Pd Genes And Cigarettes

NOS2A and the Modulating Effect of Cigarette Smoking in Parkinson’s Disease Dana B. Hancock, BS,1 Eden R. Martin, PhD,1,2 Kenichiro Fujiwara, BS,1 Mark A. Stacy, MD,1,2 Burton L. Scott, PhD, MD,1,2 Jeffrey M. Stajich, PA-C,1 Rita Jewett, RN,1 Yi-Ju Li, PhD,1,2 Michael A. Hauser, PhD,1,2 Jeffery M. Vance, PhD, MD,1,2 and William K. Scott, PhD1,2

Objective: Inducible nitric oxide synthase, a protein product of NOS2A, generates nitric oxide as a defense mechanism, but excessive levels threaten cellular survival. NOS2A is a candidate gene for Parkinson’s disease (PD) that potentially interacts with cigarette smoking. We examined NOS2A for association with PD risk and age at onset (AAO) and for interaction with smoking. Methods: We genotyped 13 NOS2A single nucleotide polymorphisms (SNPs) in 466 singleton families and in a validation set of 286 multiplex families. We tested allelic and haplotypic association using the association in the presence of linkage test, genotypic associations using the genotype pedigree disequilibrium test, AAO effects using the quantitative transmission disequilibrium test, and interactions using generalized estimating equations. Results: Among the pooled earliest onset families, rs2255929 and rs1060826 generated significant allelic (p ⫽ 0.000059 and 0.0062, respectively) and genotypic (p ⫽ 0.0039 and 0.0014, respectively) associations with risk and AAO (p ⫽ 0.00070 and 0.0073, respectively); the two-SNP haplotype generated even stronger association with PD (p ⫽ 0.000013). Significant interactions with smoking (p ⫽ 0.0015 for rs 2255929 and p ⬍ 0.0001 for rs 1060826) were detected in a subset of the families; smoking was inversely associated with PD among risk allele noncarriers, but significance diminished among carriers. Interpretation: Our findings support NOS2A as a genetic risk factor in PD, potentially by influencing AAO and by modifying the inverse association between PD and smoking. Ann Neurol 2006;60:366 –373

Parkinson’s disease (PD) is characterized by degeneration of dopaminergic neurons within the substantia nigra, which coordinates movement and balance. Affected individuals experience development of resting tremors, muscular rigidity, bradykinesia, and other clinical manifestations with severity proportional to neuronal loss. PD affects more than one million Americans, but because peak incidence occurs between ages 55 and 66 and the US population is rapidly aging, prevalence and ensuing societal costs are increasing.1,2 Multiple lines of evidence, including familial aggregation and increased sibling recurrence rate ratios (ranging from 2–14), suggest that genetic factors confer susceptibility without excluding the possibility of environmental insults.3 Identification of causal genetic variants in six genes ( parkin, ␣-synuclein, UCH-L1, DJ-1, PINK1, and LRRK2) confirms the existence of genetic factors.4,5 A number of environmental exposures have also been associated with PD, most notably the protective effect of cigarette smoking.6 Heightened inducible nitric oxide synthase (iNOS) activity leading to excessive levels of nitric oxide (NO) and its toxic metabolite, peroxynitrite, have been im-

plicated in infectious, inflammatory, and neurodegenerative diseases.7 Three NO synthesizing enzymes, iNOS, neuronal NOS (nNOS), and endothelial NOS (eNOS), have been well characterized. The constitutively expressed isoforms (nNOS and eNOS) produce low NO levels over short time intervals, whereas iNOS produces high NO levels over long intervals.8 Inflammatory mediators regulate iNOS gene (NOS2A) expression to produce enough NO to combat environmental insults. However, prolonged nitrosative stress can threaten cellular survival. Significant associations between PD and a NOS2A polymorphism (rs1060826) have been reported.9,10 In genomic screens to identify genes influencing age at onset (AAO) and risk for PD, we detected evidence for risk and AAO genes on chromosome 17 near NOS2A.11,12 Whether increased iNOS activity accompanies PD pathogenesis or contributes to disease onset is unclear. However, the toxic effects of NO suggest that NOS2A is a biologically plausible candidate gene for PD.13,14 Epidemiologic studies consistently report an inverse association between PD and cigarette smoking. Metaanalysis demonstrated that individuals with PD are half

From the 1Center for Human Genetics and 2Department of Medicine, Duke University Medical Center, Durham, NC.

Published online Jul 5, 2006, in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/ana.20915

Received Jan 19, 2006, and in revised form Apr 17. Accepted for publication May 9, 2006.

Address correspondence to Dr Scott, Center for Human Genetics, Duke University Medical Center, Box 3445, Durham, NC 27710. E-mail: [email protected]

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as likely to report ever smoking as unaffected individuals.6 We corroborated this inverse association in a subset of our sample.15 The effect of cigarette smoke on NOS2A expression is unclear, but one recent study reported that cigarette smoke condensates inhibited inflammatory induction of iNOS and reduced cytotoxic effects.16 Given the protective effect of cigarette smoking and the biological plausibility of an interaction between NOS2A and smoking, testing for this interaction may provide insight into the neurodegenerative process underlying PD. We therefore set out to investigate the main effect of NOS2A polymorphisms and NOS2Asmoking interactions on risk for PD using two familybased case–control data sets. Subjects and Methods Sample Ascertainment Affected individuals and their family members were ascertained by the Morris K. Udall PD Research Center of Excellence at the Duke Center for Human Genetics (CHG) and 13 centers of the PD Genetics Collaboration. The clinicbased ascertainment enrolled singleton families (only one sampled affected member or an affected parent–child pair) and multiplex families (nuclear families with at least two sampled affected siblings or extended pedigrees with at least two sampled affected individuals who are not siblings or a parent–child pair). Any number of unaffected relatives (depending on availability) were collected for both family types. Only multiplex families are informative for linkage, whereas both family types are informative for association. Families with mutations in the parkin, ␣-synuclein, and LRRK2 genes (20 singleton and 21 multiplex) were removed from the analysis to minimize heterogeneity. To minimize bias due to population stratification, we stratified data by self-reported race. Results for the white families, the only subset with sufficient statistical power, are reported. A total of 752 families (466 singleton and 286 multiplex families) were studied. A description of the families is presented in Table 1. A neurological examination was performed on all participants with a standard clinical evaluation, including assessment with the Unified PD Rating Scale. Affected individuals demonstrated at least two cardinal signs of PD (resting tremors, rigidity, and bradykinesia), asymmetry of symptom onset, and absence of atypical signs; unaffected individuals presented no signs of PD; and individuals who showed only one cardinal sign and/or atypical signs were classified as unclear. Unclear individuals were excluded from analysis to maximize phenotypic reliability. AAO was defined as the age at which

affected individuals recalled the development of one of the cardinal signs. AAO was missing for affected individuals in 10 families. To ensure consistency, a clinical adjudication board (M.A.S., B.L.S., and J.M.V.) reviewed all clinical examination data. A blood sample, family history, medical history, and standard cognitive status test (Blessed OrientationMemory-Concentration test, or 3MS) were collected for each participant. Institutional review boards at the respective centers approved all study protocols and consent forms, which were signed by all participants before blood and data collection.

Environmental Risk Factor Data Collection Trained interviewers administered a standard telephone questionnaire to gather environmental risk factor data. These data were collected only on families enrolled by the Duke Udall Center; consequently, the interaction analyses utilized data collected on 243 families (226 singleton and 17 multiplex families). Cigarette smoking was assessed by asking: “Have you smoked at least 100 cigarettes in your lifetime?” and “Did you ever smoke cigarettes at least once per week?” If applicable, the years of initiating and quitting smoking were recorded. Participants who responded “yes” to both questions and who initiated smoking before the reference age (derived from the population-averaged time from onset to enrollment) were classified as ever-smokers. Otherwise, participants were classified as never-smokers.

Single Nucleotide Polymorphism Selection Genotypes from 13 phase I HapMap single nucleotide polymorphism (SNPs) in NOS2A and in the 10kb regions flanking the gene were examined in 30 white European families.17 Data were input into HAPLOVIEW, a program that characterizes linkage disequilibrium (LD) patterns to identify haplotype blocks and to choose tag SNPs that uniquely identify haplotypes.18 Eight tag SNPs (one nonsynonymous, one synonymous, and six intronic) capture all common (minor allele frequency ⬎ 10%) phase I HapMap variants across NOS2A with a pairwise correlation coefficient (r2) greater than or equal to 0.8. One tag SNP located 5⬘ upstream and one tag SNP located 3⬘ downstream of NOS2A were also selected for genotyping. Three other validated coding SNPs, including the previously implicated synonymous SNP (rs1060826), were selected from the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) and Ensembl (www.ensembl.org) databases.9,10 A total of 13 NOS2A SNPs were selected for genotyping in our singleton data set of 466 families (Fig). Any SNPs significantly associ-

Table 1. Description of Pedigree Structures in 466 Singleton Families with 505 Affected Individuals and 286 Multiplex Families with 637 Affected Individuals Affected Individuals (n) Families Singleton families Multiplex families

Informative Relationships (n)

Two Parents Sampled

One Parent Sampled

No Parents Sampled

Affected Sibpairs

Discordant Sibpairs

Affected Relative Pairs

87 14

46 47

354 265

0 211

353 865

0 126

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ated with PD ( p ⬍ 0.05) were subsequently genotyped in a validation data set of 286 multiplex families.

Genotyping Blood samples were prepared and stored by the Duke CHG DNA bank. Genomic DNA was extracted from whole blood via the PUREGENE system (Gentra Systems, Minneapolis, MN). All SNPs were genotyped using the TaqMan method with probes and primers obtained through the Applied Biosystems (ABI, Foster City, CA) Assay-on-Demand or Assayby-Design services. Taqman reactions ran with 5␮l volumes, and the subsequent polymerase chain reaction (PCR) amplification, was accomplished with the GeneAmp PCR system 9700 thermocyclers (ABI) for a 50-cycle program. Fluorescence resulting from the PCR amplification was detected using the ABI PRISM 7900HT Sequence Detection Systems (SDS) and analyzed with SDS software. Stringent quality-control measures maximized genotyping accuracy. Each plate contained four controls with known genotype and four nontemplate blanks. Internal controls consisted of 24 duplicated individuals per plate to ensure consistency. Each DNA plate met 100% matching for qualitycontrol samples and at least 95% efficiency. Genotype data were then stored and managed by the PEDIGENE informatics system.

Statistical Analysis Deviations from Hardy–Weinberg equilibrium for all genotyped markers were assessed with the Genetic Data Analysis (GDA) program.19 LD across NOS2A was evaluated for all pairwise marker combinations using the Graphical Overview of LD (GOLD) program to generate both r2 and D⬘ values.20 A total of 457 affected and 299 unaffected individuals (at most one individual per family) were used in Hardy– Weinberg equilibrium and LD analyses. Family-based tests of association were used to examine the relation between PD and NOS2A SNPs. The association in the presence of linkage (APL) test captures information from nuclear families, including triads (parents and offspring), discordant siblings, and affected sibling pairs or triads, maximizing genetic information gathered from our collection of various pedigree structures.21 Parental genotype data are often missing in PD and other late-onset disease studies. Siblings can be used to infer missing parental genotypes, but when more than one sibling is affected, the correlations in transmissions from parents to multiple affected offspring due

to linkage must be taken into account.21 The APL test adjusts for these correlations by incorporating identity-bydescent parameters into inference of missing parental genotypes in nuclear families.21 Because APL uses genotypes of unaffected siblings only to infer missing parental genotypes to then derive transmitted and untransmitted allele counts to affected individuals, an age restriction for unaffected individuals is not necessary. The multilocus haplotype APL test was also applied to combinations of significant SNPs in low to moderate LD to identify SNP haplotypes associated with PD. The pedigree disequilibrium test (PDT) also tests for single-locus allelic association in nuclear pedigrees, but does not capture information from affected sibling pairs without both parents typed.22,23 An extension of this test, genotypePDT, assesses single-locus genotypic associations; such a test is not possible in APL.24 SNPs with significant APL results were analyzed with genotype-PDT to determine whether allelic association was due to an overrepresentation of a particular genotype in affected individuals. To examine genetic heterogeneity by age, we stratified by AAO. Families with at least one affected individual with onset before age 40 were classified as early-onset, and families with a minimum AAO of 40 years or older were classified as late-onset. Progressively older AAO cutoff points (45, 50, 55, and 60 years) were applied to detect association by AAO. To assess the appropriateness of stratification by minimum AAO when multiple affected individuals exist within families, we calculated Pearson’s correlation coefficient (r) between individual AAO and family reference age (minimum AAO in the family) for affected individuals in multiplex families. For SNPs significantly associated with PD in AAO strata, effects on AAO were explored with the Monks–Kaplan method of the quantitative transmission disequilibrium test (QTDT) using AAO as a quantitative trait.25 The previous association tests use affection status as the outcome, whereas the QTDT uses AAO as the outcome variable among affected individuals. To test interactions between genetic and environmental risk factors for PD, we generated models with generalized estimating equations using PROC GENMOD in SAS version 8e (SAS Institute, Cary, NC). Population-averaged generalized estimating equation models use information from all sampled family members (concordant and discordant sibling and relative pairs) and estimate parameters based on marginal case–control effects. To adjust for correlations between related individuals, we used an exchangeable correlation matrix

Fig. Gene structure of NOS2A and locations of selected single nucleotide polymorphisms (SNPs). Blocks represent the 27 exons, and lines represent flanking intronic sequences. Shaded blocks denote coding regions, and colorless blocks denote untranslated regions. Asterisks mark SNPs that show significant allelic association.

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(assuming all pairs of relatives have equal correlation) to calculate a robust estimate of the variance for each model term. Although this correlation matrix structure might be incorrect, generalized estimating equation models are quite robust to misspecification of the correlation matrix.26 GEE models assessed interactions between smoking and the risk allele of each significantly associated SNP by including the risk allele carrier status (carrier vs noncarrier), cigarette smoking history (ever vs never), an interaction term between carrier status and smoking, age at examination (AAE), and sex. For SNPs that significantly interacted with smoking, a stratified analysis was performed to assess the differential effects of smoking by risk allele carrier status. GEE models with smoking, AAE, and sex were constructed in allele carriers and noncarriers separately. Due to an excess of familial clusters containing only one individual in stratified analyses, exchangeable correlation matrices could not be estimated. Thus, an independent correlation matrix was used in stratified models. Given the robustness of GEE models to misspecification of the correlation matrix and the excess of unrelated cases and control subjects in stratified analyses (the source of the exchangeable correlation matrix estimation error), this adjustment is not likely to significantly impact the validity of the stratified analyses.26,27 Empirical p values were generated to evaluate significance for initial GEE models, and odds ratios and 95% confidence intervals were generated to assess the direction and magnitude of association for stratified analyses.

Results The 13 NOS2A polymorphisms were genotyped and analyzed in 466 nuclear singleton families. No significant deviations from Hardy–Weinberg equilibrium were detected in affected and unaffected individuals. As shown in Table 2, the APL test detected no significant allelic associations in the overall data set of 466 singleton families or in the 409 late-onset (AAO ⱖ 40 years) singleton families. However, APL detected significant association ( p ⬍ 0.05) between PD and three SNPs, rs2255929, rs1060826, and rs2248814, in the 51 early-onset families. The positions of these significantly associated SNPs in NOS2A are noted with asterisks in the Figure. SNP rs2255929 provided the most significant evidence for association (see Table 2), with moderate LD existing between this polymorphism and both rs1060826 and rs2248814 among affected individuals (Table 3). SNPs rs2248814 and rs1060826 were significantly associated with PD (see Table 2), with high LD existing between these SNPs (see Table 3). Similar levels of LD between these three markers were shown in the unaffected individuals (data not shown). Considering the LD pattern among these significantly associated markers, we selected rs2255929 and rs1060826 for analysis in a second data set. We genotyped rs2255929 and rs1060826 in a validation data set consisting of 286 nuclear and extended multiplex families. A strong correlation existed (r ⫽ 0.86) between individual AAO and family reference

Table 2. NOS2A Allelic Association Test Results Using Association in the Presence of Linkage in 466 Singleton Families APL p SNP rs2531860 rs3730014 rs3794766 rs2072324 rs8072199 rs16966563 rs3794764 rs1137933 rs2248814 rs2297518 rs1060826 rs2255929 rs4796017

Overall (N ⫽ 466)

Early-Onset (n ⫽ 51)

Late-Onset (n ⫽ 409)

0.94 0.68 0.65 0.56 0.46 0.88 0.64 0.86 0.93 0.97 0.55 0.37 0.91

0.47 0.87 0.97 0.45 0.44 0.58 0.68 0.96 0.036 0.22 0.040 0.0018 0.11

0.70 0.53 0.80 0.72 0.38 0.97 0.52 0.85 0.55 0.78 0.98 0.87 0.79

The affected member of 51 singleton families presented Parkinson’s disease symptoms before age 40, and the affected member of 409 singleton families had onset of symptoms at or beyond age 40. APL ⫽ association in the presence of linkage; SNP ⫽ single nucleotide polymorphism.

age for affected individuals in multiplex families, suggesting that stratification by minimum AAO is appropriate. Significant allelic association was replicated in the 22 multiplex families with minimum AAO less than 40 years for both rs2255929 ( p ⫽ 0.018) and rs1060826 ( p ⫽ 0.027). No significant allelic association of either polymorphism was detected in the overall multiplex data set (286 families) or in the 260 multiplex families with a minimum AAO of 40 years or older. Considering the significant allelic association of rs2255929 and rs1060826 in singleton and multiplex families, these data sets were combined for further analysis. No significant allelic association was observed for either rs2255929 or rs1060826 in the 752 combined families. Because previous analyses showed significant associations only in earlier onset families, we stratified the 742 families with AAO data available by minimum AAO using progressively increasing cutoff points of 40, 45, 50, 55, and 60 years. As shown in Table 4, the most significant association between the more frequent T allele of rs2255929 and PD was achieved in the 73 families with at least one member affected before age 40; however, association between PD and rs2255929(T) remained significant across all early-onset strata. The most significant association between PD and the less frequent A allele of rs1060826 was also achieved in the 73 earliest onset families; the association remained significant only in the families with a minimum AAO of younger than 45 and 50 years (see Table 4). Among the 73 earliest onset fami-

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Table 3. Linkage Disequilibrium between NOS2A Polymorphisms in 457 Affected and Unrelated Individuals as Measured by r2 (Above Diagonal) and D⬘ (Below Diagonal)

rs2531860 rs3730014 rs3794766 rs2072324 rs8072199 rs16966563 rs3794764 rs1137933 rs2248814 rs2297518 rs1060826 rs2255929 rs4796017

rs2531860

rs3730014

rs3794766

rs2072324

rs8072199

rs16966563

— 1a 0.858 0.421 0.845 0.794 0.403 0.774 0.434 0.722 0.435 0.353 0.642

0.003 — 0.110 1a 0.562 1a 0.999a 0.021 0.355 0.016 0.322 0.508 0.364

0.042 0.001 — 0.953a 0.470 1a 0.906 0.965a 0.641 0.824 0.652 0.679 0.605

0.127 0.005 0.077 — 0.961a 0.993a 0.985a 1a 0.937 0.696 0.956a 0.311 0.181

0.096 0.004 0.094 0.181 — 1a 0.981a 0.535 0.246 0.491 0.249 0.050 0.070

0.085 0 0.008 0.006 0.019 — 0.996a 1a 1a 0.999a 1a 1a 1a

Similar levels of linkage disequilibrium were observed in 299 unaffected and unrelated individuals. a 2

r values greater than 0.8 and D⬘ values greater than 0.95.

lies, the global test for association of all haplotypes between the two polymorphisms (rs2255929 and rs1060826) and PD risk provided stronger evidence for association than either of the individual polymorphisms ( p ⫽ 0.000013). Furthermore, among the 73 earliest onset families, the global genotype-PDT test showed a significant association for the TT genotype at rs2255929 ( p ⫽ 0.0039) and for the AA genotype at rs1060826 ( p ⫽ 0.0014). Given the AAO trend in associations of these SNPs and PD, we applied the QTDT using AAO as a quantitative trait in the combined data set. QTDT analysis demonstrated a significant association between earlier AAO of PD and the T allele at rs2255929 ( p ⫽ 0.00070) and the A allele at rs1060826 ( p ⫽ 0.0073). GEE modeling showed significant interactions between smoking and NOS2A SNPs. In a model containing rs2255929, smoking, AAE, and sex, the interactive term between rs2255929 T-allele carrier status and smoking was statistically significant ( p ⫽ 0.0015). When stratifying by rs2255929 genotype, the consistently documented significant inverse association between ever-smoking and PD was present in 90 carriers of the AA genotype; in 366 carriers of the risk allele [rs2255929(TA/TT)], this significant inverse association diminished (Table 5). The model containing rs1060826, smoking, AAE, and sex also showed a significant interaction between rs1060826 and smoking ( p ⬍ 0.0001). When stratifying by rs1060826 genotype, the inverse association between ever-smoking and PD was observed in 167 carriers of the GG genotype, but among 289 carriers of the risk allele [rs1060826(AG/AA)]; the inverse association again diminished (see Table 5).

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Discussion We identified two NOS2A polymorphisms and their haplotype to be significantly associated with PD among earlier onset singleton and multiplex families. SNP rs2255929 is an intronic variation between exons 23 and 24, and rs1060826 is a synonymous variation in exon 22. The risk alleles, rs2255929(T) and rs1060826(A), were also shown to be significantly associated with earlier AAO. We have presented a large number of statistical tests without adjustment for multiple testing. After applying the conservative Bonferroni correction for 76 tests, the significant associations of rs2255929(T) and the rs2255929-rs1060826 haplotype with early-onset PD and the significant interaction between rs1060826(A) and smoking remained statistically significant. Our findings strongly suggest that NOS2A influences the development of PD. An alteration in NOS2A regulation and/or constant environmental induction could result in prolonged iNOS activity and contribute to the depletion of dopaminergic neurons leading to PD. Mice with an ablated NOS2A resist the neurotoxic effects of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), a parkinsonism-inducing agent. The mice have a similar immunological response, yet a statistically significant increase in neuronal survival rate compared with wild-type littermates.28 –30 In rats, selective pharmacological inhibition of NOS2A attenuates inflammatoryinduced dopaminergic neuronal loss.8,31 In postmortem human brains, a substantially increased iNOS concentration has been detected in the substantia nigra of PD patients compared with matched control subjects.32 These biological data substantiate NOS2A as a strong candidate for PD susceptibility. Two previous case–control studies examined only

Table 3. Continued rs3794764 0.107 0.005 0.073 0.918a 0.195 0.007 — 1a 0.939 0.718 0.958a 0.301 0.197

rs1137933

rs2248814

rs2297518

rs1060826

rs2255929

rs4796017

0.030 0 0.825a 0.073 0.109 0.007 0.077 — 0.680 0.850 0.687 0.766 0.707

0.023 0.001 0.089 0.156 0.055 0.016 0.166 0.090 — 1a 0.976a 0.975a 0.850

0.023 0 0.535 0.031 0.080 0.006 0.035 0.640 0.174 — 1a 1a 0.948

0.023 0.001 0.094 0.158 0.055 0.016 0.169 0.093 0.953a 0.174 — 1a 0.858

0.016 0.003 0.206 0.035 0.002 0.018 0.035 0.238 0.464 0.358 0.490 — 0.837

0.053 0.002 0.156 0.011 0.005 0.018 0.014 0.193 0.369 0.304 0.371 0.675 —

Similar levels of linkage disequilibrium were observed in 299 unaffected and unrelated individuals. a 2

r values greater than 0.8 and D⬘ values greater than 0.95.

one polymorphism in this candidate gene and reported significant inverse associations between the rs1060826 AA genotype and PD.9,10 Our family-based case–control data showed a significant positive association between the A allele and AA genotype of rs1060826 and earlier onset PD. In 752 families, we examined multiple polymorphisms across NOS2A to provide comprehensive coverage of common variation and detected significant associations between PD and two SNPs: rs2255929 and rs1060826. To limit confounding due to population stratification, we restricted analyses to white families only. This is likely sufficient control for such confounding, because self-reported race has nearly perfect correlation with genetic determination of ethnic background among the major US racial groups.33

However, spurious association is possible if substantial heterogeneity exists within major racial groups.33 Numerous differences exist between our study and previous studies that may explain the opposing directions of association. First, we detected significant association between rs1060826 and PD in earlier onset families, whereas Levecque and colleagues9 examined the association in a sample consisting mostly of later onset cases and older unrelated control subjects. Although a protective effect of rs1060826 was not detected in our late-onset families, the opposing associations may be reflective of differing effects of NOS2A SNPs by AAO. Second, our family-based case–control sample consists of white individuals ascertained in the United States, whereas the two previous studies ascer-

Table 4. Allelic Association Test Results for rs1060826 and rs2255929 Using Association in the Presence of Linkage in 742 Singleton and Multiplex Parkinson’s Disease Families at Progressively Older Age-at-Onset Cutoff Points Early-Onset Families SNP Allele rs1060826(A)

rs2255929(T)

a

Minimum AAO (yr)

na

40 45 50 55 60 40 45 50 55 60

73 145 224 360 443 73 145 224 360 443

Observed Expected Transmissionsb Transmissionsc 74 150 236 366 439 96 203 315 513 613

63.5 135.1 216.2 352.3 428.1 81.8 183.2 296.6 490.2 593.0

Late-Onset Families APL p

na

0.0062 0.0063 0.0026 0.094 0.23 0.000059 0.00046 0.0059 0.0062 0.024

669 597 518 382 299 669 597 518 382 299

Observed Expected Transmissionsb Transmissionsc APL p 668 592 506 376 303 935 828 716 518 417

666.9 594.3 512.3 377.7 302.1 936.7 834.3 719.3 526.1 424.5

0.92 0.82 0.50 0.84 0.90 0.87 0.54 0.72 0.34 0.33

Number of families categorized into early- and late-onset strata at each minimum AAO cutoff point.

b c

Observed number of alleles transmitted from parents to affected offspring. Expected number of alleles transmitted from parents to affected offspring.

SNP ⫽ single nucleotide polymorphism; AAO ⫽ age at onset; APL ⫽ association in the presence of linkage.

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Table 5. Effect of Cigarette Smoking on Parkinson’s Disease Risk as Stratified by Genotypes at rs1060826 and rs2255929 SNP Genotype rs1060826(GG) rs1060826(AG/AA) rs2255929(AA) rs2255929(TA/TT)

Cases/Control Subjects (n)

OR

96/71 155/134 52/38 199/167

0.29 0.91 0.26 0.77

Lower Upper CI CI 0.14 0.57 0.11 0.51

0.59 1.44 0.66 1.19

SNP ⫽ single nucleotide polymorphism; OR ⫽ odds ratio; CI ⫽ confidence interval.

tained cases and unrelated control subjects from European populations.9,10 Haplotypic differences between these populations could result in differing associations with a polymorphism that is not considered to be a causal variant, but potentially resides in differing degrees of LD with the true variant. We detected a highly significant association between PD and haplotypes containing rs2255929 and rs1060826, but the previous studies considered only rs1060826. This illustrates the need to evaluate multiple polymorphisms across candidate genes. Finally, differences in the frequency of smoking exist between our study (46.2% of cases and 52.3% of related control subjects reported ever having smoked) and the Levecque and colleagues’ study,9 which reported smoking characteristics (33% of cases and 25% of unrelated control subjects reported ever having smoked), but did not report testing for an interactive effect. A limitation of our study is the reduced number of families in which gene–environment interactions can be examined. Even though this portion of our study has reduced power for testing marginal genetic and environmental effects, our sample size is sufficient to detect large interactions. Using parameters similar to those in the NOS2A-smoking interaction models (dominant mode of inheritance, minor allele frequency of 40%, marginal genetic effect of OR ⫽ 1.15, environmental prevalence of 50%, marginal environmental effect of OR ⫽ 0.69, and interactive effect of OR ⫽ 8.00), our sample size achieves a power of 99.68% to detect a gene–environment interaction of this magnitude as calculated by QUANTO.34 A larger sample size is required for replication if this interactive effect is overestimated. Nevertheless, in the presence of such interaction, examination of NOS2A polymorphism effects without considering the effect of smoking could lead to differing results across studies. More complete evaluation of NOS2A variants and molecular investigation into the main effect of NOS2A and its interactive effect with smoking is necessary to resolve these discrepancies. In synthesizing NO from L-arginine, the oxygenase domains of two iNOS molecules dimerize and align with the reductase domain of one subunit, which en-

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compasses a flavin adenine dinucleotide (FAD) binding domain.35 The FAD binding domain spans exons 19 through 23 of NOS2A (Ensembl). SNP rs1060826 resides in exon 22, and rs2255929 resides in the intronic region flanked by exons 23 and 24. The significantly associated markers potentially reside in strong LD with a variant that disrupts the FAD binding domain of NOS2A and alters NO synthesis. More extensive characterization of the FAD binding domain variation, its LD block, and its regulatory elements is necessary to identify this causal variant. Examination of a statistical interaction between NOS2A and smoking in relation to PD risk was previously unexplored, and limited biological evidence is available to decipher the mechanism of an interaction. One previous study reported that cigarette smoke condensates attenuate iNOS induction in vitro, ultimately reducing cytotoxic effects.16 Among noncarriers of the reported risk alleles, our results are consistent with this observed protective effect of cigarette smoking. However, a genetic alteration that disrupts the interactive mechanism could potentially hinder the inhibitory effect of cigarette smoke on iNOS induction as demonstrated by the diminished protective effect of smoking among carriers of the risk alleles. Given the strong epidemiologic evidence supporting an environmental effect by smoking and our statistical suggestion of an interaction, further investigation is warranted to establish the biological mechanism of an interaction. Our findings strongly suggest that NOS2A is a genetic risk factor for PD, and its interaction with cigarette smoking merits consideration in future studies of NOS2A and PD.

This research was supported by the NIH (National Institute on Neurological Disorders and Stroke, P50 NS39764-03, J.M.V.) and the Duke University Graduate School (D.B.H.). We are grateful to the PD families for their participation in our study. We thank the PD Genetics Collaboration members who contributed families: M. Nance, R. Watts, J. Hubble, W. Koller, K. Lyons, R. Pahwa, M. Stern, A. Colcher, B. Hiner, J. Jankovic, W. Ondo, F. Allen Jr, C. Goetz, G. Small, D. Masterman, F. Mastaglia, and J. Haines. We also thank B. Wheeler, E. Tegnell, and the CHG and Duke University Medical Center personnel for their exceptional clinical, technical, and administrative contributions.

References 1. Olanow CW, Koller WC. An algorithm (decision tree) for the management of Parkinson disease: treatment guidelines. Neurology 1998;50:S1–S57. 2. McDonald WM, Richard IH, DeLong MR. Prevalence, etiology, and treatment of depression in Parkinson’s disease. Biol Psychiatry 2003;54:363–375. 3. Gasser T. Genetics of Parkinson’s disease. Ann Neurol 1998; 44:S53–S57. 4. Vila M, Przedborski S. Genetic clues to the pathogenesis of Parkinson’s disease. Nat Med 2004;(suppl 10):S58 –S62.

5. Foroud T. LRRK2: both a cause and a risk factor for Parkinson disease? Neurology 2005;65:664 – 665. 6. Hernan MA, Takkouche B, Caamano-Isorna F, Gestal-OteroJJ. A meta-analysis of coffee drinking, cigarette smoking, and the risk of Parkinson’s disease. Ann Neurol 2002;52:276 –284. 7. Kroncke KD, Kehsel K, Kolb-Bachofen V. Inducible nitric oxide synthase in human diseases. Clin Exp Immunol 1998;113: 147–156. 8. Arimoto T, Bing G. Up-regulation of inducible nitric oxide synthase in the substantia nigra by lipopolysaccharide causes microglial activation and neurodegeneration. Neurobiol Dis 2003;12:35– 45. 9. Levecque C, Elbaz A, Clavel J, et al. Association between Parkinson’s disease and polymorphisms in the nNOS and iNOS genes in a community-based case-control study. Hum Mol Genet 2003;12:79 – 86. 10. Hague S, Peuralinna T, Eerola J, et al. Confirmation of the protective effect of iNOS in an independent cohort of Parkinson disease. Neurology 2004;62:635– 636. 11. Scott WK, Nance MA, Watts RL, et al. Complete genomic screen in Parkinson disease: evidence for multiple genes. JAMA 2001;286:2239 –2244. 12. Li YJ, Scott WK, Hedges DJ, et al. Age at onset in two common neurodegenerative diseases is genetically controlled. Am J Hum Genet 2002;70:985–993. 13. Kroncke KD, Suschek CV, Kolb-Bachofen V. Implications of inducible nitric oxide synthase expression and enzyme activity. Antioxid Redox Signal 2000;2:585– 605. 14. Ebadi M, Sharma SK. Peroxynitrite and mitochondrial dysfunction in the pathogenesis of Parkinson’s disease. Antioxid Redox Signal 2003;5:319 –335. 15. Scott WK, Zhang F, Stajich JM, et al. Family-based casecontrol study of cigarette smoking and Parkinson disease. Neurology 2005;64:442– 447. 16. Mazzio EA, Kolta MG, Reams RR, Soliman KFA. Inhibitory effects of cigarette smoke on glial inducible nitric oxide synthase and lack of protective properties against oxidative neurotoxins in vitro. Neurotoxicology 2005;26:49 – 62. 17. International HapMap Consortium. The International HapMap Project. Nature 2003;426:789 –795. 18. Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics 2005; 21:263–265. 19. Lewis PO, Zaykin D. Genetic data analysis: computer program for analysis of allelic data Version 1.0(d15). 2000, http:// hydrodictyon.eeb.uconn.edu/people/plewis/software.php

20. Abecasis GR, Cookson WOC. GOLD—graphical overview of linkage disequilibrium. Bioinformatics 2000;16:182–183. 21. Martin ER, Bass MP, Hauser ER, Kaplan NL. Accounting for linkage in family-based tests of association with missing parental genotypes. Am J Hum Genet 2003;73:1016 –1026. 22. Martin ER, Monks SA, Warren LL, Kaplan NL. A test for linkage and association in general pedigrees: the Pedigree Disequilibrium Test. Am J Hum Genet 2000;67:146 –154. 23. Martin ER, Bass MP, Kaplan NL. Correcting for a potential bias in the Pedigree Disequilibrium Test. Am J Hum Genet 2001;68:1065–1068. 24. Martin ER, Bass MP, Gilbert JR, et al. Genotype-based association tests for general pedigrees; the genotype-PDT. Genet Epidemiol 2003;23:203–213. 25. Monks SA, Kaplan NL. Removing the sampling restrictions from family-based tests of association for a quantitative-trait locus. Am J Hum Genet 2000;66:576 –592. 26. Zeger SL, Liang KY. Longitudinal data analysis for discrete and continuous outcomes. Biometrics 1986;42:121–130. 27. Hardin JW, Hilbe JM. Generalized Estimating Equations. Boca Raton, FL: Chapman & Hall/CRC, 2003. 28. Liberatore GT, Jackson-Lewis V, Vukosavic S, et al. Inducible nitric oxide synthase stimulates dopaminergic neurodegeneration in the MPTP model of Parkinson disease. Nat Med 1999; 5:1403–1409. 29. Grunewald T, Beal MF. NOS knockouts and neuroprotection. Nat Med 1999;5:1354 –1355. 30. Dehmer T, Lindenau J, Haid S, et al. Deficiency of inducible nitric oxide synthase protects against MPTP toxicity in vivo. J Neurochem 2000;74:2213–2216. 31. Herrera AJ, Castano A, Venero JL, et al. The single intranigral injection of LPS as a new model for studying the selective effects of inflammatory reactions on dopaminergic system. Neurobiol Dis 2000;7:429 – 447. 32. Hunot S, Boissiere F, Faucheux B, et al. Nitric oxide synthase and neuronal vulnerability in Parkinson’s disease. Neuroscience 1996;72:355–363. 33. Tang H, Quertermous T, Rodrigues B. Genetic structure, selfidentified race/ethnicity, and confounding in case-control association studies. Am J Hum Genet 2005;76:268 –275. 34. Gauderman JW. Sample size requirements for matched casecontrol studies of gene-environment interaction. Stat Med 2002;21:35–50. 35. Siddhanta U, Presta A, Fan B, et al. Domain swapping in inducible nitric-oxide synthase. J Biol Chem 1998;273: 18950 –18958.

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