This study reports a systematic screen of the known transcribed and core promoter regions of the human CHRM5. We identified seven variants within the screening regions, three by DHPLC and four additional variants through public sequence variation repositories. Only polymorphisms identified by DHPLC were polymorphic in our population. Of all of the markers that were polymorphic within the Australian Caucasian population, only rs7162140 was deemed to have a mior allele frequency of sufficient magnitude to be examined in our population.

Our results support a role for the rs7162140 T-allele in risk of cannabis dependence and tobacco dose. We observed a 26.8% increase in cigarette consumption in carriers of the T-allele, equating to 20.1 cigarettes per week (nominal p = 0.01). Therapeutic intervention leading to a reduction in tobacco consumption at this level would have considerable public health implications. Carriers of the T-allele were also found to have an increase in their risk of cannabis dependence by nearly 3-fold. These data complement animal models that have shown the M5R is important in modulating the reward and withdrawal processes associated with opiate and cocaine use. We did not observe association between measures of alcohol dependence or dose. Although our data for tobacco and cannabis fit a dominant mode of inheritance, we did not have sufficient power to distinguish between the dominant and heterosis-mode of inheritance for this allele. Moreover, we would exercise caution in the over interpretation of these data. We have not applied correction for multiple testing and therefore would support further studies to confirm or reject these findings.

One strength of this study is the theory-driven approach to candidate gene selection. From a purely biological perspective, the M5R is a strong candidate through its role in the control of the duration of dopamine release [1,6,7]. Mice lacking a functional M5R have reduced reward and withdrawal responses following morphine [8] and cocaine [9] administration. We examined the role of variation in the CHRM5 gene and dependence on three drugs of addiction, namely tobacco, alcohol and cannabis. The dose and dependence variables that we have examined are derived from phenotypic constructs that are common to other studies such that cross-study investigation can be facilitated.

We have attempted to improve the probability of identifying causative genetic loci that influence risk of drugs of addiction by hypothesis-based candidate polymorphism choice. Since it is not feasible to screen and study the entire genomic sequence within the gene locus, we chose to employ the VAPSE (or Variants Affecting Protein Structure or Expression) approach [10], whereby analysis is restricted to gene sequences coding for domains which are likely to affect protein structure or expression. Under the VAPSE paradigm, we predict that any functional variation is measured directly and therefore we exclude confounding associated with linkage disequilibrium. However, as a precaution analysis was restricted to individuals of European ancestry to lessen genetic and non-genetic confounders specific to ethnicity. The rs7162140 polymorphism is transcribed in the CHRM5 message. The polymorphism is located within the 5' untranslated region of CHRM5. The specific function of this polymorphism on gene expression or function is unknown and requires further investigation. We acknowledge that our approach and chosen screening region cannot account for the influence of long-range regulatory elements or that of epigenetic modifications that alter the function of the CHRM5 gene. However, consistent with our study design, a recent report indicates that polymorphisms that alter gene expression are strongly biased towards the core and proximal promoter elements [11].

Studies on rats deficient in M5R have shown that midbrain M5R mediate the duration of forebrain dopamine transmission and the maintenance of dopamine-related reward [6,7]. Moreover, mice lacking a functional M5R were shown to have reduced reward and withdrawal responses following morphine [8] and cocaine [9] administration. M5R -/- mice show reduced self administration of cocaine compared to M5R +/+ controls [9]. Our data show that polymorphisms within the CHRM5 gene locus increase tobacco use, suggesting increased reward and withdrawal following substance use in the subset containing the rs7162140 T-allele. Combined with data from animal models we would hypothesise that the rs7162140 T-allele, or an allele in linkage disequilibrium enhances the functional capacity of the M5R compared to the C-allele. Daily tobacco dose is associated with nicotine dependence in our sample (Student's T-test; mean cigarettes per week of current smokers, subsyndromal nicotine dependent = 55.7, nicotine dependent = 129.9; p < 0.0001). We observe a statistically non-significant increase in nicotine dependence in this sample (Dominant model OR = 1.6 (95%CI 0.9–2.8). One reason for the lack of association for the categorical dependence traits may be the limitation of sample size (nicotine dependence n = 273, cannabis dependence n = 85).

Our overall hypotheis was that CHRM5 would influence tobacco, alcohol and cannabis dependence. Our data does not support the role of CHRM5 as a generalised risk factor for drug dependence. Instead our data suggest specific effects for tobacco and cannabis but not alcohol. One explanation for this phenomenon can be drawn from the proposed action of the nicotine, alcohol and cannabis within the mesolimbic system. In the mesolimbic system, both nicotine (the active-compound in tobacco) and delta9-tetrahyrocannabinol (delta9-THC) (the active-compound in cannabis) act predominantly through receptor-mediated pathways. Specifically, nicotine operates through activation of nicotinic acetylcholine receptor (nAChR) complexes; delta9-THC operates through activation of the neural cannabinoid receptor, CB1R. As with M5R, the nAChR are co-expressed on dopamine neurones of the SNc and VTA [12]. The CB1R are present in the VTA and are co-expressed with dopamine neurones of the nucleus accumbens [13]. Interestingly, CB1R knockout mice show similar reduced reward and withdrawal to M5R knockout mice [14]. Clearly, the role of nicotine and delta9-THC in dopaminergic neurotransmission is complex, with receptors also expressed on neurones that are afferent to the mesolimbic dopamine-containing neurones. However, the co-localisation and direct ligand action on dopaminergic neurones is common to nicotine and delta9-THC. Conversely, although ethanol administration stimulates neurones within the VTA, ethanol is thought to elicit its effect through a non-ligand interaction with receptor proteins on mesolimbic-afferent neurones containing GABA_A (gamma-aminobutyric acid type-A) and NMDA (N-methyl-D-aspartate) receptors [4]. This mode of action, specifically the predominant role of ethanol in neurones afferent to the mesolimbic dopamine/M5R-containing neurones may in part explain the lack of association seen between CHRM5 and alcohol dependence and dose in this study.

In addition to the role in substance dependence, CHRM5 may also be important in other psychiatric disorders where dysregulation of dopaminergic neurotransmission has been implicated. A recent study examined the role of CHRM5 in schizophrenia. The marker rs623941, located within intron 3, 2.3 kb 5' of exon 4, was not associated with schizophrenia [15]. A second marker in the alpha7-cholinergic nicotinic acetylcholine receptor (CHRNA7) locus was not linked with schizophrenia. When examined together the authors reported linkage with schizophrenia. However, the CHRM5-CHRNA7 two-marker haplotype extends over 3 Mb of genomic DNA and is unlikely to describe an ancestral chromosome block. Consequently, the linkage of the CHRM5-CHRNA7 haplotype with schizophrenia is unlikely to represent evidence due to linkage disequilibrium with functional markers in either gene.