Jenni Hällfors

A Genome-Wide Association Study of a Biomarker of Nicotine Metabolism

Individuals with fast nicotine metabolism typically smoke more and thus have a greater risk for smoking-induced diseases. Further, the efficacy of smoking cessation pharmacotherapy is dependent on the rate of nicotine metabolism. Our objective was to use nicotine metabolite ratio (NMR), an established biomarker of nicotine metabolism rate, in a genome-wide association study (GWAS) to identify novel genetic variants influencing nicotine metabolism. A heritability estimate of 0.81 (95% CI 0.70–0.88) was obtained for NMR using monozygotic and dizygotic twins of the FinnTwin cohort. We performed a GWAS in cotinine-verified current smokers of three Finnish cohorts (FinnTwin, Young Finns Study, FINRISK2007), followed by a meta-analysis of 1518 subjects, and annotated the genome-wide significant SNPs with methylation quantitative loci (meQTL) analyses. We detected association on 19q13 with 719 SNPs exceeding genome-wide significance within a 4.2 Mb region. The strongest evidence for association emerged for CYP2A6 (min p = 5.77E-86, in intron 4), the main metabolic enzyme for nicotine. Other interesting genes with genome-wide significant signals included CYP2B6, CYP2A7, EGLN2, and NUMBL. Conditional analyses revealed three independent signals on 19q13, all located within or in the immediate vicinity of CYP2A6. A genetic risk score constructed using the independent signals showed association with smoking quantity (p = 0.0019) in two independent Finnish samples. Our meQTL results showed that methylation values of 16 CpG sites within the region are affected by genotypes of the genome-wide significant SNPs, and according to causal inference test, for some of the SNPs the effect on NMR is mediated through methylation. To our knowledge, this is the first GWAS on NMR. Our results enclose three independent novel signals on 19q13.2. The detected CYP2A6 variants explain a strikingly large fraction of variance (up to 31%) in NMR in these study samples. Further, we provide evidence for plausible epigenetic mechanisms influencing NMR.

Genetics and smoking

Regular smoking is the major risk factor for cardiovascular disease and cancers, and thus is one of the most preventable causes of morbidity and mortality worldwide. Intake of nicotine, its central nervous system effects, and its metabolism are regulated by biological pathways; some of these are well known, but others are not. Genetic studies offer a method for developing insights into the genes contributing to those pathways. In recent years, large genome-wide association study (GWAS) meta-analyses have consistently revealed that the strongest genetic contribution to smoking-related traits comes from variation in the nicotinic receptor subunit genes. Many other genes, including those coding for enzymes involved in nicotine metabolism, also have been implicated. However, the proportion of phenotypic variance explained by the identified genetic variants is very modest. This review intends to cover progress made in genetics and genetic epidemiology of smoking behavior in recent years, and focuses on studies revealing the nicotinic receptor gene cluster on chromosome 15q25. Evidence supporting the involvement of a novel pathway in the shared pathophysiology of nicotine dependence and schizophrenia is also briefly reviewed. A summary of the current knowledge on gene–environment interactions involved in smoking behavior is included.

Chromosome 20 Shows Linkage With DSM-IV Nicotine Dependence in Finnish Adult Smokers

INTRODUCTION Chromosome 20 has previously been associated with nicotine dependence (ND) and smoking cessation. Our aim was to replicate and extend these findings. METHODS First, a total of 759 subjects belonging to 206 Finnish families were genotyped with 18 microsatellite markers residing on chromosome 20, in order to replicate previous linkage findings. Then, the replication data were combined to an existing whole-genome linkage data resulting in a total of 1,302 genotyped subjects from 357 families. ND diagnosed by DSM-IV criteria, the Fagerström Test for Nicotine Dependence (FTND) score, and the lifetime maximum number of cigarettes smoked within a 24-hr period (MaxCigs24) were used as phenotypes in the nonparametric linkage analyses. RESULTS We replicated previously reported linkage to DSM-IV ND, with a maximum logarithm of odd (LOD) score of 3.8 on 20p11, with females contributing more (maximum LOD [MLOD] score 3.4 on 20q11) than males (MLOD score 2.6 on 20p11). With the combined sample, a suggestive LOD score of 2.3 was observed for DSM-IV ND on 20p11. Sex-specific analyses revealed that the signal was driven by females with a maximum LOD score of 3.3 (on 20q11) versus LOD score of 1.3 in males (on 20q13) in the combined sample. No significant linkage signals were obtained for FTND or MaxCigs24. CONCLUSIONS Our results provide further evidence that chromosome 20 harbors genetic variants influencing ND in adult smokers.

Metabolomic signature of angiopoietin-like protein 3 deficiency in fasting and postprandial state

Objective Loss-of-function variants in the angiopoietin-like 3 gene (ANGPTL3) have been associated with low levels of plasma lipoproteins and decreased coronary artery disease risk. We aimed to determine detailed metabolic effects of genetically-induced ANGPTL3 deficiency in fasting and postprandial state. Approach and Results We studied individuals carrying S17X loss-of-function mutation in ANGPTL3 (6 homozygous and 32 heterozygous carriers) and 38 noncarriers. Nuclear magnetic resonance metabolomics was used to quantify 225 circulating metabolic measures. We compared metabolic differences between loss-of-function carriers and noncarriers in fasting state and after a high fat meal. In fasting, ANGPTL3 deficiency was characterized by similar extent of reductions in low-density lipoprotein cholesterol (0.74 SD-units lower concentration per loss-of-function allele [95%CI 0.42–1.06]) as observed for many triglyceride-rich lipoprotein measures, including very-low-density lipoprotein cholesterol (0.75 [0.45–1.05]). Within most lipoprotein subclasses, absolute levels of cholesterol were decreased more than triglycerides, resulting in the relative proportion of cholesterol being reduced within triglyceride-rich lipoproteins and their remnants. Further, beta-hydroxybutyrate was elevated (0.55 [0.21–0.89]). Homozygous ANGPTL3 loss-of-function carriers showed essentially no postprandial increase in triglyceride-rich lipoproteins and fatty acids, without evidence for adverse compensatory metabolic effects. Conclusions In addition to overall triglyceride and low-density lipoprotein cholesterol lowering effects, ANGPTL3 deficiency results in reduction of cholesterol proportion within triglyceride-rich lipoproteins and their remnants. Further, ANGPTL3 loss-of-function carriers had elevated ketone body production, suggesting enhanced hepatic fatty acid beta-oxidation. The detailed metabolic profile in human knockouts of ANGPTL3 reinforces inactivation of ANGPTL3 as a promising therapeutic target for decreasing cardiovascular risk. HIGHLIGHTS ANGPTL3 deficiency results in similar reductions in LDL cholesterol and many triglyceride-rich lipoprotein lipids measures, such as VLDL cholesterol, with no evidence of substantial adverse effects on the comprehensive panel of circulating metabolite biomarkers tested here. In particular, ANGPTL3 deficiency results in reduction of cholesterol content in triglyceride-rich lipoproteins and their remnants, which have been highlighted as risk factor for cardiovascular disease independently of LDL levels. Homozygous ANGPTL3 loss-of-function carriers show essentially no postprandial increase in triglyceride-rich lipoproteins and fatty acids in response to a fat challenge, and display consistently elevated postprandial levels of ketone bodies and lactate when compared to noncarriers, suggesting enhanced hepatic fatty acid beta-oxidation.

Is Brain Derived Neurotrophic Factor (Bdnf) Associated With Smoking Initiation? Replication Using a Large Finnish Population Sample

Introduction Brain derived neurotrophic factor (BDNF) is a growth factor in the central nervous system. There is evidence for the involvement of BDNF in addictions and mental disorders. We aimed to replicate the earlier reported association of a functional genetic variant of BDNF with smoking initiation (SI) using a large population-based sample, and to test whether the association is independent of depression. Methods Our sample was drawn from the Finnish population-based FINRISK Surveys conducted in 1992, 1997, 2002 and 2007. We had non-missing data on the genotype BDNF Val66Met (G/A) variant (rs6265) and self-reported never (n=10,619) versus ever (n=16,028) smoking among 26,647 adults aged 25-74 years. The association between BDNF Val66Met and SI was modeled using logistic regression adjusted for age and sex, and in secondary analyses also for depression. Depression was defined as self-reported depression diagnosed or treated by physician during the past year. Results The sex and age adjusted analysis confirmed that the major (Val) allele increased the risk of being a lifetime ever smoker (per allele OR=1.07; 95%CI 1.01, 1.12; p=0.01). When depression, which itself was significantly associated with SI (OR=1.58; 95%CI 1.37, 1.82; p<0.001), was added to the model, the association of the gene with SI remained significant (per allele OR=1.06; 95%CI 1.01, 1.12; p=0.01). Exclusion of depressed individuals did not change the results (OR=1.06; 95%CI 1.01, 1.12; p=0.02). Conclusions In a Finnish population sample we replicated the earlier reported association of BDNF Val66Met with smoking initiation. Our data further suggest that this association is independent of depression.

Genome-wide association study in Finnish twins highlights the connection between nicotine addiction and neurotrophin signaling pathway: GWAS of nicotine addiction

The heritability of nicotine dependence based on family studies is substantial. Nevertheless, knowledge of the underlying genetic architecture remains meager. Our aim was to identify novel genetic variants responsible for interindividual differences in smoking behavior. We performed a genome‐wide association study on 1715 ever smokers ascertained from the population‐based Finnish Twin Cohort enriched for heavy smoking. Data imputation used the 1000 Genomes Phase I reference panel together with a whole genome sequence‐based Finnish reference panel. We analyzed three measures of nicotine addiction—smoking quantity, nicotine dependence and nicotine withdrawal. We annotated all genome‐wide significant SNPs for their functional potential. First, we detected genome‐wide significant association on 16p12 with smoking quantity (P = 8.5 × 10−9), near CLEC19A. The lead‐SNP stands 22 kb from a binding site for NF‐κB transcription factors, which play a role in the neurotrophin signaling pathway. However, the signal was not replicated in an independent Finnish population‐based sample, FINRISK (n = 6763). Second, nicotine withdrawal showed association on 2q21 in an intron of TMEM163 (P = 2.1 × 10−9), and on 11p15 (P = 6.6 × 10−8) in an intron of AP2A2, and P = 4.2 × 10−7 for a missense variant in MUC6, both involved in the neurotrophin signaling pathway). Third, association was detected on 3p22.3 for maximum number of cigarettes smoked per day (P = 3.1 × 10−8) near STAC. Associating CLEC19A and TMEM163 SNPs were annotated to influence gene expression or methylation. The neurotrophin signaling pathway has previously been associated with smoking behavior. Our findings further support the role in nicotine addiction.