Genetic basis of tobacco smoking: strong association of a specific major histocompatibility complex haplotype on chromosome 6 with smoking behavior
George Füst1,2,
Gudmundur J. Arason3,
Judith Kramer4,
Csaba Szalai5,
Jen
Duba6,
Yan Yang7,
Erwin K. Chung7,
Bi Zhou7,
Carol A. Blanchong7,
Marja-Liisa Lokki8,
Sigurdur Bödvarsson9,
Zoltán Prohászka1,2,
István Karádi1,2,
Ágnes Vatay1,
Margit Kovács1,
László Romics1,2,
Gudmundur Thorgeirsson9 and
C. Yung Yu7
1 Third Department of Internal Medicine and 2 Research Group of Metabolism and Atherosclerosis, Semmelweis University and Hungarian Academy of Sciences, Budapest, Hungary
3 Department of Immunology, Institute for Medical Laboratory Sciences, Landspítali University Hospital, Reykjavík, Iceland
4 Central Laboratory, St John Hospital, 5 Heim Pál Pediatric Hospital and 6 National Institute of Cardiology, Budapest, Hungary
7 Center for Molecular and Human Genetics, Columbus Children's Research Institute and College of Medicine and Public Health, The Ohio State University, Columbus, Ohio, USA
8 Tissue Typing Laboratory, Finnish Red Cross Blood Transfusion Service and Transplantation Laboratory, University of Helsinki, Helsinki, Finland
9 Department of Medicine, Landspitali University Hospital, Reykjavík, Iceland
Correspondence to: G. Füst; E-mail: FustGe{at}kut.sote.hu
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Abstract
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The genetic basis for addiction to tobacco smokingparticularly that of the perception of olfactory stimuli that may be important in reinforcing smoking addictionis largely unknown. A cluster of genes for olfactory receptors is in close proximity to the MHC region on chromosome 6. Polymorphisms of MHC class III genes (RCCX modules, TNFA promoter polymorphisms) were determined in 101 healthy subjects and 232 coronary artery disease (CAD) patients from Hungary with defined tobacco smoking habits. A highly significant association between ever smoking (past + current smokers) and a specific MHC haplotype was observed (odds ratios = 2.144.13; P-values = 0.012 to <0.001). This haplotype is characterized by the presence of C4A null alleles and a solitary short C4B gene linked to the TNF2 allele of the promoter for TNFA gene. This haplotype occurred more frequently in the ever smokers than in the never smokers [odds ratio: 4.97 (1.9612.62); P = 0.001], and such associations were stronger in women (odds ratio = 13.6) than in men (odds ratio = 2.79). An independent study of complement C4 protein polymorphism and smoking habits in Icelandic subjects (n = 351) yielded similar and confirmative results. Considering the documented link between olfactory stimuli and smoking in females, and the presence of a cluster of odorant receptor genes close to the MHC class I region, our findings implicate a potential role of the MHC-linked olfactory receptor genes in the initiation of smoking.
Keywords: ancestral MHC haplotype, complement component C4A deficiency, odorant receptors, tobacco smoking, tumor necrosis factor 
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Introduction
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Tobacco smoking has a high impact on the development and progression of serious diseases such as coronary artery disease and lung cancer. Dependence on nicotine is a complex trait influenced by genetic and environmental factors (1,2). Twin studies ranked the heritability estimates for smoking between 46 and 84% (3,4). In a best-fit biometrical model with data from 778 malemale and femalefemale twin pairs reared together or apart, it was shown that genetic effects accounted for 61% of the variance in liability to regular tobacco use in men; a similar value (63%) was found in women born after 1940 (4). To date, the main candidate genes associated with smoking are cytochrome P450 enzymes implicated in nicotine metabolism (5) and dopamine and dopamine receptor genes that reinforce the effect of nicotine (6). Using sib-pair linkage analysis, Bergen and colleagues found a significant association between the evernever smoking trait (EVRNVR) and four genomic regions, among which were two adjacent markers on chromosome 6 (7). These observations prompted us to investigate a possible association between smoking behavior and polymorphisms of MHC genes located on the short arm of chromosome 6. We have examined an association between coronary artery disease (CAD) and polymorphic alleles in the central region of MHC (810). In this study we employed healthy subjects and CAD patients from Hungary and Iceland with known smoking habits for genotypic and/or phenotypic analysis of complement components C4A and C4B, constituents of the RP-C4-CYP21-TNX (RCCX) modules, and single nucleotide polymorphisms at the promoter region of the tumor necrosis factor
gene (TNFA). Compelling evidence was obtained for a strong link between specific MHC haplotype(s) and cigarette smoking.
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Methods
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Study populations
The Hungarian study populations consisted of two groups. Group A consisted of 101 healthy subjects (29 males, 72 females; 44.0 ± 9.6 years old) (11). In these subjects we determined the complement C4A and C4B genotypes and analyzed the data together with C4A and C4B phenotypes that included protein polymorphisms in EDTAplasma, quantitative variations in serum protein concentrations and hemolytic activities, as well as the 308 and 238 G to A promoter polymorphisms of the TNFA gene (12). Group B consisted of 232 patients with severe CAD (182 males, 50 females; 58.7 ± 8.2 years old). All CAD patients had symptoms of severe coronary atherosclerosis that included >50% stenosis, clinical signs of stable or unstable angina pectoris, and typical ECG abnormalities. These CAD patients received aorto-coronary by-pass graft by open heart surgery. Main characteristics of these Hungarian subjects including demographic properties, laboratory markers, association of CAD with polymorphism of mannose binding lectin, seropositivity to Chlamydia pneumoniae, and high levels of antibodies to the 60 kDa human heat shock protein were reported elsewhere (1114). Only Caucasians with residence in Hungary were enrolled in the study. The Icelandic study population consisted of 246 healthy subjects (45 males, 201 females, 40.3 ± 10.6 years old) and 105 patients (55 males, 50 females, 54.3 ± 16.8 years old) who were admitted to the Landspitali University Hospital between 1995 and 1998 with chest pain which on further examination was found to be unrelated to cardiovascular diseases. Informed consents were obtained from all participants and the study was approved by the Ethic Committees of the Semmelweis University in Hungary and the Landspitali University Hospital in Iceland.
Registration of the smoking habits
Smoking behavior was registered by a physician at the study entry. Current smokers and those who had quit smoking were together defined as the group of ever smokers. Ever smokers and never smokers were distinguished in both populations according to the criteria described by Bergen et al. (7) based on previously determined epidemiological thresholds (15,16). Only cigarette smokers were considered in the analysis. In the subgroup of the 232 Hungarian CAD patients, besides ever/never smoking, other smoking traits such as age at initiation, length of smoking, maximal intensity of cigarette smoking (expressed as number of packets/day) were also available.
Preparation of genomic DNA, Southern blot analysis and DNA probes
Total genomic DNA was extracted from white blood cells using proteinase K digestion, phenol extraction and ethanol precipitation (salt out) method (17). After removal of all personal identifiers, genomic DNA samples from the study populations were sent to the laboratory in Columbus for genotyping experiments, employing a protocol approved by the Institutional Review Board of the Columbus Children's Research Institute.
Southern blot analyses of TaqI-digested or PshAI-digested genomic DNAs were performed as described previously (18,19) The TaqI genomic blots were hybridized with three specific probes corresponding to the 3' regions of the RP gene that revealed the presence and dosage of RP1-C4L (7.0 kb), RP1-C4S (6.4 kb), RP2-C4L (6.0 kb) and RP2-C4S (5.4 kb); the presence and relative dosage of CYP21B (3.7 kb) and CYP21A (3.2 kb), and the presence and relative dosage of TNXB (2.5 kb) and TNXA (2.4 kb). The number of RCCX modules was independently confirmed by PshAI RFLP using a RP 3' probe to determine the relative dosage of RP1 (7.2 kb) and RP2 (8.3 kb) genomic fragments. The relative dosages of C4A and C4B genes were determined by PshAI RFLP using a C4d specific probe corresponding to C4 gene exons 2831.
The TNFA 238 and 308 promoter polymorphisms were determined by genomic PCR using modified primer sequences and RFLP analyses, after published protocols (20).
C4 phenotyping
EDTAplasma samples digested by neuraminidase and carboxyl peptidase B were resolved by high voltage agarose gel electrophoresis, subjected to immunofixation using goat anti-human C4 serum, washed to remove diffusible proteins, and stained to reveal the C4A and C4B protein allotypes (21,22).
HLA typing
HLA class I genes A and B and class II gene DRB1 were typed by sequence-specific primer (SSP) PCR of genomic DNA, using commercial kits from One Lambda and standardized protocols.
Statistical analysis
The non-parametric MannWhitney test was used for group comparisons. Categorical data were compared using the Fisher's exact test. Multiple logistic regression was used to evaluate potential confounders. All tests were two-tailed. Statistical analysis was performed with GraphPad Prism 3.0 (GraphPad Software Inc., San Diego, CA; www.grphpad.com) and SPSS 10.0 (SPSS Inc., Chicago, IL) software.
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Results
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Relationship between some polymorphism of the central MHC genes and the ever/never smoking trait in Hungarian healthy people and CAD patients
The relationships between smoking behavior and polymorphisms of central MHC genes (TNFA, C4A and C4B; Fig. 1A) were analyzed in a Caucasian population consisting of 101 healthy individuals and 232 CAD patients residing in Hungary (12,24). Results obtained in this population are summarized in Fig. 1 (14). Among different individuals the number of complement component C4A genes present in a diploid genome varies from 0 to 5, and the size of a C4 gene may be long (20.6 kb) or short (14.2 kb) (18,23) (Fig. 1A). There were marked differences in the prevalence of smoking habits among carriers of different numbers of the C4A gene (Fig. 1B). When subjects with 0 or 1 C4A gene were compared to those with 2 or more C4A genes, 44/56 (79%) of the first group and 143/277 (52%) of the second group were ever smokers (P < 0.001) (Fig. 1C). The differences were significant in both the healthy subjects (P = 0.022) and CAD patients (P = 0.001). A higher proportion of the CAD patients (156/232, 67%) than healthy individuals (31/101, 31%) was reported to be ever smokers. We also found significantly more subjects who ever smoked among the carriers than among the non-carriers of the phenotypic C4A*Q0 allele (70% vs 52%, respectively; P = 0.003; Fig. 1D).

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Fig. 1. Strong association of smoking behaviour with markers of the central MHC in healthy individuals and patients with CAD. (A) A simplified genomic map between chromosome 6p21.33 to 6p21.31 with the human MHC and olfactory receptor genes (OR). Numbering and scale were based on data from the NCBI human genome sequence database (http://www.ncbi.nlm.nih.gov/mapview/maps.cgi?ORG=hum&MAPS=ideogr,est,loc&LINKS=ON&VERBOSE=ON&CHR=6) (43). MB, megabase; I and II, MHC class I and class II genes; RCCX, genetic module consisting of serine/threonine nuclear protein kinase RP, complement C4, steroid CYP21 and tenascin TNX (10); numbers after histones and OR represent the numbers of genes present. (B) Frequency of subjects with different gene dosages of complement component C4A among the ever smokers or among the never smokers. (C) Frequency of ever smokers and never smokers among the subjects with 01 C4A genes or among the subjects with 25 C4A genes. (D) Frequency of subjects with carriers and non-carriers of the phenotypic C4AQ0 allele among the ever smokers or among the never smokers. (E) Frequency of subjects with carriers and non-carriers of TNF2 (i.e. the A-allele at nucleotide 308 of the TNFA promoter region) among the ever smokers or among the never smokers. The healthy subjects and CAD patients were Caucasians from Hungary. The P-values for 2 test (B, C and E) or Fisher's exact test (D) are indicated.
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With respect to the A allele of 308 TNFA promoter polymorphism (TNF2), a significantly higher proportion (P < 0.001) of the carriers (69/96, 72%) than non-carriers (109/221, 49%) were smokers (Fig. 1E). The difference was detected again in both the healthy subjects (P = 0.002) and CAD patients (P = 0.019).
There are also polygenic and gene size variations for complement C4B genes in the MHC (18,23). However, no significant differences in the smoking behavior were found between the carriers and non-carriers of the C4B*Q0 allele (Fisher's exact test, P = 0.169), subjects with 01 versus 2 or more C4B genes (P = 0.859), number of short C4 genes (
2 test, P = 0.406), and homozygous, heterozygous carriers and non-carriers of the 238A TNFA promoter allele (P = 0.591). A difference of modest significance (P = 0.040) was found with the number of long C4 genes.
Relationship between a specific MHC III haplotype and the ever/never smoking trait in Hungarian healthy people and CAD patients
The absence of a C4A gene (C4A*Q0) together with the presence of a single short C4B gene (mono-S) in the MHC can be conveniently detected by a Southern blot analysis of TaqI digested genomic DNA, which appears as a distinct 6.4 kb fragment. In Caucasian populations the strong linkage disequilibrium between the mono-S haplotype with the TNF2 allele within the MHC class III region on the short arm of chromosome 6 is a characteristic feature of the Caucasian ancestral haplotype (AH) 8.1 (2527). However, as no factor B and C2 allotyping were done in this study, and HLA class I and class II typing was performed only in a limited number of subjects (see below), an individual with concurrent mono-S-RCCX (C4AQ0, mono-S C4B1) and TNF2 alleles in a diploid genome is not designated as having a probable AH8.1 but as a mono-S-RCCX-TNF2 haplotype carrier. When the frequencies of the ever/never smokers were compared between subjects with and without the mono-S-RCCX-TNF2 haplotype, we observed remarkable differences (Table 1). Mono-S-RCCX-TNF2 haplotype carriers were extremely likely to smoke (P < 0.001): all except eight (35/43, 81.4%) of the carriers but only 137/268 (51%) of the non-carriers were ever smokers. The differences were highly significant in both the healthy subjects (P = 0.008) and CAD patients (P = 0.005) (Table 1). In addition, we determined the MHC class I genes A, B and class II gene DRB1 polymorphisms in 23 (14 smokers, 9 non-smokers) Hungarian CAD patients. Six smokers had 0 or 1 C4A gene. Among them, three had TNF2 and mono-S, C4A*Q0 C4B*1. All of these three patients had at least one HLA-A*1, HLA-B*8 and HLA-DRB1*03 allele specific to AH8.1. Next, we compared the groups of ever smokers and never smokers for demographic and laboratory data that were registered in both the healthy subjects and CAD patients. Significantly more males than females smoked (P < 0.001). The smokers had significantly higher body mass index (BMI) (P = 0.033), serum total cholesterol (P = 0.027) and triglyceride (P = 0.008) levels. Therefore, we recalculated the association between smoking behavior and the tested gene polymorphisms using multiple logistic regression adjusted for these traits. Since the frequencies of smokers were markedly different in the groups of healthy subjects and CAD patients, the data were adjusted to these groups as well (Table 2). Adjusted odds ratios were significant for subjects with 01 versus 2 or more C4A alleles, carriers versus non-carriers of the C4A*Q0 allele, carriers versus non-carriers of the TNF2 allele, and the co-existence of TNF2, C4A*Q0, C4B*1-short. The highest odds ratio (95% CI) of 4.97 (1.95812.618) (P = 0.001) was found between carriers and non-carriers of the mono-S-RCCX-TNF2 haplotype.
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Table 1. Differences in the frequency of the TNF2, C4A*Q0, C4B*1 (short) haplotype between Hungarian ever smokers (ES) and never smokers (NS)
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Table 2. Logistic regression analysis* of the relationship between smoking habits and polymorphic variants of central MHC genes in 333 Hungarians (healthy individuals + patients with coronary artery disease) stratified according to sex
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Since we found highly significant interactions (P = 0.002 to 0.020) of sex with all genetic traits tested, the data were separately analyzed for males and females (Table 2). The association was stronger in the females than in the males for all traits tested, although the extent of sex-related difference was relatively low with respect to the TNF2 allele. Again, the difference was especially striking for carriers of the mono-S-RCCX-TNF2 haplotype: female carriers of this haplotype were >13-fold more likely to become smokers than non-carriers, while in males an odds ratio of borderline significance was found.
Lack of differences between the carriers and non-carriers of the TNF2, C4A*Q0, C4B*1 (short) haplotype in different smoking traits among Hungarian CAD patients
In the group of CAD patientswhere all of these data were availabledifferent traits of smoking behavior were compared between groups of the carriers and non-carriers of the mono-S-RCCX-TNF2 haplotype (Table 3). Most patients already stopped smoking at the time of by-pass operation. They initiated smoking at 20 years of age on average; used-to-smoke or active smokers had been smoking for an average of 30 years and smoked more than 20 cigarettes/day. Except the differences in the ever/never smoking already depicted above, no significant differences between the two groups (i.e. carriers and non-carriers of mono-S-RCCX-TNF2 haplotype) were found in the proportion of current and past smokers, in the average age of initiation of regular smoking, duration of smoking and number of cigarettes smoked daily (Table 3).
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Table 3. Lack of differences between the carriers and non carriers of the mono-S-RCCX-TNF2 haplotype in different smoking traits among Hungarian patients with CAD
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Differences in the proportion of the ever/never smokers between the Icelandic carriers and non-carriers of phenotypic C4A*Q0
To validate our observation we investigated the smoking behavior in 351 subjects residing in Iceland (28). This population was tested only by C4 phenotyping. The risk to become a smoker was found to be significantly higher for C4A*Q0 carriers than non-carriers and the difference was more pronounced in females than in males (Table 4). As expected, more males (64/100, 64%) than females (115/251, 46%) had ever smoked (P = 0.003). The odds ratio of smoking among C4A*Q0 carriers versus non-carriers calculated by age- and sex-adjusted multiple logistic regression was 1.801 (1.0573.070; P = 0.031). The association of the deficient C4A allele with smoking was stronger in females [OR: 1.926 (1.0143.659), P = 0.045] than in males [OR: 1.511 (0.5793.947); P = 0.399]. In contrast, there was no significant relationship (P = 0.893) between carriers and non-carriers of the C4B*Q0 allele with cigarette smoking.
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Table 4. Differences in frequencies of the apparent C4A*Q0 phenotypes in the male and female Icelandic ever smokers and never smokers.
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The lower odds ratio of the C4A*Q0 group for the increased risk of smoking in the Icelandic subjects (than those of the two Hungarian study populations) was probably due to the lower resolution of the C4 phenotyping method that sometimes does not yield accurate information on subjects with high C4 gene dosages (18).
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Discussion
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Our present findings indicate that in both healthy and CAD study populations, subjects with mono-S-RCCX-TNF2 haplotype had a significant, 5-fold higher risk of becoming smokers compared to those without the haplotype. Strikingly, such association is more pronounced in women, as a >13-fold increased risk was observed in the female cohorts.
Odds ratios of the mono-S-RCCX-TNF2 haplotype carriers to become a regular smoker found in the present study are significantly higher than any other odds ratios for candidate gene association studies with smoking behavior reported so far (1). The highest odds ratio reported previously on a candidate gene associated with smoking was on the DRD2 polymorphism of the dopaminergic receptor gene; odds ratio 2.71 [(2.053.58), P < 0.001] (30). The relatively large number of subjects investigated in this study (n = 684) allows a statistical power to demonstrate a genetic basis of cigarette smoking in Caucasians with high confidence. By contrast, no associations between these alleles and other traits of smoking behavior, such as the age of starting smoking and the duration and intensity of smoking, were found. This observation is in complete accordance with other studies indicating that genetic influences on different stages of smoking such as initiation, maintenance and cessation may not be identical (1,29).
In Caucasian populations, the presence of a solitary short C4B gene coding for a C4B1 allotype accompanied with a phenotypic deficiency of a C4A protein, together with the presence of the TNF2 allele in the MHC is a characteristic feature of the ancestral haplotype with HLA A1, B8, DR3 (AH8.1). This remarkable haplotype has a frequency between 8.6 and 11% in healthy Caucasian populations (14,18), but its frequency is significantly increased in a variety of HLA-associated diseases such as systemic lupus erythematosus (32), type 1 diabetes mellitus, IgA deficiency and common variable immunodeficiency (3335).
To our knowledge, this is the first molecular genetic study illustrating a very strong association of smoking behavior with any polymorphisms of the MHC. Several genome-wide screening studies that suggest evidence for linkage of nicotine addiction to different chromosome regions have been published (2,4,7,31) The data for linkage studies have been inconclusive so far. However, our present findings are in line with the observations of Bergen et al. (7), who found a significant association between the ever smoking trait and, among other regions, with markers on chromosome 6.
No data are available that indicate that heterozygous or complete deficiency of complement C4A, or the presence of TNF2 alleles per se contributes to higher risks of smoking. It can be assumed that carriers of 8.1AH have an increased risk of becoming smokers. On the other hand, since we did not perform factor B and C2 phenotyping and HLA class I and class II typing were performed only in a limited number of individuals, at the present time this assumption can be considered only as a hypothesis. Irrespectively of the haplotype involved, no definite explanation for our present findings can be given at the present time. It is tempting to speculate, however, that the observed association between the mono-S-RCCX-TNF2 haplotype or 8.1AH and ever smoking is related to the presence of a cluster of genes of olfactory receptors in close proximity to the MHC. One of the chemosensory systems is the main olfactory epithelium (MOE), which discriminates odors from the environment (36). There are about 5 million olfactory sensory neurons, each of which expresses one odorant receptor (OR) with a 7-transmembrane domain. The human genome contains about
1000 OR genes and one of the clusters of MOE-expressed OR genes is located telomeric to the MHC class I region (Fig. 1A) (39). Alleles from these OR genes are in strong linkage disequilibrium with each other and also with specific MHC haplotypes (37). Ehlers et al. (40) defined 13 HLA-lined OR haplotypes and demonstrated that allelic variation is a general feature of the HLA-linked OR genes.
On the other hand, Perkins and coworkers suggested that subjective hedonic and reinforcing effects of smoking behavior were influenced by olfactory/taste stimuli (38). They also noted that the olfactory/taste stimuli-dependent effects of cigarette smoking were stronger in women than in men. By contrast, nicotine itself is less reinforcing on smoking in women than in men (41).Those findings are in line with the sex-related difference found in the present study: we observed a stronger association between the mono-S-RCCX-TNF2 haplotype and smoking habit in women than in man. Therefore, it would be exciting to determine whether specific variants of the MHC-linked OR genes (40) contributed to higher risk for initiation of smoking. To this end, it is of interest to note that the MHC has also been implicated for a role in mate choice or sexual attraction (42,43), which is in keeping with the notion that MHC-linked OR genes would be relevant in olfactory-based discrimination (44) and nicotine addiction. Further studies on the genetics and functions of the MHC-linked OR genes, and the interaction of OR with other biological and environmental factors, would yield insight into genetic contributions to smoking habits, which can potentially lead to more effective policies or strategies to reduce smoking and to improve public health.
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Acknowledgements
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We are indebted to the blood donors for their participation in this study. We thank Dr Margrét Leopoldsdóttir, Dr Ragnhildur Magnúsdóttir and Professor Helgi Valdimarsson for their contribution. This work was supported by research grants from the Hungarian Academy of Sciences (2000-107 3,2), Hungarian Ministry of Health (ETT 196/2003), Hungarian National Research Fund (T032661) (GF), the ATHERNET (QLG1-CT-2002-90397) grant of the European Commission, the Icelandic Research Council grant 011330001 and the Science Fund of Landspitali University Hospital, Iceland (GJA), and the US National Institutes of Health grants NIAMS 1R01 AR050078 and NIDDK P01 DK55546 (C.Y.Y.).
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Abbreviations
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BMI | body mass index |
CAD | coronary artery disease |
MOE | main olfactory epithelium |
OR | odorant receptor |
RCCX | four consecutive genes coding for serine/threonine nuclear kinase protein RP, complement component C4, cytochrome P450 CYP21, extracellular matrix protein tenascin TNX |
SSP | sequence-specific primer |
TNF2 | 308A allele of the tumor necrosis factor gene TNFA |
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Notes
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Transmitting editor: A. Falus
Received 28 May 2004,
accepted 28 July 2004.
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