Genome-wide association study across European and African American ancestries identifies a SNP in DNMT3B contributing to nicotine dependence.

Cigarette smoking is a leading cause of preventable mortality worldwide. Nicotine dependence, which reduces the likelihood of quitting smoking, is a heritable trait with firmly established associations with sequence variants in nicotine acetylcholine receptor genes and at other loci. To search for additional loci, we conducted a genome-wide association study (GWAS) meta-analysis of nicotine dependence, totaling 38,602 smokers (28,677 Europeans/European Americans and 9925 African Americans) across 15 studies. In this largest-ever GWAS meta-analysis for nicotine dependence and the largest-ever cross-ancestry GWAS meta-analysis for any smoking phenotype, we reconfirmed the well-known CHRNA5-CHRNA3-CHRNB4 genes and further yielded a novel association in the DNA methyltransferase gene DNMT3B. The intronic DNMT3B rs910083-C allele (frequency=44-77%) was associated with increased risk of nicotine dependence at P=3.7 × 10-8 (odds ratio (OR)=1.06 and 95% confidence interval (CI)=1.04-1.07 for severe vs mild dependence). The association was independently confirmed in the UK Biobank (N=48,931) using heavy vs never smoking as a proxy phenotype (P=3.6 × 10-4, OR=1.05, and 95% CI=1.02-1.08). Rs910083-C is also associated with increased risk of squamous cell lung carcinoma in the International Lung Cancer Consortium (N=60,586, meta-analysis P=0.0095, OR=1.05, and 95% CI=1.01-1.09). Moreover, rs910083-C was implicated as a cis-methylation quantitative trait locus (QTL) variant associated with higher DNMT3B methylation in fetal brain (N=166, P=2.3 × 10-26) and a cis-expression QTL variant associated with higher DNMT3B expression in adult cerebellum from the Genotype-Tissue Expression project (N=103, P=3.0 × 10-6) and the independent Brain eQTL Almanac (N=134, P=0.028). This novel DNMT3B cis-acting QTL variant highlights the importance of genetically influenced regulation in brain on the risks of nicotine dependence, heavy smoking and consequent lung cancer.

Neuregulin signaling pathway in smoking behavior.

Understanding molecular processes that link comorbid traits such as addictions and mental disorders can provide novel therapeutic targets. Neuregulin signaling pathway (NSP) has previously been implicated in schizophrenia, a neurodevelopmental disorder with high comorbidity to smoking. Using a Finnish twin family sample, we have previously detected association between nicotine dependence and ERBB4 (a neuregulin receptor), and linkage for smoking initiation at the ERBB4 locus on 2q33. Further, Neuregulin3 has recently been shown to associate with nicotine withdrawal in a behavioral mouse model. In this study, we scrutinized association and linkage between 15 036 common, low frequency and rare genetic variants in 10 NSP genes and phenotypes encompassing smoking and alcohol use. Using the Finnish twin family sample (N=1998 from 740 families), we detected 66 variants (representing 23 LD blocks) significantly associated (false discovery rate P<0.05) with smoking initiation, nicotine dependence and nicotine withdrawal. We comprehensively annotated the associated variants using expression (eQTL) and methylation quantitative trait loci (meQTL) analyses in a Finnish population sample. Among the 66 variants, we identified 25 eQTLs (in NRG1 and ERBB4), 22 meQTLs (in NRG3, ERBB4 and PSENEN), a missense variant in NRG1 (rs113317778) and a splicing disruption variant in ERBB4 (rs13385826). Majority of the QTLs in blood were replicated in silico using publicly available databases, with additional QTLs observed in brain. In conclusion, our results support the involvement of NSP in smoking behavior but not in alcohol use and abuse, and disclose functional potential for 56 of the 66 associated single-nucleotide polymorphism.

Genome-wide association study of lifetime cannabis use based on a large meta-analytic sample of 32 330 subjects from the International Cannabis Consortium.

Cannabis is the most widely produced and consumed illicit psychoactive substance worldwide. Occasional cannabis use can progress to frequent use, abuse and dependence with all known adverse physical, psychological and social consequences. Individual differences in cannabis initiation are heritable (40-48%). The International Cannabis Consortium was established with the aim to identify genetic risk variants of cannabis use. We conducted a meta-analysis of genome-wide association data of 13 cohorts (N=32 330) and four replication samples (N=5627). In addition, we performed a gene-based test of association, estimated single-nucleotide polymorphism (SNP)-based heritability and explored the genetic correlation between lifetime cannabis use and cigarette use using LD score regression. No individual SNPs reached genome-wide significance. Nonetheless, gene-based tests identified four genes significantly associated with lifetime cannabis use: NCAM1, CADM2, SCOC and KCNT2. Previous studies reported associations of NCAM1 with cigarette smoking and other substance use, and those of CADM2 with body mass index, processing speed and autism disorders, which are phenotypes previously reported to be associated with cannabis use. Furthermore, we showed that, combined across the genome, all common SNPs explained 13-20% (P<0.001) of the liability of lifetime cannabis use. Finally, there was a strong genetic correlation (rg=0.83; P=1.85 × 10(-8)) between lifetime cannabis use and lifetime cigarette smoking implying that the SNP effect sizes of the two traits are highly correlated. This is the largest meta-analysis of cannabis GWA studies to date, revealing important new insights into the genetic pathways of lifetime cannabis use. Future functional studies should explore the impact of the identified genes on the biological mechanisms of cannabis use.

Genome-wide meta-analysis reveals common splice site acceptor variant in CHRNA4 associated with nicotine dependence.

We conducted a 1000 Genomes-imputed genome-wide association study (GWAS) meta-analysis for nicotine dependence, defined by the Fagerström Test for Nicotine Dependence in 17 074 ever smokers from five European-ancestry samples. We followed up novel variants in 7469 ever smokers from five independent European-ancestry samples. We identified genome-wide significant association in the alpha-4 nicotinic receptor subunit (CHRNA4) gene on chromosome 20q13: lowest P=8.0 × 10(-9) across all the samples for rs2273500-C (frequency=0.15; odds ratio=1.12 and 95% confidence interval=1.08-1.17 for severe vs mild dependence). rs2273500-C, a splice site acceptor variant resulting in an alternate CHRNA4 transcript predicted to be targeted for nonsense-mediated decay, was associated with decreased CHRNA4 expression in physiologically normal human brains (lowest P=7.3 × 10(-4)). Importantly, rs2273500-C was associated with increased lung cancer risk (N=28 998, odds ratio=1.06 and 95% confidence interval=1.00-1.12), likely through its effect on smoking, as rs2273500-C was no longer associated with lung cancer after adjustment for smoking. Using criteria for smoking behavior that encompass more than the single 'cigarettes per day' item, we identified a common CHRNA4 variant with important regulatory properties that contributes to nicotine dependence and smoking-related consequences.

Genome-wide association study on detailed profiles of smoking behavior and nicotine dependence in a twin sample.

Smoking is a major risk factor for several somatic diseases and is also emerging as a causal factor for neuropsychiatric disorders. Genome-wide association (GWA) and candidate gene studies for smoking behavior and nicotine dependence (ND) have disclosed too few predisposing variants to account for the high estimated heritability. Previous large-scale GWA studies have had very limited phenotypic definitions of relevance to smoking-related behavior, which has likely impeded the discovery of genetic effects. We performed GWA analyses on 1114 adult twins ascertained for ever smoking from the population-based Finnish Twin Cohort study. The availability of 17 smoking-related phenotypes allowed us to comprehensively portray the dimensions of smoking behavior, clustered into the domains of smoking initiation, amount smoked and ND. Our results highlight a locus on 16p12.3, with several single-nucleotide polymorphisms (SNPs) in the vicinity of CLEC19A showing association (P<1 × 10(-6)) with smoking quantity. Interestingly, CLEC19A is located close to a previously reported attention-deficit hyperactivity disorder (ADHD) linkage locus and an evident link between ADHD and smoking has been established. Intriguing preliminary association (P<1 × 10(-5)) was detected between DSM-IV (Diagnostic and Statistical Manual of Mental Disorders, 4th edition) ND diagnosis and several SNPs in ERBB4, coding for a Neuregulin receptor, on 2q33. The association between ERBB4 and DSM-IV ND diagnosis was replicated in an independent Australian sample. Recently, a significant increase in ErbB4 and Neuregulin 3 (Nrg3) expression was revealed following chronic nicotine exposure and withdrawal in mice and an association between NRG3 SNPs and smoking cessation success was detected in a clinical trial. ERBB4 has previously been associated with schizophrenia; further, it is located within an established schizophrenia linkage locus and within a linkage locus for a smoker phenotype identified in this sample. In conclusion, we disclose novel tentative evidence for the involvement of ERBB4 in ND, suggesting the involvement of the Neuregulin/ErbB signalling pathway in addictions and providing a plausible link between the high co-morbidity of schizophrenia and ND.

DISC1 association, heterogeneity and interplay in schizophrenia and bipolar disorder.

Disrupted in schizophrenia 1 (DISC1) has been associated with risk of schizophrenia, schizoaffective disorder, bipolar disorder, major depression, autism and Asperger syndrome, but apart from in the original translocation family, true causal variants have yet to be confirmed. Here we report a harmonized association study for DISC1 in European cohorts of schizophrenia and bipolar disorder. We identify regions of significant association, demonstrate allele frequency heterogeneity and provide preliminary evidence for modifying interplay between variants. Whereas no associations survived permutation analysis in the combined data set, significant corrected associations were observed for bipolar disorder at rs1538979 in the Finnish cohorts (uncorrected P=0.00020; corrected P=0.016; odds ratio=2.73+/-95% confidence interval (CI) 1.42-5.27) and at rs821577 in the London cohort (uncorrected P=0.00070; corrected P=0.040; odds ratio=1.64+/-95% CI 1.23-2.19). The rs821577 single nucleotide polymorphism (SNP) showed evidence for increased risk within the combined European cohorts (odds ratio=1.27+/-95% CI 1.07-1.51), even though significant corrected association was not detected (uncorrected P=0.0058; corrected P=0.28). After conditioning the European data set on the two risk alleles, reanalysis revealed a third significant SNP association (uncorrected P=0.00050; corrected P=0.025). This SNP showed evidence for interplay, either increasing or decreasing risk, dependent upon the presence or absence of rs1538979 or rs821577. These findings provide further support for the role of DISC1 in psychiatric illness and demonstrate the presence of locus heterogeneity, with the effect that clinically relevant genetic variants may go undetected by standard analysis of combined cohorts.

Replication of linkage on chromosome 7q22 and association of the regional Reelin gene with working memory in schizophrenia families.

Schizophrenia is a common and complex mental disorder. Hereditary factors are important for its etiology, but despite linkage signals reported to several chromosomal regions in different populations, final identification of predisposing genes has remained a challenge. Utilizing a large family-based schizophrenia study sample from Finland, we have identified several linked loci: 1q32.2-q42, 2q, 4q31, 5q and 7q22. In this study, an independent sample of 352 nuclear schizophrenia families (n=1626) allowed replication of linkage on 7q21-32. In a sample of 245 nuclear families (n=1074) originating from the same geographical region as the families revealing the linkage, SNP and microsatellite association analyses of the four regional candidate genes, GRM3, RELN, SEMA3A and VGF, revealed no significant association to the clinical diagnosis of schizophrenia. Instead, quantifiable trait component analyses with neuropsychological endophenotypes available from 186 nuclear families (n=861) of the sample showed significant association to RELN variants for traits related to verbal (P=0.000003) and visual working memory (P=0.002), memory (P=0.002) and executive functioning (P=0.002). Trait-associated allele-positive subjects scored lower in the tests measuring working memory (P=0.0004-0.0000000004), memory (P=0.02-0.0001) and executive functioning (P=0.001). Our findings suggest that allelic variants of RELN contribute to the endophenotypes of schizophrenia.

Linkage of nicotine dependence and smoking behavior on 10q, 7q and 11p in twins with homogeneous genetic background.

The significant worldwide health burden introduced by tobacco smoking highlights the importance of studying the genetic determinants of smoking behavior and the key factor sustaining compulsive smoking, that is, nicotine dependence (ND). We have here addressed the genetic background of smoking in a special study sample of twins, harmonized for early life events and specifically ascertained for smoking from the nationwide twin cohort of the genetically unique population of Finland. The twins and their families were carefully examined for extensive phenotype profiles and a genome-wide scan was performed to identify loci behind the smoking status, ND and the comorbid phenotype of ND and alcohol use in 505 individuals from 153 families. We replicated previous linkage findings on 10q (max logarithm of the odds (LOD) 3.12) for a smoker phenotype, and on 7q and 11p (max LOD 2.50, and 2.25, respectively) for the ND phenotype. The loci linked for ND also showed evidence for linkage for the comorbid phenotype. Our study provides confirmatory evidence for the involvement of these genome regions in the genetic etiology of smoking behavior and ND and for the first time associates drinking and smoking to a shared locus on 10q.

Frequent loss of SMAD4/DPC4 protein in colorectal cancers.

BACKGROUND AND AIMS: Loss of DNA sequences from chromosome 18q21 is a major genetic change in colorectal tumorigenesis. Multiple genes have been identified in this area. One of these, DPC4 (deleted in pancreatic cancer 4, also known as SMAD4), is mutated in a minor subset of colorectal carcinomas as well as in germlines of humans predisposed to colon tumours. PATIENTS AND METHODS: The involvement of SMAD4 in sporadic colorectal neoplasia was evaluated by immunohistochemistry in 53 unselected cases and 27 cases displaying microsatellite instability. RESULTS: SMAD4 expression was absent in 20 of 53 (38%) unselected colorectal carcinomas, and reduced in another 15 (28%) cases. However, 26 of 27 cancers displaying microsatellite instability and TGF-betaIIR mutations were positive for SMAD4 immunostaining. CONCLUSIONS: Loss of SMAD4 expression may play a more prominent role in colon cancer than anticipated based on genetic evidence, but not in mutator phenotype tumours.

A germline E-cadherin mutation in a family with gastric and colon cancer.

Inactivating mutations have been found in the cell-cell adhesion molecule E-cadherin (CDH1), which acts as a tumor suppressor gene in different kinds of cancers, e.g. primarily diffuse gastric cancer and lobular breast cancer. In this study, we screened for germline alterations in familial gastric and colon cancer cases. In total, 20 gastric and 18 colon cancer patients with both familial gastric and colon cancer were tested for germline E-cadherin alterations by using PCR/SSCP, specific restriction digestion test and sequencing. No pathogenic mutations were identified in the gastric cancer patients. In two colon cancer patients, a missense mutation in exon 12, codon 592 (Ala592Thr) was found. This alteration segregated with diffuse gastric cancer and colon cancer in one of the families. The prevalence of this alteration in the general population and colon cancer cases was almost the same. However, the fact that this alteration (Ala592Thr) segregated with colon cancer and diffuse gastric cancer in one big family, suggests that this E-cadherin missense alteration, beside predisposing to diffuse gastric cancer, also may play a role in colorectal carcinogenesis.

Extensive somatic microsatellite mutations in normal human tissue.

Microsatellite (MS) instability occurs in tumors with DNA mismatch repair (MMR) deficiencies but is typically absent in adjacent normal tissue. However, MS mutations have been observed in normal tissues from rare individuals with congenital MMR deficiencies. Autopsy tissues from a 4-year-old with congenital MMR deficiency (MLH1-/-) were examined for MS mutations. Insertions and deletions were observed in CA-repeat MS loci. Approximately 0.26 to 1.4 mutations per MS locus per cell were estimated to be present in normal heart, lymph node, kidney, and bladder epithelium. These findings illustrate that phenotypically normal MMR-deficient cells commonly accumulate MS mutations. Loss of MMR and the accumulation of some MS mutations may occur early in MMR-deficient tumor progression, even before a gatekeeper mutation.

Microsatellite marker analysis in screening for hereditary nonpolyposis colorectal cancer (HNPCC).

Hereditary nonpolyposis colorectal cancer (HNPCC) is an autosomal dominant cancer predisposition syndrome caused by germ-line mutations in DNA mismatch repair genes. It is relevant to identify HNPCC patients because colonoscopic screening of individuals with HNPCC mutations reduces cancer morbidity and mortality. Microsatellite instability (MSI) is characteristic of HNPCC tumors. A panel of five markers (BAT25, BAT26, D2S123, D5S346, and D17S250, the so-called Bethesda markers) has been proposed for screening for MSI. To test a hypothesis that the use of BAT26 alone is feasible in screening for MLH1/MSH2 mutation-positive HNPCC patients, we compared the MSI results of 494 colorectal cancer patients obtained using BAT26 with results obtained using the Bethesda markers. BAT26 was able to identify all 27 mutation-positive individuals in this series. The marker failed to identify 2 high MSI tumors and 20 low MSI tumors, all of which expressed MLH1, MSH2, and MSH6 when scrutinized by immunohistochemistry.

PPP2R1B gene in chronic lymphocytic leukemias and mantle cell lymphomas.

Deletion of chromosome bands 11q22-q23 is one of the most common structural chromosome alterations in chronic lymphocytic leukemia (CLL) and mantle cell lymphoma (MCL). The PPP2R1B gene is located very close to the minimal common deletion region of 11q22-q23 in CLL and MCL. Recently, the PPP2R1B gene was found to be mutated in human lung and colon cancers. To evaluate the role of the PPP2R1B gene in the pathogenesis of CLL and MCL, we performed RT-PCR analysis and cDNA sequencing on 10 CLL RNA samples and SSCP analysis on 26 CLL and 37 MCL genomic DNA samples. A deletion of exon 3 was found in one CLL sample. No mutation was detected in the SSCP analysis. To exclude the possibility of large genomic deletions we performed Southern blotting analysis. One MCL sample showed abnormal bands. Our results do not suggest that the PPP2R1B gene has a major pathogenic role in CLL and MCL.

Germline and somatic mutation analysis of MLH3 in MSI-positive colorectal cancer.

Microsatellite instability (MSI) is characteristic of hereditary nonpolyposis colorectal cancer, and occurs in a subset (10 to 15%) of unselected colorectal cancer cases. In hereditary nonpolyposis colorectal cancer, MSI is caused by defects in five mismatch repair genes, and in sporadic cases the main cause seems to be somatic MLH1 promoter methylation. Most likely additional hereditary nonpolyposis colorectal cancer genes remain to be discovered. Genes with simple repeats in their coding region are often targets for deletions in MSI-positive tumors. Several genes (TGFbeta RII, IGFIIR, MSH3, MSH6, BAX, MBD4) with significance in tumorigenesis harbor repeats in their coding regions and are often somatically inactivated because of deletions causing frameshifts. Recently, a novel human mismatch repair gene, MLH3, was cloned and shown to be involved in mammalian mismatch repair. To evaluate the possible role of MLH3 in hereditary cancer, we performed germline single-strand conformation polymorphism-analysis for 52 patients displaying features of inherited colorectal cancer. Forty-six of these had been diagnosed with MSI-positive tumors. No germline mutations were found. Similar to MSH3 and MSH6, MLH3 harbors mononucleotide repeats, ie, (A(6))-(A(9)), in its coding region, which makes it a putative target for somatic mutations in MSI-positive tumors. To evaluate its somatic inactivation we performed a deletion search focusing on eight exonic MLH3 mononucleotide repeats in a series of 93 MSI-positive tumors. Somatic deletions were found in 8.6% of the samples, a frequency similar to one detected in neutral noncoding mononucleotide repeats. No evidence of involvement of MLH3 in MSI tumorigenesis was obtained.

The role of hypermethylation of the hMLH1 promoter region in HNPCC versus MSI+ sporadic colorectal cancers.

INTRODUCTION: Hypermethylation of the promoter region of the hMLH1 gene is associated with absent expression of MLH1 protein in sporadic colorectal cancers with microsatellite instability (MSI+), and it has been proposed that methylation may be a mechanism of inactivation in Knudson's hypothesis. The incidence of hypermethylation of the hMLH1 promoter in hereditary non-polyposis colorectal cancer (HNPCC) versus MSI+ sporadic colorectal cancer was investigated and compared. METHODS: DNA was available from 10 HNPCC colorectal cancers (median age 58 years, range 39-67) with germline mutations in hMLH1 and 10 MSI+ sporadic colorectal cancers (mean age 79 years, range 41-85). MSI was determined by amplification of BAT26 and TGF-beta RII. The methylation status of the hMLH1 promoter was studied by the polymerase chain reaction (PCR) based HpaII restriction enzyme assay technique. Evidence of allelic loss at hMLH1 was searched for in the HNPCC colorectal cancers. RESULTS: All cases were confirmed to be MSI+. The promoter region of hMLH1 was hypermethylated in seven of 10 MSI+ sporadic cancers versus 0 of 10 HNPCC cancers (p<0.002). Evidence of loss of heterozygosity at hMLH1 was observed in eight of the 10 HNPCC colorectal cancers. CONCLUSION: While mutations and allelic loss are responsible for the MSI+ phenotype in HNPCC cancers, the majority of MSI+ sporadic cancers are hypermethylated in the promoter region of hMLH1. These data further support our argument that tumours from HNPCC patients, which almost always acquire a raised mutation rate, mostly follow a different pathway from MSI+ sporadic tumours.

Population-based molecular detection of hereditary nonpolyposis colorectal cancer.

PURPOSE: Cancer morbidity and mortality can be dramatically reduced by colonoscopic screening of individuals with the hereditary nonpolyposis colorectal cancer (HNPCC) syndrome, creating a need to identify HNPCC. We studied how HNPCC identification should be carried out on a large scale in a sensitive and efficient manner. PATIENTS AND METHODS: Colorectal cancer specimens from consecutive newly diagnosed patients were studied for microsatellite instability (MSI). Germline mutations in the MLH1 and MSH2 genes were searched for in MSI(+) individuals. RESULTS: Among 535 colorectal cancer patients, 66 (12%) were MSI(+). Among these, 18 (3.4% of the total) had disease-causing germline mutations in MLH1 or MSH2. Among these 18 patients, five were less than 50 years old, seven had a previous or synchronous colorectal or endometrial cancer, and 15 had at least one first-degree relative with colorectal or endometrial cancer. Notably, 17 (94%) of 18 patients had at least one of these three features, which were present in 22% of all 535 patients. Combining these data with a previous study of 509 patients, mutation-positive HNPCC accounts for 28 (2.7%) of 1,044 cases of colorectal cancer, predicting a greater than one in 740 incidence of mutation-positive individuals in this population. CONCLUSION: Large-scale molecular screening for HNPCC can be done by the described two-stage procedure of MSI determination followed by mutation analysis. Efficiency can be greatly improved by using three high-risk features to select 22% of all patients for MSI analysis, whereby only 6% need to have mutation analysis. Sensitivity is only slightly impaired by this procedure.

No evidence of Peutz-Jeghers syndrome gene LKB1 involvement in left-sided colorectal carcinomas.

LKB1 serine/threonine kinase is a gene for Peutz-Jeghers cancer predisposition syndrome. Most studies have detected a low frequency of LKB1 defects in sporadic cancer. A notable exception is a recent report describing frequent, mostly missense type, LKB1 mutations in Korean distal colorectal tumors. To clarify the role of LKB1 in colon cancer, we scrutinized 50 left-sided Korean and Finnish specimens. No somatic mutations were found. The seven Korean somatic missense mutations reported previously were functionally analyzed, and five were found not to alter LKB1 kinase activity. One of these changes was found to be a germ-line polymorphism. LKB1 involvement in distal colorectal cancer is not common.

Microsatellite instability in adenomas as a marker for hereditary nonpolyposis colorectal cancer.

Hereditary nonpolyposis colorectal cancer (HNPCC) is the most common of the well-defined colorectal cancer syndromes, accounting for at least 2% of the total colorectal cancer burden and carrying a greater than 80% lifetime risk of cancer. Significant reduction in cancer morbidity and mortality can be accomplished by appropriate clinical cancer screening of HNPCC patients with mutations in mismatch repair (MMR) genes. Thus, it is desirable to identify individuals who are mutation-positive. In individuals with cancer, mutation detection can be accomplished relatively efficiently by germline mutation analysis of individuals whose cancers show microsatellite instability (MSI). This study was designed to assess the feasibility of screening colorectal adenoma patients for HNPCC in the same manner. Among 378 adenoma patients, six (1.6%) had at least one MSI adenoma. Five out of the six patients (83%) had a germline MMR gene mutation. We conclude that MSI analysis is a useful method of prescreening colorectal adenoma patients for HNPCC.