Original Article
Smoking and Risk of Multiple Sclerosis Evidence of Modification by NAT1 Variants Farren B. S. Briggs,a Brigid Acuna,b Ling Shen,b Patricia Ramsay,a Hong Quach,a Allan Bernstein,c Kalliope H. Bellesis,b Ingrid S. Kockum,d Anna K. Hedström,e Lars Alfredsson,e Tomas Olsson,d Catherine Schaefer,b,* and Lisa F. Barcellosa,b,* Background: Tobacco smoke is an established risk factor for multiple sclerosis (MS). We hypothesized that variation in genes involved in metabolism of tobacco smoke constituents may modify MS risk in smokers. Methods: A three-stage gene-environment investigation was conducted for NAT1, NAT2, and GSTP1 variants. The discovery analysis was conducted among 1588 white MS cases and controls from the Kaiser Permanente Northern California Region HealthPlan (Kaiser). The replication analysis was carried out in 988 white MS cases and controls from Sweden. Results: Tobacco smoke exposure at the age of 20 years was associated with greater MS risk in both data sets (in Kaiser, odds ratio [OR] = 1.51 [95% confidence interval (CI) = 1.17–1.93]; in Sweden, OR = 1.35 [1.04–1.74]). A total of 42 NAT1 variants showed evidence for interaction with tobacco smoke exposure (Pcorrected < 0.05). Genotypes for 41 NAT1 single nucleotide polymorphisms (SNPs) were studied in the replication data set. A variant (rs7388368C>A) within a dense transcription factor-binding region showed evidence for interaction (Kaiser, OR for interaction = 1.75 [95% CI = 1.19–2.56]; Sweden, OR = 1.62 [1.05–2.49]). Tobacco smoke exposure was associated Submitted 30 May 2013; accepted 22 November 2013; posted 12 March 2014. From the aGenetic Epidemiology and Genomics Laboratory, Division of Epidemiology, School of Public Health, University of California, Berkeley, Berkeley, CA; bDivision of Research, Kaiser Permanente, Oakland, CA; c Palm Drive Hospital, Sebastopol, CA; dDepartment of Medicine, Center for Molecular Medicine, Karolinska Institute, Solna, Sweden; eInstitute of Environmental Medicine, Karolinska Institute, Solna, Sweden. *Senior authors contributed equally. The Kaiser Permanente Northern California Region Health Plan study was supported by the National Institute of Neurological Disorders and Stroke (R01 NS049510, R01 NS0495103) and the National Institute of Allergy and Infectious Diseases (R01 AI076544). Farren Briggs is a National Multiple Sclerosis Society Post-Doctoral Fellow (FG 1847A1/1). The authors report no conflicts of interest. Conflicts of interest: none declared. Supplemental digital content is available through direct URL citations in the HTML and PDF versions of this article (www.epidem.com). This content is not peer-reviewed or copy-edited; it is the sole responsibility of the author. Correspondence: Lisa F. Barcellos, Genetic Epidemiology and Genomics Laboratory, Division of Epidemiology, School of Public Health, 324 Stanley Hall, University of California, Berkeley, CA 94720. E-mail: [email protected]. Copyright © 2014 by Lippincott Williams & Wilkins ISSN: 1044-3983/14/2504-0605 DOI: 10.1097/EDE.0000000000000089
Epidemiology • Volume 25, Number 4, July 2014
with MS risk among rs7388368A carriers only; homozygote individuals had the highest risk (A/A, OR = 5.17 [95% CI = 2.17–12.33]). Conclusions: We conducted a three-stage analysis using two population-based case-control datasets that consisted of a discovery population, a replication population, and a pooled analysis. NAT1 emerged as a genetic effect modifier of tobacco smoke exposure in MS susceptibility. (Epidemiology 2014;25: 605–614)
M
ultiple sclerosis (MS) is a neuroinflammatory autoimmune disease with a multifactorial etiology characterized by inflammatory and neurodegenerative pathological phases. Strong evidence supports the contribution of both genetic and environmental factors in the pathoetiology of MS, as demonstrated by increased disease concordance among monozygotic twins (approximately 25%) compared with dizygotic twins (approximately 5%).1 MS patients are at least twice as likely to be female, with the primary age at onset between 20 and 40 years of age.2 Variation within major h istocompatibility-complex genes on chromosome 6p21 confers the greatest risk of MS, primarily within the human leukocyte antigen (HLA)-DRB1 locus (specifically, the HLA-DRB1*15:01 allele).3 Through International Consortium efforts, genome-wide association and replication studies have identified >100 MS susceptibility loci with modest effects (most odds ratios [ORs] 0.10 (based on power analyses; eAppendix; http:// links.lww.com/EDE/A777), Hardy Weinberg Equilibrium P > 0.0001, and missing genotypes 90% were extracted, resulting in 313 SNPs of which 225 passed quality control with genotyping rate = 1.
Exposure Assessment Data were collected by questionnaire on demographic variables, environmental exposures, and lifestyle behaviors. Information on current and previous smoking included duration of smoking and the average number of cigarettes/cigars/ pipes smoked per day. Study participants were considered ever-smokers of tobacco if they had smoked or currently smoke on-or-off or regularly. Participants were asked to report www.epidem.com | 607
Briggs et al
the ages at which they had started and stopped smoking cigarettes, cigars, and pipes. Two exposures were examined: (1) ever/never-smoker and (2) smoker at age 20 years. Participants were also asked to report their ethnic origin (Swedish, Scandinavian, Finnish, and non-Scandinavian). All available participants provided their year of birth, sex, and education level (bachelor’s degree). To date, the response rate for the Swedish study is 90% (of 1974 contacted) for the case group and 70% (of 4816 contacted) for controls.
Statistical Analysis Unconditional logistic regression models were used to formally test for G × E interactions on a multiplicative scale. Models included main effects (G and E) and were adjusted for year of birth, sex, and education level. We conducted a threestage analysis: (1) a discovery analysis using the Kaiser data adjusted for population ancestry; (2) a replication analysis using the Swedish data adjusted for ethnic origin; and (3) a meta-analysis to establish final measures of association. Using ever/never-smoker as the exposure of interest, we investigated 277 common variants (of which 8 correspond to specific NAT1 or NAT2 phenotypes; eTable 1A; http://links.lww.com/EDE/ A777) and an additional 5 common NAT1 and NAT2 phenotypes in the discovery analysis by using PLINK v1.07.29 Given the extensive linkage disequilibrium (eFigure 1; http://links. lww.com/EDE/A777), we corrected for the number of independent tests, as recommended, based on the Solid Spine of linkage disequilibrium as implemented in Haploview v4.2.32,33 Using a minimum D′ value of 0.75, there were 11 independent regions (eFigure 1; http://links.lww.com/EDE/A777); therefore, our corrected significance threshold was P < 0.0045. G × E interactions were investigated in the replication analysis; data for 41 of 42 variants of interest were available. A replicating variant was subsequently investigated using a random-effects meta-analysis, with cohort of origin as the random effect, and year of birth, sex, and education level as fixed effects using xtlogit. We performed a test for heterogeneity (Cochran’s Q and I2) across the two studies for the genotype-stratified analyses was performed using metan as implemented in STATA v11.2 (StataCorp, College Station, TX). We also investigated the risk of MS conferred by tobacco smoking in genotype-stratified analyses. These analyses were repeated using tobacco smoke exposure at age 20 years, in which analyses were restricted to MS cases with disease onset ≥20 years of age. Conditional logistic regression was conducted for a subset of the Kaiser study data (521 cases, 522 controls [one cases had two matched controls]), in which participants were matched on age, sex, and ZIP code. Conditional analyses investigated G × E interactions for the variant of interest using ever/never-tobacco-smoker, smoker at age 20 years, and smoking in the reference year. To determine whether the replicating variant was the primary association signal within NAT1 from among the available genetic data, linkage disequilibrium analyses were conducted. 608 | www.epidem.com
Epidemiology • Volume 25, Number 4, July 2014
All variants demonstrating r2 ≥ 0.5 with the replicating variants in either the Kaiser or Swedish datasets were identified and tested through meta-analysis. Haploreg v2 (2013.02.14) (http://www.broadinstitute.org/mammals/haploreg/haploreg. php) was used to assess the potential regulatory function of variants of interest.34 It includes an extensive library of SNPs (dbSNP 137), motif instances (ENCODE), enhancer annotations (Roadmap Epigenome Mapping Consortium), and expression quantitative trait loci (GTex eQTL browser). The study protocol was approved by the Institutional Review Boards of Kaiser Permanente of Northern California and the University of California, Berkeley. The Swedish study was approved by the Ethical Review Board at Karolinska Institutet, and all participants provided written informed consent.
RESULTS Overall, 2576 participants (1588 participants from the Kaiser study; 988 participants from the Swedish study) were studied (Table 1). On an average, study participants were in their fifties (Kaiser) and forties (Sweden); therefore, controls had aged past the highest risk period for MS (Table 1). For both crude and adjusted models, MS cases were more likely to have been ever-smokers (Kaiser study, adjusted OR = 1.27 [95% CI = 1.03–1.58]; Swedish study, 1.45 [1.12–1.88]) and smokers at age 20 years (Kaiser study, 1.51 [1.17–1.93]; Swedish study, 1.35 [1.04–1.74]) compared with controls (Table 2). When analyses were restricted to the subset of matched Kaiser cases and controls (521 MS cases, 522 controls), results were very similar (eTable 2; http://links.lww.com/EDE/A777). In addition, based on smoking status in the reference year (the year previous to onset of disease symptoms), smokers had a two-fold increased risk of developing MS compared with nonsmokers (1.97 [1.29–3.00]; eTable 2; http://links.lww.com/EDE/A777). The discovery analysis investigated G × E interactions between ever/never-smoker status and genetic variation (277 SNPs, Table 3) within three phase-2 genes by using logistic regression models. We focused on interactions of 42 NAT1 SNPs with smoking status (Pcorrected < 0.05). No variant was marginally associated with MS, and there was no association of their main effects in the interaction models (data not shown). For the replication analysis, 41 of the 42 NAT1 variants were independently tested for G × E interactions in the Swedish dataset, using logistic regression. Results for one NAT1 variant (rs7388368A) were replicated (Table 4; Kaiser study, OR of interaction = 1.75 [95% CI = 1.19–2.56]; Swedish study, 1.62 [1.05–2.49]). In the combined meta-analysis, ever-smokers had an increased risk for MS (Table 2; adjusted OR = 1.36 [95% CI = 1.16–1.60]), and evidence for an interaction with rs7388368A persisted (Table 4; OR for interaction = 1.65 [1.25–2.18]). Stratified analyses demonstrated smoking was a risk factor only for MS among persons who carried the rs7388368A minor allele (A/C or A/A genotype; Table 4). Tobacco smoking did not confer risk of MS among persons © 2014 Lippincott Williams & Wilkins
Epidemiology • Volume 25, Number 4, July 2014
NAT1 Modifies Tobacco Smoke Risk for Multiple Sclerosis
TABLE 1. Characteristics of Non-Hispanic White Case-Control Study Populations Kaiser Study Characteristic
Swedish Study
Cases (n = 1012)
Controls (n = 576)
Cases (n = 494)
Controls (n = 494)
1958 (8.7) 80 55
1957 (8.1) 84 47
1967 (10.7) 72 64
1966 (11.2) 77 60
Year of birth (no. years SD) Women; % Less than a college education; %
TABLE 2. Marginal Association Between Tobacco Smoke Exposure and MS Exposed Cases %
Kaiser study Swedish study Meta-analysis
48 60 52
Kaiser study Swedish study Meta-analysis
30 53 38
Exposed Controls %
Adjusted ORa (95% CI)
Crude OR (95% CI)
Ever-smokerb 41 51 46 Smoker at age 20 yearsc 22 46 33
1.34 (1.09–1.65) 1.40 (1.09–1.80) 1.36 (1.16–1.60)
1.27 (1.03–1.58) 1.45 (1.12–1.88) 1.35 (1.15–1.59)
1.57 (1.23–2.01) 1.32 (1.03–1.70) 1.44 (1.21–1.71)
1.51 (1.17–1.93) 1.35 (1.04–1.74) 1.42 (1.19–1.70)
a Kaiser study was adjusted for year of birth, sex, college education, and population ancestry. Swedish study was adjusted for year of birth, sex, college education, and ethnic origin. The meta-analysis included year of birth, sex, and college education as fixed effects, with cohort as the random-effect. b Kaiser study, n = 1,588 (1,012 cases and 576 controls); Swedish study, n = 988 (494 cases and 494 controls). c Analyses were restricted to MS cases with age of onset ≥20 years. Kaiser study, n = 1,474 (901 cases, 573 controls); Swedish study, n = 970 (477 cases and 493 controls).
TABLE 3. The Five Candidate Phase II Metabolism Genes and the Distribution of Variants Within the Discovery (Kaiser) Dataset Gene GSTM1 NAT1 NAT2 GSTP1 GSTT1
Chr
Start
End
SNPsa
MAF > 0.001
MAF > 0.01
MAF > 0.10
1 8 8 11 22
110,225,418 18,022,971 18,243,755 67,346,066 24,371,139
110,241,367 18,086,198 18,263,723 67,359,124 24,389,284
59 608 176 89 29
49 430 144 59 10
4 326 106 40 0
0 166 81 30 0
Chr indicates chromosome; SNPs, single nucleotide polymorphisms; MAF, minor allele frequency. a All SNPs within 5 kb of each gene with 1 × 10–4.
homozygous for the rs7388368C major/wildtype allele (C/C genotype: 893 cases and 643 controls). Among persons who were heterozygous for rs7388368A (A/C genotype: 508 cases and 358 controls), ever-smokers had a 60% increased risk of MS compared with nonsmokers (OR=1.60 [95% CI = 1.20– 2.13]). Persons who were homozygous for rs7388368A (A/A genotype, 71 cases, 48 controls) had greater than five-fold risk for MS if they were ever-smokers compared with nonsmokers (5.17 [2.17–12.33]). Results were consistent in the Kaiser and Swedish study populations (Table 4), and there was no evidence for heterogeneity for the genotype-stratified metaanalyses (I2 = 0%; Cochran’s Q P > 0.5). G × E interaction analyses were conducted for rs7388368A and smoker status at age 20 (Table 4). In the metaanalysis, there was evidence for G × E interaction (OR for interaction = 1.35 [95% CI = 1.01–1.81]). A similar relation © 2014 Lippincott Williams & Wilkins
between smoking status at age 20 and MS risk was observed when stratifying by rs7388368 genotype (C/C, OR = 1.25 [95% CI = 0.99–1.57]; A/C, 1.53 [1.13–2.08]; A/A, 3.43 [1.43–8.20]). There was no evidence for heterogeneity for the genotype-stratified meta-analyses (I2 = 0%; Cochran’s Q P > 0.5). When analyses were restricted to the subset of matched Kaiser cases and controls, results were very similar (eTable 3; http://links.lww.com/EDE/A777). When matched cases and controls were stratified by rs7388368A carrier status (C/C versus A/C and A/A), the three measures of tobacco smoke exposure were associated with MS among those with an A/C or A/A genotype only, despite very small sample sizes ( 0.65) with rs7388368A (eTable 4; http://links.lww.com/ © 2014 Lippincott Williams & Wilkins
EDE/A777). Both rs4921877 and rs6586711 showed evidence for interaction with smoking status (eTable 4; http://links.lww. com/EDE/A777), consistent with that of rs7388368 results. To further characterize the association between NAT1 and www.epidem.com | 611
Epidemiology • Volume 25, Number 4, July 2014
Briggs et al
tobacco smoke in MS, we analyzed 93 variants that had an uncorrected P < 0.05 in the discovery analysis, of which 81 SNPs were in the replication data, and we observed a total of 77 significant associations (P
Smoking and Risk of Multiple Sclerosis Evidence of Modification by NAT1 Variants Farren B. S. Briggs,a Brigid Acuna,b Ling Shen,b Patricia Ramsay,a Hong Quach,a Allan Bernstein,c Kalliope H. Bellesis,b Ingrid S. Kockum,d Anna K. Hedström,e Lars Alfredsson,e Tomas Olsson,d Catherine Schaefer,b,* and Lisa F. Barcellosa,b,* Background: Tobacco smoke is an established risk factor for multiple sclerosis (MS). We hypothesized that variation in genes involved in metabolism of tobacco smoke constituents may modify MS risk in smokers. Methods: A three-stage gene-environment investigation was conducted for NAT1, NAT2, and GSTP1 variants. The discovery analysis was conducted among 1588 white MS cases and controls from the Kaiser Permanente Northern California Region HealthPlan (Kaiser). The replication analysis was carried out in 988 white MS cases and controls from Sweden. Results: Tobacco smoke exposure at the age of 20 years was associated with greater MS risk in both data sets (in Kaiser, odds ratio [OR] = 1.51 [95% confidence interval (CI) = 1.17–1.93]; in Sweden, OR = 1.35 [1.04–1.74]). A total of 42 NAT1 variants showed evidence for interaction with tobacco smoke exposure (Pcorrected < 0.05). Genotypes for 41 NAT1 single nucleotide polymorphisms (SNPs) were studied in the replication data set. A variant (rs7388368C>A) within a dense transcription factor-binding region showed evidence for interaction (Kaiser, OR for interaction = 1.75 [95% CI = 1.19–2.56]; Sweden, OR = 1.62 [1.05–2.49]). Tobacco smoke exposure was associated Submitted 30 May 2013; accepted 22 November 2013; posted 12 March 2014. From the aGenetic Epidemiology and Genomics Laboratory, Division of Epidemiology, School of Public Health, University of California, Berkeley, Berkeley, CA; bDivision of Research, Kaiser Permanente, Oakland, CA; c Palm Drive Hospital, Sebastopol, CA; dDepartment of Medicine, Center for Molecular Medicine, Karolinska Institute, Solna, Sweden; eInstitute of Environmental Medicine, Karolinska Institute, Solna, Sweden. *Senior authors contributed equally. The Kaiser Permanente Northern California Region Health Plan study was supported by the National Institute of Neurological Disorders and Stroke (R01 NS049510, R01 NS0495103) and the National Institute of Allergy and Infectious Diseases (R01 AI076544). Farren Briggs is a National Multiple Sclerosis Society Post-Doctoral Fellow (FG 1847A1/1). The authors report no conflicts of interest. Conflicts of interest: none declared. Supplemental digital content is available through direct URL citations in the HTML and PDF versions of this article (www.epidem.com). This content is not peer-reviewed or copy-edited; it is the sole responsibility of the author. Correspondence: Lisa F. Barcellos, Genetic Epidemiology and Genomics Laboratory, Division of Epidemiology, School of Public Health, 324 Stanley Hall, University of California, Berkeley, CA 94720. E-mail: [email protected]. Copyright © 2014 by Lippincott Williams & Wilkins ISSN: 1044-3983/14/2504-0605 DOI: 10.1097/EDE.0000000000000089
Epidemiology • Volume 25, Number 4, July 2014
with MS risk among rs7388368A carriers only; homozygote individuals had the highest risk (A/A, OR = 5.17 [95% CI = 2.17–12.33]). Conclusions: We conducted a three-stage analysis using two population-based case-control datasets that consisted of a discovery population, a replication population, and a pooled analysis. NAT1 emerged as a genetic effect modifier of tobacco smoke exposure in MS susceptibility. (Epidemiology 2014;25: 605–614)
M
ultiple sclerosis (MS) is a neuroinflammatory autoimmune disease with a multifactorial etiology characterized by inflammatory and neurodegenerative pathological phases. Strong evidence supports the contribution of both genetic and environmental factors in the pathoetiology of MS, as demonstrated by increased disease concordance among monozygotic twins (approximately 25%) compared with dizygotic twins (approximately 5%).1 MS patients are at least twice as likely to be female, with the primary age at onset between 20 and 40 years of age.2 Variation within major h istocompatibility-complex genes on chromosome 6p21 confers the greatest risk of MS, primarily within the human leukocyte antigen (HLA)-DRB1 locus (specifically, the HLA-DRB1*15:01 allele).3 Through International Consortium efforts, genome-wide association and replication studies have identified >100 MS susceptibility loci with modest effects (most odds ratios [ORs] 0.10 (based on power analyses; eAppendix; http:// links.lww.com/EDE/A777), Hardy Weinberg Equilibrium P > 0.0001, and missing genotypes 90% were extracted, resulting in 313 SNPs of which 225 passed quality control with genotyping rate = 1.
Exposure Assessment Data were collected by questionnaire on demographic variables, environmental exposures, and lifestyle behaviors. Information on current and previous smoking included duration of smoking and the average number of cigarettes/cigars/ pipes smoked per day. Study participants were considered ever-smokers of tobacco if they had smoked or currently smoke on-or-off or regularly. Participants were asked to report www.epidem.com | 607
Briggs et al
the ages at which they had started and stopped smoking cigarettes, cigars, and pipes. Two exposures were examined: (1) ever/never-smoker and (2) smoker at age 20 years. Participants were also asked to report their ethnic origin (Swedish, Scandinavian, Finnish, and non-Scandinavian). All available participants provided their year of birth, sex, and education level (bachelor’s degree). To date, the response rate for the Swedish study is 90% (of 1974 contacted) for the case group and 70% (of 4816 contacted) for controls.
Statistical Analysis Unconditional logistic regression models were used to formally test for G × E interactions on a multiplicative scale. Models included main effects (G and E) and were adjusted for year of birth, sex, and education level. We conducted a threestage analysis: (1) a discovery analysis using the Kaiser data adjusted for population ancestry; (2) a replication analysis using the Swedish data adjusted for ethnic origin; and (3) a meta-analysis to establish final measures of association. Using ever/never-smoker as the exposure of interest, we investigated 277 common variants (of which 8 correspond to specific NAT1 or NAT2 phenotypes; eTable 1A; http://links.lww.com/EDE/ A777) and an additional 5 common NAT1 and NAT2 phenotypes in the discovery analysis by using PLINK v1.07.29 Given the extensive linkage disequilibrium (eFigure 1; http://links. lww.com/EDE/A777), we corrected for the number of independent tests, as recommended, based on the Solid Spine of linkage disequilibrium as implemented in Haploview v4.2.32,33 Using a minimum D′ value of 0.75, there were 11 independent regions (eFigure 1; http://links.lww.com/EDE/A777); therefore, our corrected significance threshold was P < 0.0045. G × E interactions were investigated in the replication analysis; data for 41 of 42 variants of interest were available. A replicating variant was subsequently investigated using a random-effects meta-analysis, with cohort of origin as the random effect, and year of birth, sex, and education level as fixed effects using xtlogit. We performed a test for heterogeneity (Cochran’s Q and I2) across the two studies for the genotype-stratified analyses was performed using metan as implemented in STATA v11.2 (StataCorp, College Station, TX). We also investigated the risk of MS conferred by tobacco smoking in genotype-stratified analyses. These analyses were repeated using tobacco smoke exposure at age 20 years, in which analyses were restricted to MS cases with disease onset ≥20 years of age. Conditional logistic regression was conducted for a subset of the Kaiser study data (521 cases, 522 controls [one cases had two matched controls]), in which participants were matched on age, sex, and ZIP code. Conditional analyses investigated G × E interactions for the variant of interest using ever/never-tobacco-smoker, smoker at age 20 years, and smoking in the reference year. To determine whether the replicating variant was the primary association signal within NAT1 from among the available genetic data, linkage disequilibrium analyses were conducted. 608 | www.epidem.com
Epidemiology • Volume 25, Number 4, July 2014
All variants demonstrating r2 ≥ 0.5 with the replicating variants in either the Kaiser or Swedish datasets were identified and tested through meta-analysis. Haploreg v2 (2013.02.14) (http://www.broadinstitute.org/mammals/haploreg/haploreg. php) was used to assess the potential regulatory function of variants of interest.34 It includes an extensive library of SNPs (dbSNP 137), motif instances (ENCODE), enhancer annotations (Roadmap Epigenome Mapping Consortium), and expression quantitative trait loci (GTex eQTL browser). The study protocol was approved by the Institutional Review Boards of Kaiser Permanente of Northern California and the University of California, Berkeley. The Swedish study was approved by the Ethical Review Board at Karolinska Institutet, and all participants provided written informed consent.
RESULTS Overall, 2576 participants (1588 participants from the Kaiser study; 988 participants from the Swedish study) were studied (Table 1). On an average, study participants were in their fifties (Kaiser) and forties (Sweden); therefore, controls had aged past the highest risk period for MS (Table 1). For both crude and adjusted models, MS cases were more likely to have been ever-smokers (Kaiser study, adjusted OR = 1.27 [95% CI = 1.03–1.58]; Swedish study, 1.45 [1.12–1.88]) and smokers at age 20 years (Kaiser study, 1.51 [1.17–1.93]; Swedish study, 1.35 [1.04–1.74]) compared with controls (Table 2). When analyses were restricted to the subset of matched Kaiser cases and controls (521 MS cases, 522 controls), results were very similar (eTable 2; http://links.lww.com/EDE/A777). In addition, based on smoking status in the reference year (the year previous to onset of disease symptoms), smokers had a two-fold increased risk of developing MS compared with nonsmokers (1.97 [1.29–3.00]; eTable 2; http://links.lww.com/EDE/A777). The discovery analysis investigated G × E interactions between ever/never-smoker status and genetic variation (277 SNPs, Table 3) within three phase-2 genes by using logistic regression models. We focused on interactions of 42 NAT1 SNPs with smoking status (Pcorrected < 0.05). No variant was marginally associated with MS, and there was no association of their main effects in the interaction models (data not shown). For the replication analysis, 41 of the 42 NAT1 variants were independently tested for G × E interactions in the Swedish dataset, using logistic regression. Results for one NAT1 variant (rs7388368A) were replicated (Table 4; Kaiser study, OR of interaction = 1.75 [95% CI = 1.19–2.56]; Swedish study, 1.62 [1.05–2.49]). In the combined meta-analysis, ever-smokers had an increased risk for MS (Table 2; adjusted OR = 1.36 [95% CI = 1.16–1.60]), and evidence for an interaction with rs7388368A persisted (Table 4; OR for interaction = 1.65 [1.25–2.18]). Stratified analyses demonstrated smoking was a risk factor only for MS among persons who carried the rs7388368A minor allele (A/C or A/A genotype; Table 4). Tobacco smoking did not confer risk of MS among persons © 2014 Lippincott Williams & Wilkins
Epidemiology • Volume 25, Number 4, July 2014
NAT1 Modifies Tobacco Smoke Risk for Multiple Sclerosis
TABLE 1. Characteristics of Non-Hispanic White Case-Control Study Populations Kaiser Study Characteristic
Swedish Study
Cases (n = 1012)
Controls (n = 576)
Cases (n = 494)
Controls (n = 494)
1958 (8.7) 80 55
1957 (8.1) 84 47
1967 (10.7) 72 64
1966 (11.2) 77 60
Year of birth (no. years SD) Women; % Less than a college education; %
TABLE 2. Marginal Association Between Tobacco Smoke Exposure and MS Exposed Cases %
Kaiser study Swedish study Meta-analysis
48 60 52
Kaiser study Swedish study Meta-analysis
30 53 38
Exposed Controls %
Adjusted ORa (95% CI)
Crude OR (95% CI)
Ever-smokerb 41 51 46 Smoker at age 20 yearsc 22 46 33
1.34 (1.09–1.65) 1.40 (1.09–1.80) 1.36 (1.16–1.60)
1.27 (1.03–1.58) 1.45 (1.12–1.88) 1.35 (1.15–1.59)
1.57 (1.23–2.01) 1.32 (1.03–1.70) 1.44 (1.21–1.71)
1.51 (1.17–1.93) 1.35 (1.04–1.74) 1.42 (1.19–1.70)
a Kaiser study was adjusted for year of birth, sex, college education, and population ancestry. Swedish study was adjusted for year of birth, sex, college education, and ethnic origin. The meta-analysis included year of birth, sex, and college education as fixed effects, with cohort as the random-effect. b Kaiser study, n = 1,588 (1,012 cases and 576 controls); Swedish study, n = 988 (494 cases and 494 controls). c Analyses were restricted to MS cases with age of onset ≥20 years. Kaiser study, n = 1,474 (901 cases, 573 controls); Swedish study, n = 970 (477 cases and 493 controls).
TABLE 3. The Five Candidate Phase II Metabolism Genes and the Distribution of Variants Within the Discovery (Kaiser) Dataset Gene GSTM1 NAT1 NAT2 GSTP1 GSTT1
Chr
Start
End
SNPsa
MAF > 0.001
MAF > 0.01
MAF > 0.10
1 8 8 11 22
110,225,418 18,022,971 18,243,755 67,346,066 24,371,139
110,241,367 18,086,198 18,263,723 67,359,124 24,389,284
59 608 176 89 29
49 430 144 59 10
4 326 106 40 0
0 166 81 30 0
Chr indicates chromosome; SNPs, single nucleotide polymorphisms; MAF, minor allele frequency. a All SNPs within 5 kb of each gene with 1 × 10–4.
homozygous for the rs7388368C major/wildtype allele (C/C genotype: 893 cases and 643 controls). Among persons who were heterozygous for rs7388368A (A/C genotype: 508 cases and 358 controls), ever-smokers had a 60% increased risk of MS compared with nonsmokers (OR=1.60 [95% CI = 1.20– 2.13]). Persons who were homozygous for rs7388368A (A/A genotype, 71 cases, 48 controls) had greater than five-fold risk for MS if they were ever-smokers compared with nonsmokers (5.17 [2.17–12.33]). Results were consistent in the Kaiser and Swedish study populations (Table 4), and there was no evidence for heterogeneity for the genotype-stratified metaanalyses (I2 = 0%; Cochran’s Q P > 0.5). G × E interaction analyses were conducted for rs7388368A and smoker status at age 20 (Table 4). In the metaanalysis, there was evidence for G × E interaction (OR for interaction = 1.35 [95% CI = 1.01–1.81]). A similar relation © 2014 Lippincott Williams & Wilkins
between smoking status at age 20 and MS risk was observed when stratifying by rs7388368 genotype (C/C, OR = 1.25 [95% CI = 0.99–1.57]; A/C, 1.53 [1.13–2.08]; A/A, 3.43 [1.43–8.20]). There was no evidence for heterogeneity for the genotype-stratified meta-analyses (I2 = 0%; Cochran’s Q P > 0.5). When analyses were restricted to the subset of matched Kaiser cases and controls, results were very similar (eTable 3; http://links.lww.com/EDE/A777). When matched cases and controls were stratified by rs7388368A carrier status (C/C versus A/C and A/A), the three measures of tobacco smoke exposure were associated with MS among those with an A/C or A/A genotype only, despite very small sample sizes ( 0.65) with rs7388368A (eTable 4; http://links.lww.com/ © 2014 Lippincott Williams & Wilkins
EDE/A777). Both rs4921877 and rs6586711 showed evidence for interaction with smoking status (eTable 4; http://links.lww. com/EDE/A777), consistent with that of rs7388368 results. To further characterize the association between NAT1 and www.epidem.com | 611
Epidemiology • Volume 25, Number 4, July 2014
Briggs et al
tobacco smoke in MS, we analyzed 93 variants that had an uncorrected P < 0.05 in the discovery analysis, of which 81 SNPs were in the replication data, and we observed a total of 77 significant associations (P
