ONCOGENOMICS 1997; Minna, 1993)

Linkage disequilibrium mapping of novel lung tumor susceptibility quantitative trait loci in mice

Daolong Wang1, William J Lemon1 and Ming You*,1

1Division of Human Cancer Genetics, The Ohio State University Comprehensive Cancer, 514 Medical Research Facility, 420 West 12th Avenue, Columbus, Ohio, OH 43210, USA

Linkage disequilibrium (LD) has been used to map shown to account for 69, 47 and 22% of the chromosomal regions regulating quantitative traits, also cumulative incidence of lung cancer in patients aged called quantitative trait loci (QTLs). With the increasing 50, 60 and 70 years, respectively (Sellers et al., 1990). number of available mouse polymorphic genetic markers, Several groups are currently pursuing identification of LD can be estimated for the purpose of fine-mapping a this gene. In addition to this locus, several other given QTL or in the identification of novel QTLs. A genetic factors have been linked to lung cancer whole-genome LD analysis was conducted for mapping susceptibility. For example, a K-ras intron polymorph- mouse lung tumor susceptibility QTLs in 25 strains of ism and tandem repeats in the H-ras 3’-untranslated mice with known susceptibility to lung cancer using 5638 region have been found to correlate with increased lung genetic markers. A total of 63 markers were found to be cancer incidence (Manenti et al., 1997; Sugimura et al., significantly associated with lung tumor susceptibility, 1990). Lung cancer also occurs more frequently in many of which were novel QTLs. This study demon- individuals with genetic syndromes such as Li- strates the feasibility of using LD to map QTLs on a Fraumeni syndrome (LFS), hereditary retinoblastoma, whole genome level. Further characterization of the familial breast cancer (BRCA1), and Bloom syndrome newly identified lung tumor susceptibility QTLs may lead (German, 1993; Johannsson et al., 1996; Li et al., 1988; to the identification of genes whose human homologue Sanders et al., 1989; Strong et al., 1984). Finally, may predispose some individuals to lung cancer. genetic polymorphisms in genes (e.g., CYP2D6, Oncogene (2002) 21, 6858 – 6865. doi:10.1038/sj.onc. CYP1A1, and GSTM1) responsible for metabolism of 1205886 tobacco carcinogens are implicated in susceptibility to lung cancer (Caporaso et al., 1992; Shields and Harris, Keywords: mouse; linkage disequilibrium; lung tumor; 1993; To-Figueras et al., 1996). susceptibility; gene mapping Given their phylogenetic proximity, many of the known genetic changes in lung cancer are likely common to both human and mouse lung tumors (You and Bergman, 1998). With the availability of inbred strains having marked differences in lung tumor Lung cancer is the leading cause of cancer death in susceptibility, these mice are a valuable model for men and women in the United States (Greenlee et al., studying tumor susceptibility, stages of tumor devel- 2000). Despite major therapeutic advances in recent opment, and the interaction of genetic and years, most lung cancers are disseminated at the time environmental factors which predispose to neoplasia of presentation and have a mortality rate of about (Malkinson, 1989). Several mouse lung tumor models 90% (Greenlee et al., 2000). Cancer development is have been utilized to determine the genetic basis of progressive, involving an accumulation of genetic susceptibility to chemical induction of lung tumors in mutations with time that conduce a multistage process the hope that such information may lead to the involving many regulatory genes in cells (Herzog et al., identification of human lung cancer susceptibility ONCOGENOMICS 1997; Minna, 1993). Studies of familial aggregation of gene(s) (Herzog and You, 1997). Identifying the major lung cancer suggest that genetic factors are involved in mouse lung tumor susceptibility genes has been the human lung tumor development (Goffman et al., 1982; focus of several research groups (Herzog and You, Lynch et al., 1986; McDuffie, 1991; Sellers et al., 1987; 1997). Linkage analysis is the most frequently used Shaw et al., 1991; Tokuhata and Lilienfeld, 1963). method in doing so. Recently, Tripodis et al. (2001) Specifically, segregation analyses of lung cancer through large-scale linkage analyses with crosses made proband families indicate that a Mendelian codomi- from a few congenic mouse strains reported 30 genes nant inheritance of a rare major autosomal gene is and 25 interactions affecting mouse lung tumor involved (Sellers et al., 1990). This locus has been susceptibility. While linkage analysis provides reason- able mapping resolutions for crosses of selected mouse strains using a moderate density of genetic markers, it *Correspondence: M You; E-mail: [email protected] does not take full advantage of highly dense coverage Received 10 June 2002; revised 5 July 2002; accepted 18 July 2002 of markers which has now become available because of Mapping of mouse lung tumor susceptibility loci D Wang et al 6859 the limited meiosis in the crosses. Consequently, can be applied to strains with phenotypic differences linkage analysis using F2 or backcross populations of rather than to specific crosses, since the techniques mice resolves QTL to within an average of *20 cM search for ancestral haplotypes rather than relying on resolution due to rare opportunities for recombination recent recombinations. To our knowledge, there have in one generation between closely linked loci. been no reports of genome-wide LD analysis for mouse Linkage disequilibrium (LD) is defined as the lung tumor susceptibility QTLs. Herein, we report our association of alleles at one locus with alleles at data obtained from whole-genome LD analyses based another nearby locus, which is maintained due to a on 5638 genetic markers and known lung tumor low frequency of recombinations between proximate phenotypes in 25 strains of mice. markers (Ott, 1999). LD analysis has been widely used A total of 25 mouse strains (AKR, C3H, C57, for fine-mapping genes in human complex genetic C57BR/cdJ, C57L/J, C58, DBA/2, M. spretus, SJL/J, diseases (Reich et al., 2001). The use of LD in mapping SM/J, 129, 129/Sv, BALB/c, BALB/cByJ, CBA, Lp, mouse QTLs has been reported only recently (Grupe et MA, PL/J, RF/J, RIIIS/J, ST/bJ, A/J, O20, STS, al., 2001; Manenti et al., 1999). Manenti et al. (1999) SWR) were selected for LD analysis based upon the screened for LD using dense genetic markers on a small availability of both phenotypic data defining lung region of chromosome 6 to resolve the region contain- tumor susceptibility to a lung carcinogen (i.e., ing the pulmonary adenoma susceptibility 1 (Pas1) urethane) and genotypic data on polymorphic genetic gene. Grupe et al. (2001) proposed a computational markers (Malkinson, 1989; Manenti et al., 1999; Chen method to extend LD to predict genes for complex et al., 1994). These strains fall into three phenotype murine phenotypes using SNP markers. LD methods categories: resistant (AKR, C3H, C57, C57BR/cdJ,

Figure 1 Linkage disequilibrium analysis for the whole mouse genome of 5636 markers in 26 strains of mice (AKR, C3H, C57, C57BR/cdJ, C57L/J, C58, DBA/2, M. spretus, SJL/J, SM/J, 129, 129/Sv, BALB/c, BALB/cByJ, CBA, Lp, MA, PL/J, RF/J, RIIIS/J, ST/bJ, A/J, NGP/N, O20, STS, SWR) by Fisher’s exact test. The two horizontal lines in each plot indicate the two significance levels of 7log (P)=1.3 (or P=0.05), and 7log (P)=2.0 (or P=0.01). This figure was produced with the 7log(P) value of lung tumor susceptibility for each marker against its genetic position (cM) across each of the 20 chromosomes. The P value for each marker was computed using Fisher-exact test, which is a standard statistical method for testing association of two categorical factors (here marker types and cancer susceptibilities). The P value reflects the probability of random association between the two factors. Significantly small P value indicates non-random association, or linkage disequilibrium in our case

Oncogene Mapping of mouse lung tumor susceptibility loci D Wang et al 6860 C57L/J, C58, DBA/2, M. spretus, SJL/J, SM/J), Table 1 Haplotypes at all markers showing significant to highly intermediate (129, 129/Sv, BALB/c, BALB/cByJ, significant associations with mouse lung cancer susceptibility CBA, Lp, MA, PL/J, RF/J, RIIIS/J, ST/bJ), and Marker Chr Position (cM) 7log (P) Known QTL susceptible (A/J, O20, STS, SWR) (Malkinson, 1989; D4Mit2 4 6.5 2.10 S18 Manenti et al., 1999; Chen et al., 1994). Polymorph- D6Mit26 6 74.4 2.80 P1 isms of the strains on a total of 5638 genetic markers, D8Mit205 8 30.0 2.20 including 73 biochemical markers and 5565 DNA D1Mit491 1 59.5 1.32 markers (mostly SSLP markers), were obtained from D1Mit308 1 62.1 1.45 D1Mit92 1 64.0 1.32 web sites of Mouse Genome Informatics (MGI) (http:// D1Mit141 1 73.0 1.42 www.informatics.jax.org), the Center for Inherited Es16 3 14.3 1.32 Disease Research (CIDR) (http://www.cidr.jhmi.edu/ D3Mit22* 3 33.7 1.54 mouse/mouse.html), and Whitehead Institute/MIT D3Mit352 3 83.5 1.44 D4Mit181 4 2.5 1.92 Center for Genome Research (http://www.genome.wi.- D4Mit227 4 3.2 1.58 mit.edu/ftp/distribution/mouse_sslp_releases/). These D4Mit99 4 5.0 1.42 markers were screened from an initial *7000 markers D4Mit1 4 6.3 1.83 by removing those markers with less than 6 strains D4Mit194 4 12.1 1.45 R4 typed or with no genetic mapping information. All Fv1 4 76.5 1.49 S6 D4Mit207 4 81.5 1.32 markers span the mouse genome at an average density D4Mit51 4 82.7 1.48 of 51cM. D5Mit52* 5 28.0 1.45 Fisher’s exact tests were conducted using r6c D5Mit114* 5 44.0 1.32 contingency tables (one for each marker) to detect D5Mit279* 5 68.0 1.92 D5Mit30* 5 72.0 1.62 the associations (or LD) of each marker with mouse D6Mit223 6 19.0 1.39 lung tumor susceptibility, where r (r52) stands for the D6Mit211 6 35.0 1.45 number of alleles of the marker under test, and c for D6Mit67 6 41.5 1.40 the number of categories of the tumor susceptibility D6Mit14 6 71.3 1.51 P1 (c=3 for our study) as previously described (Manenti D6Mit373 6 74.3 1.43 P1 D6Mit304 6 75.0 1.45 P1 et al., 1999). All analyses were conducted using S-Plus D7Mit152 7 1.0 1.32 S30 2000 for Windows. The probability (P) obtained for D7Mit248 7 27.8 1.92 each marker by Fisher’s exact tests was transformed to Es22 8 43.2 1.55 the corresponding negative logarithm value, 7log (P). D8Mit121 8 67.0 1.62 S9 D9Mit246 9 61.0 1.32 P4/S11 Two commonly used significance levels (P=0.05 and D9Mit121 9 71.0 1.32 P4 0.01, or 7log (P)=1.30 and 2.0) were used to select D10Mit282 10 12.0 1.45 S29 markers having reasonably strong associations with D10Mit2* 10 16.0 1.80 mouse lung tumor susceptibility. D11Mit162 11 8.0 1.45 Figure 1 shows the results from the whole-genome D11Mit80* 11 10.0 1.72 D11Mit344 11 16.0 1.45 LD analyses. A total of 63 markers were found to have D13Mit209* 13 37.0 1.92 S23 significant (P50.05) to highly significant (P50.01) D13Mit256 13 40.0 1.58 S23 association (Table 1). The first three markers, D4Mit2, D14Mit220 14 2.5 1.32 D6Mit26, and D8Mit205, showed highly significant D14Mit34 14 40.0 1.54 D17Mit226 17 8.8 1.32 LDs, among which D6Mit26 (7log (P)=2.8) was D19Mit95 19 10.0 1.45 P4 reported previously (Manenti et al., 1999). The other D19Mit49 19 51.0 1.40 S1 two markers represent novel QTLs. It is interesting to DXMit234 X 58.0 1.45 note that D4Mit2 is only 5.6 cM from D4Mit4 which DXMit178* X 65.4 1.45 was found to be involved in an interaction with First three markers are highly significant. Remaining markers are D15Mit96 (Tripodis et al., 2001). Figure 2 shows ordered by genetic distance (MGD, Jackson Laboratories, www.jax.- chromosomal regions in which the three markers org) and by chromosome. For each marker, chromosomal designa- reside. As expected, genetic markers near D4Mit2, tion, genetic map position, negative log of P-value derived from D6Mit26, and D8Mit205 have also exhibited signifi- Fisher’s exact test, and any known susceptibility or resistance QTL to which it belongs. S – Sluc, P – Pas, R – Par (Tripodis et al., 2001; cant LD. Since a gradual decrease in LD surrounding Herzog et al., 1997) each of the peak markers (D4Mit2, D6Mit26, and D8Mit205) is evident, regions can be limited to sizes of 50.2 – 0.5 cM, a higher mapping resolution compared with conventional linkage analyses. The remaining 60 gene in this region affects lung tumor susceptibility. markers in Table 1 showed significant LDs which are The three markers (D13Mit209, D13Mit43, and suggestive of loci with intermediate effects on lung D13Mit7) on chromosome 13 are located at the same tumor susceptibility. Note that, among these markers, genetic position (37 cM), and share the same degree of 8 (D4Mit181, D4Mit1, D5Mit297, D7Mit248, linkage disequilibrium and allele pattern. This also D10Mit2, D13Mit209, D13Mit43, and D13Mit7) are suggests that there may be a susceptibility gene in this nearly highly significant (7log(P) 41.80). D4Mit181 region. These results indicate that our global LD (2.5 cM) and D4Mit1 (6.3 M) are located within the analyses reveal three major QTLs and as many as 27 same region as D4Mit2 (6.5 cM). This suggests that a minor QTLs.

Oncogene Mapping of mouse lung tumor susceptibility loci D Wang et al 6861

Figure 2 Chromosomal regions each with one marker (indicated by ‘*’) showing highly signiﬁcant LD with lung cancer susceptibility. The vertical lines represent chromosomal regions; horizontal lines show the size of 7log (P) and the position (cM) of the markers. The numbers in brackets are the number of markers located at the same position, where the marker with the highest 7log (P) is shown

The location of the marker D6Mit26 with the most based on the following evidence: (a) activation of the significant LD is very close to the K-ras locus on K-ras gene is an early event in the development of both chromosome 6. K-ras has been considered a major spontaneously occurring and chemically induced lung candidate for the previously mapped Pas1 gene tumors in mice; (b) polymorphisms detected in the K- (Greenlee et al., 2000; Herzog and You, 1997; Ott, ras promoter and enhancer regions in different mouse 1999). This finding is an independent confirmation of strains correlate with their susceptibility to the the Pas1 QTL using LD mapping. At the same time, chemical induction of lung tumors; (c) these poly- our results confirm the validity of the LD method in morphisms appear to be responsible for the observed mapping mouse lung tumor susceptibility QTLs. Pas1 allele-specific expression of the K-ras allele in hybrid was initially mapped in (A/J6C3H/HeJ) F2 mice to mice; (d) allele-specific expression leads to allele-specific distal chromosome 6. This locus produced a maximum activation of the K-ras gene; and (e) genetic linkage logarithm of the likelihood ratio (LOD) score of 9 and analyses indicate a major locus only when parental accounted for approximately 45% of the observed mice have distinct K-ras genotypes (Greenlee et al., phenotypic variance in lung tumor susceptibility 2000; Grupe et al., 2001; Herzog and You, 1997). between the parental strains of mice (Gariboldi et al., Additional candidates for Pas1 based upon Celera 1993). A LOD score of 3 or greater is considered Genome Database are listed in Table 2 with all significant for linkage. Consistent results were obtained transcribed sequences (known and predicted) of known in comprehensive linkage studies using (A/J6C57BL/ or unknown genes located within the mapped Pas1 6J) F2 (60% of variance), (A/J6C57BL/6J)6C57BL/ chromosomal region. 6J (16% of variance), (A/J6M. spretus)6C57BL/6J The second locus is mapped on marker D4Mit2 (34% of variance), and A6B&B6A RI mice (51% which is only 5.6 cM from susceptibility to lung cancer of variance) (Devereux et al., 1994; Festing et al., 1994; 18 or Sluc18 (near D4Mit4) that was identified to be Lin et al., 1998; Manenti et al., 1995). Our results are involved in an interaction with Sluc26 (near D15Mit96) consistent with a recent report of genetic mapping of (Tripodis et al., 2001). Sluc genes are interactive genes the Pas1 gene to 52-Mb region of chromosome 6 that control susceptibility to lung cancer in mouse using linkage disequilibrium (Manenti et al., 1999). The recombinant congenic (RC) strains. Sluc18 synergisti- Kras gene is the primary candidate for the Pas1 locus cally interacts with Sluc26 resulting in a greater than

Oncogene Mapping of mouse lung tumor susceptibility loci D Wang et al 6862 Table 2 Genes within Celera mouse genome database extending 1 Mb proximal and distal of D6Mit26

Celera GID Description Position (bp) (cM)

mCG13312 Kras2 145, 290, 331 71.2 D6Mit15 146, 463, 639 74 mCG1027110 similar to GRANULE CELL ANTISE 146, 544, 573 mCG1027192 146, 555, 830 mCG15019 (BC003081) hypothetical protein FLJ10637 146, 592, 490 mCG15015 RIKEN cDNA 1500031J01 146, 630, 377 mCG15021 homolog to SEVEN TRANSMEMBRANE PROTEIN 146, 654, 658 D6Mit201 146, 685, 421 74.1 mCG15016 RIKEN cDNA 0610007L03 146, 694, 609 mCG15017 ribosomal protein S26 146, 699, 267 mCG1027193 146, 717, 835 mCG1027194 146, 776, 303 mCG15023 (XM_044823) KIAA0965 146, 800, 158 mCG1027195 146, 818, 547 mCG1027111 146, 819, 952 mCG15018 (U92696) ribosomal protein S2 146, 824, 023 mCG115501 brain-muscle-ARNT-like protein 2a 146, 842, 381 mCG1027196 brain-muscle-ARNT-like protein 2a 146, 874, 241 mCG15020 (AK006257) evidence:NAS*hypothetical protein*putative 146, 882, 739 mCG115504 PTPRF interacting protein, binding protein 1 (liprin beta 1) 146, 922, 960 mCG7107 NAS*hypothetical protein*putative 146, 983, 455 mCG7102 (NM_025620) RIKEN cDNA 2210417D09 147, 068, 336 mCG7108 Similar to mitochondrial ribosomal protein S35 147, 078, 693 D6Mit373 147, 088, 640 74.3 mCG7103 hypothetical protein XP_062490 147, 110, 117 mCG7099 hypothetical protein KIAA1340 147, 125, 248 mCG1027197 147, 145, 616 mCG51994 protein phosphatase 2A, B regulatory subunit delta isoform 147, 209, 780 mCG1027198 147, 279, 289 mCG7104 parathyroid hormone-like hormone 147, 289, 178 D6Mit26 147, 301, 682 74.4 mCG7101 putative*ribosomal protein L5 147, 309, 042 mCG1027199 (U83119) ORF2 consensus sequence encoding endonuclease 147, 444, 548 and reverse transcriptase minus RNaseH mCG67650 147, 476, 142 D6Mit304 147, 477, 173 75 mCG7105 (NM_025911) RIKEN cDNA 1810060J02 147, 516, 922 mCG58789 147, 517, 981 mCG1027202 (NM_025062) hypothetical protein FLJ23429 147, 538, 617 mCG1027203 147, 571, 354 mCG1027204 147, 758, 097 mCG1027205 147, 796, 965 mCG1027206 (AF318317) unknown 147, 813, 520 mCG1027207 147, 863, 210 mCG1027208 147, 961, 211 mCG115657 alpha enolase 148, 001, 974 mCG1027209 (AF318317) unknown 148, 165, 350 mCG2111 (AK027756) unnamed protein product 148, 170, 617 mCG2109 homolog to CDA14 putative 148, 211, 181 mCG1027112 148, 264, 576 mCG115656 (AK055962) unnamed protein product 148, 269, 935

The position (bp) is the physical distance based on DNA sequence assembly from the extreme proximal end of the chromosome

ﬁvefold increase in tumor size when compared with the in mice. Possible candidates for this locus are listed in mean tumor size in control animals (Tripodis et al., Table 4. In addition to the three major loci, at least 27 2001). It is highly likely that the LD-mapped locus on minor loci were also mapped to various locations chromosome 4 is the Sluc18 locus in RC strains of across the mouse genome (Table 1). Many of these mice. Candidates for this locus based upon Celera mapped loci are in the vicinity of those that have been Genome Database are listed in Table 3 with all mapped previously using F2, backcross, or recombi- transcribed sequences (known and predicted) of known nant inbred populations of mice (Table 1). Others are or unknown genes located within the mapped novel and most likely represent low-penetrance genes chromosomal region. aﬀecting lung tumor susceptibility through extensive The third major locus is located near D8Mit205 on interactions. chromosome 8. This locus has not previously been In the present study, we used LD mapping to assign reported to be associated with lung tumor susceptibility a major new QTL located on chromosome 8 and a

Oncogene Mapping of mouse lung tumor susceptibility loci D Wang et al 6863 Table 3 Genes within Celera mouse genome database extending 1 Mb proximal and distal of D4Mit2

Celera GID Description Position (bp) (cM)

D4Mit50 560 5.4 D4Mit1 14, 683, 023 6.3 mCG113552 KIAA1900 protein 21, 353, 624 mCG1043039 21, 434, 081 mCG113483 unnamed protein product 21, 481, 913 mCG1043185 ERV-L gag polyprotein-murine endogenous 21, 495, 496 mCG1043184 21, 534, 446 mCG8960 homolog to human HRPAP20 SHORT FORM 21, 633, 453 mCG61914 21, 669, 722 mCG66778 21, 705, 130 mCG8962 G protein-coupled receptor PSP24-2 21, 742, 464 mCG1043181 predicted integral membrane protein 21, 744, 470 mCG1043180 gag polyprotein (Gibbon ape leukemia virus) 21, 787, 262 mCG8955 activator of CREM in testis 21, 935, 115 mCG8956 homolog to human KIAA0776 21, 983, 091 mCG8952 1-Cys peroxiredoxin 22, 029, 779 mCG1042986 Similar to ZW10 (Drosophila) homolog 22, 075, 034 mCG49971 homolog to GLE1 22, 143, 791 mCG8954 telomerase binding protein, p23 22, 248, 794 mCG8953 fucosyltransferase 9 22, 331, 688 D4Mit2 22, 396, 241 6.5 D4Mit361 22, 396, 252 6.75 mCG1043179 probable RNA-directed DNA polymerase 22, 460, 551 mCG1043178 HYPOTHETICAL PROTEIN ORF-1137 22, 865, 712 mCG1043177 protease 22, 878, 804 mCG1043176 23, 008, 006 mCG1043175 23, 032, 561 mCG6260 endo-alpha-mannosidase 23, 035, 468 mCG16864 betaine-homocysteine methyltransferase 23, 076, 855 mCG6261 similar to RIM2 23, 151, 864 mCG50768 G protein, gamma 2 subunit 23, 218, 782

The position (bp) is the physical distance based on DNA sequence assembly from the extreme proximal end of the chromosome

Table 4 Genes within Celera mouse genome database extending 1 Mb proximal and distal of D8Mit205

Celera GID Description Position (bp) (cM)

D8Mit127 48, 205, 857 29 mCG16561 BCL1 lymphoma-derived single chain idiotype variable region 49, 610, 900 mCG1042918 49, 779, 979 mCG1042919 49, 783, 642 mCG67798 50, 123, 926 mCG1042921 50, 269, 894 mCG1042922 50, 291, 568 D8Mit205 50, 491, 199 30 mCG1042923 tyrosinase-related protein 1 50, 690, 716 mCG1042924 50, 841, 313 mCG1042925 hypothetical protein 3 50, 990, 958 mCG1042926 51, 125, 852 mCG1042927 (U83119) ORF2 consensus sequence encoding endonuclease and 51, 237, 960 reverse transcriptase minus RNaseH mCG1042928 (M13101) unknown protein 51, 268, 839 mCG1042929 (X03725) ORF 1 (280 aa) (Mus musculus) 51, 313, 661 mCG1042930 (U70935) reverse transcriptase (Peromyscus maniculatus) 51, 413, 927 mCG1042931 SRV-1 POL polyprotein 51, 464, 910 D8Mit69 56, 353, 337 31

The position (bp) is the physical distance based on DNA sequence assembly from the extreme proximal end of the chromosome number of novel minor QTLs across the mouse genetic basis of mouse lung tumor susceptibility in genome. Several QTLs are consistent with those mice and, possibly, in humans. mapped by more conventional methods and many of LD mapping has been widely used for mapping the LD-mapped markers are indicative of novel QTLs genes of outbred or natural populations with a long associated with lung cancer susceptibility. Further history; e.g., humans. The individuals from this type of characterization of these novel QTLs identiﬁed by population are all heterozygous, thus the haplotypes of LD mapping will assist greatly in the delineation of the markers and/or disease genes are not known unam-

Oncogene Mapping of mouse lung tumor susceptibility loci D Wang et al 6864 biguously. This results in a low detection power using polymorphisms, as sample size is a very important linkage disequilibrium analysis as compared with factor that affects the power and accuracy of LD parametric statistical methods. With inbred strains analysis. In addition, other events such as clustered however, each individual is actually a large haplotype sampling, gene interactions, mutations, etc., may also at all genes across the genome assuming the ideal contribute to LD, as discussed by a recent report (Ott, situation of a pure line, and thus linkage disequilibrium 1999). mapping is likely to offer a higher detection power than that for outbred populations. In fact, the present study represents one of the first attempts to conduct a Web site references genome-wide linkage disequilibrium analysis for mapping mouse lung tumor susceptibility QTLs. The Mouse Genome Informatics, http://www.informatics. mapping and identification of 63 markers showing jax.org; Center for Inherited Disease Research, http:// strong associations with lung tumor susceptibility by a www . cidr . jhmi . edu / mouse / mouse . html; Whitehead genome-wide LD analysis was made possible largely by Institute/MIT Center for Genome Research, http:// a high density (51cM) of marker coverage across the www - genome . wi.mit.edu/ftp/distribution/mouse_sslp_ mouse genome and the availability of lung tumor releases/ susceptibility data of 25 strains of mice. This study has identified three major QTLs and perhaps as many as 27 minor QTLs associated with susceptibility to the development of lung adenomas in mice following Acknowledgments We thank MRW Festing for his help in providing valuable treatment with urethane using LD analyses with 5638 information on genetic markers. We are grateful to A de la genetic markers (Figures 1 and 2 and Table 1). Chapelle and G Stoner for their critical reading of this However, we are cautious about the detected LDs manuscript and helpful discussions. This work was due to the availability of only 25 mouse strains with supported by NIH grants R01CA58554, R01CA78797 & data of both lung tumor susceptibility and marker P30CA16058.

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