In the U.S., more than 80% of African-American smokers use mentholated cigarettes, compared to less than 30% of Caucasian smokers. The reasons for these differences are not well understood. To determine if genetic variation contributes to mentholated cigarette smoking, we performed an exome-wide association analysis in a multiethnic population-based sample from Dallas, TX (N = 561). Findings were replicated in an independent cohort of African Americans from Washington, DC (N = 741). We identified a haplotype of MRGPRX4 (composed of rs7102322[G], encoding N245S, and rs61733596[G], T43T), that was associated with a 5-to-8 fold increase in the odds of menthol cigarette smoking. The variants are present solely in persons of African ancestry. Functional studies indicated that the variant G protein-coupled receptor encoded by MRGPRX4 displays reduced agonism in both arrestin-based and G protein-based assays, and alteration of agonism by menthol. These data indicate that genetic variation in MRGPRX4 contributes to inter-individual and inter-ethnic differences in the preference for mentholated cigarettes, and that the existence of genetic factors predisposing vulnerable populations to mentholated cigarette smoking can inform tobacco control and public health policies.

An exome-wide association study revealed a significant association between menthol cigarette use and coding variants in MRGPRX4, which encodes a G-protein coupled receptor expressed in sensory neurons. The variant haplotype is found only in populations of African ancestry, and encodes a receptor that displays reduced agonism by Nateglinide. Our findings indicate genetic variation contributes to the high rate of menthol cigarette use in African Americans.

Funding: This work was supported by the National Institute on Deafness and Other Communication Disorders ( https://www.nidcd.nih.gov ) awards NIHOD2013427 under subcontract HHSN263201300011C (J.K.) and Z1A-000046-16 (D.D., D.R., E.S.), by National Center for Advancing Translational Sciences/NIH ( https://ncats.nih.gov ) under award Number UL1TR001105 (J.K.), by National Institute of Mental Health Award U01MH104974 (B.R., K.L.), the Michael Hooker Distinguished Professorship (B.R.), by a Pharmaceutical Research and Manufacturer’s Association ( https://www.phrma.org ) Predoctoral fellowship (K.L.), and by National Institute of Neurological Disorders and Stroke ( https://www.ninds.nih.gov ) award F31 NS093917 (R.O.). Additional support was provided by the National Institutes of Health, Office of the Director and National Institute on Drug Abuse ( https://www.drugabuse.gov ) Grant RC1-DA028710 (T.K.), and by the U.S. Food and Drug Administration https://www.fda.gov ) through funds obtained under the Family Smoking Prevention and Tobacco Control Act (D.D., D.R.). The content was not reviewed by the Food and Drug Administration, but underwent the standard manuscript clearance process for scientific papers published from the NIH intramural research program. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

This is an open access article, free of all copyright, and may be freely reproduced, distributed, transmitted, modified, built upon, or otherwise used by anyone for any lawful purpose. The work is made available under the Creative Commons CC0 public domain dedication.

To determine whether inherited variations in the protein-coding regions of the genome contribute to menthol cigarette smoking, we performed an exome-wide association study using a population-based cohort of African Americans (AA) and European Americans (EA) from Dallas, Texas. The findings were replicated in a cohort of African-American smokers from Washington, DC.

Menthol is known to interact with transient receptor potential (TRP) channels, including TRPM8 [ 13 ] and TRPA1 [ 14 ]. Although one study found that common variants in TRPA1 were associated with menthol tobacco use among European-American smokers [ 15 ], this finding awaits replication. Variations in the TAS2R38 bitter taste receptor gene appear to have a modest effect on smoking and on menthol cigarette use [ 16 – 20 ], but no comprehensive analysis of the role of variation in these and other genes in menthol cigarette smoking has been carried out to date.

The prevalence of menthol cigarette smoking varies markedly between demographic groups, and is especially high among young adults and in African Americans [ 3 , 9 , 12 ]. In the U.S., nearly 83% of African-American smokers use menthol cigarettes, compared to 24% of white and 32% of Hispanic smokers. Whether this disparity has a genetic basis, or is attributable solely to social or cultural factors, is not known.

Cigarette smoking remains a leading cause of preventable disease and mortality in the United States, contributing to >480,000 deaths annually [ 1 ]. Although the overall rates of smoking have declined dramatically over the last 50 years [ 1 ], the use of mentholated cigarettes has not, and has actually increased in some groups [ 2 , 3 ]. Menthol is a flavoring additive commonly used in cigarettes and tobacco products. It is thought to reduce the harshness of cigarette smoke due to its cooling and anesthetic properties [ 4 – 6 ]. Menthol cigarettes currently account for about 30% of the cigarette market in the U.S. [ 7 ]. Scientific evidence suggests that the use of mentholated cigarettes leads to increased smoking initiation among youth and reduced rates of cessation [ 8 , 9 ]. This has led the FDA to conclude that menthol cigarettes likely pose a public health risk above that of nonmenthol cigarettes [ 10 , 11 ].

To provide further evidence for a role of MRGPRX4 in somatosensation, we used RT-PCR with RNA obtained from human thoracic dorsal root ganglia (DRG). The DRG serves to relay sensory information to the central nervous system, with the thoracic DRGs receiving sensory input from the lungs and airway. Using RT-PCR primers covering the length of MRGPRX4, following by sequencing of the RT-PCR products to ensure they originated from MRGPRX4 rather than any of the other closely related MRGPRX genes, we found clear expression in this tissue ( S4 Fig ), consistent with a role for MRGPRX4 in somatosensation in tissues exposed to cigarette smoke.

We also performed further studies using bioluminescence resonance energy transfer (BRET) as another measure of MRGPRX4 activity. Basal BRET in the G q/βγ dissociation assay (an index of constitutive receptor activity) was reduced at MRGPRX4 N245S relative to WT ( Fig 4A , 0.24 ± 0.025 vs 0.3 ± 0.015, t(3) = 4.986; p = 0.0155). Conversely, while basal activity of both WT and MRGPRX4 N245S was found to be increased by menthol (F(3,18) = 32.89, p < 0.0001), there was no effect of or interaction with genotype. Increasing amounts of menthol resulted in significantly elevated activity ( Fig 4B , Linear effect, F(1,28) = 42.83; p < 0.0001) at all increments except between 50 and 100 μM ( Fig 4B ). Under agonist stimulation conditions, the probe Nateglinide showed an approximate 2-fold greater potency at MRGPRX4 N245S compared to WT ( Table 5 , EC50 values of 10.49 ± 0.61 μM vs 5.25 ± 0.25 μM). This effect was significant (F(1,18) = 7.44, p = 0.0343) accounting for 43.21% of the variance between the two populations. No effect of menthol on potency nor interaction with genotype were indicated. Similarly, efficacy of the probe Nateglinide (E max ) was not affected by menthol at any concentration ( Fig 4C ), though E max as calculated by net BRET was reduced at MRGPRX4 N245S vs WT (-0.067 ± 0.001 vs -0.113 ± 0.006, F(1,6) = 15.51, p = 0.0076).

(A),(B) Average concentration response curves for Nateglinide in MRGPRX4-WT-Tango (A) or MRGPRX4-N245S+T43T-Tango (B) following 100 μM or 300 μM (-)-menthol addition, (n = 3, in triplicate, y-axis is % Nateglinide). (C),(D) Average concentration response curves for Nateglinide-induced PI hydrolysis in MRGPRX4-WT (C) or MRGPRX4- N245S+T43T (D) tetracycline inducible cells following 100 μM or 300 μM (-)-menthol addition, (n = 3, in duplicate, y-axis is % Nateglinide). (E) Plot showing the effect of agonist treatments (x-axis) Nateglinide (gray circles), Nateglinide + 100 μM (-)-menthol (gray squares) and Nateglinide + 300 μM (-)-menthol (gray triangles) between WT and N245S+T43T variant shown as ΔΔLog(E max /EC 50 ) values (y-axis). Y vales > 0 indicate increased effect for agonist at WT and values < 0 indicate increased effect for agonist at N+T variant. Bars depict 95% confidence intervals. (F) Average concentration response curves for fold change activation with Nateglinide in MRGPRX4-WT-Tango or MRGPRX4-N245S+T43T-Tango following 300 μM or 300 μM (-)-menthol addition, (n = 3, in quadruplicate). For all: NS = not significant, * p<0.05, ** p <0.01, and *** p<0.001 as calculated by F-test.

To determine whether (-)-menthol, the additive present in menthol cigarettes, alters the activity of MRGPRX4 WT or the N245S+T43T variant, we added the compound and repeated the functional assays. In the Tango assay, (-)-menthol alone showed no agonist activity at MRGPRX4 (up to 1 mM) ( S3 Fig ). We then tested whether (-)-menthol altered agonist-induced activity of the WT and N245S+T43T receptors. Increasing concentrations of (-)-menthol were added to each assay together with Nateglinide ( Fig 3 ). We observed that 100 μM and 300 μM (-)-menthol significantly reduced the E max of the agonist Nateglinide on the WT (P<0.001) and N245S+T43T (P<0.001) in the arrestin pathway ( Fig 3A and 3B ) but not in the G protein pathway as measured using the PI hydrolysis assay ( Fig 3C and 3D ). To determine whether (-)-menthol’s modulatory effect differed significantly between WT and N245S+T43T variant, we calculated ΔΔlog(E max /EC 50 ) [ 30 ] for our reference agonist Nateglinide in the presence or absence of 100 and 300 μM (-)-menthol and found that (-)-menthol modulated WT and the variant equivalently ( Fig 3E ). A comparison of the fold change activation of β-arrestin recruitment revealed that 300 μM (-)-menthol significantly reduces Nateglinide-induced activation of the N245S variant when compared to WT (P<0.001, Fig 3F ), similar to the differences in fold change for non-menthol conditions ( Fig 2C ). To test for non-specific effects of menthol on cells or cell membranes, we tested the effect of menthol on the unrelated D2 dopamine receptor in the PRESTO-Tango assay. This control showed that (-)-menthol had no modulatory effect on this receptor in this assay ( S3 Fig , panel C). Similarly, (-)-menthol and Nateglinide had no effect on PI hydrolysis in cells where tetracycline was not added (i.e., with no MRGPRX4 receptor expression) ( S3 Fig , panel D).

We then examined the effect of the agonist Nateglinide on the recruitment of β-arrestin in the PRESTO-Tango recruitment assay, which provides a quantitative measure of receptor activation and downstream signaling [ 28 , 29 ]. We found that Nateglinide had equal potency at both N245S+T43T and WT receptors, but the N245S+T43T variant displayed a dramatic reduction of fold activation (42 fold) in β-arrestin recruitment, significantly less than the WT receptor (123.9 fold) (P<0.001, Fig 2C , S6 Table ). In an independent quantitative assay, we also examined the effect of the variant on G protein signaling using the G protein-dependent phosphatidylinositol (PI) hydrolysis assay. We observed that the maximal (E max ) PI hydrolysis values following Nateglinide addition were significantly reduced in cells expressing the N245S+T43T variant compared with those expressing the WT receptor (PI Hydrolysis P<0.001, Fig 2D , S6 Table ). Together, these data demonstrate that despite an apparent increase in N245S+T43T expression, the variant has significantly reduced arrestin and G protein signaling in comparison to WT.

(A) Receptor expression as calculated by whole cell ELISA in HTLA cells transfected with N245S+T43T or WT receptor with mock transfected shown as negative control (n = 2, 64 wells per experiment; y-axis is fold expression normalized to WT). (B) Receptor expression as calculated by whole cell ELISA in tetracycline inducible WT or N245S+T43T cells with non-tetracycline-induced cells shown as negative control (n = 2, 64 wells per experiment; y-axis depicts fold expression normalized to WT). (C) Average concentration response curves for Nateglinide in PRESTO-Tango arrestin assay with WT and N245S+T43T receptors, (n = 4, in quadruplicate; y-axis is in fold response over basal signaling). (D) Average concentration response curves for Nateglinide in PI hydrolysis assay with WT and N245S+T43T tet-inducible cell lines, (n = 3, in duplicate; y-axis is in fold response over basal signaling). * indicates P<0.05, *** indicates P<0.001 as calculated by F-test.

MRGPRX4 is an orphan G protein-coupled receptor (GPCR) expressed in mammalian sensory neurons [ 22 , 23 ]. Although the endogenous ligand(s) for this receptor are not known, the potassium channel modulator Nateglinide has been identified to be a highly efficacious agonist and was used to demonstrate that this receptor couples predominantly to G αq [ 26 ]. We used a cell-based approach to determine if the N245S variant affected the responsiveness of either MRGPRX4 β-arrestin or G αq downstream signaling in response to Nateglinide. We first generated FLAG-tagged, codon-optimized wild-type (WT) and N245S variant constructs for the PRESTO-Tango β-arrestin recruitment assay and then generated stable, tetracycline-inducible cell lines for the FLAG-tagged WT MRGPRX4 and the N245S variant. Notably, all N245S variant constructs in this study also included the synonymous variant T43T. To measure membrane expression levels of WT MRGPRX4 and N245S+T43T, we used an established whole-cell ELISA assay [ 27 ] in HTLA cells. Using a 1-way ANOVA, we determined that N245S+T43T was expressed slightly but significantly more than the WT receptor in HTLA cells and in the tetracycline-inducible stable cells ( Fig 2A and 2B ).

To identify whether additional coding variants in MRPGRX4 were associated with menthol cigarette smoking, we sequenced all exons of MRPGRX4 in a subset of Dallas cohort participants (N = 389, Table 4 ). This analysis confirmed that the rs7102322 (N245S) variant was in complete LD with rs61733596 (T43T), which showed an equivalent association with menthol smoking (OR = 3.3, P = 0.007). No other coding variant in MRGPRX4 was in linkage disequilibrium with N245S or associated with menthol cigarette smoking in this group.

To evaluate whether rs7102322 SNP was in linkage disequilibrium (LD) with another functional variant in the MRGPRX4 locus, we examined data from the 1000 Genomes Project [ 25 ]. Consistent with our observations, the rs7102322 variant was observed solely in African-ancestry populations (MAF = 11.5% in Africans and 8% in African Americans in Southwest U.S.). The rs7102322 variant was in LD with the SNP rs61733596[A/G], which encodes a synonymous substitution at codon 43 (T43T) in MRGPRX4. Genotyping the rs61733596 variant in the Schroeder cohort confirmed that this variant is in complete linkage disequilibrium (R 2 = 1) with rs7102322.

The MRGPRX4 gene encodes a Mas-related G-protein coupled receptor member X4, which is expressed in nociceptive neurons of the dorsal root ganglia and trigeminal neurons, and may regulate pain and somatosensation [ 22 – 24 ]. The MRGPRX4 rs7102322 variant encodes an asparagine-to-serine substitution at codon 245 (N245S). The residue is conserved in chimpanzees, and resides immediately 5’ to a region highly conserved across primates ( Fig 1 ).

The rs7102322 variant was seen exclusively in African-American participants (minor allele frequency [MAF] = 8% in the Dallas cohorts, 5% in Schroeder) and was not observed in European Americans (0% in DHS EA). Among the AA participants in the Dallas cohorts, the allele frequency of the variant was five-to-eight fold higher in menthol smokers compared to non-menthol smokers (10.4% vs 1.3%, odds ratio (OR) = 8.5, P = 5.6x10 -5 ( Table 3 ). A similar magnitude of difference was seen in the Schroeder cohort (7.0% vs 1.3%, OR = 6.3, P = 2.1x10 -6 , Table 3 ). Although limited by low power, our analyses found highly similar differences in the MRGPRX4 allele frequencies between menthol and non-menthol smokers in males and females ( S1 Table ). To determine whether the lower frequency of the rs7102322 variant in the Schroeder cohort (5%) was influenced by admixture, we estimated the percentage of African and European ancestry in a subset of this cohort. This indicated that Schroeder cohort participants indeed have a higher degree of European admixture compared to West Africans and African Americans from other regions of the U.S. ( S2 Fig ). Further analyses that included ancestry informative markers and an inferred proportion of African ancestry at this locus maintained strong support for association with menthol smoking (P<1e-5), indicating that the observed association is unlikely to be due to differential admixture ( S4 and S5 Tables).

A total of 52,298 variants were tested for association with menthol cigarette smoking in the Dallas cohort. Genomic control [ 21 ] value was acceptable (λ gc = 1.05) and QQ-plot of P-values showed no systematic inflation of association results ( S1 Fig ). No variant met our exome-wide significance threshold (9.6x10 -7 ). We therefore decided to investigate the top variants with a suggestive level of significance (P<1x10 -4 ) in greater detail. A total of three variants reached this level of significance in our exome-wide screen ( Table 2 ), and these were genotyped in an additional cohort of 741 AA smokers from Washington DC (Schroeder cohort). While no association was found with two of the three variants ( Table 2 ), the third variant, rs7102322 in the gene MRGPRX4, was strongly associated with menthol smoking in the replication cohort (P = 2.1x10 -6 ). Meta-analysis of the two samples together revealed an even lower P-value that exceeded criteria for genome-wide significance (P = 1.6x10 -8 , Table 2 ).

The discovery cohort included 561 participants (394 AA and 167 EA) from the Dallas Heart Study (DHS) and the Dallas Biobank ( Table 1 ). The average age of participants was 55±11.0 (SD) years, and 60% were women. Nearly 78% of DHS AA and 86% of Biobank AA subjects reported smoking mentholated cigarettes, compared to 33% of European Americans (P<0.001), consistent with national trends [ 3 ]. Menthol smokers were younger than non-menthol smokers among African Americans (P<0.05), but there was no difference in age among European-American smokers. The prevalence of menthol smoking was not significantly different between DHS and Biobank AA after adjusting for age (P = 0.59). In the replication cohort (Schroeder), most of the participants (N = 424, 57.2%) were menthol smokers ( Table 1 ). A higher percentage of menthol smokers than non-menthol smokers were female (39.6% versus 24.3%, P<0.001) consistent with previous literature [ 12 ]. No differences were found in the mean age of menthol smokers and non-menthol smokers (P = 0.41).

Discussion

We have identified a variant haplotype of MRGPRX4 that is associated with increased prevalence of menthol cigarette smoking. This variant is found solely in individuals of African ancestry, and increases the odds of menthol use 5-to-8 fold among cigarette smokers. Cell-based assays of MRGPRX4 receptor function identified menthol as a novel negative modulator for this receptor, acting to reduce the responsiveness of this G protein-coupled receptor to its only known agonist at the WT and African-specific coding variant further.

While our understanding of MRGPRX4 gene function is limited, the members of this gene family are expressed in primary sensory neurons and are believed to be involved in somatosensation and nociception, including pruritus [22–24]. Although the natural ligand(s) for MRGPRX4 have not yet been identified, it is of interest that the MRGPRX4 agonist Nateglinide, a drug used to treat Type 2 diabetes, has been reported to have pruritus as a side effect [31]. Together, this suggests that menthol may act outside of the taste sensory system and may exert an anesthetic effect, which is further enhanced by the African-specific form of this receptor, which has dampened signaling capacity.

The MRGPRX4 variant associated with menthol cigarette smoking is relatively uncommon, with a MAF 8% in African Americans. Therefore, this variant alone cannot account for all of the difference in menthol cigarette smoking prevalence between African Americans and other ethnic groups. Thus, it is likely that other factors contribute to these differences. Surprisingly, we did not observe any consistent association at loci previously reported to be associated with menthol cigarette smoking (such as TRPA1 and TAS2R38), or the gene encoding the TRPM8 channel, which has been shown to be the target for menthol action in the somatosensory system. This may be due to the small size of our discovery cohort, which was powered to discover only large effect sizes (OR >2–3). The TRPA1 variants previously linked to menthol smoking had more modest effect sizes (odds ratios 1.3–1.4); thus, our study may have had insufficient power to detect their effects. We also genotyped the TRPA1 SNPs in our Schroeder cohort (N = 741) and could not replicate these associations, suggesting that factors other than power may be responsible for the difference in the results. The previously described association of TRPA1 variants with menthol smoking was restricted to heavy smokers, and was not observed in lighter smokers. Although our discovery cohort likely included a substantial proportion of light smokers, our replication cohort included mostly heavy smokers, which suggests that the lack of association in TRPA1 is unlikely to be explained entirely by the difference in phenotype.

Another possibility is that menthol smoking preferences are regulated by TRPA1 or TRPM8 non-coding variants that were not captured by the Exome chip or whole-exome sequencing. However, we have previously sequenced the exons and adjacent intronic regions of TRPM8 and TRPA1 in the Schroeder and Dallas cohorts and could not find variants with a consistent association with menthol smoking. Nevertheless, these genes remain plausible candidates and further studies, including larger samples of precisely phenotyped individuals, are warranted.

There are currently no crystal structures of the MRGPRX4 receptor available to conclusively determine the location and role of the N245S variant uncovered in this study. Based on a sequence alignment of MRGPRX4 with a published computational model of the related receptor MRGPRX2 [32], N245 (D in MRGPRX2) appears to be located in the third extracellular loop (EL3) of the receptor. Here, N245S reduces Nateglinide-induced agonism in both arrestin and G protein signaling pathways despite an apparent increase in membrane expression. EL3 has been demonstrated in the serotonin receptor 5HT2B to be involved in sterics of ligand binding and the kinetics of ligand and receptor interactions [28]. Thus, it is possible that the N245S variant changes the steric or kinetic properties of Nateglinide binding that influence arrestin and G protein signaling, though further studies will be needed to dissect the mechanism of this effect.

The strengths of our study are the use of a population-based sample including both African-American and European-American smokers, and a replication in a large independent cohort of African Americans. Unlike previous studies that looked at candidate polymorphisms, we performed a hypothesis-free exome-wide screen that provided broad and dense coverage of variation in the coding regions of the genome.

One limitation of our study, as mentioned above, is the relatively small size of our discovery cohort, which provided adequate power to discover only variants with large effect sizes (OR>2–3), and may have missed other genetic variants with lower allele frequencies or smaller effect sizes. Nevertheless, our approach represents an unbiased investigation into the genetic determinants of menthol cigarette smoking in a multiethnic cohort. Another potential limitation is that our data on menthol cigarette use was based on self-report, thus some individuals may have been misclassified with regard to their phenotype. However, our estimates of prevalence of menthol cigarette smoking among ethnic groups were consistent with national estimates, suggesting that misclassification error, if present, is likely small. Likewise, if such misclassification in the Schroeder population led to an overestimate of the association between MRGPRX4 and menthol smoking due to hidden population substructure, this is likely to be small because we found minimal evidence for heterogeneity within this group by large-scale SNP genotyping. Finally, not all participants responded to our questionnaire, thus results may not generalize to other populations. However, responders were similar to non-responders in terms of age and ethnicity (see Materials and Methods).

Menthol is known to exert its effects through transient receptor potential (TRP) channels TRPM8 [33, 34], and to a lesser extent TRPA1 [14]. TRPM8 is also known to mediate menthol-induced analgesia [35–37], and studies have shown that even low levels of menthol in tobacco, below those required to produce mint-like taste or aroma in tobacco, can activate TRPM8 [38]. Although our study was underpowered to detect variants with small effects, we found no evidence of association between variants at the TRPM8 and TRPA1 loci and menthol use, suggesting that variation in these menthol receptors is not a major contributor to the differential use of menthol cigarettes among African Americans.

Menthol cigarettes have been identified as a major threat to public health that have a disproportionate effect on ethnic minorities [39]. Our data suggest that ancestry-specific variants in genes involved in nociception contribute to both inter-individual and inter-ethnic differences in menthol cigarette smoking. The existence of population-specific genetic variants presents a new risk factor for menthol cigarette use, and suggests that the existence of this risk factor can inform health policies and tobacco regulatory actions designed to reduce health disparities in the United States.