Genotype Components as Predictors of Phenotype in Model Gene Regulatory Networks.

Models of gene regulatory networks (GRN) have proven useful for understanding many aspects of the highly complex behavior of biological control networks. Randomly generated non-Boolean networks were used in experimental simulations to generate data on dynamic phenotypes as a function of several genotypic parameters. We found that predictive relationships between some phenotypes and quantitative genotypic parameters such as number of network genes, interaction density, and initial condition could be derived depending on the strength of the topological (positional) genotype on specific phenotypes. We quantitated the strength of the topological genotype effect (TGE) on a number of phenotypes in multi-gene networks. For phenotypes with a low influence of topological genotype, derived and empirical relationships using quantitative genotype parameters were accurate in phenotypic outcomes. We found a number of dynamic network properties, including oscillation behaviors, that were largely dependent on genotype topology, and for which no such general quantitative relationships were determinable. It remains to be determined if these results are applicable to biological gene regulatory networks.

Quantitative analysis of DNA methylation after whole bisulfitome amplification of a minute amount of DNA from body fluids.

Cell-free circulating DNA isolated from the plasma of individuals with cancer has been shown to harbor cancer-associated changes in DNA methylation, and thus it represents an attractive target for biomarker discovery. However, the reliable detection of DNA methylation changes in body fluids has proven to be technically challenging. Here we describe a novel combination of methods that allows quantitative and sensitive detection of DNA methylation in minute amounts of DNA present in body fluids (quantitative Methylation Analysis of Minute DNA amounts after whole Bisulfitome Amplification, qMAMBA). This method involves genome-wide amplification of bisulphite-modified DNA template followed by quantitative methylation detection using pyrosequencing and allows analysis of multiple genes from a small amount of starting DNA. To validate our method we used qMAMBA assays for four genes and LINE1 repetitive sequences combined with plasma DNA samples as a model system. qMAMBA offered high efficacy in the analysis of methylation levels and patterns in plasma samples with extremely small amounts of DNA and low concentrations of methylated alleles. Therefore, qMAMBA will facilitate methylation studies aiming to discover epigenetic biomarkers, and should prove particularly valuable in profiling a large sample series of body fluids from molecular epidemiology studies as well as in tracking disease in early diagnostics.

Early effects of low benzene exposure on blood cell counts in Bulgarian petrochemical workers.

OBJECTIVES: Only few studies have examined early hematological effects in human populations exposed to low benzene levels and their findings are controversial. We evaluated hematological outcomes (WBC, neutrophils, lymphocytes, monocytes, eosinophils, basophils, RBC, Hb, HCT MCV, platelets and MPV) in a population of 153 Bulgarian petrochemical workers exposed to benzene (range 0.01-23.9 ppm) and 50 unexposed subjects. METHODS: Written informed consent was obtained and a self-administered questionnaire used to collect information on current smoking habits, lifestyle, and occupational activities. Exposure assessment was based on personal monitoring sampling the day before phlebotomy. Urinary trans-trans-muconic acid (t,t-MA) was determined at the beginning and end of the work shift. Based on individual airborne benzene measurements, study subjects were categorized in three exposure categories (referents, <1 and > or =1 ppm). Mean values of each hematologic outcomes in each exposure category were compared with the referent group using a multiple linear regression model adjusted for age, gender, current smoking habits and environmental toluene level. The influence of the CYP2E1 (RsaI and DraI) and NQO1 609C>T genetic polymorphisms on differential hematological parameters was also investigated. RESULTS: No dose-response effect was observed for most of the examined hematological outcomes (WBC, lymphocytes, neutrophils, monocytes, RBC, Hb, HCT, MCV, platelets and MPV). The eosinophil count was inversely related to benzene exposure only among smokers. Conversely, basophils increased with increasing exposure. No effect on benzene hematotoxicity was found for any of the investigated polymorphisms. CONCLUSION: In our study we did not find a decline in WBC and lymphocytes related to benzene exposure. A myeloproliferative effect of benzene is highly unlikely to explain the observed reduction in eosinophils and increase in basophils as it would lead to a concordant depression in all granulocyte subpopulations. Whether benzene effects at low doses are present in Caucasian populations remains uncertain, thus warranting further investigations.

Effect of vitamin levels on biomarkers of exposure and oxidative damage-the EXPAH study.

DNA adducts are markers of carcinogen exposure and of their biological effect; they have been shown to be related to mutagenesis, and therefore they could be a predictive biomarker of human cancer. The objective of this study was to assess if there is a relationship between vitamins A, C, and E, which are known to play a significant role as free radical scavengers and antioxidant agents, and biomarkers of genotoxicity and oxidative stress. Three hundred and fifty-six subjects from Czech Republic, Slovak Republic and Bulgaria, who completed a questionnaire on dietary information and had a measurement of plasma A, C, E vitamins, DNA adduct levels (benzo[a]pyrene (B[a]P) and bulky (DNA-Tot) DNA adducts) and oxidative damage (cyclic pyrimidopurinone N-1,N2 malondialdehyde-2 deoxyguanosine (M1dG) and 8-oxo-7,8-dihydro-2_deoxyguanosine (8-oxodG)) were analyzed. A significant inverse correlation was observed between plasma vitamin levels and both benzo[a]pyrene (B[a]P) and bulky DNA adducts. Vitamin A was also significantly inversely correlated with M1dG, a marker of oxidative damage. The associations were stronger in non-smokers than in smokers. Dietary intake of certain antioxidants such as vitamins is associated with reduced levels of markers of DNA damage (B[a]P and DNA-Tot) and oxidation (M1dG and 8-oxodG) measured in peripheral white blood cells. This could contribute to the protective role of such a dietary pattern on cancer risk. The protective effect of dietary vitamins is less evident in smokers.

Meat intake and bladder cancer in a prospective study: a role for heterocyclic aromatic amines?

BACKGROUND: The suspect carcinogens, heterocyclic amines (HAAs), found in well-done meat require host-mediated metabolic activation before inducing DNA mutations. The role of SULT1A1 and of NAT2 on the activation of HAAs suggests that NAT2 rapid acetylator genotype and SULT1A1 allele variants can have an effect on HAA carcinogenicity. METHODS: Data were collected as part of a case-control study nested within the EPIC cohort, the Gen Air investigation. EPIC is a prospective study designed to investigate the relationship between nutrition and cancer. Information was collected through a non-dietary questionnaire on lifestyle variables and through a dietary questionnaire. The subjects were restricted to non-smokers. We calculated the matched odds ratio for bladder cancer risk using logistic regression, controlling for potential confounders. RESULTS: There were 227 bladder cases and 612 controls matched 1:3. Meat intake and NAT2 genotype were not independently associated with bladder cancer risk. A significant relationship was observed between bladder cancer risk and consumption of meat only among subjects with the rapid NAT2 genotype (odds ratios [OR] 2.9, 95% CI 1.0-7.9 for the 2nd quartile of meat intake; 3.6, 95% CI 1.3-9.7 for the 3rd quartile; and 3.5, 95% CI 1.2-9.7 for the 4th quartile), and was not present among subjects with the slow genotype. An interaction between NAT2 and meat intake was found in logistic regression (P = 0.034). No association was observed for SULT1A *1/2 genotype (1.0; 95% CI 0.7-1.5) and for SULT1A1 *2/2 genotype (0.9; 95% CI 0.5-1.7). CONCLUSIONS: These results are suggestive of a role of meat intake and NAT2 on bladder cancer risk. They support the hypothesis that among subjects with the rapid NAT2 acetylation genotype higher levels of HAAs exposure are a bladder cancer risk factor. We did not observe an effect of SULT1A1 allele variants on this cancer. The present study adds new information on the possible long-term adverse effects of diets with high meat intake.

Genetic susceptibility according to three metabolic pathways in cancers of the lung and bladder and in myeloid leukemias in nonsmokers.

BACKGROUND: We chose a set of candidate single nucleotide polymorphisms (SNPs) to investigate gene-environment interactions in three types of cancer that have been related to air pollution (lung, bladder and myeloid leukemia). PATIENTS AND METHODS: The study has been conducted as a nested case-control study within the European Prospective Investigation into Cancer and Nutrition cohort (409 cancer cases and 757 matched controls). We included never and ex-smokers. SNPs were in genes involved in oxidative stress, phase I metabolizing genes, phase II metabolizing genes and methylenetetrahydrofolate reductase (MTHFR). RESULTS: The most notable findings are: GSTM1 deletion and bladder cancer risk [odds ratio (OR) = 1.60; 95% confidence interval 1.00-2.56]; CYP1A1 and leukemia (2.22, 1.33-3.70; heterozygotes); CYP1B1 and leukemia (0.47, 0.27-0.84; homozygotes); MnSOD and leukemia (1.91, 1.08-3.38; homozygotes) and NQO1 and lung cancer (8.03, 1.73-37.3; homozygotes). Other statistically significant associations were found in subgroups defined by smoking habits (never or ex-smokers), environmental tobacco smoke or gender, with no obvious pattern. When gene variants were organized according to the three main pathways, the emerging picture was of a strong involvement of combined phase I enzymes in leukemia, with an OR of 5 (1.63-15.4) for those having three or more variant alleles. The association was considerably stronger for leukemias arising before the age of 55.

Multi-factor dimensionality reduction applied to a large prospective investigation on gene-gene and gene-environment interactions.

It is becoming increasingly evident that single-locus effects cannot explain complex multifactorial human diseases like cancer. We applied the multi-factor dimensionality reduction (MDR) method to a large cohort study on gene-environment and gene-gene interactions. The study (case-control nested in the EPIC cohort) was established to investigate molecular changes and genetic susceptibility in relation to air pollution and environmental tobacco smoke (ETS) in non-smokers. We have analyzed 757 controls and 409 cases with bladder cancer (n=124), lung cancer (n=116) and myeloid leukemia (n=169). Thirty-six gene variants (DNA repair and metabolic genes) and three environmental exposure variables (measures of air pollution and ETS at home and at work) were analyzed. Interactions were assessed by prediction error percentage and cross-validation consistency (CVC) frequency. For lung cancer, the best model was given by a significant gene-environment association between the base excision repair (BER) XRCC1-Arg399Gln polymorphism, the double-strand break repair (DSBR) BRCA2-Asn372His polymorphism and the exposure variable 'distance from heavy traffic road', an indirect and robust indicator of air pollution (mean prediction error of 26%, P<0.001, mean CVC of 6.60, P=0.02). For bladder cancer, we found a significant 4-loci association between the BER APE1-Asp148Glu polymorphism, the DSBR RAD52-3'-untranslated region (3'-UTR) polymorphism and the metabolic gene polymorphisms COMT-Val158Met and MTHFR-677C>T (mean prediction error of 22%, P<0.001, mean CVC consistency of 7.40, P<0.037). For leukemia, a 3-loci model including RAD52-2259C>T, MnSOD-Ala9Val and CYP1A1-Ile462Val had a minimum prediction error of 31% (P<0.001) and a maximum CVC of 4.40 (P=0.086). The MDR method seems promising, because it provides a limited number of statistically stable interactions; however, the biological interpretation remains to be understood.

Meta- and pooled analysis of GSTT1 and lung cancer: a HuGE-GSEC review.

Lung cancer is the most common malignancy in the Western world, and the main risk factor is tobacco smoking. Polymorphisms in metabolic genes may modulate the risk associated with environmental factors. The glutathione S-transferase theta 1 gene (GSTT1) is a particularly attractive candidate for lung cancer susceptibility because of its involvement in the metabolism of polycyclic aromatic hydrocarbons found in tobacco smoke and of other chemicals, pesticides, and industrial solvents. The frequency of the GSTT1 null genotype is lower among Caucasians (10-20%) than among Asians (50-60%). The authors present a meta- and a pooled analysis of case-control, genotype-based studies that examined the association between GSTT1 and lung cancer (34 studies, 7,629 cases and 10,087 controls for the meta-analysis; 34 studies, 7,044 cases and 10,000 controls for the pooled analysis). No association was observed between GSTT1 deletion and lung cancer for Caucasians (odds ratio (OR) = 0.99, 95% confidence interval (CI): 0.87, 1.12); for Asians, a positive association was found (OR = 1.28, 95% CI: 1.10, 1.49). In the pooled analysis, the odds ratios were not significant for either Asians (OR = 0.97, 95% CI: 0.83, 1.13) or Caucasians (OR = 1.09, 95% CI: 0.99, 1.21). No significant interaction was observed between GSTT1 and smoking on lung cancer, whereas GSTT1 appeared to modulate occupational-related lung cancer.

Role of simian virus 40 in cancer incidence in solid organ transplant patients.

Transplant recipients have an increased risk of developing cancer in comparison with the general population. We present here data on cancer development in transplanted subjects who received organs from donors whose DNA was previously examined for the genomic insertion of Simian Virus 40 (SV40). Active follow-up of 387 recipients of solid organs donated by 134 donors, not clinically affected by cancer, was performed through the National Transplant Center (NTC). The average length of follow-up after transplant was 671+/-219 days (range 0-1085 days). Out of 134 proposed donors, 120 were utilised for organ donation. Of these, 12 (10%) were classified as positive for SV40 genomic insertion. None of the 41 recipients of organs from SV40 positive donors developed a tumour during the follow-up. In all, 11 recipients of organs given by SV40 negative donors developed a tumour (cancer incidence: 0.015 per year). In conclusion, cancer rates observed in our study are comparable to what reported by the literature in transplanted patients. Recipients of solid organs from SV40 positive donors do not have an increased risk of cancer after transplant. The role of SV40 in carcinogenesis in transplanted patients may be minimal.

MTHFR C677T polymorphism, GSTM1 deletion and male infertility: a possible suggestion of a gene-gene interaction?

Methylenetetrahydrofolate reductase (MTHFR) is a gene involved in the process of DNA synthesis and methylation. The MTHFR C677T polymorphism has been associated with male infertility. A prospective study was conducted on men seeking care at the infertility clinic in Milano to determine if the MTHFR C677T polymorphism is associated with infertility, and if such an association is modified by a common deletion of one of the glutathione transferases, GSTM1. One year after enrolment, 46 subjects reported having had a child, while 59 were still childless. Subjects carrying the MTHFR C677T homozygous variant polymorphism were at increased risk of being infertile after 1-year follow-up (OR 3.7, 95% CI?=?1.4-10.4); carriers of the homozygous variant MTHFR genotype and of a functional copy of GSTM1 appear to have a significantly higher risk of infertility (n=11; OR?=?22.0 95% CI?=?3.8-127.9) than subjects who carry the wild-type genotype for both genes. Such risk becomes non-significant when the GSTM1 deletion is also present (n=5; OR?=?1.1 95% CI?=?0.2-5.1). A possible explanation of this unexpected result could lie in the known involvement of glutathione transferases in the metabolic pathways of both methylation and transulfuration. The interaction found deserves confirmation and replication in a larger population, since it may be relevant to several chronic diseases such as cardiovascular diseases and cancer.

DNA repair polymorphisms and cancer risk in non-smokers in a cohort study.

Environmental carcinogens contained in air pollution, such as polycyclic aromatic hydrocarbons, aromatic amines or N-nitroso compounds, predominantly form DNA adducts but can also generate interstrand cross-links and reactive oxygen species. If unrepaired, such lesions increase the risk of somatic mutations and cancer. Our study investigated the relationships between 22 polymorphisms (and their haplotypes) in 16 DNA repair genes belonging to different repair pathways in 1094 controls and 567 cancer cases (bladder cancer, 131; lung cancer, 134; oral-pharyngeal cancer, 41; laryngeal cancer, 47; leukaemia, 179; death from emphysema and chronic obstructive pulmonary disease, 84). The design was a case-control study nested within a prospective investigation. Among the many comparisons, few polymorphisms were associated with the diseases at the univariate analysis: XRCC1-399 Gln/Gln variant homozygotes [odds ratios (OR) = 2.20, 95% confidence intervals (CI) = 1.16-4.17] and XRCC3-241 Met/Met homozygotes (OR = 0.51, 95% CI = 0.27-0.96) and leukaemia. The recessive model in the stepwise multivariate analysis revealed a possible protective effect of XRCC1-399Gln/Gln in lung cancer (OR = 0.22, 95% CI = 0.05-0.98), and confirmed an opposite effect (OR = 2.47, 95% CI = 1.02-6.02) in the leukaemia group. Our results also suggest that the XPD/ERCC1-GAT haplotype may modulate leukaemia (OR = 1.28, 95% CI = 1.02-1.61), bladder cancer (OR = 1.38, 95% CI = 1.06-1.79) and possibly other cancer risks. Further investigations of the combined effects of polymorphisms within these DNA repair genes, smoking and other risk factors may help to clarify the influence of genetic variation in the carcinogenic process.

Methodology of laboratory measurements in prospective studies on gene-environment interactions: the experience of GenAir.

Several large prospective investigations are under way or are planned in different parts of the world, aiming at the investigation of gene-environment interactions for chronic diseases. Technical, practical and ethical issues are raised by such large investigations. Here we describe how such issues were approached within a case-control study nested in EPIC, a large European cohort, and the kind of validation studies that have been set up. The GenAir investigation aimed at measuring the effects of air pollution and environmental tobacco smoke on human health in EPIC with a nested design and with biological measures. Validation studies included (a) comparisons between cotinine measurements, hemoglobin adducts and questionnaire data; (b) an analysis of the determinants of DNA adduct concentration; (c) comparison among different genotyping methods; (d) an analysis of the determinants of plasma DNA amounts. We also describe how the ethical issues were dealt with in our investigation.

Environmental tobacco smoke and risk of respiratory cancer and chronic obstructive pulmonary disease in former smokers and never smokers in the EPIC prospective study.

OBJECTIVES: To investigate the association between environmental tobacco smoke, plasma cotinine concentration, and respiratory cancer or death. DESIGN: Nested case-control study within the European prospective investigation into cancer and nutrition (EPIC). PARTICIPANTS: 303,020 people from the EPIC cohort (total 500,000) who had never smoked or who had stopped smoking for at least 10 years, 123,479 of whom provided information on exposure to environmental tobacco smoke. Cases were people who developed respiratory cancers or died from respiratory conditions. Controls were matched for sex, age (plus or minus 5 years), smoking status, country of recruitment, and time elapsed since recruitment. MAIN OUTCOME MEASURES: Newly diagnosed cancer of lung, pharynx, and larynx; deaths from chronic obstructive pulmonary disease or emphysema. Plasma cotinine concentration was measured in 1574 people. RESULTS: Over seven years of follow up, 97 people had newly diagnosed lung cancer, 20 had upper respiratory cancers (pharynx, larynx), and 14 died from chronic obstructive pulmonary disease or emphysema. In the whole cohort exposure to environmental tobacco smoke was associated with increased risks (hazard ratio 1.30, 95% confidence interval 0.87 to 1.95, for all respiratory diseases; 1.34, 0.85 to 2.13, for lung cancer alone). Higher results were found in the nested case-control study (odds ratio 1.70, 1.02 to 2.82, for respiratory diseases; 1.76, 0.96 to 3.23, for lung cancer alone). Odds ratios were consistently higher in former smokers than in those who had never smoked; the association was limited to exposure related to work. Cotinine concentration was clearly associated with self reported exposure (3.30, 2.07 to 5.23, for detectable/non-detectable cotinine), but it was not associated with the risk of respiratory diseases or lung cancer. Frequent exposure to environmental tobacco smoke during childhood was associated with lung cancer in adulthood (hazard ratio 3.63, 1.19 to 11.11, for daily exposure for many hours). CONCLUSIONS: This large prospective study, in which the smoking status was supported by cotinine measurements, confirms that environmental tobacco smoke is a risk factor for lung cancer and other respiratory diseases, particularly in ex-smokers.

Association of metabolic gene polymorphisms with alcohol consumption in controls.

The objectives were to study the association between metabolic genes involved in alcohol metabolism (CYP2E1 RsaI, CYP2E1 DraI, ADH1C, NQO1) and alcohol consumption in a large sample of healthy controls. Healthy subjects were selected from the International Collaborative Study on Genetic Susceptibility to Environmental Carcinogens (GSEC). Subjects with information on both alcohol consumption and at least one of the studied polymorphisms were included in the analysis (n=2224). Information on the amount of alcohol consumption was available for a subset of subjects (n=844). None of the studied genes was significantly associated with drinking habits. A significant heterogeneity with age was observed when studying the association between CYP2E1 RsaI and alcohol drinking. CYP2E1 RsaI polymorphism was significantly associated with being a never drinker at older ages (odds ratio [OR] 2.4, 95% confidence interval [CI] 1.2-4.8; at ages above 68 years), while the association was reversed at ages below 47 years (OR 0.5, 95% CI 0.2-1.4). For subjects with detailed information on alcohol intake, no association between alcohol quantity and polymorphisms in metabolic genes was observed; subjects carrying the NQO1 polymorphism tended to drink more than subjects carrying the wild-type alleles. Therefore, no significant association between CYP2E1 RsaI, CYP2E1 DraI, ADH1C, NQO1 polymorphisms and alcohol consumption was observed in healthy controls.

[European multicenter cross-sectional study on exposure to low doses of benzene]. / Studio trasversale multicentrico europeo su esposizione a basse dosi di benzene.

A cross-sectional multicenter european study has been carried out to evacuate the relations between exposure to low level of benzene and biological markers of internal dose (t,t-MA, S-PMA) and early biological effect (DNA-SSB). The research has shown significantly increased levels (adjusted for smoking habits) of the urinary excretion of t,t-MA, S-PMA and DNA-SSB in petrochemical workers (mean benzene level = 5,694 micrograms/m3) but not in filling station attendants, traffic police officers, and bus drivers compared to referent workers. Dose-response relations were detected between benzene air levels, t,t-MA, S-PMA and DNA-SSB in petrochemical workers, with significantly increased levels of DNA-SSB detected for benzene exposure levels in the range 391-1,800 micrograms/m3 (0.12-0.58 ppm).

Polymorphisms in CYP1A1, GSTM1, GSTT1 and lung cancer below the age of 45 years.

BACKGROUND: A genetic component of early-onset lung cancer has been suggested. The role of metabolic gene polymorphisms has never been studied in young lung cancer cases. Phase 1 and Phase 2 gene polymorphisms are involved in tobacco carcinogens' metabolism and therefore in lung cancer risk. METHODS: The effect of metabolic gene polymorphisms on lung cancer at young ages was studied by pooling data from the Genetic Susceptibility to Environmental Carcinogens (GSEC) database. All primary lung cancer cases of both sexes who were Caucasian and

Metabolic gene polymorphisms and p53 mutations in healthy centenarians and younger controls.

To obtain insights into the genetic mechanisms of ageing, we studied the frequency of the simultaneous presence of polymorphisms in phase I and phase II genes and of several p53 germline mutations in a group of 66 nonagenarians and centenarians in good health, selected from a larger sample of a multicentre Italian study in Northern Italy, and in a sample of 150 young healthy volunteers of the same ethnic group. We found a statistically significant difference in the frequency of 1the GSTT1 deletion and the p53 genotypes: the absence of any p53 polymorphisms and of GSTT1 deletion, and the simultaneous presence of the three p53 polymorphisms and of GSTT1 deletion, were much more frequent in young subjects than in centenarians (41.5% versus 26.9% and 8.8% versus 3.8%, respectively). One hypothesis to explain this difference is that subjects with both GSTT1 deletion and p53 polymorphisms may accumulate carcinogens and may have reduced DNA repair ability, and thus are more at risk for cancer. Another possible explanation is that both metabolic genes and p53 act on pathways related to cell ageing and death, and therefore certain composite genetic patterns could represent a generic mechanism of protection against ageing, not just against the development of chronic diseases. It is likely that longevity is related to a complex genetic trait as well as to certain environmental exposures.

Metabolic susceptibility genes as cancer risk factors: time for a reassessment?

Polymorphisms in metabolic cancer susceptibility genes have not shown a consistent role as cancer risk factors, when only main effects are examined. This is actually to be expected given the limited and specific biochemical role such genes play in the carcinogenic process. However, when particular groups of case populations are examined separately, the importance of these genetic polymorphisms may often become quite clear. Examples from the literature and a hypothetical model are presented to support the view that metabolic gene risk alleles should be studied in subgroups of large case control studies with sound biochemical hypotheses related to the action of the gene product as a function of demographic, environmental, or other genetic variables.

Metabolic gene polymorphism frequencies in control populations.

Using the International Project on Genetic Susceptibility to Environmental Carcinogens (GSEC) database containing information on over 15,000 control (noncancer) subjects, the allele and genotype frequencies for many of the more commonly studied metabolic genes (CYP1A1, CYP2E1, CYP2D6, GSTM1, GSTT1, NAT2, GSTP, and EPHX) in the human population were determined. Major and significant differences in these frequencies were observed between Caucasians (n = 12,525), Asians (n = 2,136), and Africans and African Americans (n = 996), and some, but much less, heterogeneity was observed within Caucasian populations from different countries. No differences in allele frequencies were seen by age, sex, or type of controls (hospital patients versus population controls). No examples of linkage disequilibrium between the different loci were detected based on comparison of observed and expected frequencies for combinations of specific alleles.