Rare coding variants in CHRNB2 reduce the likelihood of smoking.

Human genetic studies of smoking behavior have been thus far largely limited to common variants. Studying rare coding variants has the potential to identify drug targets. We performed an exome-wide association study of smoking phenotypes in up to 749,459 individuals and discovered a protective association in CHRNB2, encoding the ß2 subunit of the α4ß2 nicotine acetylcholine receptor. Rare predicted loss-of-function and likely deleterious missense variants in CHRNB2 in aggregate were associated with a 35% decreased odds for smoking heavily (odds ratio (OR) = 0.65, confidence interval (CI) = 0.56-0.76, P = 1.9 × 10-8). An independent common variant association in the protective direction ( rs2072659 ; OR = 0.96; CI = 0.94-0.98; P = 5.3 × 10-6) was also evident, suggesting an allelic series. Our findings in humans align with decades-old experimental observations in mice that ß2 loss abolishes nicotine-mediated neuronal responses and attenuates nicotine self-administration. Our genetic discovery will inspire future drug designs targeting CHRNB2 in the brain for the treatment of nicotine addiction.

Genome-wide association study and functional validation implicates JADE1 in tauopathy.

Primary age-related tauopathy (PART) is a neurodegenerative pathology with features distinct from but also overlapping with Alzheimer disease (AD). While both exhibit Alzheimer-type temporal lobe neurofibrillary degeneration alongside amnestic cognitive impairment, PART develops independently of amyloid-ß (Aß) plaques. The pathogenesis of PART is not known, but evidence suggests an association with genes that promote tau pathology and others that protect from Aß toxicity. Here, we performed a genetic association study in an autopsy cohort of individuals with PART (n = 647) using Braak neurofibrillary tangle stage as a quantitative trait. We found some significant associations with candidate loci associated with AD (SLC24A4, MS4A6A, HS3ST1) and progressive supranuclear palsy (MAPT and EIF2AK3). Genome-wide association analysis revealed a novel significant association with a single nucleotide polymorphism on chromosome 4 (rs56405341) in a locus containing three genes, including JADE1 which was significantly upregulated in tangle-bearing neurons by single-soma RNA-seq. Immunohistochemical studies using antisera targeting JADE1 protein revealed localization within tau aggregates in autopsy brains with four microtubule-binding domain repeats (4R) isoforms and mixed 3R/4R, but not with 3R exclusively. Co-immunoprecipitation in post-mortem human PART brain tissue revealed a specific binding of JADE1 protein to four repeat tau lacking N-terminal inserts (0N4R). Finally, knockdown of the Drosophila JADE1 homolog rhinoceros (rno) enhanced tau-induced toxicity and apoptosis in vivo in a humanized 0N4R mutant tau knock-in model, as quantified by rough eye phenotype and terminal deoxynucleotidyl transferase dUTP nick end-labeling (TUNEL) in the fly brain. Together, these findings indicate that PART has a genetic architecture that partially overlaps with AD and other tauopathies and suggests a novel role for JADE1 as a modifier of neurofibrillary degeneration.

Integration of Alzheimer's disease genetics and myeloid genomics identifies disease risk regulatory elements and genes.

Genome-wide association studies (GWAS) have identified more than 40 loci associated with Alzheimer's disease (AD), but the causal variants, regulatory elements, genes and pathways remain largely unknown, impeding a mechanistic understanding of AD pathogenesis. Previously, we showed that AD risk alleles are enriched in myeloid-specific epigenomic annotations. Here, we show that they are specifically enriched in active enhancers of monocytes, macrophages and microglia. We integrated AD GWAS with myeloid epigenomic and transcriptomic datasets using analytical approaches to link myeloid enhancer activity to target gene expression regulation and AD risk modification. We identify AD risk enhancers and nominate candidate causal genes among their likely targets (including AP4E1, AP4M1, APBB3, BIN1, MS4A4A, MS4A6A, PILRA, RABEP1, SPI1, TP53INP1, and ZYX) in twenty loci. Fine-mapping of these enhancers nominates candidate functional variants that likely modify AD risk by regulating gene expression in myeloid cells. In the MS4A locus we identified a single candidate functional variant and validated it in human induced pluripotent stem cell (hiPSC)-derived microglia and brain. Taken together, this study integrates AD GWAS with multiple myeloid genomic datasets to investigate the mechanisms of AD risk alleles and nominates candidate functional variants, regulatory elements and genes that likely modulate disease susceptibility.

Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders.

Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3' untranslated regions (3'UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3'UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD.

Exome Chip Meta-analysis Fine Maps Causal Variants and Elucidates the Genetic Architecture of Rare Coding Variants in Smoking and Alcohol Use.

BACKGROUND: Smoking and alcohol use have been associated with common genetic variants in multiple loci. Rare variants within these loci hold promise in the identification of biological mechanisms in substance use. Exome arrays and genotype imputation can now efficiently genotype rare nonsynonymous and loss of function variants. Such variants are expected to have deleterious functional consequences and to contribute to disease risk. METHODS: We analyzed ∼250,000 rare variants from 16 independent studies genotyped with exome arrays and augmented this dataset with imputed data from the UK Biobank. Associations were tested for five phenotypes: cigarettes per day, pack-years, smoking initiation, age of smoking initiation, and alcoholic drinks per week. We conducted stratified heritability analyses, single-variant tests, and gene-based burden tests of nonsynonymous/loss-of-function coding variants. We performed a novel fine-mapping analysis to winnow the number of putative causal variants within associated loci. RESULTS: Meta-analytic sample sizes ranged from 152,348 to 433,216, depending on the phenotype. Rare coding variation explained 1.1% to 2.2% of phenotypic variance, reflecting 11% to 18% of the total single nucleotide polymorphism heritability of these phenotypes. We identified 171 genome-wide associated loci across all phenotypes. Fine mapping identified putative causal variants with double base-pair resolution at 24 of these loci, and between three and 10 variants for 65 loci. Twenty loci contained rare coding variants in the 95% credible intervals. CONCLUSIONS: Rare coding variation significantly contributes to the heritability of smoking and alcohol use. Fine-mapping genome-wide association study loci identifies specific variants contributing to the biological etiology of substance use behavior.

A common haplotype lowers PU.1 expression in myeloid cells and delays onset of Alzheimer's disease.

A genome-wide survival analysis of 14,406 Alzheimer's disease (AD) cases and 25,849 controls identified eight previously reported AD risk loci and 14 novel loci associated with age at onset. Linkage disequilibrium score regression of 220 cell types implicated the regulation of myeloid gene expression in AD risk. The minor allele of rs1057233 (G), within the previously reported CELF1 AD risk locus, showed association with delayed AD onset and lower expression of SPI1 in monocytes and macrophages. SPI1 encodes PU.1, a transcription factor critical for myeloid cell development and function. AD heritability was enriched within the PU.1 cistrome, implicating a myeloid PU.1 target gene network in AD. Finally, experimentally altered PU.1 levels affected the expression of mouse orthologs of many AD risk genes and the phagocytic activity of mouse microglial cells. Our results suggest that lower SPI1 expression reduces AD risk by regulating myeloid gene expression and cell function.

Are genetic variants for tobacco smoking associated with cannabis involvement?

BACKGROUND: Cannabis users are highly likely to also be tobacco cigarette smokers and a proportion of this comorbidity is attributable to shared genetic influences. Three large meta-analyses of genomewide association studies (GWAS) of tobacco smoking have identified multiple genomewide significant (p<5×10(-8)) single nucleotide polymorphisms (SNPs). We examine whether these SNPs are associated with tobacco smoking and with cannabis involvement in an independent sample. METHOD: Eleven SNPs associated with cigarettes per day (CPD), ever versus never smoking and current smoking/smoking cessation at p<5×10(-8) were selected from three published meta-analyses. Association analyses were conducted with similar tobacco smoking measures in 2716 European-American subjects from the Study of Addictions Genes and Environment (SAGE) and with lifetime and current cannabis use and DSM-IV cannabis abuse/dependence. RESULTS: Cannabis use and tobacco smoking correlated at 0.54. Rs16969968 in CHRNA5 (and its proxy, rs1051730 in CHRNA3) and rs1451240, a proxy for rs13280604 in CHRNB3, were associated with CPD after Bonferroni correction (p<0.006). rs1451240 was also associated with DSM-IV cannabis abuse/dependence. Rs6265 in BDNF was associated with smoking initiation, as in the original meta-analysis and also with lifetime cannabis use. Associations with cannabis involvement were no longer significant upon adjustment for the tobacco smoking measures. CONCLUSIONS: The modest associations between cannabis involvement and SNPs for tobacco smoking were not independent of the comorbidity between tobacco and cannabis involvement. Larger samples of individuals might be required to articulate the specific genetic architecture of cannabis involvement.

Rare missense variants in CHRNB3 and CHRNA3 are associated with risk of alcohol and cocaine dependence.

Previous findings have demonstrated that variants in nicotinic receptor genes are associated with nicotine, alcohol and cocaine dependence. Because of the substantial comorbidity, it has often been unclear whether a variant is associated with multiple substances or whether the association is actually with a single substance. To investigate the possible contribution of rare variants to the development of substance dependencies other than nicotine dependence, specifically alcohol and cocaine dependence, we undertook pooled sequencing of the coding regions and flanking sequence of CHRNA5, CHRNA3, CHRNB4, CHRNA6 and CHRNB3 in 287 African American and 1028 European American individuals from the Collaborative Study of the Genetics of Alcoholism (COGA). All members of families for whom any individual was sequenced (2504 African Americans and 7318 European Americans) were then genotyped for all variants identified by sequencing. For each gene, we then tested for association using FamSKAT. For European Americans, we find increased DSM-IV cocaine dependence symptoms (FamSKAT P = 2 × 10(-4)) and increased DSM-IV alcohol dependence symptoms (FamSKAT P = 5 × 10(-4)) among carriers of missense variants in CHRNB3. Additionally, one variant (rs149775276; H329Y) shows association with both cocaine dependence symptoms (P = 7.4 × 10(-5), ß = 2.04) and alcohol dependence symptoms (P = 2.6 × 10(-4), ß = 2.04). For African Americans, we find decreased cocaine dependence symptoms among carriers of missense variants in CHRNA3 (FamSKAT P = 0.005). Replication in an independent sample supports the role of rare variants in CHRNB3 and alcohol dependence (P = 0.006). These are the first results to implicate rare variants in CHRNB3 or CHRNA3 in risk for alcohol dependence or cocaine dependence.

A meta-analysis of two genome-wide association studies to identify novel loci for maximum number of alcoholic drinks.

Maximum number of alcoholic drinks consumed in a 24-h period (maxdrinks) is a heritable (>50 %) trait and is strongly correlated with vulnerability to excessive alcohol consumption and subsequent alcohol dependence (AD). Several genome-wide association studies (GWAS) have studied alcohol dependence, but few have concentrated on excessive alcohol consumption. We performed two GWAS using maxdrinks as an excessive alcohol consumption phenotype: one in 118 extended families (N = 2,322) selected from the Collaborative Study on the Genetics of Alcoholism (COGA), and the other in a case-control sample (N = 2,593) derived from the Study of Addiction: Genes and Environment (SAGE). The strongest association in the COGA families was detected with rs9523562 (p = 2.1 × 10(-6)) located in an intergenic region on chromosome 13q31.1; the strongest association in the SAGE dataset was with rs67666182 (p = 7.1 × 10(-7)), located in an intergenic region on chromosome 8. We also performed a meta-analysis with these two GWAS and demonstrated evidence of association in both datasets for the LMO1 (p = 7.2 × 10(-7)) and PLCL1 genes (p = 4.1 × 10(-6)) with maxdrinks. A variant in AUTS2 and variants in INADL, C15orf32 and HIP1 that were associated with measures of alcohol consumption in a meta-analysis of GWAS studies and a GWAS of alcohol consumption factor score also showed nominal association in the current meta-analysis. The present study has identified several loci that warrant further examination in independent samples. Among the top SNPs in each of the dataset (p ≤ 10(-4)) far more showed the same direction of effect in the other dataset than would be expected by chance (p = 2 × 10(-3), 3 × 10(-6)), suggesting that there are true signals among these top SNPs, even though no SNP reached genome-wide levels of significance.

The genetics of substance dependence.

A large segment of the population suffers from addiction to alcohol, smoking, or illicit drugs. Not only do substance abuse and addiction pose a threat to health, but the consequences of addiction also impose a social and economic burden on families, communities, and nations. Genome-wide linkage and association studies have been used for addiction research with varying degrees of success. The most well-established genetic factors associated with alcohol dependence are in the genes encoding alcohol dehydrogenase (ADH), which oxidizes alcohol to acetaldehyde, and aldehyde dehydrogenase (ALDH2), which oxidizes acetaldehyde to acetate. Recently emerging genetic studies have linked variants in the genes encoding the α3, α5, and ß4 nicotinic acetylcholine receptor subunits to smoking risk. However, the influence of these well-established genetic variants accounts for only a small portion of the heritability of alcohol and nicotine addiction, and it is likely that there are both common and rare risk variants yet to be identified. Newly developed DNA sequencing technologies could potentially advance the detection of rare variants with a larger impact on addiction risk.

Variants located upstream of CHRNB4 on chromosome 15q25.1 are associated with age at onset of daily smoking and habitual smoking.

Several genome-wide association and candidate gene studies have linked chromosome 15q24-q25.1 (a region including the CHRNA5-CHRNA3-CHRNB4 gene cluster) with alcohol dependence, nicotine dependence and smoking-related illnesses such as lung cancer and chronic obstructive pulmonary disease. To further examine the impact of these genes on the development of substance use disorders, we tested whether variants within and flanking the CHRNA5-CHRNA3-CHRNB4 gene cluster affect the transition to daily smoking (individuals who smoked cigarettes 4 or more days per week) in a cross sectional sample of adolescents and young adults from the COGA (Collaborative Study of the Genetics of Alcoholism) families. Subjects were recruited from families affected with alcoholism (either as a first or second degree relative) and the comparison families. Participants completed the SSAGA interview, a comprehensive assessment of alcohol and other substance use and related behaviors. Using the Quantitative trait disequilibrium test (QTDT) significant association was detected between age at onset of daily smoking and variants located upstream of CHRNB4. Multivariate analysis using a Cox proportional hazards model further revealed that these variants significantly predict the age at onset of habitual smoking among daily smokers. These variants were not in high linkage disequilibrium (0.28