Genome-wide association studies (GWAS) have laid the foundation for investigations into the biology of complex traits, drug development and clinical guidelines. However, the majority of discovery efforts are based on data from populations of European ancestry 1-3. In light of the differential genetic architecture that is known to exist between populations, bias in representation can exacerbate existing disease and healthcare disparities. Critical variants may be missed if they have a low frequency or are completely absent in European populations, especially as the field shifts its attention towards rare variants, which are more likely to be population-specific 4-10. Additionally, effect sizes and their derived risk prediction scores derived in one population may Reprints and permissions information is available at http://www.nature.com/reprints.

Nicotine metabolism influences smoking behavior and differences in metabolism probably contribute to ethnic variability in lung cancer risk. We report here on the proportion of nicotine metabolism by cytochrome P450 2A6-catalyzed C-oxidation, UDP-glucuronosyl transferase 2B10 (UGT2B10)-catalyzed N-glucuronidation and flavin monooxygenase 3-catalyzed N-oxidation in five ethnic/racial groups and the role of UGT2B10 genotype on the metabolic patterns observed. Nicotine and its metabolites were quantified in urine from African American (AA, n = 364), Native Hawaiian (NH, n = 311), White (n = 437), Latino (LA, n = 453) and Japanese American (JA, n = 674) smokers. Total nicotine equivalents, the sum of nicotine and six metabolites, and nicotine metabolism phenotypes were calculated. The relationship of UGT2B10 genotype to nicotine metabolic pathways was determined for each group; geometric means were computed and adjusted for age, sex, creatinine, and body mass index. Nicotine metabolism patterns were unique across the groups, C-oxidation was lowest in JA and NH (P < 0.0001), and N-glucuronidation lowest in AA (P < 0.0001). There was no difference in C-oxidation among Whites and AA and LA. Nicotine and cotinine glucuronide ratios were 2- and 3-fold lower in AA compared with Whites. Two UGT variants, a missense mutation (Asp67Tyr, rs61750900) and a splice variant (rs116294140) accounted for 33% of the variation in glucuronidation. In AA, the splice variant accounted for the majority of the reduced nicotine glucuronidation. UGT2B10 variant allele carriers had increased levels of C-oxidation (P = 0.0099). Our data indicate that the relative importance of nicotine metabolic pathways varies by ethnicity, and all pathways should be considered when characterizing the role of nicotine metabolism on smoking behavior and cancer risk.

Background We previously found that African Americans and Native Hawaiians were at highest lung cancer risk compared with Japanese Americans and Latinos; whites were midway in risk. These differences were more evident at relatively low levels of smoking intensity, fewer than 20 cigarettes per day (CPD), than at higher intensity. Methods We apportioned lung cancer risk into three parts: age-specific background risk (among never smokers), an excess relative risk term for cumulative smoking, and modifiers of the smoking effect: race and years-quit smoking. We also explored the effect of replacing self-reports of CPD with a urinary biomarker—total nicotine equivalents—using data from a urinary biomarker substudy. Results Total lung cancers increased from 1979 to 4993 compared to earlier analysis. Estimated excess relative risks for lung cancer due to smoking for 50 years at 10 CPD (25 pack-years) ranged from 21.9 (95% CI = 18.0 to 25.8) for Native Hawaiians to 8.0 (95% CI = 6.6 to 9.4) for Latinos over the five groups. The risk from smoking was higher for squamous cell carcinomas and small cell cancers than for adenocarcinomas. Racial differences consistent with earlier patterns were seen for overall cancer and for cancer subtypes. Adjusting for predicted total nicotine equivalents, Japanese Americans no longer exhibit a lower risk, and African Americans are no longer at higher risk, compared to whites. Striking risk differences between Native Hawaiians and Latinos persist. Conclusions Racial differences in lung cancer risk persist in the Multiethnic Cohort study that are not easily explained by variations in self-reported or urinary biomarker-measured smoking intensities.

Genetic variation in cytochrome P450 2A6 (CYP2A6) gene is the primary contributor to the intraindividual and interindividual differences in nicotine metabolism and has been found to influence smoking intensity. However, no study has evaluated the relationship between CYP2A6 genetic variants and the CYP2A6 activity ratio (total 3-hydroxycotinine/cotinine) and their influence on smoking intensity [total nicotine equivalents (TNE)], across five racial/ethnic groups found to have disparate rates of lung cancer. This study genotyped 10 known functional CYP2A6 genetic or copy number variants in 2115 current smokers from the multiethnic cohort study [African Americans (AA) = 350, Native Hawaiians (NH) = 288, Whites = 413, Latinos (LA) = 437 and Japanese Americans (JA) = 627] to conduct such an investigation. Here, we found that LA had the highest CYP2A6 activity followed by Whites, AA, NH and JA, who had the lowest levels. Adjusting for age, sex, race/ethnicity and body mass index, we found that CYP2A6 diplotypes were predictive of TNE levels, particularly in AA and JA (P trend < 0.0001). However, only in JA did the association remain after accounting for cigarettes per day. Also, it is only in this population that the lower activity ratio supports lower TNE levels, carcinogen exposure and thereby lower risk of lung cancer. Despite the association between nicotine metabolism (CYP2A6 activity phenotype and diplotypes) and smoking intensity (TNE), CYP2A6 levels did not correlate with the higher TNE levels found in AA nor the lower TNE levels found in LA, suggesting that other factors may influence smoking dose in these populations. Therefore, further study in these populations is recommended.

Metabolism of nicotine by cytochrome CYP2A6 is a suspected determinant of smoking dose and, consequently, lung cancer risk. We conducted a genome-wide association study (GWAS) of CYP2A6 activity, as measured by the urinary ratio of trans-3′-hydroxycotinine and its glucuronide conjugate over cotinine (total 3HCOT/COT), among 2,239 smokers in the Multiethnic Cohort (MEC) study. We identified 248 CYP2A6 variants associated with CYP2A6 activity (p<5×10−8). CYP2A6 activity was correlated (r=0.32, p<0.0001) with total nicotine equivalents (a measure of nicotine uptake). When we examined the effect of these variants on lung cancer risk in the Transdisciplinary Research in Cancer of the Lung (TRICL) consortium GWAS dataset (13,479 cases, 43,218 controls), we found that the vast majority of these individual effects were directionally consistent and associated with an increased lung cancer risk. 226 of the 248 variants associated with CYP2A6 activity in the MEC were available in TRICL. Of them, 81% had directionally consistent risk estimates and six were globally significantly associated with lung cancer. When conditioning on nine known functional variants and two deletions, the top two SNPs (rs56113850 in MEC and rs35755165 in TRICL) remained significantly associated with CYP2A6 activity in MEC and lung cancer in TRICL. The present data support the hypothesis that a greater CYP2A6 activity causes smokers to smoke more extensively and be exposed to higher levels of carcinogens, resulting in an increased risk for lung cancer. Although the variants identified in these studies may be used as risk prediction markers, the exact causal variants remain to be identified.

Rare variation in protein coding sequence is poorly captured by GWAS arrays and has been hypothesized to contribute to disease heritability. Using the Illumina HumanExome SNP array, we successfully genotyped 191,032 common and rare non-synonymous, splice site, or nonsense variants in a multiethnic sample of 2,984 breast cancer cases, 4,376 prostate cancer cases, and 7,545 controls. In breast cancer, the strongest associations included either SNPs in or gene burden scores for genes LDLRAD1, SLC19A1, FGFBP3, CASP5, MMAB, SLC16A6, and INS-IGF2. In prostate cancer, one of the most associated SNPs was in the gene GPRC6A (rs2274911, Pro91Ser, OR = 0.88, P = 1.3×10−5) near to a known risk locus for prostate cancer; other suggestive associations were noted in genes such as F13A1, ANXA4, MANSC1, and GP6. For both breast and prostate cancer, several of the most significant associations involving SNPs or gene burden scores (sum of minor alleles) were noted in genes previously reported to be associated with a cancer-related phenotype. However, only one of the associations (rs145889899 in LDLRAD1, p = 2.5×10−7 only seen in African Americans) for overall breast or prostate cancer risk was statistically significant after correcting for multiple comparisons. In addition to breast and prostate cancer, other cancer-related traits were examined (body mass index, PSA level, and alcohol drinking) with a number of known and potentially novel associations described. In general, these findings do not support there being many protein coding variants of moderate to high risk for breast and prostate cancer with odds ratios over a range that is probably required for protein coding variation to play a truly outstanding role in risk heritability. Very large sample sizes will be required to better define the role of rare and less penetrant coding variation in prostate and breast cancer disease genetics.

Background The lung cancer risk of smokers varies by race/ethnicity even after adjustment for smoking. Evaluating the role of genetics in nicotine metabolism is likely important in understanding these differences, as disparities in risk may be related to differences in nicotine dose and metabolism. Methods We conducted a genome-wide association study in search of common genetic variants that predict nicotine and cotinine glucuronidation in a sample of 2,239 smokers (437 European Americans, 364 African Americans, 453 Latinos, 674 Japanese Americans and 311 Native Hawaiians) in the Multiethnic Cohort Study. Urinary concentration of nicotine and its metabolites were determined. Results Among 11,892,802 variants analyzed, 1,241 were strongly associated with cotinine glucuronidation, 490 of which were also associated with nicotine glucuronidation (p<5×10−8). The vast majority were within chromosomal region 4q13, near UGT2B10. Fifteen independent and globally significant SNPs explained 33.2% of the variation in cotinine glucuronidation, ranging from 55% for African Americans to 19% for Japanese Americans. The strongest single SNP association was for rs115765562 (p=1.60×10−155). This SNP is highly correlated with a UGT2B10 splice site variant, rs116294140, which together with rs6175900 (Asp67Tyr) explain 24.3% of the variation. The top SNP for nicotine glucuronidation (rs116224959, p=2.56×10−43) was in high LD (r2=.99) with rs115765562. Conclusions Genetic variation in UGT2B10 contributions significantly to nicotine and cotinine glucuronidation but not to nicotine dose. Impact The contribution of genetic variation to nicotine and cotinine glucuronidation varies significantly by racial/ethnic group, but is unlikely to contribute directly to lung cancer risk.

1,3-Butadiene (BD) is an important industrial and environmental chemical classified as a known human carcinogen. Occupational exposure to BD in the polymer and monomer industries is associated with an increased incidence of lymphoma. BD is present in automobile exhaust, cigarette smoke, and forest fires, raising concern about potential exposure of general population to this carcinogen. Following inhalation exposure, BD is bioactivated to 3,4-epoxy-1-butene (EB). If not detoxified, EB is capable of modifying guanine and adenine bases of DNA to form nucleobase adducts, which interfere with accurate DNA replication and cause cancer-initiating mutations. We have developed a nanoLC/ESI+-HRMS3 methodology for N7-(1-hydroxy-3-buten-2-yl) guanine (EB-GII) adducts in human urine (limit of detection: 0.25 fmol/mL urine, limit of quantitation: 1.0 fmol/mL urine). The new method was successfully used to quantify EB-GII in urine of F344 rats treated with 0 – 200 ppm BD, occupationally exposed workers, and smokers belonging to two different ethnic groups. EB-GII amounts increased in a dose-dependent manner in urine of laboratory rats exposed to 0, 62.5, or 200 ppm BD. Urinary EB-GII levels were significantly increased in workers occupationally exposed to 0.1–2.2 ppm BD (1.25 ± 0.51pg/mg creatinine) as compared to administrative controls exposed to < 0.01 ppm BD (0.22 ± 0.08 and pg/mg creatinine) (p = 0.0024), validating the use of EB-GII as a biomarker of human exposure to BD. EB-GII was also detected in smokers’ urine, with European American smokers excreting significantly higher amounts of EB-GII than African American smokers (0.48 ± 0.09 vs 0.12 ± 0.02 pg/mg creatinine, p = 3.1 × 10−7). Interestingly, small amounts of EB-GII were observed in animals and humans with no known exposure to BD, providing preliminary evidence for its endogenous formation. Comparison of EB-GII adduct levels in urine to urinary mercapturic acids of BD (MHBMA, DHBMA) and genetic polymorphisms were also attempted in the genotyped smoker ethnic groups cohort.