**3.1 A dominant modifier on Chr 1,** *Hcs7***, maps to a 3.3 Mb region between 175.35 and 178.64 Mb**

To map the Chromosome 1 modifier to a smaller region, we crossed B6 mice with B6.C3H-Ch1 congenic mice carrying *Hcs7* susceptibility alleles on Chromosome 1. Mice that had undergone recombination in the susceptibility region were selected and then bred again with B6 mice to generate heterozygous progeny cohorts for phenotyping. Male progeny were injected with DEN at 12 days and sacrificed at 32 weeks of age. The median number of tumors developed by males of each recombinant line is shown in Figure 1, below.

Most of the susceptibility conferred by *Hcs7* can be mapped to the distal end of the chromosome. Lines carrying C3H alleles only distal of *D1Mit143* at 165 Mb developed 5- to 12-fold more tumors than B6 (line 1R28: *P* < 10-7; line 1R33: *P* < 10-6; line 1R42: *P* < 10-5). For lines 1R28 and 1R33, the number of tumors developed was not significantly different from the number developed by the homozygous B6.C3H-Ch1 parental congenic line (1R28: *P* > 0.30; line 1R33: *P* > 0.11).

Six of the seven recombinant lines that carry C3H from *D1Mit15* at 170 Mb to *D1Mit17* at 191 Mb near the telomere were highly susceptible. These six lines developed an average of eight times as many tumors as B6. The exception, line 1R5, carries this susceptibility region but was resistant (1R5 vs B6: *P* > 0.35). Comparative genomic hybridization revealed a potential explanation. In the distal susceptibility region, line 1R5 differs from C3H at the 5' end of the *Ifi202b* gene (Figure 2). While line 1R5 and C3H both carry polymorphisms that suggest duplication of part of this region relative to B6, line 1R5 also carries polymorphisms that reduce hybridization to the CGH probes relative to B6 and C3H. This effect could be due to deletion, rearrangement, or novel sequences.

Genomic DNA from male spleens was prepared using a standard Proteinase K/ammonium acetate/isopropyl alcohol precipitation protocol as described (Bilger et al., 2004), except DNA was treated with RNase A, which was then precipitated with ammonium acetate before the final DNA precipitation. Genomic DNA of either line 1R5 or inbred C3H mice was then labeled and mixed with control genomic B6 DNA, for dual-color CGH, and hybridized to the MM 8\_WG\_CGH\_1of8 chip. This array has 50-75 nucleotide probes spaced at an average of 650 bp, covering all of Chr 1 and Chr 2 through position 123959568. Results were analyzed by Nimblegen CGH Services, using the CGH-segMNT algorithm (Roche

The significance of differences between tumor multiplicity data sets was determined by the Wilcoxon rank-sum test, using Mstat software (version 5.4, McArdle Laboratory for Cancer Research; URL, http://www.mcardle.wisc.edu/mstat). All *P-*values were calculated on the basis of a two-sided test, except in the case of spontaneous tumorigenesis, for which we were testing the one-sided hypothesis that C3H alleles that increase DEN-induced tumor multiplicity also increase spontaneous tumor multiplicity. Differences between RNA expression data sets were evaluated with the limma software package (Smyth, 2004), using a

**3.1 A dominant modifier on Chr 1,** *Hcs7***, maps to a 3.3 Mb region between 175.35 and** 

To map the Chromosome 1 modifier to a smaller region, we crossed B6 mice with B6.C3H-Ch1 congenic mice carrying *Hcs7* susceptibility alleles on Chromosome 1. Mice that had undergone recombination in the susceptibility region were selected and then bred again with B6 mice to generate heterozygous progeny cohorts for phenotyping. Male progeny were injected with DEN at 12 days and sacrificed at 32 weeks of age. The median number of

Most of the susceptibility conferred by *Hcs7* can be mapped to the distal end of the chromosome. Lines carrying C3H alleles only distal of *D1Mit143* at 165 Mb developed 5- to 12-fold more tumors than B6 (line 1R28: *P* < 10-7; line 1R33: *P* < 10-6; line 1R42: *P* < 10-5). For lines 1R28 and 1R33, the number of tumors developed was not significantly different from the number developed by the homozygous B6.C3H-Ch1 parental congenic line (1R28:

Six of the seven recombinant lines that carry C3H from *D1Mit15* at 170 Mb to *D1Mit17* at 191 Mb near the telomere were highly susceptible. These six lines developed an average of eight times as many tumors as B6. The exception, line 1R5, carries this susceptibility region but was resistant (1R5 vs B6: *P* > 0.35). Comparative genomic hybridization revealed a potential explanation. In the distal susceptibility region, line 1R5 differs from C3H at the 5' end of the *Ifi202b* gene (Figure 2). While line 1R5 and C3H both carry polymorphisms that suggest duplication of part of this region relative to B6, line 1R5 also carries polymorphisms that reduce hybridization to the CGH probes relative to B6 and C3H. This effect could be

tumors developed by males of each recombinant line is shown in Figure 1, below.

**2.5 Comparative genomic hybridization** 

moderated t-statistic followed by FDR correction.

due to deletion, rearrangement, or novel sequences.

Nimblegen, Madison, WI).

**2.6 Statistical analysis** 

**3. Results** 

**178.64 Mb** 

*P* > 0.30; line 1R33: *P* > 0.11).

Fig. 1. C3H liver cancer susceptibility alleles map to distal Chromosome 1.

Male mice carrying C3H alleles on Chromosome 1 on a B6 genetic background were injected with DEN and liver tumors were counted at 32 weeks. The line designated B6.C31 refers to B6.C3H-Ch1. Regions inherited from B6 are shown in white; C3H regions are shown in black. Regions carrying a breakpoint between B6 and C3H alleles are shown in grey. The median tumor multiplicity and number of mice tested are shown to the right of each line. Lines that were significantly more susceptible than B6 are marked with a "+." The positions of markers along Chromosome 1 are shown as tics below the position axis at the top of the figure.

Two additional lines, derived from the susceptible 1R14 and 1R33 lines during the breeding of experimental progeny, suggested that *Hcs7* lies in a 6.4 Mb region carrying *Ifi202b*. Although few mice were phenotyped (five per line), the results for each line were significant. Line 33a, like its parent, was significantly more susceptible than B6 (*P* < 10-3) and not significantly different from B6.C3H-Ch1 (*P* > 0.55). The distal breakpoint for line 1R33a is proximal of 179.3 Mb, suggesting that the minimal susceptibility region lies proximal of 179.3 Mb. Line 1R14a, unlike its parental line 1R14, was resistant to hepatocarcinogenesis. The difference between the lines is the distal breakpoint between 172.9 and 173.0 Mb in line 1R14a, suggesting that the minimal susceptibility region is distal of 172.9 Mb. Together, the two sublines suggest that *Hcs7* lies between 172.9 and 179.3 Mb.

Fig. 2. The homozygous line 1R5 genome differs from C3H at *Ifi202b*.

Genomic DNA of either line 1R5 or inbred C3H was labeled and mixed with control genomic B6 DNA before hybridization to a chip carrying Chromosome 1 probes spaced 650 bp apart. The log2 ratio of the mean of the intensity of the 1R5 or C3H signal (Test) relative to B6 is plotted. C3H is shown as a dashed grey line; the 1R5 recombinant is shown as a solid black line.

Several other congenic lines that lacked the distal susceptibility region were significantly more susceptible than B6, developing three- to four-fold more tumors by 32 weeks (lines marked with plus signs, Figure 1; *P* < 10-3 for all). These lines and the closely related congenic lines that are as resistant as B6 reveal a complex pattern of possibly interacting modifiers along proximal Chromosome 1. Resistant line 1R50, for example, carries more C3H than sensitive line 1R12, but it carries less C3H than sensitive line 1R54 (1R50 vs B6: *P* > 0.06; vs R12: p < 10-2; vs 1R54: p < 0.02). Similarly, the C3H region carried by resistant line 1R7 extends farther distally than the C3H region carried by sensitive line 1R43. However, the resistance of line 1R7 might be explained by a unique B6 genotype at the proximal end of Chromosome 1 (proximal of 74 Mb).

To localize the distal Chromosome 1 liver cancer modifier further, we bred line 1R33 with B6 and selected males with breakpoints in or near the region between 172.9 and 179.3 Mb (Figure 3). These mice were bred with B6 females to generate experimental progeny that were injected with DEN at 12 days; tumors were counted at 32 weeks.

1R14a, suggesting that the minimal susceptibility region is distal of 172.9 Mb. Together, the

175 176 177

Position (Mb)

Genomic DNA of either line 1R5 or inbred C3H was labeled and mixed with control genomic B6 DNA before hybridization to a chip carrying Chromosome 1 probes spaced 650 bp apart. The log2 ratio of the mean of the intensity of the 1R5 or C3H signal (Test) relative to B6 is plotted. C3H is shown as a dashed grey line; the 1R5 recombinant is shown

Several other congenic lines that lacked the distal susceptibility region were significantly more susceptible than B6, developing three- to four-fold more tumors by 32 weeks (lines marked with plus signs, Figure 1; *P* < 10-3 for all). These lines and the closely related congenic lines that are as resistant as B6 reveal a complex pattern of possibly interacting modifiers along proximal Chromosome 1. Resistant line 1R50, for example, carries more C3H than sensitive line 1R12, but it carries less C3H than sensitive line 1R54 (1R50 vs B6: *P* > 0.06; vs R12: p < 10-2; vs 1R54: p < 0.02). Similarly, the C3H region carried by resistant line 1R7 extends farther distally than the C3H region carried by sensitive line 1R43. However, the resistance of line 1R7 might be explained by a unique B6 genotype at the

To localize the distal Chromosome 1 liver cancer modifier further, we bred line 1R33 with B6 and selected males with breakpoints in or near the region between 172.9 and 179.3 Mb (Figure 3). These mice were bred with B6 females to generate experimental progeny that

two sublines suggest that *Hcs7* lies between 172.9 and 179.3 Mb.

Fig. 2. The homozygous line 1R5 genome differs from C3H at *Ifi202b*.

proximal end of Chromosome 1 (proximal of 74 Mb).

were injected with DEN at 12 days; tumors were counted at 32 weeks.


as a solid black line.

0

0.5

log2 Test/B6

1

Fig. 3. C3H liver cancer susceptibility alleles map to a 3.3 Mb region between 175.35 and 178.64 Mb.

Male mice were injected with DEN and liver tumors were counted at 32 weeks. "B6" and "C3" mice were inbred; lines beginning with a "1" or "2" were B6.C3 congenic mice carrying C3H Chromosome 1 alleles on a B6 genetic background; "B6.BR." lines carry BR Chromosome 1 alleles on a B6 genetic background; and the C3H.B6-Ch1 (C3.B6-Ch1) line carries B6 Chromosome 1 alleles on a C3H background. Regions inherited from B6 are shown in white; C3H and BR regions are shown in black. Regions carrying a breakpoint between B6 and C3H alleles, or between B6 and BR alleles, are shown in grey. The median tumor multiplicity and number of mice tested are shown to the right of each line. Lines that were significantly more susceptible than B6 are marked with a "+." The positions of markers along Chromosome 1 are shown as tics below the position axis at the top of the figure.

Mice from line 2R16, which carries the smallest susceptibility region, developed 7-fold more tumors than B6 (*P* < 10-5; Figure 3). This line carries C3H from 172.9 to 178.64 Mb, confirming and narrowing the location of the distal modifier. Line 2R16 was derived from line 2R3, which carries an additional ~14 Mb of C3H alleles distal of 178.64 Mb. Like 2R16, 2R3 was highly susceptible, developing 10-fold more tumors than B6 (line 2R3 vs. 2R16: *P* > 0.12; vs. B6: *P* < 10-8). Line 2R8 has a larger congenic region than 2R16, extending up to 7.5 Mb farther proximally and ~ 700kb farther distally. This line was also highly susceptible, developing more than 8-fold more tumors than B6 (*P* < 10-5). Together with the parental line 1R33 (again highly significantly different from B6), these lines establish that the *Hcs7* locus confers a 7- to 10-fold increase in tumor multiplicity and lies between 172.9 and 178.64 Mb. This region carries 132 genes (www.ensembl.org; 8/2011). Line 2R7, which extends distally from 175.35 Mb, was also highly susceptible relative to B6, developing almost 9-fold more tumors (*P* < 10-5). In addition, line 2R7 is approximately 3-fold more susceptible than line 2R5, which carries C3H alleles distal of 178.9 Mb (*P* < 0.02). Together with the 2R16 results that place *Hcs7* between 172.9 and 178.64 Mb, these data suggest that *Hcs7* lies in the 3.3 Mb between 175.35 and 178.64 Mb. This region carries 44 genes (www.ensembl.org; 8/2011).

Again, the remaining lines suggest that the pattern of modifiers along the chromosome is complex. Lines 2R9 and 2R7 both carry C3H alleles to near the telomere and line 2R9 carries more C3H alleles than 2R7 proximally (their breakpoints differ by about 100 Kb), but line 2R9 is resistant (2R9 vs B6: *P* > 0.24). Similarly, the resistance of line 2R11 (2R11 vs. B6: *P* > 0.09), which, like line 2R9, was derived from sensitive line 2R3, together with the susceptibility of lines 2R2, 2R10, 2R15, 2R7, and 2R5, suggests there are additional modifiers both proximal to and distal of the 175.4 to 178.64 Mb minimal region. Alternatively, some lines may have undergone rearrangement in the susceptibility region.

Recombination between B6 and C3H was suppressed in the minimal susceptibility region, between 175.4 and 178.4 Mb. No recombinants were observed among approximately 1350 segregating progeny, although 24 would be expected. This difference is highly significant (*P* < 10-6). Recombination is frequently suppressed by chromosomal rearrangements such as inversions (Kirkpatrick, 2010).
