In this study, we investigated the distribution of rare variants in CHRNA4 in more than 2000 ND cases and comparison subjects. Compared to cases, controls had a significantly higher frequency of rare nonsynonymous variants located in the exon 5 region encoding the cytoplasmic loop of the α4 nAChR (FET p=0.009; association test p=0.009, OR=0.43, 95%CI=0.23-0.81; WSM p=0.014). Functional in-silico analysis (PolyPhen) further demonstrated that the comparison subjects carried significantly more potentially damaging rare nonsynonymous variants than the cases (FET p=0.005; association test p=0.008, OR= 0.29, 95%CI=0.11-0.72; WSM p=0.005), suggesting that some rare functional variants exert a protective effect against ND. In addition, although we observed that comparison subjects had a higher frequency of rare nonsynonymous variants in the exon 5 cytoplasmic loop region in both EAs and AAs, the difference was significant only in AAs.
The basic structure of different nAChR subunits is quite similar, but the large cytoplasmic loop is highly divergent, which accounts in part for the functional differences and differences in the distribution (39
), assembly (40
), and other characteristics of nAChR subunits. This cytoplasmic loop is also important for interaction with intercellular proteins, which could increase the stability of α4β2 nAChRs (41
), as well as post-translational modification of the α4 subunit (42
). Our imaging study showed that for nonsmokers, specific rare variants in the CHRNA5
gene region encoding the cytoplasmic loop could increase the availability of α4β2 nAChRs in multiple brain regions. In addition, a nonsynonymous rare variant seems to have a bigger effect than the synonymous rare variants (). One possible explanation for the difference in nAChR availability observed in the subject with the nonsynonymous rare variant is that increased availability of α4β2 nAChRs compensates for damaged protein function. Alternatively, an increase in stability of the mutated α4 protein could increase baseline nAChR function, providing protection against further increases by the nicotine in tobacco. The results from this imaging study are preliminary, and these hypotheses remain to be tested. For smokers carrying nonsynonymous rare variants, Chronic exposure to nicotine up-regulates α4β2 nAChRs (43
), and this may mask the effect of rare variants. It is hard to generalize based on these findings however, because it is plausible that each specific variant could have a unique effect on function. These findings should therefore be viewed as illustrative of possible effects rather than predictive of all.
Animal experiments have demonstrated that activation of α4β2 nAChRs is involved in the development of ND (5
). Therefore, current understanding is consistent with the notion that nonsynonymous rare variants s in the cytoplasmic loop of the α4 subunit may have a protective effect against ND. Evaluation of the role of these rare variants in the α4 subunit in nAChR function by making point mutations in the functional consequence using in vitro
or in vivo
models will help identify the structure-activity changes resulting from these changes.
Recently, Wessel et al. resequenced 10 nAChR subunit genes and found a significant association between rare variants in CHRNA4
and FTND score (44
). However, due to the small sample size in that study (N=430), they observed a total of only 21 rare variants, including 5 nonsynonymous rare variants in CHRNA4
. Because they were unable to perform more detailed analyses on nonsynonymous variants, the results of their study were not conclusive. In contrast, we used a two stage case-control design. When results from the two stages were combined, a total of 169 rare variants of 59 types were observed in exon 5. Among them, we found a total of 63 nonsynonymous rare variants of 27 types. This sample size proved to be adequate to provide significant evidence that functional nonsynonymous rare variants located in the cytoplasmic loop of the α4 subunit play a protective role in ND, especially in the AA population.
Among the 20 types of missense rare variants we detected in the exon 5 cytoplasmic loop region, 12 were observed only once (MAF=0.025%), with the most frequent missense rare variants having an MAF of 0.25%. Although the joint analysis of all rare variants provided sufficient power to detect differences between ND cases and controls, this approach assumes the same direction of effect (protective, deleterious or neutral) for all of the variants on ND risk, otherwise it could adversely affect power (45
). To address this concern, we conducted a bioinformatic analysis to predict the effect of each nonsynonymous rare variant on protein function. This analysis suggested that eight of the 20 types of rare variants were predicted to be damaging to α4 subunit function. Restricting the comparisons to the aggregate effect of these eight variants confirmed the results of the analysis of all 20 nonsynonymous rare variants.
Even though the proportion of nonsynonymous variants in the exon 5 cytoplasmic region did not differ significantly between EA ND cases and comparison subjects, we identified a missense rare variant, p.Pro451Leu, that was significantly more frequent in controls and may have a protective effect with respect to ND risk in that population. p.Pro451Leu is predicted by PolyPhen to be damaging, and was exclusively observed in EA subjects. In the discovery sample, this variant was found in 3 controls and 1 case; in the test sample, it was observed in 5 comparison subjects and 1 case. The difference is marginally significant (FET p=0.05).
A characteristic of our sample is a high level of comorbidity with other kinds of substance dependence and ND, a consequence of our recruitment methods and also of our detailed phenotypic assessment protocol. Notwithstanding this fact, we note that we have replicated ND findings first reported by other groups (for example, regarding the CHRNA3/A5/B4
gene cluster (46
)), and our findings have been replicated by others (for example, ANKK1/TTC12
)). This holds not only for association findings, but for linkage findings as well. Thus, existing evidence supports our hypothesis that ND in this sample is similar to ND in other samples. Of the 33 subjects from the comparison group who carried missense rare variants in the exon 5 cytoplasmic loop region (two of whom had double missense rare variants at different loci), two were alcohol dependent, and one was cocaine dependent. All of the other individuals were healthy, with no psychiatric disorders or substance dependence. All ND cases who carried missense rare variants in the cytoplasmic loop region were comorbid with alcohol, cocaine or opioid dependence.
In conclusion, we observed that ND cases had significantly fewer rare nonsynonymous variants in the cytoplasmic loop of the α4 nAChR subunit than comparison subjects, especially among AAs. We hypothesize that these rare variants have protective effects against ND. Further genetic and functional studies are needed to test this hypothesis.