Localization of a human lung adenocarcinoma susceptibility locus, possibly syntenic to the mouse Pas1 locus, in the vicinity of the D12S1034 locus on chromosome 12p11.2-p12.1
Noriko Yanagitani1,2,
Takashi Kohno1,
Noriaki Sunaga1,2,
Hideo Kunitoh3,
Tomohide Tamura3,
Satoshi Tsuchiya2,
Ryusei Saito2 and
Jun Yokota1,4
1 Biology Division, National Cancer Center Research Institute, 1-1,Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan,
2 First Department of Internal Medicine, Gunma University School of Medicine, 39-15, Showa-machi 3-chome, Gunma 371-8511, Japan and
3 Division of Thoracic Oncology, National Cancer Center Hospital, 1-1, Tsukiji 5-chome, Chuo-ku, Tokyo 104-0045, Japan
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Abstract
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Pulmonary adenoma susceptibility 1 (Pas1) is a major locus affecting inherited predisposition to the development of lung adenocarcinoma in mice, and is mapped to chromosome 6q near the Kras2 gene. However, it is still unclear whether the PAS1 locus on human chromosome 12p11.2-p12.1, the region showing synteny to the mouse Pas1 region, is involved in susceptibility to human lung adenocarcinoma development. Thus, we conducted a case-control study of 100 lung adenocarcinoma cases and 100 controls using 20 highly polymorphic microsatellite markers dispersed in a 13 cM region covering a putative PAS1 locus. The differences in the allele and genotype distributions were observed at several loci, and the difference was at a maximum at the D12S1034 locus (P = 0.034 and P = 0.036, respectively). The differences in the allele and genotype distributions at D12S1034 remained significant in the 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 = 0.031 and P = 0.027, respectively). The D12S1034 locus was located 8001350 kb proximal to the KRAS2 locus, and in the region syntenic to the core Pas1 region of ~1.5 Mb in size where a single haplotype is shared by several mouse-inbred strains susceptible to lung adenocarcinoma development. These results indicate that the PAS1 locus is located in the vicinity of D12S1034 and a genetic variation(s) at this locus is involved in susceptibility to human lung adenocarcinoma.
Abbreviations: EST, expressed sequence tag; GPCC, Gunma Prefectural Cancer Center; HWE, HardyWeinberg equilibrium; LD, linkage disequilibrium; NCC, National Cancer Center; NGH, National Nishigunma Hospital; OR, odds ratio; Pas1, Pulmonary adenoma susceptibility 1; PCR, polymerase chain reaction; PCRRFLP, PCRrestriction fragment length polymorphism; SNP, single nucleotide polymorphism.
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Introduction
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Lung cancer is the leading cause of cancer-related deaths in the world. Lung cancer consists of three major histological subtypes: adenocarcinoma, squamous cell carcinoma and small cell carcinoma. In recent years, adenocarcinoma has replaced squamous cell carcinoma as the most frequent histological subtype in lung cancer (1,2). Thus, identification of genetic factors responsible for susceptibility to lung adenocarcinoma is indispensable to establishing novel and efficient ways of preventing the disease (3,4). Genetic linkage studies using the crosses among inbred strains of mice that differ in susceptibility to spontaneous and chemically induced lung adenocarcinoma have been conducted to identify loci related to susceptibility to lung adenocarcinoma (5). Up to the present, multiple (>20) loci have been identified as being involved in susceptibility, and their interactions in murine lung carcinogenesis have been elucidated (69). Pulmonary adenoma susceptibility 1 (Pas1) is a major locus affecting inherited predisposition to lung adenocarcinoma development in mice, and it maps near the Kras2 locus on mouse chromosome 6q (6). Linkage of Pas1 with both tumor multiplicity and tumor volume has been confirmed by several studies using different strains and carcinogens (6,1013). Therefore, a genetic variation(s) in the Pas1 locus is considered to play a central role in the genetic predisposition to lung adenocarcinoma in mice (14). A recent study indicates that a region of ~1.5 Mb in size, which contains the Kras2 and Krag (K-ras oncogene-associated gene) loci, is the core region of the Pas1 locus, as a single haplotype is shared in this region among several mouse-inbred strains susceptible to lung adenocarcinoma development (13). However, a genetic variation(s) responsible for lung adenocarcinoma susceptibility has not been identified yet.
The human KRAS2 and KRAG genes have been mapped within 1 Mb of each other on chromosome 12p, indicating that this region is syntenic to the core region of the mouse Pas1 locus (1316). Fluorescence in situ hybridization analysis revealed that the KRAS2 and KRAG genes are mapped in 12p12.1 and 12p11.2, respectively (The Genome Database, http://gdbwww.gdb.org/). Thus, it was indicated that this syntenic region is located on human chromosome 12p11.2-p12.1. A case-control study of lung adenocarcinoma in the Italian population showed that several genetic polymorphisms in this region, including a single nucleotide polymorphism (SNP) in the KRAS2 gene, are associated with the risk for lung adenocarcinoma (17). This result has lead us to hypothesize that these genetic polymorphisms are in linkage disequilibrium (LD) with the PAS1 mutation responsible for susceptibility to human lung adenocarcinoma. In the following case-control studies in the populations of Japan and several European countries, allele distributions of the SNPs in the syntenic region were also different between cases and controls. However, the differences did not reach statistical significance (14,15). Therefore, it remains unclear whether the human chromosome 12p11.2-p12.1 region contains the PAS1 locus, whose polymorphism(s) is associated with susceptibility to lung adenocarcinoma development.
In the present study, we conducted a case-control study of 100 lung adenocarcinoma cases and 100 controls in the Japanese population to investigate the involvement of the PAS1 locus in human lung adenocarcinoma susceptibility. We used microsatellite markers distributed in a 13 cM region in the 12p12-12q12 region as a tool to detect differences in the allele and genotype distributions between the cases and controls. Because of the fact that microsatellite loci have multiple alleles and usually show higher frequencies of heterozygosity than SNP loci, microsatellite markers are more informative than SNPs for the analysis of allele and genotype distributions (18). Furthermore, the length of LD detected by microsatellite markers has been shown to often extend out
1 cM, while that detected by SNPs is still unclear (1922). In fact, microsatellite markers have been shown to be a powerful tool in case-control studies to detect and map the loci associated with susceptibility to several common diseases, including sporadic Alzheimer's disease and diffuse panbronchiolitis (19,22,2426). Therefore, microsatellite markers were thought to be more suitable than SNPs in searching for loci from a wide area that show differences in the allele and genotype distributions between cases and controls due to LD with the PAS1 mutation. Then, the differences in the allele and genotype distributions were observed at several loci, and the difference was at a maximum at the D12S1034 locus (P = 0.034 and P = 0.036, respectively). These differences were confirmed in the populations in which 239 lung adenocarcinoma cases and 63 controls were added to the 100 cases and 100 controls used for the initial screening. This locus was mapped in the region syntenic to the core Pas1 region of ~1.5 Mb, and was located 8001350 kb proximal to the KRAS2 locus. These results strongly indicate the presence of a locus associated with lung adenocarcinoma susceptibility in the vicinity of the D12S1034 locus and proximal to the KRAS2 locus. This locus could be the human PAS1 locus.
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Materials and methods
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Subjects
A total of 339 pathologically documented lung adenocarcinoma cases were enrolled at the National Nishigunma Hospital (NGH), Gunma, the National Cancer Center Hospital (NCC), Tokyo and Gunma Prefuctural Cancer Center, Gunma (GPCC). The cases were 66 from NGH, 271 from NCC and two from GPCC. A sample of 163 unrelated control subjects was established to estimate the allele genotype distributions in the Japanese population, from non-cancer patients and healthy individuals consisting of 141 from NGH and 22 from NCC. All the cases and controls were Japanese. The distribution of clinical diagnoses among the controls was as follows: chronic obstructive pulmonary disease, 21 cases; pulmonary tuberculosis, 17 cases; bronchitis or pneumonia, 16 cases; pulmonary non-tuberculous mycobacteriosis, 10 cases; pneumoconiosis, nine cases; old pulmonary tuberculosis, nine cases; pulmonary abscess, nine cases; interstitial pneumonia, six cases; pulmonary aspergillosis, five cases; diabetes mellitus, three cases; sarcoidosis, three cases; other respiratory disease, 21 cases; pancreatitis, two cases; gastric ulcer, two cases; uterine myoma, one case; hypertension, one case; angina pectoris, one case; pericarditis, one case; rheumatoid arthritis, one case; posterior mediastinal tumor, one case; and 24 healthy individuals.
From the case and control subjects, 10 ml of whole-blood samples were obtained. Genomic DNAs were isolated using the Blood Maxi Kit (Qiagen, Tokyo, Japan) according to the supplier's instruction protocol. Smoking history was obtained via interview using a questionnaire. Smoking habit was represented by cigarette-years, which was defined as the number of cigarettes smoked daily multiplied by years of smoking, both in current smokers and former smokers. Non-smokers were defined as those who had never smoked. Informed consent was obtained before blood sampling, and the study was approved by the ethical committees of NGH, NCC and GPCC.
Microsatellite markers
Fourteen known microsatellite markers, D12S1606, D12S1591, D12S1057, D12S1617, D12S1596, D12S1034, D12S1042, D12S1292, D12S333, D12S345, D12S1692, D12S2080, D12S331 and D12S823, were examined in this study. Information on these markers was obtained from the Genome Database (http://gdbwww.gdb.org/). To identify polymorphic microsatellite loci near the D12S1034 locus, genomic sequences deposited in GenBank, AC019209, AC087256, AC024225, AC025637, AC055707 and AC022509, were processed by the SEQUENCHER program Version 3.1.1 (GENECODES, Ann Arbor, MI), and a sequence of 526 588 bp was obtained. Microsatellite loci in the 330 kb sequence containing the D12S1034 locus were searched for by using the RepeatMasker program (http://ftp.genome.washington.edu/cgi-bin/RepeatMasker). Six sets of primer pairs, used to amplify six novel microsatellite loci, were as follows: NY-int-4B, 5'-AGATTCCCACA-GCACTCATG-3' and 5'-GTGGGAAGATTGCTTGAAGAT-3'; NY-int-2E, 5'-TCTGCAAAGTGATAAATCTTCTAC-3' and 5'-CTTTCTTATGGCTCCCAGAAC-3'; NY-1034-2A, 5'-CTTAGGTTTAAGTGCGCGTC-3' and 5'-AAACCTGTAACTCCTAATCAAG-3'; NY-1034-2C, 5'-GCACTGCAGGAGGACTGTA-3' and 5'-CACCATAAGTGACTTGTCTTG-3'; NY-1034-2G, 5'-GGATGTTTAACTAGACCTGAC-3' and 5'-CTTACTCCCCACG-CCACATT-3'; and NY-RAG-2, 5'-AATGGGTCAATTCTGCTGAGA-3' and 5'-TGGCCGCCATACCTAAAATG-3'.
Genotyping for microsatellite polymorphisms
A unilateral primer of each primer set was 5'-end labeled with fluorescent reagents, 6-FAM, HEX or NED (PE Biosystems, Tokyo, Japan). Ten nanograms of genomic DNA were suspended in a total volume of 16 µl polymerase chain reaction (PCR) buffer, containing 1.5 mM MgCl2, 7 pmol specific primer pairs, including one labeled with a fluorescent reagent, 200 µM dNTPs, and 0.5 U Taq polymerase (PE Biosystems). The reactions were carried out in a thermal cycler for 10 cycles under the conditions of 94°C for 15 s, 55°C for 15 s and 72°C for 30 s, followed by 25 cycles under the conditions of 89°C for 15 s, 55°C for 15 s and 30 s at 72°C with a final extension of 10 min at 72°C. Amplified products were denatured for 2 min at 95°C, mixed with deionized-formamide dye, applied with a size standard marker of 400HD (PE Biosystems) to each lane, and run on a GeneScan Polymer (POP4) in an ABI PRISM 310 DNA Sequencer (PE Biosystems). The size of each fragment was determined automatically using the GeneScan software (PE Biosystems).
Genotyping for SNPs
DNA fragments containing two SNPs, KRAS2/RsaI and M4/BstXI, were amplified by PCR. The sequences of paired primers used for amplification (identification) of the KRAS2/RsaI and M4/BstXI alleles were described previously (15,17). Ten nanograms of genomic DNA were suspended in a total volume of 20 µl PCR buffer containing 1.5 mM MgCl2, 20 pmol specific primer pairs, 200 µM dNTPs, 0.25 U Taq polymerase (PE Biosystems, Tokyo, Japan), and 35 PCR cycles were performed. The genotype of the KRAS2/RsaI polymorphism was determined by a PCRrestriction fragment length polymorphism (PCRRFLP) method. DNA fragments of 990 bp in size containing the RsaI restriction site were amplified from the pcDNA3.1() plasmid DNA (Invitrogen, Tokyo, Japan) by PCR using the forward 5'-TGATCAAGAGACAGGATGAG-3' and reverse 5'-CGAAGAACTCCAG-CATGAGA-3' primers, and were used as controls to confirm the complete digestion with the RsaI restriction enzyme. A mixture of PCR products for KRAS2/RsaI and control DNA fragments was digested with RsaI (New England Biolabs, Tokyo, Japan), and electrophoresed through 3% agarose gel, followed by staining with ethidium bromide. The genotype of the M4/BstXI polymorphism was determined by direct sequencing of individual PCR products. The PCR products were purified using a QIAquickspin 96 PCR purification kit (Qiagen) and directly sequenced in the mono direction with a BigDye Terminator Cycle Sequencing Ready Reaction Kit and ABI PRISM 310 DNA Sequencer (PE Biosystems, CA).
Statistical analysis
Differences in the allele frequencies for each locus between the cases and controls were analyzed by the 2xn
2 test (n is the number of microsatellite alleles) using StatView software Version 5.0 (SAS Institute, NC). The exact test of HardyWeinberg proportion for multiple alleles and the genotypic differentiation test were performed by the Markov chain method with the GENEPOP program with the following parameters: dememorization number = 1000; number of batches = 400; iteration per batch = 8000 (http://wbiomed.curtin.edu.au/genepop/genepop_op1.html) (2428). A level of P < 0.05 was considered as statistically significant.
Differences in the allele frequencies for the D12S1034 locus between the cases and controls were analyzed by the 2x2
2 test using StatView software Version 5.0 (SAS Institute). The strength of association between lung adenocarcinoma risk and D12S1034 genotypes was measured as odds ratios (ORs). ORs adjusted for age, gender and smoking habit with 95% confidence intervals were calculated using an unconditional logistic regression analysis (29).
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Results
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We first selected 100 lung adenocarcinoma cases and 100 control subjects, matching the distributions of gender, age and smoking habit to each other, from 339 cases and 163 control subjects, respectively (Table I
). The distributions of gender, age and smoking habit were not significantly different between the cases and controls (P = 0.655, P = 0.889 and P = 0.271, respectively, by the 2xn
2 test).
Genotypes for two SNPs, KRAS2/RsaI and M4/BstXI, which showed biased allele distributions between lung adenocarcinoma cases and controls in previous studies (14,15,17), were determined in this study population (Table II
). Distributions of alleles and genotypes for these SNPs were almost the same between the cases and controls. The two SNPs did not deviate from the HardyWeinberg equilibrium (HWE) either in the cases or controls (P > 0.5).
We selected 13 microsatellite markers distributed in a 13 cM region in chromosome 12p12-12q12 with intervals of 02.8 cM (30) to compare the allele and genotype distributions between the cases and controls. A recently published physical map of human chromosome 12 indicated that the KRAS2 gene is located between the D12S1617 and D12S1596 loci (16). The cases and controls were genotyped individually. Three microsatellite markers that were contiguously set in a 1.9 cM region containing the KRAS2 gene, showed greater differences in distributions of both alleles and genotypes between the cases and controls (P < 0.2) than 10 other microsatellite markers (Table III
, Figure 1
). Among these three markers, the difference in the distribution of alleles at the D12S1034 locus was the greatest and statistically significant between the cases and controls (P = 0.034). The distribution of genotypes at this locus was also significantly different between the cases and controls (P = 0.036). The exact test of HardyWeinberg proportion was carried out for these 13 microsatellite markers in terms of deviation from HWE (Table III
). The D12S1034 locus deviated greatly from HWE in the cases (P = 0.007). The D12S2080 locus also deviated from HWE in the cases (P = 0.039), while the remaining 11 loci did not. In the controls, one of the 13 loci, D12S1591, deviated from HWE (P = 0.048).
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Table III. Genotype distributions of chromosome 12p12-q12 microsatellite markers in lung adenocarcinoma cases and controls
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Fig. 1. P values for the differences in the allele and genotype distributions in patients with lung adenocarcinoma obtained by the 2 test (closed circle) and the genotypic differentiation test (closed square). P values are shown with the locations of microsatellite markers. These markers were genetically mapped within a 13 cM segment at the 12p12-12q12 region based on the Marshfield chromosome 12 sex-averaged linkage map (30).
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To define the genomic region showing the greatest difference in the allele and/or genotype distributions, we further attempted to examine additional microsatellite loci in the vicinity of the D12S1034 locus in the same cases and controls. As there were no other microsatellite markers mapped in this region (16), we searched for novel microsatellite markers in the region surrounding the D12S1034 locus. For this purpose, the sequence of 526 588 bp covering the D12S1034 locus was determined by the re-construction of genomic sequences deposited in GenBank. The sequence contained the D12S1034 and D12S1596 loci, but not the other 11 loci used for the initial genotyping above. The distance between the D12S1034 and D12S1596 loci was 350 kb. A BLAST search revealed that a known microsatellite locus, D12S823, is located 79 kb proximal to the D12S1034 locus. Next, the RepeatMasker computer program was employed to identify microsatellite loci in the 330 kb sequence composed of the 123 kb sequence proximal to and the 207 kb sequence distal to the D12S1034 locus, respectively. Then, 13 microsatellite loci with uninterrupted di- to tetra-nucleotide runs with more than five repeat units were identified in addition to the D12S823 locus. We therefore determined the lengths of the 13 microsatellites in DNAs from eight of the 100 controls to verify whether these loci are polymorphic in the Japanese population. Nine of the 13 microsatellite loci were successfully amplified by PCR, while the remaining four loci were not amplified by PCR, probably because they were surrounded by interspersed repetitive sequences. Six of these nine loci, were considered to be highly polymorphic, because more than five different sizes of alleles were detected among the eight individuals. In contrast, the remaining three loci were poorly polymorphic, as less than four different sizes of alleles were detected among them. Thus, the six microsatellite markers with more than five different sizes of alleles and the D12S823 marker were used for further genotype analysis of the region surrounding the D12S1034 locus. These seven polymorphic microsatellite loci were distributed in a 260 kb region including the D12S1034 locus with intervals of 1188 kb (Figure 2
).

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Fig. 2. P values for the differences in the allele and genotype distributions in patients with lung adenocarcinoma obtained by the 2 test (closed circle) and the genotypic differentiation test (closed square). P values are shown with the locations of microsatellite markers. Locations of microsatellite markers are expressed by the physical distance (kb) from the D12S1034 locus to each microsatellite marker.
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The allele and genotype distributions for these additional seven microsatellite loci were compared between the same 100 cases and 100 controls (Table III
, Figure 2
). Two loci, NY-1034-2A and NY-1034-2C, near the D12S1034 locus showed greater differences in the distribution of both alleles and genotypes between the cases and controls (P < 0.2) than the five other loci. The NY-1034-2A and NY-1034-2C loci were located at 37 and 15 kb distal to the D12S1034 locus, respectively. However, the differences in the allele and genotype distributions were the greatest at the D12S1034 locus among the eight loci in the 260 kb region surrounding D12S1034. Among the seven loci additionally tested, the NY-1034-2A locus showed a statistically significant deviation from HWE (P = 0.014) and the NY-1034-2C was marginal in the cases (P = 0.065), while the remaining five loci did not. In the controls, none of the seven loci deviated from HWE (Table III
, Figure 2
).
To confirm the differences in allele and genotype distribution at the D12S1034 locus, the remaining 239 cases and 63 controls, which were not analyzed in the initial screening, were further genotyped individually for the D12S1034 locus. In total, 339 cases and 163 controls were genotyped for the D12S1034 locus in the present study (Table I
). The distributions of gender and age, but not of smoking habit, were significantly different between the cases and controls (P = 0.042, P = 0.024 and P = 0.748, respectively, by the 2xn
2 test) (Table I
). The allele and genotype distributions for the D12S1034 locus remained significantly different between the total of 339 cases and 163 controls (P = 0.031 and P = 0.027, respectively) (Table III
). The exact test of HardyWeinberg proportion was carried out for all the populations in terms of deviation from HWE. The D12S1034 locus deviated significantly from HWE in the 339 cases (P = 0.018), but not in the 163 controls (Table III
).
As the mouse Pas1 locus has been suggested to be inherited in an autosomal dominant manner (6,1013), we further examined the differences in the distributions of nine polymorphic alleles (A1A9) at the D12S1034 locus. The frequencies of the A2 and A4 alleles in the cases were significantly higher than those in the controls, whereas the frequencies of the other seven alleles in the cases were lower than or almost equal to those in the controls (Table IV
). Therefore, if we assume that the A2 and/or A4 alleles co-segregate with a putative PAS1 mutation, we were able to calculate the relative risks (ORs) of the genotypes, A2/others + A2/A2 and/or A4/others + A4/A4, as shown in Table V
. ORs of genotypes with the A2 or A4 allele and of combined genotypes with either or both of the A2 and A4 alleles were 2.29, 1.33 and 1.38, respectively, but such increases were not statistically significant. This result may imply that the A2 and/or A4 alleles were not the risk alleles, or that the number of cases examined was too small to show statistical significance for their risks. However, as it is presently totally unknown which alleles co-segregate with the PAS1 mutation, further analyses are necessary to define the relative risk of the PAS1 mutation.
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Discussion
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Allele and genotype distributions for 13 microsatellite loci distributed in a 13 cM region in 12p12-12q12 were compared between 100 lung adenocarcinoma cases and 100 controls to investigate whether or not this chromosomal region harbors a locus with genetic variation involved in lung adenocarcinoma susceptibility. The D12S1034 locus showed significant differences in both allele and genotype distributions between the cases and controls (P = 0.034 and P = 0.036, respectively). Seven additional microsatellite loci in the vicinity of D12S1034 were further examined, and the P values of two nearby loci, NY-1034-2A and NY-1034-2C, which are 37 and 15 kb apart from the D12S1034 locus, were relatively small among them in both allele and genotype distributions between the cases and controls (P < 0.2). However, the differences were at a maximum at the D12S1034 locus. The differences in the allele and genotype distributions at D12S1034 remained significant in the 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 = 0.031 and P = 0.027, respectively). Thus, it was strongly suggested that a lung adenocarcinoma susceptibility locus is in the vicinity of the D12S1034 locus.
Statistically significant deviation from HWE was observed at the D12S1034 locus in the 100 cases used in the initial screening as well as in the total of 339 cases, while it was not observed in controls. Such a deviation was also observed at a nearby locus, NY-1034-2A (P = 0.014), and it was marginal at another nearby locus, NY-1034-2C (P = 0.065). Biased allele distributions at polymorphic loci in a population due to LD with a genetic variation linked to a disease often lead to deviation from HWE (2426,31,32). Therefore, the present result indicates that the differences in the allele and genotype distributions between the cases and controls were caused by the biased allele distribution in the cases, suggesting that the cases share the risk allele(s) for lung adenocarcinoma susceptibility.
In previous case control studies of lung adenocarcinoma patients in Italy and other countries, including Japan, differences in the genotype distribution have been observed at the KRAS2/RsaI and M4/BstXI loci (14,15,17). Therefore, it was suggested that the human PAS1 mutation is harbored in the region containing these loci. Our analysis of the genomic sequences deposited in GenBank indicated that the KRAS2/RsaI locus is located <200 kb proximal to D12S1617 and >400 kb distal to D12S1596, while M4/BstXI is located 30 kb proximal to the NY-RAG-2 locus, and the distance between these two SNPs is 9001350 kb. Therefore, the D12S1034 locus is flanked by these two SNPs in the 12p11.2-12p12.1 region. The present result indicates that the difference in the allele and genotype distributions was at a maximum at the D12S1034 locus, and the differences at D12S1617 and D12S1596 near KRAS2/RsaI, and at NY-RAG-2 near M4/BstXI, were smaller than that at D12S1034. Thus, it was strongly suggested that a putative lung adenocarcinoma susceptibility locus in this chromosomal region is closer to the D12S1034 locus than the KRAS2 or M4 locus. In the studies of mouse Pas1 locus, a region of ~1.5 Mb in size that contains the Kras2 and Krag loci was defined as the critical region, where a single haplotype was shared among several mouse-inbred strains susceptible to lung adenocarcinoma development. Our analysis of genomic sequences deposited in GenBank and the mapping data in the YAC contig map (33) indicates that D12S1034 is present between the KRAS2 and KRAG loci, and its location is 8001350 kb proximal to the KRAS2 locus and 100 kb distal to the KRAG locus. Therefore, the D12S1034 locus is located in the region syntenic to the core Pas1 region. Thus, it is probable that the region in the vicinity of the D12S1034 locus harbors a human PAS1 mutation that is responsible for human lung adenocarcinoma susceptibility.
In the Italian population, the genotype distribution for both the KRAS2/RasI and M4/BstXI loci was significantly different between the cases and controls, suggesting that the region of 9001350 kb in size between these two loci is in LD in the Italian cases. In contrast, the difference in the genotype distribution for these two SNPs was less evident in the Japanese subjects (15), and this finding was confirmed in the present study. This could be due to the lower frequencies of minor alleles for these two SNPs in the Japanese population than in the Italian population (14,15,17). The number of Japanese subjects might have been too small to detect differences in the allele and genotype distributions due to LD with PAS1 mutation in the vicinity of D12S1034 by these poorly informative SNPs. However, at present, the allele and genotype distributions for the D12S1034 markers in lung adenocarcinoma patients of Italy and other European countries are unknown. Genotype analyses of D12S1034 and nearby microsatellite markers in these subjects are needed to clarify this possibility.
BLAST program analysis revealed that KRAG is the only characterized gene located within the 260 kb region surrounding the D12S1034 locus (Figure 2
), and it extends in the region 100138 kb proximal to D12S1034. The KRAG gene encodes a protein of the transmembrane-4 superfamily members, whose physiological function is unknown (34). A recent study showed that expressed sequence tag (EST) clones were mapped at several sites in this 260 kb region (35). Therefore, it is probable that several unknown genes are present in this region. The PAS1 mutation is probably present in one of these genes or in the KRAG gene. However, at present, the mode of the PAS1 mutation is unknown. We should next identify the haplotype(s) specifically shared in adenocarcinoma cases. Genotyping as well as searching for SNPs in the region surrounding the D12S1034 locus are in progress in our laboratory. The KRAS2 gene is known to be mutated in a subset of lung adenocarcinoma (36). Furthermore, recent studies indicated that the wild-type Kras2 gene can inhibit lung carcinogenesis in mice (37). Thus, although the present result indicated that the PAS1 mutation was not present in the KRAS2 locus, it will be also necessary to define haplotypes covering the KRAS2 locus to conclude whether or not the KRAS2 locus carries the PAS1 mutation. Further analyses should enable us to identify the human PAS1 mutation responsible for individual susceptibility to lung adenocarcinoma.
In terms of histological appearance and clinical features, lung adenocarcinoma is known to have a greater diversity than other histological subtypes of lung cancer, suggesting that it is etiologically also heterogeneous (38). However, the fact that no etiological factors have been identified inhibited the elucidation of molecular mechanisms underlying the susceptibility to this disease. Therefore, studies on the association of the PAS1 mutation with the risks of each histological subtype of adenocarcinoma, such as bronchioloalveolar carcinoma, will provide valuable information to elucidate the possible etiological heterogeneity of lung adenocarcinoma. In addition, we should consider that this study included considerable numbers of individuals with respiratory and other diseases associated with smoking as control subjects, since this fact could lead to underestimation or overestimation for the risk assessment of each genotype, if some genotypes were related to the risks for the diseases observed in the control subjects. Thus, associations of the D12S1034 polymorphisms with lung adenocarcinoma risks should be further examined in different populations of adenocarcinoma cases as well as controls to elucidate the pathogenetic significance of the PAS1 mutation.
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Notes
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4 To whom correspondence should be addressed Email: jyokota{at}gan2.ncc.go.jp 
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Acknowledgments
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This work was supported in part by Grants-in-Aid from the Ministry of Health, Labour and Welfare for the 2nd-term Comprehensive 10-Year Strategy for Cancer Control, the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Pharmaceutical Safety and Research of Japan, and the Ministry of Education, Culture, Sports, Science and Technology of Japan. We thank Dr Kouichi Minato and Dr Shinichi Ishihara of Gunma Prefectural Cancer Center for their collection of blood samples from adenocarcinoma patients. N.Yanagitani and N.Sunaga are awardees of Research Resident Fellowship from the Foundation for Promotion of Cancer Research in Japan. We also thank Dr Masatomo Mori of the Gunma University School of Medicine for encouragement throughout this study. We also greatly thank Ms Mina Takahashi for technical assistance.
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Received January 2, 2002;
revised March 22, 2002;
accepted April 5, 2002.