Romain Icick

Benoît Forget

Isabelle Cloëz-Tayarani

Stéphanie Pons

Uwe Maskos

Morgane Besson
email: morgane.besson@pasteur.fr

Isabelle Tayarani

Genetic susceptibility to nicotine addiction: Advances and shortcomings in our understanding of the CHRNA5/A3/B4 gene cluster contribution

Keywords: nicotine addiction, nicotinic receptor, human genetics, polymorphisms, animal models nicotine addiction, nicotinic receptor, human genetics, polymorphisms, animal models

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Introduction

Nicotine addiction

Tobacco smoking is still responsible for more than 8 million deaths each year (WHO, https://www.who.int/news-room/fact-sheets/detail/tobacco). Tobacco-related morbidity and mortality represents one of the most dramatic, yet preventable, public health tragedies. There is a huge need for more effective therapeutic approaches considering the low success rates of the currently available psychosocial and pharmaco-therapies to assist in quitting tobacco use [START_REF] Prochaska | Current advances in research in treatment and recovery: Nicotine addiction[END_REF]. Similar to other substance use disorders (SUDs), tobacco addiction is a chronic relapsing psychiatric disorder in which drug compulsive use and seeking behaviors persist despite negative consequences and a strong motivation to quit [START_REF] Babb | Quitting Smoking Among Adults -United States, 2000-2015[END_REF][START_REF] Chaiton | Estimating the number of quit attempts it takes to quit smoking successfully in a longitudinal cohort of smokers[END_REF][START_REF] Yucel | A transdiagnostic dimensional approach towards a neuropsychological assessment for addiction: an international Delphi consensus study[END_REF]. Among the thousands of compounds found in tobacco smoke, nicotine is the primary psychoactive substance responsible for its reinforcing and addictive properties [START_REF] Caille | Modeling nicotine addiction in rats[END_REF][START_REF] Clemens | Behavioral and neural substrates of habit formation in rats intravenously self-administering nicotine[END_REF][START_REF] Cohen | Robust escalation of nicotine intake with extended access to nicotine self-administration and intermittent periods of abstinence[END_REF][START_REF] Dome | Smoking, nicotine and neuropsychiatric disorders[END_REF][START_REF] Gilpin | Nicotine vapor inhalation escalates nicotine self-administration[END_REF]. Given the inconstant efficacy of currently available treatments, and since products of tobacco combustion are the main cause of smoking-induced diseases, governmental and economical control of smoking behavior has led to the development of devices for smokeless nicotine delivery such as heated and electronic nicotine vaping products [START_REF] Prochaska | Current advances in research in treatment and recovery: Nicotine addiction[END_REF]. However, the health consequences of the use, especially the chronic long-term use, of these devices are uncertain and more investigation will be required during the next decade to ascertain their safety. Moreover, while such devices are very likely to reduce the risk for lethal diseases among individuals suffering from nicotine addiction (NA), under a "harm reduction" goal, [START_REF] Chatterjee | Is vaping a gateway to smoking: a review of the longitudinal studies[END_REF][START_REF] Etter | Gateway effects and electronic cigarettes[END_REF]. For recent insights on the topic, see https://www.nejm.org/vaping.

For now, there is still an urgent need to provide a better understanding of the aetiopathogeny of NA to develop alternative prevention and intervention strategies. Importantly, although the prevalence of individuals diagnosed as dependent is higher for tobacco than that for any other SUD, not all smokers become nicotine dependent [START_REF] Anthony | Comparative Epidemiology of Dependence on Tobacco, Alcohol, Controlled Substances, and Inhalants: Basic Findings From the National Comorbidity Survey[END_REF][START_REF] Birge | What Proportion of People Who Try One Cigarette Become Daily Smokers? A Meta-Analysis of Representative Surveys[END_REF][START_REF] Kandel | Prevalence and demographic correlates of symptoms of last year dependence on alcohol, nicotine, marijuana and cocaine in the U.S. population[END_REF]. Addiction is a complex disorder, and there are individual differences in the vulnerability to develop this pathology that can be conceptualized at different levels interacting with each other, including environmental and genetic components. It is of particular importance to better understand the genetic contribution of addiction vulnerability, which can account for more than 70% of an individual's risk for this pathology [START_REF] Ducci | The genetic basis of addictive disorders[END_REF]. The information obtained from research into addiction vulnerability has the potential to help preventing and treating addictive disorders in several ways [START_REF] Morrow | Neuroscience of resilience and vulnerability for addiction medicine: From genes to behavior[END_REF]. For instance, a better understanding of the genetic predisposition factors could lead to the discovery of biomarkers to identify individuals who are at high risk of developing addiction. Individuals who already have addiction may also benefit from knowledge of genetic vulnerability factors, notably through personalized pharmacotherapy during specific aspects of the addiction cycle, such as drug seeking reinstatement which often occurs in 'occasion setter' contexts [START_REF] Crombag | Review. Context-induced relapse to drug seeking: a review[END_REF].

The CHRNA5-A3-B4 gene cluster and NA vulnerability

The identification of specific genes and loci contributing to addiction vulnerability has been challenging, likely because of the genetic complexity of psychiatric diseases such as addictive disorders. Such complexity includes multiple risk alleles, gene x gene interactions and epigenetic effects [START_REF] Gelernter | Genetics of complex traits in psychiatry[END_REF]. In this context, genome-wide association studies (GWAS) have the advantages of covering the entire genome, implementing a 'hypothesis-free' approach, and having the power for detecting relatively common alleles. The potential of GWAS in the addiction field will most likely increase in the future with more extensive data sharing, larger samples, and the optimization of phenotyping and refinement of statistical analytic methods. So far, the strongest and widely reproduced GWAS finding for addiction has been the identification of the CHRNA5-A3-B4 gene cluster on chromosome 15q25, in association with a propensity to NA [START_REF] Berrettini | Alpha-5/alpha-3 nicotinic receptor subunit alleles increase risk for heavy smoking[END_REF][START_REF] Bierut | Novel genes identified in a high-density genome wide association study for nicotine dependence[END_REF][START_REF] Thorgeirsson | A variant associated with nicotine dependence, lung cancer and peripheral arterial disease[END_REF]. This was initially discovered [START_REF] Saccone | Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs[END_REF] and further confirmed [START_REF] Sherva | Association of a single nucleotide polymorphism in neuronal acetylcholine receptor subunit alpha 5 (CHRNA5) with smoking status and with 'pleasurable buzz' during early experimentation with smoking[END_REF][START_REF] Stevens | Nicotinic receptor gene variants influence susceptibility to heavy smoking[END_REF] by candidate gene approaches, and extensive meta-analysis (Liu et al., 2010;[START_REF] Tobacco | Genome-wide meta-analyses identify multiple loci associated with smoking behavior[END_REF]. Interestingly, the same hits were identified in GWAS for lung cancer [START_REF] Amos | Genome-wide association scan of tag SNPs identifies a susceptibility locus for lung cancer at 15q25.1[END_REF][START_REF] Hung | A susceptibility locus for lung cancer maps to nicotinic acetylcholine receptor subunit genes on 15q25[END_REF]. As for other phenotypes, human genetics of NA have mostly been studied in populations of European ancestry.

However, it has been more and more acknowledged that the study of samples with diverse geographic ancestries addresses both a scientific and an ethical challenge [START_REF] Peterson | Genome-wide Association Studies in Ancestrally Diverse Populations: Opportunities, Methods, Pitfalls, and Recommendations[END_REF]. As a result, the CHRNA5-A3-B4 gene cluster has been increasingly studied in African and East-Asian populations.

Notably, meta-analytical evidence showed that the functional CHRNA5 rs16969968 is associated with smoking heaviness across European, African and Asian populations [START_REF] Chen | Smoking and genetic risk variation across populations of European, Asian, and African American ancestry--a meta-analysis of chromosome 15q25[END_REF]. Other variants affecting CHRNA5 expression were also associated with heavy smoking in European and Asian, but not African samples.

The CHRNA5-A3-B4 gene cluster encodes for the α5, α3, and β4 subunits of the nicotinic acetylcholine (ACh) receptors (nAChRs). nAChRs are homo-and hetero-pentameric cationic ligandgated ion channels composed of α and β subunits that can co-assemble in various combinations of distinct brain distribution and functional properties. There are six α (2-7) and three β (2-4) subunits expressed in the nervous system of mammals [START_REF] Changeux | Nicotine addiction and nicotinic receptors: lessons from genetically modified mice[END_REF][START_REF] Picciotto | Molecular mechanisms underlying behaviors related to nicotine addiction[END_REF][START_REF] Taly | Nicotinic receptors: allosteric transitions and therapeutic targets in the nervous system[END_REF]. , but not β, subunits have adjacent cysteines, analogous to cysteines 192-193 of the muscle 1 subunit [START_REF] Sine | Recent advances in Cys-loop receptor structure and function[END_REF]. Rodent studies have established that the 3 subunit mostly combines with 4, and that 34* (* stands for containing) nAChRs show a discrete expression profile in the brain, mainly in the medial habenula (mHb) and the interpeduncular nucleus (IPN) [START_REF] Grady | Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the αβ4* and αβ3β4* subtypes mediate acetylcholine release[END_REF]Salas et al., 2003b), where they play an important role for cholinergic modulation. The 5 subunit is considered as 'accessory', as it does not contribute to ACh binding sites, nor does it form functional nAChRs by itself or in combination with only one other type of subunit, though it significantly influences the receptor's functional properties [START_REF] Exley | Striatal α5 nicotinic receptor subunit regulates dopamine transmission in dorsal striatum[END_REF][START_REF] Kuryatov | Roles of accessory subunits in alpha4beta2(*) nicotinic receptors[END_REF][START_REF] Ramirez-Latorre | Functional contributions of alpha5 subunit to neuronal acetylcholine receptor channels[END_REF][START_REF] Tapia | Ca2+ permeability of the (alpha4)3(beta2)2 stoichiometry greatly exceeds that of (alpha4)2(beta2)3 human acetylcholine receptors[END_REF]. The 5 subunit is expressed in several brain areas important for addiction-like processes, including the ventral tegmental area (VTA), which is crucial for nicotine rewarding properties [START_REF] Klink | Molecular and physiological diversity of nicotinic acetylcholine receptors in the midbrain dopaminergic nuclei[END_REF], but mostly in the IPN where it can combine with 42* or 34*nAChRs [START_REF] Grady | Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the αβ4* and αβ3β4* subtypes mediate acetylcholine release[END_REF][START_REF] Hsu | Medial habenula output circuit mediated by alpha5 nicotinic receptor-expressing GABAergic neurons in the interpeduncular nucleus[END_REF]. Alterations in nAChRs containing these subunits may impact the risk for NA by modifying the affinity for nicotine but also by altering the basal activity of the complex brain circuitry involved in addiction-like processes, as suggested by the implication of the CHRNA5-A3-B4 cluster in the abuse of other substances in addition to nicotine [START_REF] Besson | Profound alteration in reward processing due to a human polymorphism in CHRNA5: a role in alcohol dependence and feeding behavior[END_REF][START_REF] Haller | Rare missense variants in CHRNB3 and CHRNA3 are associated with risk of alcohol and cocaine dependence[END_REF][START_REF] Sherva | Variation in nicotinic acetylcholine receptor genes is associated with multiple substance dependence phenotypes[END_REF].

Importantly, the genetic risk linked to this cluster for a complex trait like NA is likely to be mediated by several sets of variant combinations. The present review focuses on how the CHRNA5-A3-B4 genomic region has been implicated in clinical and preclinical phenotypes related to NA, and aims at examining the limitations of the studies conducted so far. It also discusses future approaches for better understanding and modelling of the interplay of the multiple variants of this cluster in NA-like processes.

CHRNA5

State of the art of the associations identified between CHRNA5 variants, NA, and brain function in humans

General considerations for human genetic analysis

The sub-phenotypes of tobacco addiction implicate different biological and genetic correlates [START_REF] Lassi | The CHRNA5-A3-B4 Gene Cluster and Smoking: From Discovery to Therapeutics[END_REF]. In the present review, we will focus on associations between NA and genetic variants in the genes coding for the α5 (CHRNA5), α3 (CHRNA3) and β4 (CHRNB4) nAChR subunits, considering all kinds of genetic variants: rare and frequent single nucleotide variants (SNVs), insertions and deletions (InDels) and copy number variations (CNVs). We will report data from both candidate SNV/gene association studies and GWAS. The functional impact of these variants will be discussed based on direct evidence from preclinical models or from in silico databases describing the impact of both exonic (synonymous or not) and intronic/intergenic variants at multiple levels, from protein structure changes to close-and long-range promoter interactions (Table 1).

Gene variants associated with changes in brain function measured experimentally by neuropsychological testing or brain imaging (functional and structural) and the possible relationship of these changes with NA will also be discussed. Finally, in order to provide a more global and relevant genetic architecture of NA, these results will be discussed in light of multigenic models and evidence, taking into account linkage disequilibrium (LD) and genetic correlation issues.

Genetic associations between CHRNA5 and NA

The CHRNA5 gene (HGNC: 1959, ENSG00000169684) has a relative low expression in the human brain (Fig. 1). It harbors ~7000 SNVs and Indels and three CNVs (one deletion, one duplication and one inversion, identified by SNV array). The gene has a pLI score of 2.5 x 10 -6 (higher scores indicate poorer tolerance to mutations) with 43% haploinsufficiency, indicating an overall high tolerance to mutation. The missense mutation rs16969968 (RefSeqGene NG_023328.1:g.30064G>A), a frequent SNV (called single nucleotide polymorphism (SNP)), changing one amino acid in the α5 protein at position 398, from aspartate to asparagine, has been the focus of extensive clinical and preclinical research due to robust and repeated evidence for association of its risk allele (A in populations with Caucasian ancestry) with NA. rs16969968 has been repeatedly associated with NA, including in a first GWAS in 2008 [START_REF] Thorgeirsson | A variant associated with nicotine dependence, lung cancer and peripheral arterial disease[END_REF], an observation further strengthened by meta-analyses (Liu et al., 2010;[START_REF] Marees | Exploring the role of low-frequency and rare exonic variants in alcohol and tobacco use[END_REF]. The presence of this SNP doubles the risk to develop NA for AA compared to GG genotypes [START_REF] Saccone | Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs[END_REF]. In the latest and largest GWAS to date, NA was not assessed but the proxy "cigarettes per day" (CPD) was used (Erzurumluoglu et al., 2019). It did not evidence association with rs16969968, suggesting this SNP may be rather associated with the complete NA phenotype (measured by the Fagerström Test for Nicotine Dependence (FTND), a stringent, yet composite measure of NA; or by DSM criteria) than with the number of CPD. This could be linked to the fact that (i) rs16969968 modulates the aversive properties of nicotine in humans [START_REF] Jensen | A CHRNA5 Smoking Risk Variant Decreases the Aversive Effects of Nicotine in Humans[END_REF] (as also suggested in preclinical studies, see part 2.2.), which is not properly represented by CPD measure, (ii) this measure is highly variable as regards its association with NA, especially since the strength of smoke inhalation has high inter-and intra-individual variability [START_REF] Scherer | Smoking behaviour and compensation: a review of the literature with meta-analysis[END_REF]. Associations between CHRNA5, including with the rs16969968, and NA are further supported by a GWAS finding regarding another important smoking phenotype, exhaled carbon monoxide level, a reliable biomarker of current cigarette exposure [START_REF] Bloom | Beyond cigarettes per day. A genome-wide association study of the biomarker carbon monoxide[END_REF]. Other CHRNA5 SNPs have been associated with NA in candidate-SNP studies. Intronic SNPs rs17486278 and rs569207 have been associated with NA in smokers who initiated smoking before the age of 16 (along with rs16969968) [START_REF] Weiss | A candidate gene approach identifies the CHRNA5-A3-B4 region as a risk factor for age-dependent nicotine addiction[END_REF]. Overall, in this study, all associations between CHRNA5-A3-B4 variants and FTND score were significant in early-onset smokers only. This might suggest that more severe smoking phenotypes such as those combining NA and early onset could have a stronger genetic basis than less severe phenotypes, as was previously tested in other psychiatric conditions such as bipolar disorder [START_REF] Kennedy | The genetics of early-onset bipolar disorder: A systematic review[END_REF].

CHRNA5 and human brain function

There have been three functional neuroimaging reports published to date regarding CHRNA5dependent alterations in brain circuitry in humans, all testing rs16969968. First, in a rather large functional magnetic resonance imaging (fMRI) study of 184 subjects [START_REF] Hong | A genetically modulated, intrinsic cingulate circuit supports human nicotine addiction[END_REF], rs16969968 risk allele was associated with eight neuronal circuits originating from the dorsal anterior cingulate cortex (dACC, which was the seed for the study). This association was independent from other psychiatric diagnoses, age, gender, level of smoking exposure, and two other SNPs that modulate CHRNA5 expression (see below). The strongest effect was a decreased resting state connectivity in a circuit including the dACC, the ventral striatum and the extended amygdala. This finding can be considered robust given the large study sample, the independence of associations from key possible confounders, and the relevance of the top circuit reported regarding smoking, affective and cognitive phenotypes. In a much smaller study [START_REF] Janes | Association between CHRNA5 genetic variation at rs16969968 and brain reactivity to smoking images in nicotine dependent women[END_REF], rs16969968 AA genotype was associated with increased reactivity of female smokers' hippocampus and dorsal striatum compared to GG genotype. Both samples were matched on age and smoking outcomes. However, another study found that chronic smokers who carried the low-risk genotype had no increase in brain activation when presented with smoking-related vs. neutral images during the task [START_REF] Franklin | Limbic Activation to Cigarette Smoking Cues Independent of Nicotine Withdrawal: A Perfusion fMRI Study[END_REF]. This discrepancy might reflect the existence of spurious associations due to the small sample sizes. Finally, the IMAGEN consortium has recently reported that both smoking tobacco and being homozygote for rs16969968 high-risk genotype were associated with a reduction in cortical thickness of the ventromedial prefrontal cortex (PFC), with additive smoking + genotype effects (Chaarani et al., 2019).

Two other neuroimaging studies are worth mentioning, although they did not focus on tobacco smokers. First, an association between increased habenular connectivity and rs16969968 has been reported in opiate users [START_REF] Curtis | Increased habenular connectivity in opioid users is associated with an alpha5 subunit nicotinic receptor genetic variant[END_REF]. Unfortunately, their clinical status was somewhat unclear, likely mixing people with light, mild, and severe opiate use disorders, making it difficult to further interpret this finding. Second, there was a negative correlation between dorsolateral PFC activation and accuracy at the 2-Back working memory test in double heterozygotes for rs16969968 and the rs1076560 from the gene encoding the dopamine (DA) receptor type 2 (DRD2) [START_REF] Giorgio | DRD2/CHRNA5 Interaction on Prefrontal Biology and Physiology during Working Memory[END_REF].

Although smoking status did not interfere with the main result, the fact that significant differences were observed in risk allele heterozygotes compared to risk allele homozygotes for both SNPs is unexpected (unless one can make specific hypotheses regarding the genetic model of the risk conferred by rs16969968, which would make plausible that the risk would not necessarily increase with the number of risk alleles). It may indicate either power issues (i.e. too few people homozygotes for the risk allele) or unmeasured bias.

In parallel with functional imaging studies, cognitive function in humans has also been studied as a function of the occurrence of CHRNA5 variants. In an attempt to replicate the GWAS association between schizophrenia and rs16969968 (Schizophrenia Working Group of the Psychiatric Genomics et al., 2014), researchers reported that, in patients with early-onset schizophrenia, the minor allele was associated with poorer performance at all subscales of the Wisconsin Card Sorting Test (WCST), which assesses working memory and executive functions related to the prefrontal lobe. In tobacco smokers without schizophrenia, the effect of five tag CHRNA5 SNPs was tested [START_REF] Evans | CHRNA5 variants moderate the effect of nicotine deprivation on a neural index of cognitive control[END_REF] after nicotine deprivation by quantification of the P300 wave (an event-related brain potential (ERP), considered a biomarker of cognitive control [START_REF] Polich | Neuropsychology and neuropharmacology of P3a and P3b[END_REF]. Minor allele carriers of the noncoding rs588765 and rs17408276 SNPs showed reductions in P300 amplitude, suggesting weaker cognitive control, without significant association with rs16969968. This was in contrast with two previous studies reporting associations between rs16969968 minor allele and poorer cognitive function [START_REF] Giorgio | DRD2/CHRNA5 Interaction on Prefrontal Biology and Physiology during Working Memory[END_REF][START_REF] Winterer | Risk gene variants for nicotine dependence in the CHRNA5-CHRNA3-CHRNB4 cluster are associated with cognitive performance[END_REF]. Moreover, poorer performance at the WCST was also associated with CHRNA5 rs3841324 (an insertion) and rs615470 [START_REF] Zhang | Variation in the Nicotinic Acetylcholine Receptor Gene Cluster CHRNA5-CHRNA3-CHRNB4 and Its Interaction with Recent Tobacco Use Influence Cognitive Flexibility[END_REF], specifically regarding pervasive errors. Finally, in female tobacco smokers, CHRNA5 rs680244 was further associated with reduced accuracy of familiar face recognition [START_REF] Rigbi | Why do young women smoke? V. Role of direct and interactive effects of nicotinic cholinergic receptor gene variation on neurocognitive function[END_REF].

Several of the CHRNA5 SNPs associated with altered cognitive/brain function may have functional impacts according to various mechanisms. rs615470 is associated with reduced CHRNA5 expression in the brain. rs588765 and rs680244 are associated with reduced expression of CHRNA3 and CHRNA5, but also of the CHRNA5 antisense RNA RP11-650L12.2 in the brain (see example for rs680244 in Fig. 2). Significantly increased cis-methylation of CHRNA5 promoter region by these SNPs is also observed, albeit with small to very small effect sizes. Importantly, LD between all those SNPs -rs16969968 exceptedis above 0.8 (and mostly =1), meaning that all those associations indicate the same genetic information (Fig. 3).

Overall, there is consistent GWAS evidence supporting some direct effect of the CHRNA5 nonsynonymous SNP rs16969968 in NA. However, the strength of evidence rather comes from candidate gene studies and from the observation of a functional impact of this variant on the receptor function.

Associations between other CHRNA5 SNPs were scarce and limited to candidate SNP studies.

Overall, CHRNA5 SNPs were rather associated with FTND scores than with the number of CPD.

There were repeated associations between SNPs in CHRNA5 and poorer performance in a range of cognitive tasks, especially those related to cognitive flexibility, with possible modulation of brain function by rs16969968, although to a lesser extent. Importantly enough, evidence from neuroimaging and cognitive studies were consistent toward altered prefrontal function depending on rs16969968 genotype. Some limitations in the methodology implemented across the various studies that we reviewed must be acknowledged. A relative discrepancy in the outcome of the investigations is observed depending on the variables chosen to measure smoking or NA severity. Such discrepancy likely reflects methodological issues, notably as regards brain function studies which tended to rely on few (if not single) numbers of SNPs, possibly overlooking the functional and/or linkage interplay between many SNPs in the 15q25 region. Furthermore, several of these studies were performed after nicotine deprivation (minutes to weeks), limiting our ability to disentangle between vulnerability issues and the effects of alleviating withdrawal symptoms. Finally, to date, standardized methods that take into account LD to moderate the associations between genotypes and phenotypes require large GWAS datasets to run properly [START_REF] Kranzler | Genome-wide association study of alcohol consumption and use disorder in 274,424 individuals from multiple populations[END_REF], leaving researchers with only assumptions or empirical/intuition-based methods to account for LD when characterizing the effects of a selected set of genetic variants in the CHRNA5-A3-B4 cluster.

Preclinical refinement of the impact of CHRNA5 in NA-like processes

NA-like phenotypes in preclinical models of CHRNA5 alterations

Most preclinical studies investigating the impact of CHRNA5 in NA-like processes used knockout (KO) mice for this gene. α5KO mice chronically injected with nicotine exhibit reduced somatic signs of nicotine withdrawal after administration of the nicotinic antagonist mecamylamine [START_REF] Jackson | Differential role of nicotinic acetylcholine receptor subunits in physical and affective nicotine withdrawal signs[END_REF][START_REF] Salas | Nicotinic receptors in the habenulointerpeduncular system are necessary for nicotine withdrawal in mice[END_REF]. They self-administer more nicotine at high doses that are considered aversive in WT mice [START_REF] Fowler | Habenular alpha5 nicotinic receptor subunit signalling controls nicotine intake[END_REF][START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]. They also present a conditioned place preference (CPP) for high doses of nicotine, which are not rewarding in wild-type (WT) mice, but they are able to discriminate nicotine as well as WTs [START_REF] Jackson | Role of alpha5 nicotinic acetylcholine receptors in pharmacological and behavioral effects of nicotine in mice[END_REF].

Only one polymorphism in the CHRNA5 gene associated with tobacco dependence in humans has been preclinically studied: the non-synonymous SNP rs16969968 (hereafter called α5SNP in the context of preclinical modelling). First, a recent study took advantage of the facts that cynomolgus monkeys naturally express the α5SNP at a frequency similar to humans and that there is great homology of nAChR protein sequences between human and non-human primates (98%) [START_REF] Shorey-Kendrick | Nicotinic receptors in non-human primates: Analysis of genetic and functional conservation with humans[END_REF]. WT (n=5) and α5SNP cynomolgus monkeys (n=2) had a choice to drink at a bottle containing a nicotine solution with sweetener or at a bottle containing water, and the concentration of nicotine was progressively increased over time (from 25 to 750 µg/ml; 6 days per concentration). Surprisingly, α5SNP monkeys drank less of the nicotine solution than WTs at low doses (100-200 µg/ml) and did not differ from WTs at high doses (400-750 µg/ml), suggesting a decreased sensitivity to nicotine. The authors also observed a difference in the pattern of nicotine consumption between genotypes, with a tendency to consume more nicotine during the nocturnal period for the α5SNP monkeys compared to WTs, and a drinking of nicotine unrelated to food consumption, contrary to WTs [START_REF] Shorey-Kendrick | Nicotinic receptors in non-human primates: Analysis of genetic and functional conservation with humans[END_REF]. However, it is difficult to draw clear conclusions on the impact of the α5SNP on nicotine sensitivity from this study considering the low number of monkeys tested and the fact that the WT group included monkeys that were heterozygotes for the α5SNP.

Another approach is to artificially insert the α5SNP in species that do not naturally express it. It has first been done in a study where pregnant WT and homozygous α5SNP mice were forced to drink a nicotine solution and their offspring were subsequently tested for their nicotine consumption in a twobottle choice paradigm [START_REF] O'neill | The interaction of the Chrna5 D398N variant with developmental nicotine exposure[END_REF]. Interestingly, the α5SNP offspring consumed more nicotine at the highest concentration (400 µg/ml) than WT offspring, a result consistent with the increased SA of nicotine at high doses observed in α5KO mice [START_REF] Fowler | Habenular alpha5 nicotinic receptor subunit signalling controls nicotine intake[END_REF][START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]. Our laboratory inserted the α5SNP in the genome of the Rat using zinc finger nuclease (ZFN)directed genome editing and tested them in long-term nicotine SA [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF]. We demonstrated that α5SNP rats acquire nicotine SA at a training dose as well as WT rats but selfadminister more nicotine at high doses in both fixed-and progressive-ratio schedules, a result in line with the performance of α5KO mice in nicotine SA [START_REF] Fowler | Habenular alpha5 nicotinic receptor subunit signalling controls nicotine intake[END_REF][START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]. In addition, while the α5SNP rats did not differ from WT rats on extinction or cueinduced reinstatement of nicotine seeking, they exhibited a strong increase in reinstatement of nicotine seeking induced by a priming dose of nicotine, a model of relapse induced by tobacco re-exposure after abstinence in humans. This last result is particularly important considering the high relapse rate in NA in humans. Taken together, these preclinical results fit well with the human situation where the α5SNP is associated with an increased risk for NA, lower ratings of aversive effect, and delayed smoking cessation [START_REF] Bierut | Variants in Nicotinic Receptors and Risk for Nicotine Dependence[END_REF][START_REF] Chen | CHRNA5 risk variant predicts delayed smoking cessation and earlier lung cancer diagnosis--a meta-analysis[END_REF][START_REF] Jensen | A CHRNA5 Smoking Risk Variant Decreases the Aversive Effects of Nicotine in Humans[END_REF][START_REF] Saccone | Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs[END_REF].

Brain circuits involved

The α5 nAChR subunit is expressed in discrete regions of the rodent central nervous system (CNS), including the striatum where it regulates DA transmission [START_REF] Exley | Striatal α5 nicotinic receptor subunit regulates dopamine transmission in dorsal striatum[END_REF], the cerebral cortex, cerebellum, thalamus, hippocampus, substantia nigra (SN), VTA and mHb, with the highest concentration found in the IPN [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF][START_REF] Hsu | Medial habenula output circuit mediated by alpha5 nicotinic receptor-expressing GABAergic neurons in the interpeduncular nucleus[END_REF] Some studies contributed to a better understanding of the neuronal impact of the lack of α5 or the presence of the α5SNP. In particular, the re-expression of the α5 subunit in the mHb of α5KO mice restored a control-like nicotine SA behavior while a down regulation in the same structure in rats induced an increase in nicotine SA at high doses [START_REF] Fowler | Habenular alpha5 nicotinic receptor subunit signalling controls nicotine intake[END_REF], suggesting an important contribution of the α5 subunit in the mHb in nicotine consumption. However, other structures are important in the effect of the α5 subunit on behaviors related to NA. Thus, re-expression of the α5 subunit into DAergic neurons of the VTA of α5KO mice also restored nicotine SA behavior, while the expression of the α5SNP in the same VTA neurons only partially restored SA behavior, leading to the consumption of more nicotine than WT mice [START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]. The IPN has recently attracted a lot of attention in studies on nicotine effects due to its high concentration in α5*nAChRs. In particular, stimulation of α5 containing neurons in the IPN induced aversion after nicotine exposure [START_REF] Morton | Chrna5-Expressing Neurons in the Interpeduncular Nucleus Mediate Aversion Primed by Prior Stimulation or Nicotine Exposure[END_REF], and the lack of α5 subunit induced a significant decrease in nicotine-induced currents in the IPN [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF][START_REF] Morton | Chrna5-Expressing Neurons in the Interpeduncular Nucleus Mediate Aversion Primed by Prior Stimulation or Nicotine Exposure[END_REF] but not in the mHb [START_REF] Morton | Chrna5-Expressing Neurons in the Interpeduncular Nucleus Mediate Aversion Primed by Prior Stimulation or Nicotine Exposure[END_REF]. Furthermore, inhibition of GABAergic neurons of the IPN projecting to the laterodorsal tegmentum (LDTg) blocked nicotine conditioned place aversion, suggesting that this pathway is a critical part of the circuitry that mediates the aversive effects of nicotine. In addition, a recent study identified a cell population containing the α5 subunit in the IPN (α5-Amigo1) that appeared to be essential for nicotine-induced CPP [START_REF] Ables | Retrograde inhibition by a specific subset of interpeduncular alpha5 nicotinic neurons regulates nicotine preference[END_REF].

Finally, the α5SNP induced a reduction in nicotine-induced current in the IPN of rats and the neuronal activity in the IPN was negatively correlated with nicotine-induced reinstatement of nicotine seeking, pointing to a potentially important role of the α5 subunit of nAChRs in the IPN in this behavior [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF].

In conclusion, the α5 subunit in the IPN appears essential for nicotine aversion, withdrawal and relapse while the α5 subunit in the VTA and the mHb seems important to limit nicotine SA at high doses.

Recommendation for future research

The preclinical studies described above helped to understand more specifically which behaviors related to NA are dependent on the α5 subunit of nAChRs and altered by the presence of the α5SNP rs16969968, and the neuronal pathways potentially involved. These results can thus orientate future clinical studies to specific directions, such as investigating the impact of the rs16969968 on relapse to tobacco smoking and the brain structures to image. Considering the deleterious effects of the lack of α5 subunit or the presence of the α5SNP in preclinical models related to NA, together with the restricted expression of this subunit in the brain, it would be of great interest to develop a class of therapeutic compounds designed to specifically enhance the activity of α5*nAChRs. For instance, positive α5*nAChR allosteric modulators may have clinical potential to prevent excessive tobacco smoking and relapse. However, there is more preclinical work to do to unravel the circuits underlying the role of the α5 subunit and the impact of the α5SNP on NA-related processes. For example, it would be interesting to investigate if the α5-containing neurons in the IPN projecting to the LDTg, which are key actors of nicotine aversion [START_REF] Morton | Chrna5-Expressing Neurons in the Interpeduncular Nucleus Mediate Aversion Primed by Prior Stimulation or Nicotine Exposure[END_REF], are also involved in behaviors more directly related to NA such as nicotine-SA and relapse to nicotine seeking. More generally, the use of neuron-and pathway-specific opto-or chemo-genetic techniques would be of great help to decipher the specific neurons implicated in the phenotypes previously described. It would also be important to clarify the involvement of the mHb neurons in these phenotypes. Indeed, the fact that the lack of the α5 subunit does not modify nicotine-induced current in the ,mHb [START_REF] Morton | Chrna5-Expressing Neurons in the Interpeduncular Nucleus Mediate Aversion Primed by Prior Stimulation or Nicotine Exposure[END_REF]. Fowler et al. used a viral re-expression of the α5 subunit in the mHb that also induced α5 subunit expression in neuron terminals in the IPN via transport trough the fasciculus retroflexus [START_REF] Fowler | Habenular alpha5 nicotinic receptor subunit signalling controls nicotine intake[END_REF]. Thus, the effect observed on nicotine intake might be due to the expression of α5*nAChRs on mHb axon terminals in the IPN that affect glutamatergic transmission. However, recent studies indicate that the α5 subunit within the mHb-IPN pathway are rather expressed postsynaptically in GABAergic neurons of the IPN [START_REF] Hsu | Medial habenula output circuit mediated by alpha5 nicotinic receptor-expressing GABAergic neurons in the interpeduncular nucleus[END_REF][START_REF] Morton | Chrna5-Expressing Neurons in the Interpeduncular Nucleus Mediate Aversion Primed by Prior Stimulation or Nicotine Exposure[END_REF]. Such apparent discrepancies might be reconciled if one assumes that these two circuits mediate distinct behavioral components, such as nicotine satiety for the glutamatergic neurons of the mHb, as suggested recently [START_REF] Tuesta | GLP-1 acts on habenular avoidance circuits to control nicotine intake[END_REF] and nicotine aversion for the GABAergic neurons of the IPN projecting to the LDTg [START_REF] Morton | Chrna5-Expressing Neurons in the Interpeduncular Nucleus Mediate Aversion Primed by Prior Stimulation or Nicotine Exposure[END_REF].

The α5SNP is the only polymorphism in the CHRNA5 gene associated with tobacco dependence in humans that was preclinically studied in details, generating important novel insights. Yet, as indicated above (see part 2.1), other CHRNA5 SNPs have been associated with tobacco dependence in humans, such as the intronic rs17486278 and rs569207. It would thus be interesting to further study those SNPs in preclinical models of NA to understand their mechanisms of action. As a first approach, non-human primates represent a good model organism for such investigations considering the high conservation and homology of nAChRs between humans and non-human primates and the natural presence of some SNPs homologous to humans ones [START_REF] Shorey-Kendrick | Nicotinic receptors in non-human primates: Analysis of genetic and functional conservation with humans[END_REF]. In particular, there are ten conserved SNPs in the CHRNA5 gene between Rhesus macaques and humans and 6 conserved SNPs in CHRNA5 exons between cynomolgus macaques and humans, including the rs16969968 (α5SNP). However, the low number of monkeys available for such studies limits their power. It would thus be important to insert SNPs of interest into the genome of rodents as a second approach to study their impact in more details. For example, intronic CHRNA5 SNPs have been associated with NA, alterations in the expression of several nicotinic subunits and alterations in cognitive function (see part 2.1), but the mechanisms mediating these alterations are unknown. In particular it is difficult to determine whether they have a direct functional impact or act through LD with other(s) functional SNP(s) in the cluster [START_REF] Mccauley | SNPs in Multi-species Conserved Sequences (MCS) as useful markers in association studies: a practical approach[END_REF]. Recent genome editing tools, such as ZFNs and CRISPR/Cas9 techniques allow the insertion of SNPs in non-coding region, thus representing a great opportunity to study model organisms in detail without the potential influence of other SNPs in LD, as it was done recently in rats to study the α5SNP [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF]. However, the degree of conservation of intron sequences, structure and length between humans and rodents varies greatly among genes with an overall lower degree of homology than exonic sequences [START_REF] Batzoglou | Human and mouse gene structure: comparative analysis and application to exon prediction[END_REF][START_REF] Roy | Large-scale comparison of intron positions in mammalian genes shows intron loss but no gain[END_REF]. It would thus be necessary to verify the similarity of CHRNA5 intronic sequences between humans and the organism model of choice before performing such investigations. If the degree of homology is not sufficient, an interesting alternative would be to perform the same gene editing in human to investigate the functional impact of intronic SNPs, directly in the cells or in more complex environments such as organoid culture models or cell grafts in the rodent brain for in vivo investigations.

CHRNA3

Current state of the art of the associations identified between CHRNA3 variants, NA and brain function in humans

The CHRNA5 has been recognized as an important candidate gene for NA as initially reported [START_REF] Saccone | Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs[END_REF], this nicotinic subunit may represent one of the best validated targets for the development of drugs which can regulate nicotine uptake [START_REF] Lassi | The CHRNA5-A3-B4 Gene Cluster and Smoking: From Discovery to Therapeutics[END_REF]. Furthermore, a genomic region tagged by CHRNA3 synonymous exonic variant, rs1051730, has consistently shown the strongest association to CPD within all polymorphisms in the cluster [START_REF] Tobacco | Genome-wide meta-analyses identify multiple loci associated with smoking behavior[END_REF], although this may be due to multiple haplotypic effects rather than to functional impact of this variant has also been linked to sensorimotor gating in a clinical study of schizophrenics [START_REF] Petrovsky | Sensorimotor gating is associated with CHRNA3 polymorphisms in schizophrenia and healthy volunteers[END_REF]. Several genetic studies have then been conducted more specifically on the CHRNA3 gene (ENSG00000080644, HGNC 1957), which has a low to moderate expression in the human brain (Fig. 1) CHRNA3 harbors ~7700 SNVs and InDels and three CNVs (one deletion, one duplication and one inversion, identified by SNV array). The gene has a pLI score of 0.01 (higher scores indicate poorer tolerance to mutations) with 33% haploinsufficiency, indicating an overall high tolerance to mutation. In one recent study, the authors have evaluated the association between a CHRNA3 (rs578776-NG_016143.1) polymorphism with head and neck cancer (HCN) development and the intensity of cigarette smoking by using a TaqMan genotyping method by real-time PCR, in a cohort of 1,067 patients from Sao Paulo Hospital [START_REF] Silva | Polymorphisms of CHRNA3 and CHRNA5 : Head and neck cancer and cigarette consumption intensity in a Brazilian population[END_REF]. Results from this study did not reveal any statistically significant association between the identified SNP with both HCN and smoking risk as compared to control groups [START_REF] Silva | Polymorphisms of CHRNA3 and CHRNA5 : Head and neck cancer and cigarette consumption intensity in a Brazilian population[END_REF]. The variant of CHRNA3 (C>T) is found in the 3' untranslated region (3'-UTR) of the gene and contains regulatory sequences [START_REF] Bierut | Variants in Nicotinic Receptors and Risk for Nicotine Dependence[END_REF][START_REF] Conlon | Single Nucleotide Polymorphisms in CHRNA5 rs16969968, CHRNA3 rs578776, and LOC123688 rs8034191 Are Associated With Heaviness of Smoking in Women in Northeastern Ontario, Canada[END_REF]. The 3'-UTR sequence is located immediately after the translation termination codon and plays an important role in gene expression through various specific sequences including microRNA elements, AU-rich elements and the poly A tail. As a consequence, one cannot exclude a role for this polymorphism in some yet undefined functional effects [START_REF] Conlon | Single Nucleotide Polymorphisms in CHRNA5 rs16969968, CHRNA3 rs578776, and LOC123688 rs8034191 Are Associated With Heaviness of Smoking in Women in Northeastern Ontario, Canada[END_REF]. A previous study had suggested that the C allele of CHRNA3 (rs 578776) may be considered as a risk allele for NA [START_REF] Robinson | The CHRNA3 rs578776 Variant is Associated with an Intrinsic Reward Sensitivity Deficit in Smokers[END_REF]. These authors used ERPs methods to evaluate reward sensitivity in smokers. Their results support the hypothesis that the T allele may have a protective effect. Importantly, they are in accordance with results from several other studies which show the association of CHRNA3 rs578776 with reduced risk for NA [START_REF] Bierut | Variants in Nicotinic Receptors and Risk for Nicotine Dependence[END_REF] and a protective effect of this SNP against the risk of heavy smoking [START_REF] Conlon | Single Nucleotide Polymorphisms in CHRNA5 rs16969968, CHRNA3 rs578776, and LOC123688 rs8034191 Are Associated With Heaviness of Smoking in Women in Northeastern Ontario, Canada[END_REF][START_REF] Pérez-Morales | CHRNA3 rs1051730 and CHRNA5 rs16969968 polymorphisms are associated with heavy smoking, lung cancer, and chronic obstructive pulmonary disease in a mexican population[END_REF]. Another study of Hong and collaborators identified two CHRNA3 variants, namely the rs578776 and rs1051730 [START_REF] Hong | A genetically modulated, intrinsic cingulate circuit supports human nicotine addiction[END_REF]. The former variant corresponds to the one described above and the latter represents a synonymous variant previously associated strongly with smoking and lung cancer [START_REF] Bierut | Variants in Nicotinic Receptors and Risk for Nicotine Dependence[END_REF][START_REF] Saccone | Cholinergic nicotinic receptor genes implicated in a nicotine dependence association study targeting 348 candidate genes with 3713 SNPs[END_REF][START_REF] Thorgeirsson | A variant associated with nicotine dependence, lung cancer and peripheral arterial disease[END_REF]. Zhang and collaborators have recently reported the existence of 69 single variants (SNP) for CHRNA3 associated to NA [START_REF] Zhang | Considering Genetic Heterogeneity in the Association Analysis Finds Genes Associated With Nicotine Dependence[END_REF]. A CHRNA3-rs2869546-T-allele was found associated to higher perseverative responses in cognitive flexibility tasks performed in two cohorts of European and Africans individuals, recruited for genetic association studies of drug or alcohol dependence [START_REF] Zhang | Variation in the Nicotinic Acetylcholine Receptor Gene Cluster CHRNA5-CHRNA3-CHRNB4 and Its Interaction with Recent Tobacco Use Influence Cognitive Flexibility[END_REF]. However, as discussed by the authors, since no data on nicotine exposure on the day of cognitive evaluation is available [START_REF] Zhang | Variation in the Nicotinic Acetylcholine Receptor Gene Cluster CHRNA5-CHRNA3-CHRNB4 and Its Interaction with Recent Tobacco Use Influence Cognitive Flexibility[END_REF].

So far, the mechanisms by which CHRNA3 variants mediate their effects on NA within brain regions have not been fully evaluated. By using fMRI studies, Hong and collaborators demonstrated in their initial studies that the G risk allele of CHRNA3 3′UTR (rs578776) was selectively associated with an increase in the connectivity between the dorsal anterior cingulate-ventral striatal circuit and the left thalamus in humans, but did not correlate with NA severity [START_REF] Hong | A genetically modulated, intrinsic cingulate circuit supports human nicotine addiction[END_REF]. Interestingly, CHRNA3 is particularly expressed in the thalamus in humans (https://www.proteinatlas.org/). Further studies are needed to fully understand the precise role of the different CHRNA3 alleles.

Lack of preclinical studies for the impact of CHRNA3 in NA-like processes

In the nervous system, the α3 subunit is highly expressed in the periphery with a more restricted expression profile in the CNS [START_REF] Gotti | Neuronal nicotinic receptors: From structure to pathology[END_REF][START_REF] Greenbaum | Differential contribution of genetic variation in multiple brain nicotinic cholinergic receptors to nicotine dependence: Recent progress and emerging open questions[END_REF][START_REF] Zoli | Diversity of native nicotinic receptor subtypes in mammalian brain[END_REF]. In the rodent brain, the α3 subunit is predominantly expressed in the IPN [START_REF] Grady | Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the αβ4* and αβ3β4* subtypes mediate acetylcholine release[END_REF][START_REF] Perry | Measuring nicotinic receptors with characteristics of α4β2, α3β2 and α3β4 subtypes in rat tissues by autoradiography[END_REF][START_REF] Winzer-Serhan | Codistribution of nicotinic acetylcholine receptor subunit α3 and β4 mRNAs during rat brain development[END_REF], mHb [START_REF] Grady | Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the αβ4* and αβ3β4* subtypes mediate acetylcholine release[END_REF][START_REF] Quick | Alpha3beta4 subunitcontaining nicotinic receptors dominate function in rat medial habenula neurons[END_REF][START_REF] Zoli | Developmental regulation of nicotinic ACh receptor subunit mRNAs in the rat central and peripheral nervous systems[END_REF], pineal gland [START_REF] Zoli | Developmental regulation of nicotinic ACh receptor subunit mRNAs in the rat central and peripheral nervous systems[END_REF] and olfactory bulb [START_REF] Dineley-Miller | Gene transcripts for the nicotinic acetylcholine receptor subunit, beta4, are distributed in multiple areas of the rat central nervous system[END_REF]. It is also present in the SN [START_REF] Azam | Expression of neuronal nicotinic acetylcholine receptor subunit mRNAs within midbrain dopamine neurons[END_REF] and the VTA [START_REF] Greenbaum | Differential contribution of genetic variation in multiple brain nicotinic cholinergic receptors to nicotine dependence: Recent progress and emerging open questions[END_REF][START_REF] Perry | Measuring nicotinic receptors with characteristics of α4β2, α3β2 and α3β4 subtypes in rat tissues by autoradiography[END_REF]. Finally, a weak expression of α3 is found in the brainstem [START_REF] Morley | The embryonic and post-natal expression of the nicotinic receptor α3-subunit in rat lower brainstem[END_REF], cerebellum [START_REF] Turner | Nicotinic cholinergic receptors in the rat cerebellum: Multiple heteromeric subtypes[END_REF], hippocampus [START_REF] Gahring | Mouse Strain-Specific Nicotinic Acetylcholine Receptor Expression by Inhibitory Interneurons and Astrocytes in the Dorsal Hippocampus[END_REF][START_REF] Winzer-Serhan | Codistribution of nicotinic acetylcholine receptor subunit α3 and β4 mRNAs during rat brain development[END_REF], and thalamus [START_REF] Perry | Measuring nicotinic receptors with characteristics of α4β2, α3β2 and α3β4 subtypes in rat tissues by autoradiography[END_REF] The Chrna3 gene was one of the first neuronal nAChR subunits to be cloned [START_REF] Boulter | Isolation of a cDNA clone coding for a possible neural nicotinic acetylcholine receptor alpha-subunit[END_REF]), yet the contribution of the 3 subunit in cognitive functions, in particular in processes related to NA, remains poorly understood. The role of most nicotinic subunits in NA has been studied by using various genetic tools to modify or regulate the expression of the subunit gene in animal models. In the case of the α3 subunit, this approach has proven to be more challenging so far. Indeed, a mouse line carrying a null mutation in the α3 gene (Xu et al., 1999a) has been generated, but the homozygous KO animals are affected by multi-organ dysfunction and die before adulthood.

Salas and collaborators, using heterozygous α3 WT/KO mice, were able to show that these animals are resistant to nicotine-induced seizures, but do not show any alterations in nicotine-induced hypolocomotion, compared to their WT littermates (Salas et al., 2004a). Yet, this model did not allow further significant investigation on the role of this subunit. Interestingly, the authors demonstrated, by in situ hybridization, that the lack of 4 subunit decreases the level of expression of 3 mRNA in some specific regions of the brain like the olfactory bulb or colliculi but not in structures implied in NA like the mHb, the SN and the VTA.

No other 3 mRNA expression modification was observed in the 4 or 5 KO lines even though the Chrna3, Chrnb4 and Chrna5 genes belong to same genetic locus and share some common regulatory elements [START_REF] Improgo | The nicotinic acetylcholine receptor CHRNA5/A3/B4 gene cluster: dual role in nicotine addiction and lung cancer[END_REF]. KO animal models for Chrna3, the majority of studies interested in the role of this subunit in NA-related processes mostly focused on the role of α3β4*containing nAChR receptors using pharmacological approaches. As describe above, in the CNS, the mesolimbic DA pathway contains some α3β4*nAChRs but these receptors are mostly found in the mHb and IPN.

Interestingly, α3β4*nAChRs have been reported to control ACh release in the fasciculus retroflexus, the tract of cholinergic fibers projecting from the mHb to the IPN [START_REF] Grady | Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the αβ4* and αβ3β4* subtypes mediate acetylcholine release[END_REF]. In these brain regions, an important role for the α5 subunit, which can co-assemble with α3 and β4 to form functional nAChRs, has been highlighted in NA-like processes. However, in rodents, it has been shown that only a part of α3β4*nAChRs expressed in these areas include an α5 subunit in their composition (15 and 35% in the mHb and IPN of mice, respectively, and only 10% and 15% in rats). Using intracerebroventricular injections of the α3β4*nAChR selective antagonist conotoxin AuIB combined to behavioral studies on mouse models, Jackson et al (2013) showed that these receptors contribute to nicotine reward and somatic nicotine withdrawal, but do not seem to mediate the acute nicotineinduced antinociception, decrease in body temperature and hypolocomotion, nor the affective manifestation of nicotine withdrawal in mice (Jackson et al., 2013b). [START_REF] Toll | AT-1001: A high affinity and selective α3Β4 nicotinic acetylcholine receptor antagonist blocks nicotine self-administration in rats[END_REF], using a high affinity and more selective antagonist for α3β4*nAChRs, the AT-1001 (N-(2-bromophenyl)-9-methyl-9azabicyclo[3.3.1]nonan-3-amine), were able to demonstrate an implication of these receptors in nicotine SA in rats, a dose-dependent and reversible effect [START_REF] Toll | AT-1001: A high affinity and selective α3Β4 nicotinic acetylcholine receptor antagonist blocks nicotine self-administration in rats[END_REF]. This effect seems specific to nicotine since AT-1001 injections did not affect food SA in the same study. These pharmacological studies support a significant role for α3β4*nAChRs in NA but did not allow to understand the specific role of CHRNA3 and its polymorphisms in the mechanisms of addiction. For this, novel approaches are now needed as discussed below.

Key possible approaches to model CHRNA3 alterations in future studies

A comprehensive analysis of the role of each nAChR subtype in addictive behaviors has been initiated in animal models. As stated above, transgenic rodent models for several nAChR subunits have been developed. Such models mostly reside in the deletion of selective nAChR genes but also on targeted knock-in (KI) gene mutations. Further experiments were carried out either by re-expressing a deleted gene into the mouse brain using stereotaxic injection of a lentiviral vector which carries the missing gene, or by injecting a short hairpin RNA (shRNA) to repress a selective gene expression [START_REF] Avale | Interplay of beta2* nicotinic receptors and dopamine pathways in the control of spontaneous locomotion[END_REF][START_REF] Changeux | Nicotine addiction and nicotinic receptors: lessons from genetically modified mice[END_REF][START_REF] Fowler | Habenular alpha5 nicotinic receptor subunit signalling controls nicotine intake[END_REF][START_REF] Picciotto | Molecular mechanisms underlying behaviors related to nicotine addiction[END_REF]. In this context, several approaches to better understand the role of the specific role of the α3 subunit in NA-like processes could now be implemented. One possible approach would be to use the Gensat's transgenic mice Tg(Chrna3-cre)NO196Gsat, generated with the BAC clone RP23-336O6, where the expression of the Cre-recombinase is driven by the regulatory sequences of the α3 promoter, to over-express this subunit in brain regions where it is constitutively present by injecting viral vectors expressing a floxed copy of Chrna3 and evaluate the consequences of such over-expression on NA phenotypes and mechanisms. Another possibility would be to combine the injections of viral vectors (lentiviruses or AAVs) expressing the Cre-recombinase in target brain areas of conditional mice developed by the European Conditional Mouse Mutagenesis (EUCOMM) program, wherein the Chrna3 sequence is flanked by lox sites [START_REF] Smedley | Cre recombinase resources for conditional mouse mutagenesis[END_REF]. This would result in the local inactivation of α3 expression in specific brain areas or pathways of interest, such as the Hb-IPN system. Then, the expression of specific CHRNA3 variant(s), identified in the human genetic studies, could be locally targeted through lentiviral gene delivery within these pathways on top on the inactivation of native α3 subunit expression, as it has been done for the α5SNP rs16969968 in α5KO mice [START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF].

Another way to achieve a comprehensive study of the role of the α3 subunit could be to generate local silencing of CHRNA3, using lentival vectors expressing either shRNA or, as previously shown [START_REF] Koukouli | Nicotine reverses hypofrontality in animal models of addiction and schizophrenia[END_REF], specific CRISPR against CHRNA3 [START_REF] Peng | Gene editing vectors for studying nicotinic acetylcholine receptors in cholinergic transmission[END_REF].

However, as the CHRNA3 SNP most robustly associated to CPD is a synonymous exonic variant [START_REF] Tobacco | Genome-wide meta-analyses identify multiple loci associated with smoking behavior[END_REF], a KI mouse or rat model could potentially provide important clues.

To our knowledge, studies with human cells have been so far exclusively focused on the CHRNA5 polymorphism. Modeling CHRNA3 alterations would be possible by using the same experimental approaches. Some authors have even been able to develop a kind of transchromosomic mice expressing selective gene clusters [START_REF] Abe | Modification of single-nucleotide polymorphism in a fully humanized CYP3A mouse by genome editing technology[END_REF]. Such humanized models have been obtained by performing SNP modification using genome editing technology in mouse ES cells and fertilized eggs [START_REF] Abe | Modification of single-nucleotide polymorphism in a fully humanized CYP3A mouse by genome editing technology[END_REF]. A similar approach could be applied to model the CHRNA5-A3-B4 gene cluster.

Gene editing in iPSC-derived cells enhances their potential both for basic research and render in vivo studies possible. The CRISPR/Cas9 genome engineering has been recently demonstrated in vivo through the use of engraftable human brain-derived neural stem cells [START_REF] Dever | CRISPR/Cas9 Genome Engineering in Engraftable Human Brain-Derived Neural Stem Cells[END_REF]. The CRISPR/Cas9-genome engineering has also been applied to three-dimensional organoid culture models [START_REF] Bian | Genetically engineered cerebral organoids model brain tumor formation[END_REF]. This approach offers new technical options to model CHRNA3 polymorphism in human neurons in near future. Furthermore, it could provide a unique opportunity to investigate the possibilities of crossregulation between CHRNA3 and CHRNA5, which have been shown to interact at the mRNA level through the formation of RNA-RNA duplexes in humans but not in rodents [START_REF] Solda | In vivo RNA-RNA duplexes from human alpha3 and alpha5 nicotinic receptor subunit mRNAs[END_REF].

CHRNB4

State of the art of the associations identified between CHRNB4 variants, NA and brain function in humans

Unlike the 5 subunit, the nAChR 4 subunit participates in forming ACh binding sites in nAChR pentamers [START_REF] Changeux | Nicotine addiction and nicotinic receptors: lessons from genetically modified mice[END_REF]. CHRNB4, the gene encoding the 4 subunit (HGNC 1964, ENSG00000117971), is also located at 15q25. It harbors ~23400 SNVs, including ~2600 InDels, and is encompassed by 54 CNVs ranging from short tandem repeats to large mosaic rearrangements (see Table 1 for database references). Those larger CNVs have been associated with severe neurodevelopmental delay, although the CHRNB4 gene itself is tolerant to mutations (pLI =0.019). Several CHRNB4 SNPs have been associated with NA. Recently, it was made clear that some of these variants act as enhancers toward other genes, notably through increased methylation of cis CpG (cytosine nucleotide followed by a guanine nucleotide) sites, rather than directly affecting 4*nAChR function. In particular, the CHRNB4 variant rs1948, which was associated with NA, showed one of the largest effect on CHRNA5 expression, and the effect of CHRNA5 rs16969968 was found strongly dependent on rs1948. In that study, the CHRNB4 SNP rs7178270 exhibited similar associations and properties as rs1948. Strikingly, another CHRNB4 variant, rs11636753, was the most significantly associated SNP with NA across five independent cohorts (Hancock et al., 2015).

Haplotype analyses showed that the simultaneous presence of rs11636753-G and rs16969968-A alleles together increased the risk for NA more than each variant alone [p = 3.1 × 10 -12 , OR (95% CI) = 1.32 (1.22-1.43)]. rs11636753, rs7178270 and rs1948 are in moderate LD (r 2 =0.56), suggesting that they are inherited quite independently. rs4887074, which is associated with reduced CHRNB4 expression, has been associated with a reduced NA risk, both alone and in the context of diplotype/haplotype analysis of the cluster [START_REF] Lee | Combined genetic influence of the nicotinic receptor gene cluster CHRNA5/A3/B4 on nicotine dependence[END_REF]. Using the TAD browser ((http://dna.cs.miami.edu/TADKB/) applied to the 15q25 region, we evidenced a close-range loop within CHRNB4. Interestingly, this loop spatially bonds positions 78920000-78930000, which harbor two SNPs associated with NA, and 79000000-79010000, which contain three transcription factor binding sites, one open chromatin region, and one enhancer. These are very likely to further regulate CHRNB4 expression, although the precise effects of these elements on 4*nAChRs remain unclear.

As regards brain function, CHRNB4 rs11637890 has been associated with perseverative responses in general, and errors in particular, in the WCST [START_REF] Zhang | Variation in the Nicotinic Acetylcholine Receptor Gene Cluster CHRNA5-CHRNA3-CHRNB4 and Its Interaction with Recent Tobacco Use Influence Cognitive Flexibility[END_REF]. Unexpectedly, the major allele was responsible for this association. Elements regulating gene expression in the cluster are also found in intergenic regions. In 403 patients with ADHD, vocabulary and planning abilities were tested using the relevant subscales from the Wechsler Adult Intelligence Scale-Revised (WAIS-R) [START_REF] Schuch | Pleiotropic effects of Chr15q25 nicotinic gene cluster and the relationship between smoking, cognition and ADHD[END_REF]. Among eight SNPs from the CHRNA5-A3-B4 cluster analyzed individually and as possible haplotypes, stratified by current smoking status, only rs8023462 (an intergenic SNP between CHRNA3 and CHRNB4) was associated with poorer vocabulary abilities, solely in current tobacco smokers. In line with this finding, CHRNB4 rs12914008 was associated with intra-individual variability in attention (i.e. "unstable attention during the test") in a GWAS of 479 adults suffering from ADHD [START_REF] Alemany | New suggestive genetic loci and biological pathways for attention function in adult attention-deficit/hyperactivity disorder[END_REF]. We could not find any neuroimaging study in humans involving CHRNB4 SNPs. CHRNB4 variants that were associated with either NA or cognitive function were all intron variants, which elicit major changes in gene expression in the 15q25 region. The minor allele of rs11637890 and rs7178270 are both associated with increased CHRNA5 and CHRNA3 expression in the brain. These SNPs have been further associated with methylation changes at trans CpG sites between positions 78856949 and 79053858, thus encompassing the entire CHRNA5-A3-B4 cluster. Methylation was decreased for both CHRNA5 and CHRNA3 CpG sites, in line with the observation of enhanced gene expression, and increased for intergenic CpG sites located downwards the cluster (reverse strand), the impact of which remaining to be elucidated. Conversely, rs1948 and rs8023462 were associated with reduced brain expression of CHRNA3 and CHRNA5, which is consistent with their association to a mostly increased methylation of their CpG sites. rs12914008 is also associated with increased methylation of a CHRNA3 CpG site.

Taken altogether, association studies and GWAS evidence followed by assessment of gene expression and methylation uncovered a key functional role of several CHRNB4 SNPs in the relationship between the CHRNA5-A3-B4 cluster and NA. Along with CHRNA3 (see above) and intergenic variants, the cluster has been legitimately described as a regulome containing multigenic haplotypes, including rs16969968 or not, showing complex associations with NA [START_REF] Barrie | The CHRNA5/CHRNA3/CHRNB4 Nicotinic Receptor Regulome: Genomic Architecture, Regulatory Variants, and Clinical Associations[END_REF].

Two sets of CHRNB4 SNPs showing opposite regulatory features can be identified from gene expression (Fig. 2 for rs8023462) and methylation databases, some data being redundant due to high LD (Fig. 3). All CHRNB4 SNPs reviewed here form a haplotype block (Fig. 3, bottom-right) and, thus, carry the same statistical information although they show distinct regulatory characteristics. This recent line of evidence should be more taken into account in future clinical and preclinical studies, in order to better characterize the pathophysiology of NA and the neurobiology of the nicotinic system.

Preclinical studies of the role of CHRNB4 in NA-like processes

The β4 nAChR subunit can combine with several other subunits to form functional nAChRs but mostly assembles with the α3 subunit. Co-expression of β4 and α3 has been observed for many brain areas where β4 is strongly expressed, notably the mHb and the IPN [START_REF] Gotti | Structural and functional diversity of native brain neuronal nicotinic receptors[END_REF][START_REF] Grady | Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the αβ4* and αβ3β4* subtypes mediate acetylcholine release[END_REF]. β4*nAChRs display specific properties such as slow-rate desensitization and low affinity for nicotine [START_REF] Cachelin | Beta subunits determine the time course of desensitization in rat alpha 3 neuronal nicotinic acetylcholine receptors[END_REF][START_REF] Parker | Neuronal nicotinic receptor beta2 and beta4 subunits confer large differences in agonist binding affinity[END_REF]. Several studies have attempted to study the role of α3β4*nAChRs in NA-like behaviors through pharmacological agonism or antagonism of these receptors (as discussed above, see part 3.2). Notably, the 18-Methoxycoronaridine (18-MC), a potent antagonist which is relatively selective of α3β4*nAChRs, has been proposed as a promising antiaddictive compound as it was shown to decrease SA of several drugs including nicotine in rodents [START_REF] Maisonneuve | Anti-addictive actions of an iboga alkaloid congener: a novel mechanism for a novel treatment[END_REF]. These studies do not bring insights into the discrete role of the β4 subunit in the phenotypes observed.

In this way, the use of mice KO for the β4 subunit gene has provided a good opportunity to better characterize the role of this subunit in nicotine properties.

Amongst several processes related to NA, rodent nicotine withdrawal is associated with the occurrence of somatic signs and hyperalgesia, together with affective manifestations such as elevations in brain reward thresholds measured through intracranial self-stimulation, considered as a form of anhedonia. Interestingly, somatic symptoms, hyperalgesia and reward threshold elevation were found decreased in β4KO mice during spontaneous or mecamylamine-induced nicotine withdrawal (Salas et al., 2004b;[START_REF] Stoker | Role of alpha7-and beta4-containing nicotinic acetylcholine receptors in the affective and somatic aspects of nicotine withdrawal: studies in knockout mice[END_REF], indicating an important role of β4*nAChRs in the several aspects of nicotine withdrawal.

The fact that similar phenotypes were observed in α5KO mice (see part 2.2) suggests that nAChRs containing both α5 and β4 subunits in the mHb-IPN axis are key modulators of nicotine aversion signalling. Yet, a subtler role of these receptors in the balance between rewarding versus aversive nicotine signalling is supported by studies that examined nicotine rewarding effects in several models of transgenic mice for the β4 subunit. First, increased nicotine-evoked mHb currents, stronger aversive responses to nicotine and reduced nicotine intake were observed in transgenic mice overexpressing the β4 subunit ('Tabac' mice) [START_REF] Frahm | Aversion to nicotine is regulated by the balanced activity of beta4 and alpha5 nicotinic receptor subunits in the medial habenula[END_REF]. Interestingly, the latter phenotype was reversed upon viral-mediated expression, in the mHb, of the α5SNP previously associated with heavy smoking in humans, further supporting the interplay of α5 and β4 subunits in mediating nicotine signalling and intake. Moreover, lentiviral-mediated overexpression of β4 or expression of the β4 T374I gain of function variant (associated with decreased risk for smoking [START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF]), in the mHb, was found sufficient to produce nicotine aversion [START_REF] Slimak | Habenular expression of rare missense variants of the beta4 nicotinic receptor subunit alters nicotine consumption[END_REF]. Alterations in the balance between the rewarding and the aversive effects of nicotine were also observed in another model of transgenic mice overexpressing the human CHRNA5/A3/B4 cluster with increased functional α3β4*nAChRs [START_REF] Gallego | Overexpression of the CHRNA5/A3/B4 genomic cluster in mice increases the sensitivity to nicotine and modifies its reinforcing effects[END_REF]. These mice displayed an increased sensitivity to the pharmacological effects of nicotine including higher nicotine-induced hypolocomotion, seizures and mHb activation, but reduced nicotine-induced activation of VTA DA neurons, together with increased nicotine intravenous SA under a progressive ratio schedule.

Thereafter, the role of β4*nAChRs in nicotine signalling and intake was further elucidated in β4KO mice [START_REF] Harrington | Role of beta4* Nicotinic Acetylcholine Receptors in the Habenulo-Interpeduncular Pathway in Nicotine Reinforcement in Mice[END_REF]. In these mice, nicotine intravenous SA was found decreased at low and high doses, and DA neurons of the mesolimbic system showed enhanced sensitivity to nicotine while NAcb basal extracellular levels of DA were increased. Lentivirus-mediated re-expression of β4*nAChRs in the IPN, but not in the mHb, rescued nicotine SA and VTA DA neuron sensitivity to nicotine. These observations suggest a role for IPN β4*nAChRs in regulating the response to nicotine of the reward system despite a lack of direct efferent connection of the IPN with the VTA [START_REF] Antolin-Fontes | The habenulo-interpeduncular pathway in nicotine aversion and withdrawal[END_REF]. They further indicate a distinct contribution of mHb and IPN β4*nAChRs to nicotine signalling. Such a dichotomy between mHb and IPN β4*nAChRs had previously been suggested in rats, with the observation of decreased and increased nicotine intravenous SA following the local blockade of α3β4*nAChRs by the antagonist 18-MC in the mHb and the IPN, respectively [START_REF] Glick | Brain regions mediating alpha3beta4 nicotinic antagonist effects of 18-MC on nicotine self-administration[END_REF]. Recently, β4KO mice were found to show decreased and increased intra-VTA nicotine SA at low and high doses, respectively, in a paradigm particularly suitable for the assessment of both reinforcing and aversive of nicotine. By contrast, Tabac mice displayed increased aversion for high doses of nicotine in the same paradigm. These data further support an important role for β4*nAChRs in the modulation of nicotine sensitivity, and confirm that the strength of nicotine aversive effects varies with β4*nAChR availability. The same study also reports that lentivirus-mediated reexpression of β4*nAChRs in the VTA of β4KO mice enhanced intra-VTA nicotine SA at all doses, while β4*nAChR re-expression in either the MH or the IPN limited intra-VTA nicotine SA at high doses.

Taken together with the data obtained from studies examining the effects of systemic nicotine [START_REF] Frahm | Aversion to nicotine is regulated by the balanced activity of beta4 and alpha5 nicotinic receptor subunits in the medial habenula[END_REF][START_REF] Glick | Brain regions mediating alpha3beta4 nicotinic antagonist effects of 18-MC on nicotine self-administration[END_REF][START_REF] Harrington | Role of beta4* Nicotinic Acetylcholine Receptors in the Habenulo-Interpeduncular Pathway in Nicotine Reinforcement in Mice[END_REF][START_REF] Slimak | Habenular expression of rare missense variants of the beta4 nicotinic receptor subunit alters nicotine consumption[END_REF], these recent observations indicate that MHb and IPN β4*nAChRs play distinct roles in controlling nicotine intake when directly mediating nicotine signalling, but drive a similar control of the VTA response to nicotine when mediating endogenous ACh signalling. Such a comprehensive and subtle role of β4*nAChRs in both ACh and nicotine signalling may explain the complexity of the preclinical information gathered so far about the involvement of these receptors in NA-like behaviors, and may also substantially contribute to the strong clinical implication of the CHRNA5-A3-B4 gene cluster in tobacco smoking.

Recommendation for future research

As described above, preclinical data strongly suggest that the β4 nAChR subunit plays a central role in the nicotine reward/aversion balance, which likely represents a key element for better understanding inter-individual variability in the risk to develop NA [START_REF] De Biasi | Reward, addiction, withdrawal to nicotine[END_REF]. Surprisingly, there is only a very limited number of studies specifically investigating the role of this subunit in NA-related processes, despite the fact that β4 KO mice have been available for many years (Xu et al., 1999b).

The novel insights obtained from recent preclinical work highlight the importance of further deciphering the role of the β4 nAChR subunit in the circuits directly and indirectly connecting the VTA, mHb and IPN. It will be also important to further investigate the role of β4 in the mediation of nicotine action on the brain beyond its rewarding and aversive properties. For this, behavioral models capturing features of NA with stronger face and construct validities should be implemented. A crucial role of the α5 subunit has notably been established in the mediation of nicotine seeking relapse after abstinence in transgenic rats [START_REF] Forget | A Human Polymorphism in CHRNA5 Is Linked to Relapse to Nicotine Seeking in Transgenic Rats[END_REF]. It would be of interest to generate a line of rats in order to assess how the β4 nAChR subunit may also significantly influence different steps implicated in long-term nicotine SA. In particular, β4 may impact the levels of perseverative nicotine seeking during extinction since human genetic studies have linked CHRNB4 to perseverative behavior [START_REF] Zhang | Variation in the Nicotinic Acetylcholine Receptor Gene Cluster CHRNA5-CHRNA3-CHRNB4 and Its Interaction with Recent Tobacco Use Influence Cognitive Flexibility[END_REF].

Additional studies at the cellular level of investigation will also be helpful to better characterize the interplay between the β4 subunit and both the α3 and the α5 subunits in modulating the functional properties of nAChR, especially in the different cell populations of the IPN, where α5 is mostly expressed and where α3 is the main partner of β4 [START_REF] Grady | Rodent habenulo-interpeduncular pathway expresses a large variety of uncommon nAChR subtypes, but only the αβ4* and αβ3β4* subtypes mediate acetylcholine release[END_REF]. This could be achieved by the combination of several technologies, including synaptosome analysis, single cell RT PCR, slice patch clamp and in vivo electrophysiological recording, both in drug naïve conditions, i.e. in response to endogenous cholinergic stimulation, and in response to nicotine.

To our knowledge, there is no preclinical model attempting to model CHRNB4 genetic variation so far.

Considering that all the CHRNB4 variants which have been associated with NA in human studies concern non-exonic regions, it seems complicated to propose straightforward animal models to further investigate their impact on NA. The use of viral vectors for targeted expression of mutated nAChR subunit gene, in specific brain areas of KO mice, has proven to be very efficient to unravel key mechanisms of action of the α5SNP rs16969968 on NA-like behaviors (Besson et al., 2016;[START_REF] Frahm | Aversion to nicotine is regulated by the balanced activity of beta4 and alpha5 nicotinic receptor subunits in the medial habenula[END_REF][START_REF] Morel | Nicotine consumption is regulated by a human polymorphism in dopamine neurons[END_REF]. However, implementing a similar approach may be compromised in the case of non-exonic SNPs which would require to encompass the gene in its entirety. Other methodologies are conceivable, like the use of efficient genome editing tools in rodents, including in rats. Yet, several limitations, such as a possible lack of similarity in the CHRNA5, CHRNA3, and CHRNB4 gene cross-regulation mechanisms between humans and rodents, will first need to be assessed and overcome whenever it will be feasible, as discussed above for the modelling of CHRNA5 intronic variants (see part 2.3).

General discussion and conclusion

General considerations

We are providing here a comprehensive summary of the human genetic and preclinical findings linking polymorphisms in a cluster on chromosome 15q to NA. The genetic architecture of this cluster, encompassing multiple SNPs in the CHRNA5, CHRNA3 and CHRNB4 genes, is complex, and includes substantial LD. This leads to the presence of haplotypes in many human populations. Recent advances in human genomics indicate that the CHRNA5-A3-B4 cluster lies in a particularly rich regulatory region. Most SNPs associated with NA are synonymous and/or non-exonic variants. The role of the coding polymorphism rs16969968 has received a lot of attention, and is reviewed [START_REF] Maskos | The nicotinic receptor alpha5 coding polymorphism rs16969968 as a major target in disease: Functional dissection and remaining challenges[END_REF] . The magnitude of association between this SNP and NA has been shown to depend strongly on other variants reviewed here, in line with the fact that SNPs showing significant eQTLs in the brain mostly modulate CHRNA5 expression, whichever their harboring gene is. Thus, in the cluster, sources of variability include haplotypes, enhancers/repressors of gene expression acting across genes, and changes in chromatin states (which can either facilitate or limit gene transcription). Using the TAD browser (http://dna.cs.miami.edu/TADKB/) applied to the 15q25 region (based on very recent evidence about the 3D organizational structure of DNA, at the kilobasepair level [START_REF] Liu | TADKB: Family classification and a knowledge base of topologically associating domains[END_REF])), we observed that the CHRNA5, CHRNA3 and CHRNB4 are clustered in a region accounting for 25 possible variations of chromatin states and expressing over 50 long non-coding RNAs, which are important regulators of gene expression. There was no evidence of long-range DNA interactions in this region, but a close-range loop within CHRNB4, this gene appearing to fulfil the strongest regulatory role in the cluster according to the information available so far. Interestingly enough, SNPs that alter CHRNA5 expression also commonly alter that of the mRNA antisense gene RP11-650L12.2 (which represses CHRNA5 expression), in the same direction, evoking a negative feedback loop. Repeated associations between rs16969968 and NA may imply several parameters of the NA phenotype, which is often measured by the FTND. The FTND notably measures the "delay between wake-up and first cigarettes", a proxy for withdrawal avoidance, and CPD, along with other variables reflecting other aspects of NA including psychosocial ones. Such repeated associations between rs16969968 and NA could also reflect alterations in the aversive effects of nicotine [START_REF] Jensen | A CHRNA5 Smoking Risk Variant Decreases the Aversive Effects of Nicotine in Humans[END_REF], which may impair the ability to self-regulate/titrate nicotine intake [START_REF] Macqueen | Variation in the α 5 nicotinic acetylcholine receptor subunit gene predicts cigarette smoking intensity as a function of nicotine content[END_REF].

Overall, the wide range of genetic associations between the CHRNA5-B4-A3 cluster and NA suggests that different SNPs and SNP combinations are associated with different components of the NA syndrome. Component of the different stages of the NA cycle is only partially overlapping [START_REF] Sharp | Neurogenetic determinants and mechanisms of addiction to nicotine and smoked tobacco[END_REF]. A similar complexity has been observed in the preclinical literature, where rs16969968 has been associated with various functional consequences at the molecular and behavioral levels. The current review focused on frequent genetic polymorphisms. However, rare variants (i.e. with minor allele frequencies <5%), such as the missense mutations CHRNB4 T375I and T91I [START_REF] Haller | Rare missense variants in CHRNB4 are associated with reduced risk of nicotine dependence[END_REF] and CHRNA3 RH7 [START_REF] Clark | Deep Sequencing of Three Loci Implicated in Large-Scale Genome-Wide Association Study Smoking Meta-Analyses[END_REF], have been associated with reduced NA risk. Since part of these associations were found at the gene set level, meaning that these variants were involved in statistically significant association in combination with other variants in the same gene, and since they are rare, they likely mediate only a low fraction of NA variability in human populations, even though they may leverage significant functional consequences (see part 4.2).

Refinement and broadening of the neuropsychological phenotyping implemented to assess NA is now needed to better characterize the impact of CHRNA5, CHRNA3 and CHRNB4 polymorphisms on constructs even more relevant to addiction [START_REF] Yucel | A transdiagnostic dimensional approach towards a neuropsychological assessment for addiction: an international Delphi consensus study[END_REF], especially since the most robust genetic associations with NA only explain ~1% variance of the phenotypeleaving much NA heritability missing [START_REF] Sharp | Neurogenetic determinants and mechanisms of addiction to nicotine and smoked tobacco[END_REF]. Along with the use of polygenic risk scoring [START_REF] Belsky | Polygenic risk and the developmental progression to heavy, persistent smoking and nicotine dependence: evidence from a 4-decade longitudinal study[END_REF] and data mining [START_REF] Barrie | The CHRNA5/CHRNA3/CHRNB4 Nicotinic Receptor Regulome: Genomic Architecture, Regulatory Variants, and Clinical Associations[END_REF][START_REF] Civelek | Systems genetics approaches to understand complex traits[END_REF], future human genetic studies should notably feed on preclinical insights and, for instance, assess the associations between the cluster and relapse-related parameters more carefully. In addition, various functions strongly linked to the development of addiction should deserve more attention, including reward-processing, stress sensitivity, self-and cognitive-control.

There is also a strong need to develop novel preclinical models. First, the cluster should be considered as a whole to evaluate its impact with greater face validity, finally taking into consideration the crossregulation between the three nicotinic genes. For this, a combination of several approaches can be envisaged, as discussed throughout the review, including the use of iPSC. Such novel approaches should also be considered to finally provide a model for CHRNA3 alterations, which has been crucially lacking so far. Finally, as for clinical studies, future animal investigations should consider implementing behavioral models with stronger translational value for NA, as it has started to be developed in a few studies which have made efforts to assess behaviors reflecting a loss of control over nicotine intake [START_REF] Caille | Modeling nicotine addiction in rats[END_REF][START_REF] Clemens | Behavioral and neural substrates of habit formation in rats intravenously self-administering nicotine[END_REF][START_REF] Cohen | Robust escalation of nicotine intake with extended access to nicotine self-administration and intermittent periods of abstinence[END_REF][START_REF] Gilpin | Nicotine vapor inhalation escalates nicotine self-administration[END_REF].

Gender background considerations

Gender effects have consistently been evidenced regarding NA, with men exhibiting more persistent and heavier smoking than women, although the sex ratio in the prevalence of current smoking has been closing to one in the recent years [START_REF] Peters | Do smoking habits differ between women and men in contemporary Western populations? Evidence from half a million people in the UK Biobank study[END_REF]. Such gender effects are especially important in terms of mental health comorbidity usually associated with NA, gender, or both. Furthermore, men and women do not show the same sensitivity to the toxic effects of tobacco. Yet, a few studies have specifically examined the gender impact on the influence of nAChR genetics on smoking outcomes. First evidence came from a twin study, showing that the genetic vulnerability in smoking initiation was higher for women than men, with an opposite pattern regarding smoking persistence; along with further gender differences in environmental influences on smoking [START_REF] Li | A meta-analysis of estimated genetic and environmental effects on smoking behavior in male and female adult twins[END_REF]. In line with these findings, CHRNA5 rs16969968 has been shown to interact with childhood adversity in risk to develop NA in males only, even if both genders had increased NA prevalence in the case of childhood adversity [START_REF] Xie | Childhood adversity increases risk for nicotine dependence and interacts with alpha5 nicotinic acetylcholine receptor genotype specifically in males[END_REF]. Finally, using recent polygenic risk scoring and genetic correlation methods, a significant interaction between CHRNA5 and NR3C2, a gene encoding glucocorticoid receptors (which play a key role in the stress response mediated by the hypothalamopituitary axis) has become evident [START_REF] Schmitz | Examining sex differences in pleiotropic effects for depression and smoking using polygenic and gene-region aggregation techniques[END_REF]. Overall, it seems that the gender variable, in genetic studies on NA, has been more considered as a cofactor rather than a significant parameter for refining genetic associations in specific subpopulations, despite the clinical and biological relevance to do so. Further research, both in humans and in preclinical models, should consider analysing males and females separately.

Conclusion

Associations between the chromosome 15 CHRNA5-A3-B4 cluster and NA are robust. However, the current review emphasizes recent evidence which highlighted that these associations rely on a more complex, interdependent pattern of SNPs in high LD, mostly affecting gene expression levels, especially those of CHRNA5. In our opinion, methodological limitations of the studies conducted so far, at both the preclinical and clinical level, reflect the need to better define and model the genetic context in which the role of nAChR subunit genes and their variants are studied. Refined neuropsychological assessment for the different stages of the NA cycle should also be considered in future studies, both in patient cohorts and animal models. Table 2: Single Nucleotide Polymorphisms (SNPs) reliably associated with nicotine addiction and the width of their functional impact on gene expression, known modulation of DNA transcription. SNP id, SNP name according to the dbSNP database v.153 ; eQTL, expression quantitative trait loci (i.e. associations with altered gene expression) ; mQTL, methylation quantitative trait loci (i.e. associations with altered gene methylation) in samples from middle-aged individuals. All mQTL sites were cis, meaning that increased methylation is associated with decreased DNA transcription. RegulomeDB scores read as listed. N/A, not available. a cluster refers to CHRNA5-A3-B4 genes and RP11-650L12.2 and brain to brain regions as defined by GTEx portal (https://gtexportal.org/home/), except for the spinal chord. b CpG islands located 50 kbp up-or downstream CHRNA5, CHRNA3 or CHRNB4 were considered. Polymorphisms associated with Nicotine Addiction in the cluster. LD is expressed as minor alleles correlation (r 2 ). The more red the color, the higher the correlation.

Figure 1 :Figure 2 :

 12 Figure 1: Scheme representing the expression of CHRNA5-B4-A3 cluster genes in the brain (GTEx database V. 8) Figure 2: Scheme representing the brain eQTLs for rs680244, rs1051730 and rs8023462 Single Nucleotide Polymorphisms (SNPs) from the cluster. All normalized effect sizes (NES) are below zero, indicating downregulation of gene expression. RP11-650L12.2 encodes an antisense RNA that represses CHRNA5 expression. Only significant associations are displayed.

Figure 3 :

 3 Figure 3: Linkage Disequilibrium (LD) map in European populations of Single Nucleotide

Figure

  Figure(s) 1Click here to download high resolution image

Table ( s)

 (

	Database	URL	Information provided
			National Institutes of Health (NIH)
	ENSEMBL v. 98	http://grch37.ensembl.org/Homo_sapiens	information hub for gene structure,
			mapping, function and variants
	Single Nucleotide Polymorphism Database (dbSNP) v. 151	https://www.ncbi.nlm.nih.gov › snp	ENSEMBL database for SNPs
	Genotype-Tissue (GTEx) portal v. 8 (Baran et al., Expression 2015)	https://gtexportal.org/	Tissue-and variant-specific gene expression data
	Methylation quantitative trait loci database (mQTLdb) (Gaunt et al., 2016)	http://www.mqtldb.org	Genome-wide DNA methylation analysis of 1,000 mother-child pairs at serial time points across the life-course (ARIES)
	Regulome database (regulomeDB) (Boyle et al., 2012)	http://www.regulomedb.org	DNA features and regulatory elements in non-coding regions
	Topologically Associating Domain Knowledge Base (TADKB) (Liu et al., 2019)	http://dna.cs.miami.edu/TADKB/	Exploring topologically associating domains (TADs)
	DatabasE of genomiC varIation and		Gene tolerance to mutations
	Phenotype in Ensembl Resources (DECIPHER) v. Humans using	https://decipher.sanger.ac.uk	[score of tolerance to loss-of-function mutations (pLI), level of
	9.30 (Lek et al., 2016)		haploinsufficiency]

Table 1 :

 1 Main databases used in the present review for gene function and properties and variant frequency and impact.

Table 2 :

 2 Single Nucleotide Polymorphisms (SNPs) reliably associated with nicotine addiction and the width of their functional impact on gene expression, known and modulation of DNA transcription. SNP id, SNP name according to the dbSNP database v.153 ; eQTL, expression quantitative trait loci (i.e. associations with altered gene expression) ; mQTL, methylation quantitative trait loci (i.e. associations with altered gene methylation) in samples from middleaged individuals. All mQTL sites were cis, meaning that increased methylation is associated with decreased DNA transcription. RegulomeDB scores read as listed. N/A, not available. a cluster refers to CHRNA5-A3-B4 genes and RP11-650L12.2 and brain to brain regions as defined by GTEx portal (https://gtexportal.org/home/), except for the spinal chord. b CpG islands located 50 kbp up-or downstream CHRNA5, CHRNA3 or CHRNB4 were considered.

	.6123	0.7511	0.8284	19	9	N/A

Table 3 :

 3 RegulomeDB score classification (from http://www.regulomedb.org). eQTL, expression quantitative trait loci; TF, transcription factor.

All were trans methylation sites. c rs11637890 and rs7178270 were the only ones to be associated with upregulation of CHRNA3, CHRNA5 and RP11-650L12.2.

Author contribution

All authors listed have made substantial, direct and intellectual contribution to the work, and approved it for publication.

Conflict of interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Romain ICICK has given a lecture for Pierre Fabre Inc. in 2019.

Credit author statement

All authors listed have made substantial, direct and intellectual contribution to the work, and approved it for publication.