.

<p>(A, B) RNA immunoprecipitation (RIP) followed by qRT-PCR demonstrating <i>ANRIL</i> binding to PRC but not to CoREST/REST proteins in (A) ANRIL2 and (B) ANRIL4 cells. Copies of ANRIL relative to input control are given in (A) blue and (B) red, nuclear ncRNA <i>U1</i> (white) was used as negative control. rIgG/mIgG/gIgG- rabbit/mouse/goat IgG controls. Error bars indicate s.e.m. (C,D) SUZ12 binding in promoters of <i>ANRIL</i> up-(green), down-(red), and not (black) regulated genes in (C) vector control cell line and (D) in BGO3 cells (GSM602674). TSS- transcription start site. (E, F) Effect of <i>ANRIL</i> over-expression on (E) SUZ12 and (F) CBX7 binding in promoters of up-regulated genes (vector control- dotted line vs. ANRIL2- straight line). (G) Reversal of <i>ANRIL trans</i>-regulation by RNAi against SUZ12 and CBX7 in ANRIL2 cells. SCR- scrambled siRNA control.</p

Pivotal role for Alu motif in <i>ANRIL</i> RNA function.

<p>(A) Cell lines over-expressing variants of <i>ANRIL2</i> (ANRIL2a, 2b, 2c) and <i>ANRIL4</i> (ANRIL4a, 4b) devoid of Alu motif sequences highlighted by boxes. Validation of over-expression using qRT-PCR assays. (B) Reversal of up-regulation (<i>TSC22D3</i>) and (C) down-regulation (<i>COL3A1</i>) of <i>ANRIL</i> target-gene mRNA expression in ANRIL2a-2c and ANRIL4a,4b compared to ANRIL2 and ANRIL4. Reversal of (D) cell adhesion, (E) apoptosis, and (F) proliferation in cell lines over-expressing mutant <i>ANRIL</i> isoforms. (G) Stable cell lines containing mutated forms of the Alu motif in <i>ANRIL2</i>. Positions of nucleotide exchanges (0, 25%, 33%, and 100%) in the 48 base-pair Alu motif are indicated in red. (H,I) Reversal of <i>trans</i>-regulation in mutant cell lines compared to ANRIL2. (J) Cell adhesion, (K) apoptosis, and (L) proliferation in mutant cell lines. (B,C,H,I,F,L) 3–4 biological replicates/isoform.(D,E,J,K) quadruplicate measurements per pool of 2–3 biological replicates. Error bars indicate s.e.m.</p

Annotated <i>ANRIL</i> transcripts in the Chr9p21 region, transcript structure and association of <i>ANRIL</i> isoforms with Chr9p21 genotype.

<p>(A) Chr9p21 haplotype structure (HapMap CEU, r²) and core atherosclerosis region (between rs12555547 and rs1333050). (B) Exons of initially discovered <i>ANRIL</i> transcripts and their relative position in the Chr9p21 region. *A full list of currently annotated <i>ANRIL</i> isoforms is given in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003588#pgen.1003588.s001" target="_blank">Figure S1B</a>. (C) <i>ANRIL</i> transcripts identified by RACE and PCR amplification (L1-L17). 4 major isoform groups with 4 distinct transcriptions ends were identified. Frequency of exon occurrence/isoform group is color-coded and given in %. <i>ANRIL1-4</i> denote highly expressed consensus transcripts harbouring exons found in >50% transcripts of the respective isoform group (red). Dotted lines indicate positions of transcript-specific qRT-PCR assays. (D) Study design of <i>ANRIL</i> expression and association with Chr9p21. (E) Association of <i>ANRIL</i> isoforms with Chr9p21 in human PBMC, whole blood and vascular tissue (effect, % change/risk allele defined by rs10757274, rs2383206, rs2383297, and rs10757278).</p

Validation of <i>ANRIL</i>-associated cellular effects in primary cells and schematic of molecular scaffolding by <i>ANRIL</i>.

<p>(A) PBMC from carriers of the Chr9p21 CAD-risk allele defined by rs10757274, rs2383206, rs2383297, and rs10757278 (n = 8) showed increased adhesion (<i>P</i> = 0.001) and (B) decreased apoptosis (<i>P</i> = 0.008) compared to cells of carriers of the protective allele (n = 8). (C) Schematic of molecular scaffolding by <i>ANRIL</i> mediated through potential chromatin-RNA interaction by Alu motifs.</p

Identification of Alu motifs in promoters of <i>ANRIL trans</i>-regulated genes and <i>ANRIL</i> RNA and their spatial relation to PcG protein binding.

<p>(A) DNA motif in promoters (5 kb) of <i>trans</i>-regulated genes representing an Alu-DEIN repeat <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003588#pgen.1003588-Deininger1" target="_blank">[33]</a>. (B) Number of Alu motifs in promoters of <i>trans</i>- and not regulated transcripts (n per 5 kb). (C) ChIP-seq enrichment of SUZ12 and CBX7 binding distal to Alu motif, demonstrating a specific spatial relation of motif occurrence to PcG protein binding. RPM- reads per million mapped reads, CTR- random DNA control sequence. (D) Motif-associated SUZ12 signal peaks in an independent data set from BGO3 cells. The actual signal peak only becomes apparent, if a multiple matching policy is adopted. (E) Using a strict unique-matches only policy, a substantial signal reduction is seen downstream of the motif. Please note differences in y-axis scaling in (D, E). (F) Secondary RNA structure prediction for ANRIL2 using the Vienna RNA package <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003588#pgen.1003588-Tacker1" target="_blank">[58]</a>. Within the minimum free energy structure, the Alu-DEIN motif is located in a stem-loop structure (arrow). (G) RIP demonstrating <i>ANRIL</i> binding to histone H3 (H3) and trimethylated lysine 27 of histone 3 (H3K27me3) in ANRIL2 (blue) and ANRIL4 (red) cells. <i>U1</i> was used as negative control. mIgG/rIgG- mouse/rabbit IgG control. Error bars indicate s.e.m.</p