Identification of D 19 S 246 as a Novel Lung Adenocarcinoma Susceptibility Locus by Genome Survey with 10-cM Resolution Microsatellite Markers 1

Adenocarcinoma is now the most common histological subtype of lung cancer; however, genetic factors that affect cancer susceptibility are largely unknown. In this study, we performed a systematic survey of the human genome with an average resolution of 10 cM to identify loci that could help us target novel risk genes for lung adenocarcinoma using linkage disequilibrium. Genotyping of DNA “pools” from 100 lung adenocarcinoma cases and 100 controls, respectively, for 322 microsatellite loci dispersed in the human genome led us to identify 5 loci at which allele distribution was significantly (P < 0.05) or marginally (0.05 < P < 0.1) different between the cases and controls. One of the 5 loci, D19S246 at chromosome 19q13.3, showed significant differences both in the allele and genotype distributions in the subsequent analysis in which 239 lung adenocarcinoma cases and 63 controls were added to the 100 cases and 100 controls used for the initial screening (P 0.037 and P 0.026, respectively), whereas the remaining 4 loci did not. These results suggest that the chromosome 19q13.3 region encompassing D19S246 contains a gene(s) of which the genetic polymorphisms are associated with lung adenocarcinoma risk and are in linkage disequilibrium with the D19S246 locus. Introduction Lung cancer is a common cause of cancer death, and consists of three major histological subtypes: adenocarcinoma, squamous cell carcinoma, and small cell carcinoma (1–3). It is well known that the development of squamous and small cell carcinomas is strongly associated with smoking. Up to the present, genes responsible for susceptibility to lung cancer have been searched for by candidate gene approaches. The association of cancer risk with polymorphisms in genes that encode enzymes involved in the metabolism of tobacco carcinogens has been extensively studied, and several genes, such as CYP1A1-encoding cytochrome P450-1A1, CYP2A6-encoding cytochrome P450-2A6, and GSTM1 encoding glutathione S-transferase M1 have been recognized as being involved in susceptibility to squamous cell lung carcinoma (4–7). Of the three types of lung cancer, adenocarcinoma is now the most common, and its proportion to the other two is increasing (8). Thus, identification of genetic factors responsible for susceptibility to lung adenocarcinoma is indispensable to establishing novel and efficient ways of preventing the disease. Smoking is considered to be responsible for the majority of lung adenocarcinomas; however, its development is less associated with smoking compared with squamous cell lung carcinoma and small cell carcinoma (2, 3, 8). In addition, genetic polymorphisms associated with the risk of squamous cell lung carcinoma have not been clearly associated with the risk of lung adenocarcinoma (4, 5, 7, 9, 10). These facts lead us to think that genetic factors involved in susceptibility are different among the histological subtypes of lung cancer. Recent candidate gene studies by us and others have identified several candidates for lung adenocarcinoma susceptibility genes. They are drug/carcinogen metabolism genes, such as NQO1 (NAD(P)H:quinone oxidoreductase) and GSTT1 (glutathione S-transferase T1), and DNA repair genes, such as XRCC1 (X-ray cross-complementary group 1; Ref. 11–13). However, an alternative approach, such as genome scanning using LD in the human genome (14), will be also effective in identifying genes associated with the risk of lung adenocarcinoma, considering that a number of such genes is predicted to be harbored in the human genome (15, 16). We conducted a case-control study of lung adenocarcinoma recently using 20 highly polymorphic microsatellite markers dispersed in a 13-cM region of chromosome 12p12-q12 containing the region syntenic to the mouse Pas1 region (17). Microsatellite markers were used because they usually show high frequencies of heterozygosity, which enhance the sensitivity to detect allelic associations, and have been shown to be powerful tools in Received 8/20/02; revised 12/30/02; accepted 1/5/03. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported in part by Grants-in-Aid from the Ministry of Health, Labor and Welfare for the Second Term Comprehensive 10-Year Strategy for Cancer Control and from the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan. N. Y. is an awardee of a Research Resident Fellowship, and J-G. K. is an awardee of an invitation program for foreign researchers from the Foundation for Promotion of Cancer Research in Japan. 2 To whom requests for reprints should be addressed, at Biology Division, National Cancer Center Research Institute, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan. Phone: 81-3-3547-5272; Fax: 81-3-3542-0807; E-mail: jyokota@gan2.ncc.go.jp. 3 The abbreviations used are: LD, linkage disequilibrium; Pas1, pulmonary adenoma susceptibility 1; OR, odds ratio. 366 Vol. 12, 366–371, April 2003 Cancer Epidemiology, Biomarkers & Prevention Research. on October 28, 2017. © 2003 American Association for Cancer cebp.aacrjournals.org Downloaded from case-control studies to detect and map the loci associated with susceptibility to several common diseases (18–24). The D12S1034 locus mapped in the region syntenic to the core Pas1 region of 1.5-Mb in size was found to be associated with lung adenocarcinoma risk, indicating that the human PAS1 mutation is located in the vicinity of the D12S1034 locus and in LD with D12S1034 polymorphisms. This result indicated that a genome survey using microsatellite markers is useful for the identification of loci responsible for lung adenocarcinoma susceptibility. Here, we performed a systematic survey of the human genome to find novel lung adenocarcinoma susceptibility loci by genotyping 322 microsatellite loci that span 22 autosomes at approximate intervals of 10 cM. In this study, pools of DNA from 100 lung adenocarcinoma cases and 100 controls were first genotyped for 322 microsatellite loci, because DNA pooling has been shown to be a potential tool for the efficient analysis of large numbers of samples and DNA markers that are necessary for genome-wide association studies (19, 24–28). The genome survey led us to the identification of 5 candidate loci showing significant or marginal differences in the allele distribution between the cases and controls. One of the 5 loci, D19S246 at chromosome 19q13.3 was defined as the strongest candidate after analysis with a larger number of cases and controls. Thus, the risk-associated alleles of D19S246 as well as the genes located in the vicinity of the locus were defined for the identification of a novel lung adenocarcinoma susceptibility gene. Materials and Methods Subjects. The study population consisted of 339 lung adenocarcinoma cases and 163 control subjects recruited from the National Nishigunma Hospital and the National Cancer Center Hospital from 1999 to 2001 (Table 1). These 339 cases and 163 controls are the same as the ones used in our previous study (17). All of the cases and control subjects were Japanese. All of the lung adenocarcinoma cases, from whom informed consent as well as blood samples were obtained, were consecutively included in this study without any particular exclusion criteria. The participation rate was nearly 80%. All of the cases were diagnosed by cytological and/or histological examinations according to the WHO classification (29). Diagnoses of primary lung cancers but not of metastases of other cancers were made by pathological examinations for these cases. Controls were randomly selected from inpatients and outpatients with no history of cancer at the hospitals during the study periods. The case distribution of clinical diagnoses among the controls was as follows: chronic obstructive pulmonary disease (21), pulmonary tuberculosis (17), bronchitis or pneumonia (16), pulmonary nontuberculous mycobacteriosis (10), pneumoconiosis (9), old pulmonary tuberculosis (9), pulmonary abscess (9), interstitial pneumonia (6), pulmonary aspergillosis (5), diabetes mellitus (3), sarcoidosis (3), other respiratory disease (21), pancreatitis (2), gastric ulcer (2), uterine myoma (1), hypertension (1), angina pectoris (1), pericarditis (1), rheumatoid arthritis (1), posterior mediastinal tumor (1), and 24 healthy individuals. The ages of the participants were computed from their date of birth. Smoking history was obtained via interview using a questionnaire. Smoking habit was represented by cigaretteyears, which was defined as the number of cigarettes smoked daily multiplied by years of smoking, both in current smokers and former smokers. Nonsmokers were defined as those who had never smoked. Smokers were defined as those who had smoked at least 1 cigarette a day for 1 year. Informed consent was obtained from all of the participants before blood sampling, and the study was approved by the ethical committees of the Nishigunma Hospital and the National Cancer Center. From each subject, 10 ml of whole-blood sample was obtained. Genomic DNA was isolated using a Blood Maxi kit (Qiagen, Tokyo, Japan) according to the supplier’s instructions. Microsatellite Markers. A genome-wide scan was performed using the human genome-wide screening set version 9 consisting of 387 microsatellite markers placed at an average intermarker distance of 10 cM (Research Genetics, Inc., Huntsville, AL). A unilateral primer of each primer set was labeled with the ABI dyes, 6-FAM, HEX, or TED. To identify polymorphic microsatellite loci near the D19S246 locus, the genomic sequence of 200-kb containing the D19S246 sequence was obtained from the Celera Genome Database. Microsatellite loci in the 200-kb sequence was searched for using the RepeatMasker program. Three sets of p