Association of the phosphatase and tensin homolog gene (PTEN) with smoking initiation and nicotine dependence.код для вставкиСкачать
American Journal of Medical Genetics Part B (Neuropsychiatric Genetics) 141B:10 – 14 (2006) Association of the Phosphatase and Tensin Homolog Gene (PTEN) With Smoking Initiation and Nicotine Dependence Lan Zhang,1,2 Kenneth S. Kendler,1 and Xiangning Chen1* 1 Department of Psychiatry, Virginia Institute of Psychiatric and Behavioral Genetics, Virginia Commonwealth University, Richmond, Virginia 2 Department of Psychiatry, West China Hospital, Sichuan University, Chengdu, China Since PTEN (phosphatase and tensin homolog) has elevated expression in rat brain (amygdala) after chronic administration of nicotine and the PTEN gene is located in the vicinity of the chromosome 10q23 linkage peak in a genome-scan study of nicotine dependence, PTEN seems a plausible candidate gene for smoking behavior. To test this hypothesis, we use a three-group casecontrol design and genotype five SNPs in the PTEN gene. We compare allele and genotype frequencies between the smokers and nonsmokers and between the low nicotine dependent and high nicotine dependent subjects. Three SNPs in the PTEN gene are significantly associated with smoking initiation (rs1234221, P ¼ 0.0311; rs1234213, P ¼ 0.0002; and rs2735343, P ¼ 0.0028). Rs1234213 also shows association with nicotine dependence (P ¼ 0.0278). Haplotype analyses indicate that a major haplotype, 1-1-2-2-1 for rs1234221-rs2299939-rs1234213-rs2735343-rs70184, is associated with smoking initiation. A minor haplotype (about 3%), 1-2-2-2-1 for the same five SNPs, is observed only in the high nicotine dependence group. These results suggest that the PTEN gene may be involved in the etiology of both smoking initiation and nicotine dependence. ß 2005 Wiley-Liss, Inc. KEY WORDS: PTEN; smoking initiation; nicotine dependence; association study INTRODUCTION Tobacco smoking is a complex and addictive behavior. Earlier epidemiologic and genetic studies have established that both environmental and genetic factors play a significant role in smoking [True et al., 1997; Kendler et al., 1999; Li et al., 2003; Munafo et al., 2004]. Other studies indicate that the course from the start of smoking to becoming addicted is an evolving process that includes at least two major stages: smoking initiation (SI) and progression to nicotine dependence [U.S. Department of Health and Human Services, 1989; Kendler et al., 1999]. Genetic factors play important roles in both stages [Sullivan and Kendler, 1999]. These findings have lead to the search for genes predisposing to tobacco smoking and nicotine dependence. Despite some successful results [Tyndale, 2003; Feng et al., 2004], however, the search in general has not been very productive. In addition to other factors such as sample size and power, lack of robust strategies to identify biologically plausible candidate genes may be a contributing factor. We recently have explored a strategy that uses microarray profiling as a screening tool to identify candidates for association study [Chen et al., 2004]. This report is a continuation of our efforts to test more candidate genes identified through microarray studies and other approaches. The PTEN (phosphatase and tensin homolog) gene is involved in inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) turnover as well as intracellular calcium homeostasis [Berridge, 2005]. PTEN negatively regulates intracellular levels of Ins(1,4,5)P3 in cells and also functions as a tumor suppressor by negatively regulating Akt (also known as protein kinase B, PKB) signaling pathway [Roth, 2004; Sansal and Sellers, 2004; Kim et al., 2005]. In a microarray study, the expression of the PTEN gene was elevated in the amygdala of rat brain [Konu et al., 2001]. The PTEN gene is located at 10q23, a region implicated in a linkage study of nicotine dependence [Straub et al., 1999]; the peak marker D10S2469 is about16 million basepairs telomeric to the PTEN gene. Another linkage study with illegal drugs also produced a linkage peak in the same region [Uhl et al., 2001]. Given the fact that both smoking initiation (SI) and nicotine dependence (ND) are complex traits, relatively low penetrance and a small effect are expected of individual genes, the distance between the PTEN gene and the linkage peak is well within the confidence interval of the linkage region [Roberts et al., 1999]. Combining the information of its elevated expression upon nicotine exposure and chromosomal location under a linkage peak, we hypothesize that PTEN gene may be involved in tobacco smoking and ND. Based on this rationale, we conduct this study. MATERIALS AND METHODS Subjects Grant sponsor: NIH; Grant numbers: MH-40828, MH/AA/DA49492, AA-09095; Grant sponsor: NIDA INVEST International Fellowship; Grant sponsor: Virginia Tobacco Settlement Foundation; Grant number: 8520012. *Correspondence to: Xiangning Chen, Virginia Institute for Psychiatric and Behavioral Genetics and Department of Psychiatry, Virginia Commonwealth University, 800 Leigh Street, Suite 1-110, Richmond, VA 23298. E-mail: firstname.lastname@example.org Received 1 February 2005; Accepted 18 July 2005 DOI 10.1002/ajmg.b.30240 ß 2005 Wiley-Liss, Inc. A total of 688 subjects used in this study were selected from two large, population-based twin studies [Kendler and Prescott, 1999; Kendler et al., 1999]. We randomly chose one subject from a twin pair who were all of European ancestry and unrelated. Smoking data collected included smoking history, the Fagerstrom tolerance questionnaire (FTQ) reported for the time of maximal tobacco consumption [Fagerstrom, 1978] and the severity of withdrawal symptoms. The FTQ was used as the phenotype to measure the degree of ND. Based on their smoking history and the FTQ scores, the subjects were divided into three groups: lifetime nonsmokers (NS) who reported 0.741 0.486 0.671 0.045 0.829 11 7 39 23 31 82 53 112 113 100 124 153 70 72 75 0.093 0.934 0.621 0.174 0.054 18 5 22 16 49 68 50 90 90 88 122 145 77 74 71 0.143 0.278 0.886 0.872 0.357 26 7 20 16 54 87 51 92 91 108 119 173 115 114 72 — 40671 32171 16108 21316 A/C, A C/A, C G/A, G G/C, G T/C, T rs1234221 rs2299939 rs1234213 rs2735343 rs701848 22 P-value of HWE test 11 12 22 P-value of HWE test 11 12 High-ND Low-ND NS 22 12 11 Distance between SNPs (bp) Polymorphism, major allele Marker name Data Analysis The genotypes were checked for deviation from Hardy– Weinberg equilibrium. To examine SI, we compared the nonsmokers with both low-ND and high-ND smokers. For ND, we compared the Low-ND group with the High-ND group. We used w2 tests to compare the allele and genotype frequencies for each of the five markers typed for the sample. Linkage disequilibrium (LD), as assessed by D0 and r2, was calculated by the FAMHAP software [Becker and Knapp, 2004] and the haplotype blocks were estimated using the confidence-interval Numbers of genotypes in the three subject groups SNP Selection and Genotyping The PTEN gene is about 102 kb in length. Five haplotypetagged SNPs spanning the gene were selected using the software SNP Browser designed by Applied Biosystems Incorporated (ABI, Foster City, CA). The haplotype tagged SNPs were selected based on Caucasian genotype data. None of the five SNPs were in exons and no functions of these SNPs were known. They were selected solely on the basis that they tagged and differentiated major haplotypes of the PTEN gene. SNPs were genotyped by the 50 nuclease cleavage assay (also called TaqMan method) [Livak, 1999]. Reactions were performed in 384-well plates with 5 ml reaction volume containing 0.25 ml of 20X Assays-on-DemandTM SNP assay mix, 2.5 ml of TaqMan universal PCR master mix and 5 ng of genomic DNA. The conditions for PCRs were initial denaturizing at 958C for 10 min, followed by 40 cycles of 928C for 15 sec and 558C for 1 min. After the reaction, fluorescence intensities for reporter 1 (VIC, exercitation ¼ 520 10 nm, emission ¼ 550 10 nm) and reporter 2 (FAM, exercitation ¼ 490 10 nm, emission ¼ 510 10 nm) were read by the Analyst fluorescence plate reader (LJL Biosytems, Sunnyvale, CA). Genotypes were scored by a Euclidian clustering algorithm developed in our laboratory [van den Oord et al., 2003]. TABLE I. Characteristics of the Five Genotyped SNPs never having smoked an entire cigarette in their lives; regular smokers with low nicotine dependence (low-ND) who smoked regularly (five cigarettes per week) for at least 5 years but whose FTQ scores were 0–2; and regular smokers with high nicotine dependence (high-ND) who smoked regularly for at least 5 years with FTQ scores of 7–11 during their lifetime period of maximum use. In this study, we intended to test the genetic factors separately for SI and ND. To accomplish this goal, we used the 3-group study design. To access genetic factors involved in SI, we compared the nonsmokers with regular smokers, which includes both the Low- and High-ND groups. To identify factors involved in ND, we compared the Low-ND and High-ND groups. In classifying the subjects, we used a stringent definition for low-ND smokers. We realized that this definition excluded a fraction of smokers (some of whom might be called light social smokers or ‘‘chippers’’). For the two population samples used in this study, this very light smoker group who did not meet the inclusion criteria for current study accounted for about 23–25% of the total samples. We did not include these subjects because we were not confident that they all had a low liability to ND due to insufficient nicotine exposure. For this reason, our test for ND was more stringent than most other two-group case-control studies. The sample in this study included 244 nonsmokers, 215 low-ND smokers, and 229 high-ND smokers. All subjects provided informed consent and used standard cytology brushes to obtain buccal epithelial cells for DNA extraction. The method for DNA extraction from the cytology brushes was described previously [Straub et al., 1999]. The population stratification was evaluated by typing 16 unlinked microsatellite makers and no significant stratification was found [Sullivan et al., 2001b]. P-value of HWE test The PTEN Gene and Smoking 11 12 Zhang et al. TABLE II. Allelic and Genotypic Associations Between the Genotyped SNPs and SI and ND Genotype Allele SI Marker name rs1234221 rs2299939 rs1234213 rs2735343 rs701848 ND SI ND w2 P w2 P w2 P w2 P 4.82 0.67 14.44 11.28 1.49 0.0898 0.7165 0.0007 0.0036 0.4749 2.82 0.23 5.00 1.88 4.92 0.2437 0.8929 0.0821 0.3908 0.0856 4.65 0.40 13.85 8.93 2.07 0.0311 0.5279 0.0002 0.0028 0.1503 0.12 0.08 4.84 1.57 2.47 0.7252 0.7720 0.0278 0.2105 0.1161 method implemented in the HAPLOVIEW program [Gabriel et al., 2002; Barrett et al., 2005]. FAMHAP software was also used for haplotype analyses. P-values for haplotype associations were obtained by simulations of the FAMHAP program. RESULTS Six hundred eighty-eight subjects were genotyped for five SNPs in the region of the PTEN gene. Of these SNPs, rs2299939, rs1234213, and rs2735343 were within the PTEN gene, rs1234221 and rs701848 were located within 10 kb distance centromeric and telomeric to the PTEN gene. Of the five typed SNPs, one (rs2735343) slightly deviated from HWE (w2 ¼ 4.98, P ¼ 0.026) when all subjects were combined. To examine if the deviation was caused by genotyping errors, we computed the deviations (P-values) for the three groups separately. As shown in Table I, the deviation was restricted to high-ND smokers only. Two more markers, rs1234221 and rs701848, showed marginal deviations in the Low-ND group. In contrast, none of the five typed SNPs showed significant deviations in the nonsmokers (controls). This information, by itself, was supportive of the association between the PTEN gene and tobacco smoking, because modest deviations in HWE in the affected subjects can be due to association [Hoh et al., 2001]. The average scoring rate for the five SNPs was 93% (from 89 to 95%). To investigate the associations for SI and ND, we compared the genotype and allele frequencies amongst the nonsmokers, the low-ND and high-ND smokers (Table II). Of the five SNPs typed, three of them (rs1234221, rs1234213, and rs2735343) showed significant differences in allele frequencies between the nonsmokers and regular smokers (P ¼ 0.0311, 0.0002, and 0.0028 respectively). Genotypically, rs1234213 and rs2735343 also showed significant differences between the nonsmokers and regular smokers (P ¼ 0.0007 and 0.0036). Rs1234221 showed a trend (P ¼ 0.0898). When the low-ND and high-ND smokers were compared, rs1234213 showed significant allelic association, where the high-ND smokers had a higher frequency (43% vs. 34.5%) for the minor allele (allele A). To understand the haplotype structure of the PTEN gene, we calculated pairwise LDs (D0 and r2) for the five typed SNPs (Table III). There were substantial LD between these SNPs, especially between rs1234212 and rs2735343, where the LD was very strong (D0 ¼ 0.95, r2 ¼ 0.90). When analyzed by the HAPLOVIEW program, the five SNPs were partitioned into three LD blocks by the confidence interval algorithms. Rs1234221 and rs2299939 formed the first block, rs1234213 and rs2735343 formed the second block and rs701848 stood alone. This was slightly different from the structure of the Caucasian sample typed by the HapMap project where rs2299939, rs1234213, and rs2735343 were all in the same LD block and rs1234221 was located in the inter-block region (rs701948 was not typed by the HapMap project, but by its chromosomal position, it belonged to the same block as rs2299939, rs1234213, and rs2735343). In haplotype association analyses, we tested 2- , 3- and 4marker haplotypes for both SI and ND (Table IV). In 2-marker analyses, all combinations, except 1-2, 1-5, and 2-5, reached global significance for SI. In 3- and 4-marker analyses, all haplotypes that carried the minor alleles for both rs1234213 and rs2735343 were significant despite some combinations were not globally significant. Combining the risk haplotype information observed from these 2-, 3- and 4-marker haplotype tests, we found that a major haplotype, 1-1-2-2-1 for the five tested markers, was consistently overrepresented in smokers compared to nonsmokers. This was confirmed by direct testing of 5-marker haplotypes where haplotype 1-1-2-2-1 had a Pvalue of 0.0221 while the global P-value was not significant (P ¼ 0.1236) (data not shown). The frequency of this haplotype was about 7–9% higher in the smokers than the nonsmokers regardless how many markers were used for the analyses (Table IV), giving a relatively constant odds ratio of 1.3. These results were consistent with the haplotype structure observed in the gene, where the tight LD between rs1234213 and rs2735343 constituted the core of this haplotype. It was the minor alleles of these two markers that separated this risk haplotype from other haplotypes. This was also consistent with the single marker association results where rs1234213 and rs2735343 produced the most significant associations. For ND, many marker combinations did not yield significant results. In two 2-marker combinations, that is, 2-3 and 2-4, a minor haplotype (about 3–4%) was detected only in the HighND group. These results were consistent with multi-marker analyses (combinations 1-2-3, 2-3-4, and 2-3-4-5) despite that TABLE III. Pairwise LDs of the SNPs in the PTEN Gene* rs1234221 rs1234221 rs2299939 rs1234213 rs2735343 rs701848 0 0.96 0.68 0.64 0.75 2 rs2299939 rs1234213 rs2735343 rs701848 0.06 0.09 0.08 0.08 0.06 0.90 0.25 0.17 0.26 0.24 0.83 0.71 0.82 *D , below the diagonal; r ,above the diagonal. 0.95 0.76 0.74 The PTEN Gene and Smoking 13 TABLE IV. Haplotype Associations Between the PTEN Gene and SI and ND Marker combination* Global P-value 1-3 1-4 2-3 3-4 3-5 1-2-3 1-3-4 2-3-4 3-4-5 1-3-5 1-4-5 1-2-3-4 2-3-4-5 0.0053 0.0289 0.0162 0.0100 0.0078 0.0158 0.0231 0.0526 0.1323 0.0431 0.2026 0.0308 0.0647 2-3 2-4 3-4 3-5 1-2-3 2-3-4 2-3-4-5 0.0348 0.0369 0.2183 0.0504 0.1022 0.1241 0.2386 Risk haplotype Haplotype frequency (case:control) Smoking initiation 1-2 0.36:0.27 1-2 0.33:0.25 1-2 0.38:0.29 2-2 0.38:0.29 2-1 0.35:0.26 1-1-2 0.35:0.27 1-2-2 0.35:0.27 1-2-2 0.37:0.29 2-2-1 0.35:0.26 1-2-1 0.32:0.24 1-2-1 0.30:0.23 1-1-2-2 0.34:0.27 1-2-2-1 0.34:0.26 Nicotine dependence 2-2 0.03:0.00 2-2 0.04:0.00 2-2 0.41:0.35 2-1 0.40:0.31 1-2-2 0.03:0.00 2-2-2 0.03:0.00 2-2-2-1 0.02:0.00 Odds ratio Haplotype P-value 1.33 1.33 1.31 1.32 1.38 1.27 1.29 1.27 1.34 1.34 1.31 1.25 1.28 0.0017 0.0032 0.0011 0.0007 0.0006 0.0057 0.0061 0.0042 0.0021 0.0037 0.0071 0.0167 0.0105 N/A N/A 1.19 1.30 N/A N/A N/A 0.0445 0.0195 0.0369 0.0058 0.0425 0.0324 0.0360 *Key to marker combinations: 1, rs1234221; 2, rs2299939; 3, rs1234213; 4, rs2735343; 5, rs701848. the global tests did not reach significance (Table IV). When all five markers were included, this minor haplotype observed only in the High-ND group was 1-2-2-2-1. Rs2299939 was the marker that differentiated the 1-1-2-2-1 haplotype, which was overrepresented in regular smokers, from the 1-2-2-2-1 haplotype, which was only observed in high-ND smokers. DISCUSSION In this study, we use a three-group case-control design to investigate if the PTEN gene is involved in smoking behaviors, that is, smoking initiation and nicotine dependence. We have genotyped five SNPs in the PTEN gene and compared the allelic and genotypic frequencies amongst the nonsmokers, low-ND and high-ND smokers. In single marker analyses, we find that three SNPs (rs1234221, rs1234213, and rs2735343) are associated with SI and one SNP (rs1234213) is associated with ND. Two-, 3- and 4-marker haplotype analyses indicate that there is a major haplotype (1-1-2-2-1) over-represented in the smokers and there is a minor haplotype (1-2-2-2-1) observed only in the high-ND smokers. These results indicate that haplotype 1-1-2-2-1 might elevate the risk to smoking and haplotype 1-2-2-2-1 correlates with high nicotine dependence. Twin studies [Kendler et al., 1999; Maes et al., 2004] suggest that the genetic factors impacting on smoking initiation and nicotine dependence are correlated, albeit not identical. It is interesting that we find different haplotypes in the PTEN gene that are specifically associated with smoking initiation and nicotine dependence. There are several factors that can potentially cause false positive results in association studies. The most common ones include population stratification, lack of priori knowledge of the candidates and sample size and power. The subjects used in this study are of European descent and not related. We have evaluated potential stratification by typing 16 unlinked microsatellite markers and no significant evidence of population stratification is found [Sullivan et al., 2001a]. This reduces the probability that results obtained in this study are caused by population stratification. The PTEN gene is not randomly selected for this study. It has been shown to be upregulated in the amygdala of rat brain after chronic exposure of nicotine. This upregulation is confirmed by real-time RT-PCR. Functionally, PTEN is involved in the regulation of inositol triphosphatase, a secondary messenger that regulates many cellular processes, including the Caþþ signal pathway that is involved in cellular responses to external stimuli in many different cell types [Berridge, 2005]. In addition, PTEN is located under a linkage peak found by previous genome scan studies [Straub et al., 1999; Uhl et al., 2001]. Taking all together, this information suggests that PTEN is likely to have a functional role in cellular response to nicotine and may contribute to tobacco smoking in humans. The priori knowledge increases the likelihood that findings in this study are truly positive. Furthermore, our relatively large sample size gives us reasonable power to detect genes with modest effects. In conclusion, our results indicate that PTEN gene is associated with cigarette smoking and possibly involved in both the initiation of smoking and progression to nicotine dependence. However, given our poor understanding of the pathophysiology of smoking and nicotine dependence, further replications are necessary before definitive conclusions can be made. ACKNOWLEDGMENTS This study was supported by Virginia Tobacco Settlement Foundation through the Virginia Youth Tobacco Project to Virginia Commonwealth University. We thank Dr. Linda Corey for assistance with the ascertainment of twins from the Virginia Twin Registry, now part of the Mid-Atlantic Twin Registry (MATR), currently directed by Dr. Judy Silberg. The MATR has received support from the National Institutes of Health, the Carman Trust and the WM Keck, John Templeton and Robert Wood Johnson Foundations. REFERENCES Barrett JC, Fry B, Maller J, Daly MJ. 2005. Haploview: Analysis and visualization of LD and haplotype maps. Bioinformatics 21:263–265. Becker T, Knapp M. 2004. A powerful strategy to account for multiple testing in the context of haplotype analysis. Am J Hum Genet 75:561–570. Berridge MJ. 2005. Unlocking the secrets of cell signaling. Annu Rev Physiol 67:1–21. 14 Zhang et al. Chen X, Wu B, Kendler KS. 2004. Association study of the Epac gene and tobacco smoking and nicotine dependence. Am J Med Genet 129B: 116–119. Roberts SB, MacLean CJ, Neale MC, Eaves LJ, Kendler KS. 1999. Replication of linkage studies of complex traits: An examination of variation in location estimates. Am J Hum Genet 65:876–884. Fagerstrom KO. 1978. Measuring degree of physical dependence to tobacco smoking with reference to individualization of treatment. Addict Behav 3:235–241. Roth MG. 2004. Phosphoinositides in constitutive membrane traffic. Physiol Rev 84:699–730. Feng Y, Niu T, Xing H, Xu X, Chen C, Peng S, Wang L, Laird N, Xu X. 2004. A common haplotype of the nicotine acetylcholine receptor alpha 4 subunit gene is associated with vulnerability to nicotine addiction in men. Am J Hum Genet 75:112–121. Gabriel SB, et al. 2002. The structure of haplotype blocks in the human genome. Science 296:2225–2229. Hoh J, Wille A, Ott J. 2001. Trimming, weighting, and grouping SNPs in human case-control association studies. Genome Res 11:2115–2119. Sansal I, Sellers WR. 2004. The biology and clinical relevance of the PTEN tumor suppressor pathway. J Clin Oncol 22:2954–2963. Straub RE, et al. 1999. Susceptibility genes for nicotine dependence: A genome scan and followup in an independent sample suggest that regions on chromosomes 2, 4, 10, 16, 17 and 18 merit further study. Mol Psychiatry 4:129–144. Sullivan PF, Kendler KS. 1999. The genetic epidemiology of smoking. Nicotine Tob Res 1(Suppl 2):S51–S57. Kendler KS, Prescott CA. 1999. A population-based twin study of lifetime major depression in men and women. Arch Gen Psychiatry 56:39–44. Sullivan PF, Jiang Y, Neale MC, Kendler KS, Straub RE. 2001a. Association of the tryptophan hydroxylase gene with smoking initiation but not progression to nicotine dependence. Am J Med Genet B 105:479–484. Kendler KS, Neale MC, Sullivan P, Corey LA, Gardner CO, Prescott CA. 1999. A population-based twin study in women of smoking initiation and nicotine dependence. Psychol Med 29:299–308. Sullivan PF, et al. 2001b. An association study of DRD5 with smoking initiation and progression to nicotine dependence. Am J Med Genet B 105:259–265. Kim D, et al. 2005. AKT/PKB signaling mechanisms in cancer and chemoresistance. Front Biosci 10:975–984. True WR, Heath AC, Scherrer JF, Waterman B, Goldberg J, Lin N, Eisen SA, Lyons MJ, Tsuang MT. 1997. Genetic and environmental contributions to smoking. Addiction 92:1277–1287. Konu O, et al. 2001. Region-specific transcriptional response to chronic nicotine in rat brain. Brain Res 909:194–203. Li MD, Cheng R, Ma JZ, Swan GE. 2003. A meta-analysis of estimated genetic and environmental effects on smoking behavior in male and female adult twins. Addiction 98:23–31. Tyndale RF. 2003. Genetics of alcohol and tobacco use in humans. Ann Med 35:94–121. Livak KJ. 1999. Allelic discrimination using fluorogenic probes and the 50 nuclease assay. Genet Anal 14:143–149. U.S. Department of Health and Human Services. 1989. Reducing the health consequences of smoking: 25 years of progress. Rockville, MD: Office on Smoking and Health, Center for Chronic Disease Prevention and Health Promotion, Centers for Disease Control, Public Health Sevice, U.S. Department of Health and Human Services. Maes HH, Sullivan PF, Bulik CM, Neale MC, Prescott CA, Eaves LJ, Kendler KS. 2004. A twin study of genetic and environmental influences on tobacco initiation, regular tobacco use and nicotine dependence. Psychol Med 34:1251–1261. Uhl GR, Liu QR, Walther D, Hess J, Naiman D. 2001. Polysubstance abusevulnerability genes: Genome scans for association, using 1,004 subjects and 1,494 single-nucleotide polymorphisms. Am J Hum Genet 69: 1290– 1300. Munafo M, Clark T, Johnstone E, Murphy M, Walton R. 2004. The genetic basis for smoking behavior: A systematic review and meta-analysis. Nicotine Tob Res 6:583–597. van den Oord EJ, Jiang Y, Riley BP, Kendler KS, Chen X. 2003. FP-TDI SNP scoring by manual and statistical procedures: A study of error rates and types. Biotechniques 34:610–620, 622.