For the field of ND genetics, rare variant investigation started with the nAChR subunit genes, which not only are biologically important but also have yielded the most replicable results in both GWASs and candidate gene association studies, as presented above. Wessel et al.133 first examined the contribution of common and rare variants in 11 nAChR genes to FTND in 448 EA smokers, which revealed significant effects of common and rare variants combined in CHRNA5 and CHRNB2, as well as of rare variants only in CHRNA4. Xie et al.134 followed up on the CHRNA4 finding by sequencing exon 5, where most of the nonsynonymous rare variants were detected, in 1,000 ND cases and 1,000 non-ND controls with equal numbers of EAs and AAs. They discovered that functional rare variants within CHRNA4 may reduce ND risk. Also, Haller et al.135 detected protective effects of missense rare variants at conserved residues in CHRNB4. They examined in vitro the functional effects of the three major association signal contributors (i.e., T375I and T91I in CHRNB4 and R37H in CHRNA3), finding that the minor alleles of
