Rare coding variants in CHRNB3 associate with reduced daily cigarette smoking across ancestries

Abstract

Rare coding variants that alter protein function and confer beneficial health effects can suggest potential drug targets. CHRNB3 encodes the β3 subunit of nicotinic acetylcholine receptors that bind nicotine and mediate its action in the brain. Here we report an exome-wide association study of number of cigarettes smoked per day (cig per day) in 37,897 current smokers from the Mexico City Prospective Study. We identify a deleterious missense variant in CHRNB3, p.Glu284Gly, that associates with a significant reduction in daily cigarette consumption. The missense variant is enriched in people of Indigenous Mexican ancestry but rare in other ancestries. We further identify a predicted loss-of-function variant in CHRNB3 that significantly associates with reduction in number of smoked cigarettes per day in participants of Japan Biobank. This variant is enriched in people of East Asian ancestry but is rare in other ancestries. Finally, we find that rare deleterious missense and predicted loss-of-function variants in aggregate associate with a reduction in the number of smoked cigarettes per day in individuals of European ancestry from the UK Biobank. Our results suggest that loss of function of CHRNB3 significantly associates with daily cigarette smoking, proposing β3 inhibition as a potential therapeutic strategy for nicotine addiction.

Fig. 1. Study design.

The flow chart summarizes the study design. The primary study is a genetic association analysis of cigarettes per day in 37,897 individuals from the MCPS cohort. The secondary studies include genetic association analyses of cigarettes per day in MCPS (independent sample), UK Biobank, and Japan Biobank. In addition to cigarettes per day, other smoking phenotypes (ever smoker and heavy smoker) were analyzed, which are described in the manuscript. ExWAS Exome-wide association study, GWAS Genome-wide association study, MCPS Mexico City Prospective Study.

Fig. 2. ExWAS Manhattan plots.

Manhattan plots showing results from exome-wide rare-variant association studies of cigarettes per day in the MCPS (a) and UKB (b). Number of individuals analyzed: MCPS – 37,897; UK Biobank – 133,133. Only aggregate level associations are plotted. The X axes correspond to chromosomal base pair start positions of individual genes and Y axes correspond to P values (expressed in negative log scale) from burden association tests performed using REGENIE. The dotted line corresponds to a statistical significance threshold of P = 2.6e-6. The direction of the triangle represents the direction of association (downward–cig per day decreasing; upward–cig per day increasing); MCPS Mexico City Prospective Study. Statistical test used: burden association test implemented in REGENIE software. Two-sided tests were performed with adjustment for multiple tests using Bonferroni method adjusting for the number of unique genes tested (n genes = 18,955).

Fig. 3. CHRNB3 p.Glu284Gly characterization.

a Illustrates the distribution of rare pLOF, deleterious missense and other missense variants (differentiated using colors) in CHRNB3 identified in the MCPS cohort. The X axis corresponds to the amino acid position on canonical transcript. The Y axis corresponds to P values of variant associations with cigarettes per day in the MCPS. The thin grey bar represents the full protein, and the thick colored bars correspond to protein domains. M1 to M4 are parts of the transmembrane domain of the β3 subunit. The dotted grey line corresponds to P = 0.05 and the red dotted line corresponds to P = 5e-7, exome-wide association threshold, multiple-tests-adjusting for number of unique rare variants tested across the whole exome. The size of the circles corresponds to different minor allele frequency (MAF) thresholds. b displays the MAF of Glu284Gly in MCPS and other databases (UKB, gnomAD, and All of Us) in five continental ancestries: Europeans (EUR), admixed Americans (AMR), Africans (AFR), East Asians (EAS), and South Asians (SAS). In the MCPS, using fine-scale ancestry estimation, we further report the Indigenous Mexican (IMX) specific MAF. c displays box plots overlaid on a scatter plot of cigarettes per day distribution across three genotype groups of p.Glu284Gly. Data are presented as individual data points with box plots showing median (center line), first and third quartiles (box bounds), and whiskers extending to 1.5 times the interquartile range. n = 37,206 participants (homozygous reference), n = 687 participants (heterozygous), n = 4 participants (homozygous variant). Statistical test used: linear regression adjusted for ancestry, age, and sex. Two-sided test. No multiple tests adjustment.

Fig. 4. CHRNB3 summary forest plots.

The figure displays forest plots summarizing the rare (a) and common variant associations (b) of CHRNB3 with cigarettes per day in diverse ancestries. The rare-variants plot (a) displays an allelic series of associations of rare missense and pLOFs from MCPS, UKB, and BBJ. N carriers represent total number of heterozygous and homozygous carriers for effect allele. The effect size in standard deviation units along with 95% confidence intervals are plotted. The dashed vertical line corresponds to null effect (β = 0). IMX Indigenous Mexicans, EUR Europeans, EAS East Asians, UKB UK Biobank, MCPS Mexico City Prospective Study, BBJ Biobank Japan. Statistical tests used: linear or logistic regression models, testing association with individual variant or aggregate of variants, adjusted for ancestry, age, and sex for MCPS; Two-sided tests. No multiple tests adjustment.

Fig. 5. CHRNB2 vs CHRNB3.

The figure displays forest plots contrasting the rare-variant associations of CHRNB3 vs CHRNB2 for binary (a) and quantitative (b) smoking phenotypes. Aggregate level associations based on the rare deleterious missense variants for CHRNB3 (which showed the strongest association with its primary phenotype, cigarettes per day, in the MCPS) and rare pLOFs plus deleterious missense variants for CHRNB2 (which showed the strongest association with its primary phenotype, heavy smoker, in the UKB). The “Ref/Het/Hom” counts refer to number of homozygous for reference allele, heterozygous, and homozygous for effect allele respectively. The effect size in standard deviation units along with 95% confidence intervals are plotted. The dashed vertical line corresponds to null effect (β = 0). IMX Indigenous Mexicans, EUR Europeans, UKB UK Biobank, MCPS Mexico City Prospective Study. Statistical tests used: linear regression for cigarettes per day; logistic regression for smoking status phenotypes. All models adjusted for ancestry, age, and sex. Two-sided tests. No multiple tests adjustment.

Fig. 6. GWAS Manhattan plots.

Manhattan plots showing results from genome-wide common variant association studies of cigarettes per day in the MCPS (a) and UKB (b). The X axes correspond to chromosomal base pair start positions of individual variants and Y axes correspond to P values (expressed in negative log scale) from variant association tests performed using REGENIE. The dotted line corresponds to the genome-wide statistical significance threshold of P = 5e-8. UKB UK Biobank, MCPS Mexico City Prospective Study. Statistical test used: linear regression. Two-sided tests with no multiple tests adjustment, beyond genome-wide significant threshold.