Proc. Natl. Acad. Sci. USAVol. 93, pp. 1082-1086, February 1996Genetics

Fine mapping of colon tumor susceptibility (Scc) genes in themouse, different from the genes known to be somaticallymutated in colon cancer

(genetics/recombinant congenic strains of mice/linkage analysis/recombinant analysis)

CORINA J. A. MOEN*, PETER C. GROOT*, AUGUSTINUS A. M. HARTt, MARGRIET SNOEK*, AND PETER DEMANT*Departments of *Molecular Genetics and tRadiotherapy, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands

Comnmlnicated by George Klein, Karolinska Institute, Stockholm, Sweden, October 10, 1995 (received for review March 28, 1995)

ABSTRACT The predisposition to colon cancer is multi-genetically controlled in animals and probably also in hu-mans. We have analyzed the multigenic control of suscepti-bility to 1,2-dimethylhydrazine-induced colon tumors in miceby using a set of 20 homozygous CcS/Dem recombinantcongenic strains, each of which contains a different randomsubset of 12.5% of genes from the susceptible strain STS/Aand 87.5% of genes from the relatively resistant strainBALB/cHeA. Some CcS/Dem strains received the allelesfrom the susceptible strain STS/A at one or more of themultiple colon tumor susceptibility loci and are susceptible,whereas others are resistant. Linkage analysis shows thatthese susceptibility genes are different from the mouse ho-mologs of the genes known to be somatically mutated inhuman colon cancer (KRAS2, TP53, DCC, MCC, APC, MSH2,and probably also MLHJ). Different subsets of genes controltumor numbers and size. Two colon cancer susceptibilitygenes, Sccl and Scc2, map to mouse chromosome 2. The Sccllocus has been mapped to a narrow region of 2.4 centimorgans(90% confidence interval).

Colon cancer is influenced by environmental factors as well asmultiple genetic factors (1). A number of genes have beenfound somatically mutated in colon cancer, including theoncogene KRAS2 and the tumor suppressor genesAPC, TP53,DCC, and possibly MCC (2).

Germ-line mutation in theAPC gene causes familial adeno-matous polyposis (3, 4). Some families of hereditary nonpol-yposis colon cancer have been found to carry mutations in theMSH2 gene (5). Subsequently, it turned out that germ-linemutations in mismatch repair genes including also MLHJ,PMSJ, and PMS2 are responsible for most cases of hereditarynonpolyposis colon cancer (6), and somatic mutations in thesegenes were found also in sporadic colon cancer (7). Interest-ingly, the Li-Fraumeni syndrome, caused by germ-line TP53mutations, is only weakly associated with colon tumors (8). As>90% of cases of colon cancer are sporadic and theoreticalconsiderations suggest that a large part of them may occur ingenetically susceptible individuals without obvious familial oc-currence (9, 10), the genes possibly influencing the susceptibilityto sporadic colon cancer may be of considerable interest.

Inbred strains of mice differ widely in susceptibility to1,2-dimethylhydrazine (DMH)-induced colon tumors (11),due to multiple nonlinked genes (12). To map these genes weused the CcS/Dem (CcS) series of 20 homozygous recombi-nant congenic strains, derived from the parental strainsBALB/cHeA (BALB/c) (relatively resistant to DMH-inducedcolon adenomas) and STS/A (STS) (susceptible) (13). EachCcS strain contains a random subset of about 12.5% of genesfrom STS, whereas the rest of the genome comes from

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BALB/c. This led to separation of nonlinked colon tumorsusceptibility genes of STS in individual CcS strains (14). As aresult, the CcS strains that received such susceptibility gene(s)from STS are highly susceptible to colon adenomas, whereasothers are relatively resistant (13). The BALB/c- and STS-derived segments in each of the 20 CcS strains were identified(13, 15-17).

Previously we found linkage of a colon tumor susceptibilitygene, Sccl (ausceptibility to colon cancer 1), to mouse chro-mosome 2 (18). In this study we tested whether the differencein tumor susceptibility between the strains BALB/c and STSis caused by oncogenes, tumor suppressor genes, or mismatchrepair genes, presently known to be mutated in colon cancer.In addition, we demonstrated a second colon tumor suscep-tibility gene, Scc2, linked to Sccl, and tested whether thesetumor susceptibility genes can be mapped precisely.

MATERIALS AND METHODSMouse Strains and Genetic Crosses. The CcS strains (14)

and their genetic composition have been described (13, 15-17).BALB/c x (BALB/c x CcS)Fl backcross mice were pro-

duced by crossing several (BALB/c x CcS)Fl males withBALB/c females. For each backcross population, the F1 maleshad one common male parent of the particular CcS strain.Recombinants in the Sccl region were produced as follows.

BALB/c females were mated with a CcS-19 male, and the F1hybrids were used to produce (BALB/c x CcS-19)F2 mice.The F2 mice with a recombination in the Hc-Ill segment ofchromosome 2 were crossed with BALB/c to produce F2N1mice. F2N2 and F2N3 generations were produced by furtherbackcrossing with BALB/c females.

Genetic Markers. The DNAs were isolated from tails ortumors as described (19). The oligonucleotides for the micro-satellite loci (20) used in the simple sequence length polymor-phism analyses were purchased from Research Genetics(Huntsville, AL). For markers and methods, see refs. 13, 15,16, 18, 21, and 22.Carcinogen Treatment. The mice received s.c. injections of

DMH at 15 mg/kg of body weight in 1 mM EDTA (pH 6.8)weekly for 26 weeks (13). They were killed 32 weeks after thefirst injection, with the exception of the females from thebackcrosses derived from strains CcS-7, -16, and -17 and fromthe (BALB/c x CcS-19)F2, which were killed 4 weeks later. Atthis time interval, a better distinction between susceptible andresistant females was obtained. At autopsy the colons wereexamined using a dissection microscope, and the number andsize of the adenomas were recorded. The adenomas were snapfrozen in liquid nitrogen and stored at -80°C.

Statistical Analyses. Linkage in backcross mice was testedby ANOVA, taking into account possible sex differences, after

Abbreviations: BALB/c, BALB/cHeA; CcS, CcS/Dem; cM, centi-morgan; DMH, 1,2-dimethylhydrazine; STS, STS/A; LOD, logarithmof odds.

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Proc. Natl. Acad. Sci. USA 93 (1996) 1083

a variance stabilizing power transformation using the exponentof 0.43 for the number of tumors and after the logarithmictransformation of the mean tumor size in individual mice.

Fine mapping of the colon tumor susceptibility loci onchromosome 2 was based on the power-transformed number oftumors (power, 0.43) as dependent variable and the genotypesfor the markers as independent variables. The analysis con-sisted of three steps:

(i) A preliminary ordinary linear regression analysis, ignor-ing the genetic relationships between different animals (23). Aforward stepwise selection process, followed by backwardelimination was used to identify which markers might beclosely linked to one of possibly multiple colon tumor suscep-tibility genes on chromosome 2. Program 2R of the statisticalpackage BMDP-PC90 was used.

(ii) Starting with all candidate markers resulting from theprevious analysis (markers between D2Mit7 and D2NdsJ onchromosome 2), a backward elimination was performed usinga series of mixed model ANOVAs with the ancestors belongingto the F2 and F2N1 generation as random factors and one ormore markers as covariates. The maximum likelihood methodwas used for the estimation of (a) the environmental variation,(b) the genetic variations explained by an identical F2 or F2N1ancestor, and (c) the strength of the Scc genes. The P valueswere calculated using the Wald test in program 3V of thestatistical package BMDP-386.

(iii) Logarithm of odds (LOD) scores were calculated foreach marker in three situations: (a) no a priori assumptionsabout location of a single Scc gene, (b) fixing one Scc gene atthe most important marker as indicated by step ii, and (c) fixingone additional Scc locus on chromosome 2 with positionderived from step ii. LOD scores were calculated using themethod of maximum likelihood as implemented in the pro-gram 3v of the statistical package BMDP-386.

RESULTSSomatic mutations of KRAS2, TP53, APC, MCC, DCC, MLHJ,and MSH2 have frequently been found in colon cancer. Table

I shows the strain distribution patterns of these genes in theCcS strains, in comparison with their susceptibility to colontumors. Linkage analysis on backcrosses of seven susceptibleCcS strains (CcS-3, -5, -7, -11, -16, -17, and -19) was used todetermine whether the chromosomal regions where thesegenes are located were associated with susceptibility to colontumors. The loci whose STS alleles are present in more thanthree susceptible CcS strains were tested in three susceptiblestrains for effects on susceptibility or microsatellite mutations.If no involvement could be found, we considered this locus notresponsible for susceptibility. In cases where one or twosusceptible CcS strains carry the STS alleles for a particularlocus, all these strains were tested. The susceptible strainsCcS-5, -11, and -17 received the alleles of all of these genesfrom the resistant parent BALB/c. Hence, these genes couldnot be responsible for the susceptibility of these strains. Thesusceptible strains CcS-3, -4, -7, -16, -18, and -19 received thealleles of one or more of these genes from the susceptible strainSTS. However, in backcrosses of the strains CcS-3, -7, -16, and-19 with BALB/c, no association was found between tumormultiplicity and the presence of the region containing thesegenes from STS (Table 2). In addition to data in Table 2, nolinkage was found between tumor susceptibility and D17Mitl(near Msh2) in CcS-7 derived backcrosses (A. P. M. Stassen,personal communication). The mouse homologs of the humanmismatch repair genes MLHJ, PMSJ, and PMS2, which havealso been found to be mutated in colon cancer, have not yetbeen mapped. None of the susceptible CcS strains carries STSalleles of D9MitJ5 and D9Mitl8, which mark the mousehomologous region of human 3p21-p23 (24, 25) carryingMLHJ. Therefore, if the mouse homolog of MLHI is locatedin this region, it is not likely to affect tumor susceptibility in theCcS strains. In addition to the above genes, which becomesomatically mutated in colon cancer, we also rule out the genefor secretory type II phospholipase A2 (Pla2s), which is acandidate for the Mom] locus (26) on mouse chromosome 4

Table 1. Strain distribution patterns in the CcS strains of genes known to be somatically mutated in colon cancercompared with susceptibility to colon cancer

Kras2 MlhI MlhI Trp53 Msh2 Mcc Dcc Apc Mean no. 95%CcS 6 9(a) 9(b) 11 17 18 18 18 of tumors confidenceno. Onco Mmr Mmr Onco Mmr Ts ? Ts Ts ? per mouse interval1 S C C C C C C C 5.3 3.4-6.42 S C C C C C C C 5.2 3.5-7.43 S C C S C C C C 4* 2.2-6.54 C C C S C C C C 11.3 7.8-15.65 C C C C C C C C 9.7 6.7-13.36 C C C C C C C C 0.9 0).4-1.87 C C C C S C C C 13.5 10.5-16.98 C C C Z C C C C 0.8 0.1-2.19 C S C S C C C C 3.8 2.3-5.9

10 C C C C C C C C 0.3 0.1-0.711 C C C C C C C C 17.6 13.7-22.112 C C C C S C C C 2.5 1.3-4.213 C C C C C C C C 5 2.9-7.814 C C C C C C C C 7.9 5.3-11.215 C C C S C C S C 0.5 0.1-1.()16 C C C S S S S S 24.4 19.9-29.417 C C C C C C C C 19 14.2-24.518 C C C C S C C C 9.9 7.2-13.219 C C C S S C S S 26.3 20-33.620 C C C C C C C C 0.7 0.3-1.3

Kras2 was typed using the marker D6MitJ4, located near Kras2, the Mlhl region using (a) D9Mit]5 and (b) D9Mit]8; Msh2was typed using DI 7Mit; and Apc was typed using D18Mitl4. S, alleles inherited from the susceptible parent STS; C, allelesinherited from the resistant parent BALB/c; Z, heterogeneous; Onco, oncogene; Ts, tumor suppressor gene; Mmr, mismatchrepair gene. Boldfaced italic type indicates that these CcS strains differed significantly in the number of tumors from BALB/cand were considered susceptible. The mean number of tumors per mouse in the parental strain BALB/c was 1.6 (95%confidence interval, 0.7-2.7).*CcS-3 females were relatively susceptible (mean, 8.3; 4.8-12.7), whereas the CcS-3 males were resistant (mean, 1.5; 0.4-3.6).

Genetics: Moen et al.

Proc. Natl. Acad. Sci. USA 93 (1996)

Table 2. Linkage analysis of number of colon tumors

Marker CcS Marker CcS Marker CcS

Chromosome 1 D4NdsJ 19 TrpS3 16, 19DJMitJ6 19 D4MitJl 19 DJJMit8 19

Chromosome 2 D4MitJ7 19 Achrb 3Hc 19 Chromosome 6 Chromosome 16D2Mit61 19 D6MitJ4 3 D16Nds2 3D2Mit9 19 Chromosome 7 Chromosome 17D2Mit56 17 Gpil 3 D17Mitl 19D2Mit66 17,19 D7Mit9 3 Chromosome 18D2NdsJ 1X, 17, 19 D7MitJO 3, 19 D18Mitl9 19Cd44 16, 17, 19 Chromosome 8 D18Mit62 3D2Mit42 169,17,19 D8MitJ2 3 D18Mitl4 16, 19D2Mitl7 16, 17, 19 Chromosome 10 D18Mit57 16,19mil 16,19 DiONdsl 19 Mcc 16

Chromosome 3 DJOMitJ4 3 D18Mit24 16, 19D3MitJ8 3 cMyb 19 D18Mit4O 3, 16

Chromosome 4 Chromosome 11 D18Mit9 16, 19D4Mit7 3 DllMit4 3, 19 Dcc 16, 19

The number of mice tested in the strains CcS-3, -16, -17, and -19 was 31, 39, 42, and 32, respectively.Boldfaced italic type indicates that in these strains strong linkage has been found between that markerand the number of tumors (P < 0.0001). Underlining indicates a distortion in the frequency ofheterozygosity for these markers in CcS-16 (D2Ndsl, Cd44: 74%; D2Mit42: 72%). Nevertheless, there isstrong evidence for a tumor susceptibility locus near D2Ndsl: P value is 0.0001, using Bonferroni'scorrection method, taking into account all markers and ignoring correlations. No linkage was foundbetween any of these markers and the size of the tumors. Cd44 was typed using a 0.4-kb fragment encodingrat Cd44 transmembrane region detecting a HindIII restriction fragment length polymorphism (RFLP)(BALB/c, 5.1 kb; STS, 2.5 kb) or using the polymerase chain reaction with the primer pair D2Nki1F(5'-ATGGACAAGTTTTGGTGGCA-3') and D2Nki1R (5'-AATCGATCTGCTGATGTGGA-3'mixed with 5'-AATCGATCTGCTGATGTGCC-3') to detect a sequence within the Cd44 gene (21),polymorphic between BALB/c and STS (77 and 83 nucleotides, respectively). Trp53: probe pM8 (22)detected a Rsa I RFLP (STS: 1 kb, constant fragments of 0.9, 1.4, and 1.5 kb); Mcc: probe MB56-Adetected a Rsa I RFLP (BALB/c: 1.5 kb, STS: 1.6 kb, constant fragments of 0.3, 0.6, 0.8, and 1.4 kb).

influencing susceptibility in mice with ApcMin mutations (27),because the susceptible CcS strains tested here carry theBALB/c alleles of markers located in this region. Moreover,the only strain (CcS-10) that carries the STS alleles of themarkers D4Mit7O and D4Mitl3 (17) flanking Pla2s is highlyresistant to colon adenomas.

Microsatellite Analysis in Colon Adenomas. We analyzed 42adenomas from 23 (BALB/c x CcS-16)Fl mice for mutationsin five SSR (simple sequence repeat) loci (D2Ndsl, D4Mit9,DllMitS, D18Mit9, and D18Mitl4). We found a single mu-tation (6-bp deletion) in the BALB/c allele of D2Ndsl in onetumor. In tests of six SSR loci (D2Ndsl, D4Mit9, DllMitSl,D14MitJ, Dl5Mit37, and D18Mitl4) in 15 adenomas from 15CcS-17 mice and 16 adenomas from 10 CcS-19 mice, themarker Dl5Mit37 showed in two tumors from CcS-17 a slightmodification in the ladder pattern, possibly due to a 1-bpdeletion.Mapping of Colon Tumor Susceptibility Genes. Using the

strain CcS-19, we have previously mapped a colon tumorsusceptibility gene, Sccl, on mouse chromosome 2 to a 31centimorgan (cM) region between Hc and Ill (Sccl region)(18). Two other susceptible CcS strains (CcS-16 and -17) alsoreceived a part of this segment from STS (Fig. 1). The part ofchromosome 2 that they inherited from STS is not the same,but they share a region of about 8.6-12.4 cM. The STS-derivedfragment in CcS-19 is interrupted by a BALB/c-derived seg-ment of unknown length located between D2Mit9 and D2Mit94,containing D2Mit56, -35, and -37.

Linkage analysis in CcS-16 and -17 showed clear associationbetween tumor multiplicity and the presence of chromosome2 markers from STS (Table 2). In CcS-16, linkage was foundto the markers D2Mit7 and Ill. Due to a distortion in thefrequency of heterozygosity of other markers in the Scc region(74% and 69% heterozygosity for D2Ndsl and D2Mit58,respectively), we detected no significant linkage to thesemarkers, but the data were compatible with the location of

Sccl near D2Ndsl. No evidence was found for a susceptibilitylocus on other chromosomes tested (Table 2).Tumor Size. We found no linkage between the size of the

colon tumors and markers in the Sccl region or with Kras2,

CcS- CcS- CcS-Chr.2 16 17 19

Hc 1.27

7.4

613.6

956,35,37

6

9414,66,126 4c186

Ndsl 0'7130 1.4

134~ 0.4Fshp:'58 1.4101,131/1 2.2p2M,17 2.9

*STS ELBALB/c a region of recomb.

FIG. 1. Part of mouse chromosome 2 of the three susceptiblestrains CcS-16, -17, and -19. The distances between the markers wereestimated according to the recombination frequency in the back-crosses of these strains and the (BALB/c X CcS-19)F2 mice. Theposition of D2Mit56, -35, and -37 is based on recombinations withD2Mit94 because no CcS strain carries the STS alleles of both thesethree markers and D2Mit9.

1084 Genetics: Moen et al.

Proc. Natl. Acad. Sci. USA 93 (1996) 1085

Markers on mouse chromosome 2 Tot. no. of

Class ab d e h kil oorecf Recombinants F2N1N2N3

1 k 1 56 18_2 5 08,41,58,81,90 54133 i I 3 33A,62A87A 22 9

4 11 . 3 12,14,76 24 2220

5 I I I 3 06A2835 20 12

6 1 2 23,60 2193 06133B377 17.12116

8 11 11 I 3 13,20,54 26 121239 [d I IIUI I 7 07,10,25,45,59,61,69 54 45

1i0 - 11 I 1 70 1711 ---J zuIt1 1A 1 11 5 39A,39B,63,67,87B 4 5 9

12-. ---: _-t _ 1 55 6 9

13 _ W-1 11 2 62B,75 18_

Total no. of independent haplotypes: 39

BALB/c STS --Region of recombination

FIG. 2. Genetic composition of recombinants between Hc and Il]on chromosome 2: a, Hc; b, Mit7; c, Mit61; d, Mit9; e, Mit94; f, Mit66;g, Nds]; h, Mitl30; i, Cd44; j, Mit43; k, Mit42; 1, Mit58; m, MitlOl; n,

Mitl7; o, III. The distances are given in Fig. 1. The numbers of theindividual recombinants, 16 females (numbers 06-45) and 18 males(numbers 54-90), the classes of the recombinant haplotypes accordingto the recombination region, and the total number of progeny testedfor tumor multiplicity are given. Recombinants 07, 10, 12, 28, 55, 56,58,59, 63, 67, 75, and 77 carry the STS alleles on the other chromosome2, and the recombinants 08, 13, 14, 20, 23, 25, 35, 41, 45, 54, 60, 61, 69,70, 76, 81, and 90 carry only BALB/c alleles on the other chromosome2. The recombinants 06, 33, 39, 62, and 87 have a recombination in bothhaplotypes (A and B).

Trp53, Dcc, Mcc, Apc, and DI7Mitl near Msh2 (Table 2). Thesize and the number of the colon tumors were not found to becorrelated in backcrosses derived from the strains CcS-3, -5, -7,-11, -16, -17, and -19 (the correlation coefficientR ranged from0.01 to 0.34).

Fine Mapping of Sccl. To map Sccl to a narrow interval, 68(BALB/c x CcS-19)F? hybrids were genotyped, and 34 micecarrying a recombination between Hc and Ill were identified(Fig. 2). The corresponding Hc-lll segment on the otherchromosome 2 was in 12 recombinants from STS and in 17recombinants from BALB/c, and 5 mice had a recombinationin both chromosomes. Hence, a total of 39 independentrecombinant haplotypes were found, which could be dividedinto 13 classes according to the crossover sites within the Scclregion (Fig. 2). Each recombinant was crossed with BALB/c,and their progeny were tested for susceptibility to DMH-induced colon tumors. Approximately half of the mice fromthe offspring of each individual recombinant carried therecombinant chromosome 2. The progeny of the 5 micecarrying a recombination in both chromosomes all carry one

recombinant chromosome 2 and one chromosome 2 from theresistant parent BALB/c.

In the first step of fine mapping Sccl, the tumor multiplicityof the offspring carrying a particular recombinant chromo-some 2 was compared with that of their littermates that did not

carry a recombinant chromosome and should be either sus-

ceptible (heterozygous for the whole Sccl region) or resistant(only BALB/c alleles present). This analysis indicated that therecombinants of the classes 4, 6, 8, 9, and 11 were susceptible.In most recombinants of the classes 5, 7, and 10, the nonre-

combinant chromosome 2 was of STS origin; the progeny tests

revealed no difference in susceptibility between the recombi-nant and the nonrecombinant chromosome, which is compat-ible with these recombinants being susceptible as well.To map Sccl to a narrow interval, we determined the

genotype and the susceptibility to colon tumors of two subse-quent generations of potentially informative recombinants(Fig. 2). Since in the course of backcrossing further recombi-nation occurred, more detailed information about the role ofindividual regions of chromosome 2 was obtained. Ordinary

15-

o 14-00

13-0

° 12-

11 -

10-17 I-IT

-16 -12 -8 -4 0centiMorgan

4 8 12

FIG. 3. Trace A is a LOD score plot showing the maximumlikelihood for the position of Sccl on chromosome 2. The 99% and90% confidence intervals are given (7, D2Mit7; 9, D2Mit9; etc. NdsJ,D2Ndsl). The plot line is dashed between D2Mit9 and D2Mit94,because CcS-19 carries a BALB/c-derived segment of unknown lengthbetween these markers in which Sccl cannot be located. The LODscores were calculated from the combined data of the CcS-17- andCcS-19-derived backcrosses and the CcS-19-derived recombinants.Trace B is the maximum likelihood estimate of the position of Scc2 on

chromosome 2. LOD score calculations were done using the data ofthe CcS-19-derived recombinants, after fixing Sccl at D2Mit66 andassuming one additional Scc locus. The distances (in cM) are givenwith respect to D2MitI86.

linear regression analysis provided no evidence of a Scc gene

telomerically from D2NdsJ. Subsequent ANOVAs showedthat there is at least one Scc gene (Sccl) near D2Mit66 (P <0.0001) and one additional Scc gene (Scc2) near D2Mit7 (P0.0029), centromerically from D2Mit66.The data on all the progeny of the (BALB/c x CcS-19)F2

recombinants, together with the data from CcS-17 and CcS-19derived backcrosses, were used to construct a LOD score plot(Fig. 3, trace A). The 90% confidence interval for the locationof Sccl covers a region of about 2.4 cM around D2Mit66. TheLOD-2 support interval (99% confidence interval) covers a

region of about 7.7 cM located between D2Mit9 and Cd44.LOD score calculations on the data from the recombinants,

after fixing Sccl at D2Mit66, also indicated the second Sccgene, Scc2, more centromerically from Sccl, near D2Mit7 (Fig.3, trace B). There was no evidence for a third Scc locus betweenHc and Ill.

The strength of the Scc loci was assessed by determining theircontribution to the genetic variation, which accounted for 38% ofthe overall variation between unrelated animals in the F2N1-3populations. Twenty-nine percent of the genetic variation couldbe explained by Sccl and 7% could be explained by Scc2.

DISCUSSIONMost genes controlling tumor susceptibility are not known.The germ-line mutations of oncogenes, tumor suppressor

genes, and mismatch repair genes are the basis of many knownfamilial cancer syndromes. On the other hand, the occurrence

of sporadic cancer might be strongly influenced by genes withlow penetrance, resulting in an excess of carriers of these genes

among sporadic cancer patients but low relative risk for otherfamily members (9, 10). In the experiments reported here, no

linkage has been found between the multiplicity of DMH-induced colon tumors and Kras2, Trp53, Apc, Mcc, Dcc, Msh2,and probably Mlhl. As tumors from hereditary nonpolyposiscolon cancer patients almost always exhibit microsatellitemutations (28-30), we analyzed tumors of the strains CcS-17,-19, and (BALB/c x CcS-16)FI hybrids for such mutations.We found one mutation (6-bp deletion) in a tumor from a

(BALB/c x CcS-16)FI mouse and two possible mutations(1-bp deletion) in only one microsatellite in two tumors fromCcS-17 (total frequency of mutations: 0.8%). This also indi-cates that the mismatch repair genes are not likely involved in

66. A: Sccl

/ Ndsl

7 B: Scc29

........8..Cd44.'ls6d 17

131i . , , . . ~~~~ ~ ~ ~ ~~~~I1..

Genetics: Moen et al.

Proc. Natl. Acad. Sci. USA 93 (1996)

the susceptibility of these strains to colon tumors. These datatogether indicate that genes other than those known to besomatically mutated in colon tumors are responsible for thedifference in colon tumor susceptibility between BALB/c andSTS. Although these data are based on two inbred strains only,the fact that not a single one of these seven genes has asignificant role in tumor susceptibility indicates that thesegenes as a group either are not polymorphic or do not have ameasurable effect. It is possible that in the future one or severalof these genes may be found to influence colon tumor sus-ceptibility in other strains. However, it is very likely that thepresently largely unknown Scc genes, such as the ones segre-gating in the BALB/c-STS cross, will account for a large partof genetic differences in colon tumor susceptibility in mice.Recently, using other strains, a colon tumor susceptibility locushas been mapped to mouse chromosome 12 (31). This locationis also different from that of the genes listed above. The Sccgenes can operate through a variety of mechanisms, includingmetabolism of carcinogen, size and proliferation rate of thetarget cell population, signal transduction, epithelium-mesenchyme interactions, antitumor immune response, etc.They may be analogous to the putative low penetrance genesaffecting susceptibility to sporadic cancer in humans.No significant linkage was found between the size of the

colon adenomas and Sccl or any of the oncogenes, tumorsuppressor genes, and mismatch repair genes tested. Further-more, the number and size of the colon tumors were not foundto be correlated in the CcS strains (13) and their backcrosses,and hence these two components of tumor susceptibility arelikely to be controlled by different subsets of genes.

Linkage analysis in three independent backcross popula-tions, derived from CcS-16, CcS-17, and CcS-19, confirmed thelocation of Sccl on chromosome 2 between Hc and Ill (31 cM)(Table 2).For the fine mapping of Sccl, we used recombinants within

this region, derived from CcS-19. ANOVA as well as LODscore calculations revealed two Scc genes within this region:Sccl near D2Mit66 and Scc2 near D2Mit7. The 99% confidenceinterval for the location of Sccl has been reduced to a regionof 7.7 cM between D2Mit9 and Cd44. At present, a total of 99DNA markers and 28 genes (between rh and Crabp2) [MITGenome Date Base (mapbase@genome.wi.mit.edu); MouseGenome Database (http://www.informatics.jax,org)] areknown to be located in this region.

In the strain CcS-19 the fragment inherited from STS isinterrupted by a BALB/c-derived segment between D2Mit9and D2Mit94. The LOD-2 support interval probably includesthis segment, and hence the Sccl region may be <7.7 cM.Within this region, the most likely location of Sccl (90%confidence interval) is a 2.4 cM region around D2Mit66. Scclis also present in the strain CcS-16, which only inherited theSTS alleles of the markers located telomerically from D2Mit66.Although this strain may carry a very short undetected STSsegment centromerically from D2Mit66, it is more likely thatSccl maps telomerically from D2Mit66. This implies that theestimated Sccl region is <1 cM long.

In conclusion, we have shown that susceptibility to DMH-induced colon tumors in mice is largely determined by genesother than those found somatically mutated in colon cancerand that these genes can be mapped with high precision. Thisopens the way to their positional cloning, as well as to the studyof their function in the carcinogenic process.

We thank J. de Moes, D. van Bokhoven, B. Boeser, C. Koomen, R.Klompmaker, J. Krijger, and H. van Vugt for expert technical assis-tance; Dr. A. Stassen for information about D] 7MitJ in CcS-7-derivedbackcrosses; and Drs. R. Nusse, P. Herrlich, and B. Vogelstein forproviding the Trp53, Cd44, and Mcc probes, respectively. This researchwas supported by grants from the Dutch Cancer Foundation.

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