Although several familial cancer genes with high-penetrance mutations have been identified, the major genetic component of susceptibility to sporadic cancers is attributable to low-penetrance alleles. These ‘weak’ tumor susceptibility genes do not segregate as single Mendelian traits and are therefore difficult to find in studies of human populations. Previously, we have proposed that a combination of germline mapping and analysis of allele-specific imbalance in tumors may be used to refine the locations of susceptibility genes using mouse models of cancer. Here, we have used linkage analysis and congenic mouse strains to map the major skin tumor susceptibility locus Skts1 within a genetic interval of 0.9 cM on proximal chromosome 7. This interval lies in an apparent recombination cold spot, and corresponds to a physical distance of about 15 Mb. We therefore, used patterns of allele-specific imbalances in tumors from backcross and congenic mice to refine the location of Skts1. We demonstrate that this single tumor modifier locus has a dramatic effect on the allelic preference for imbalance on chromosome 7, with at least 90% of tumors from the congenics showing preferential gain of markers on the chromosome carrying the susceptibility variant. Importantly, these alterations enabled us to refine the location of Skts1 at higher resolution than that attained using the congenic mice. We conclude that low-penetrance susceptibility genes can have strong effects on patterns of allele-specific somatic genetic changes in tumors, and that analysis of the directionality of these somatic events provides an important and rapid route to identification of germline genetic variants that confer increased cancer risk.
The identification of the specific genetic variants responsible for increased susceptibility to familial or sporadic cancers remains an important but challenging goal with major implications for prediction of individual cancer risk, as well as for development of strategies for prevention or targets for therapy (Peto, 2001; Ponder, 2001; Balmain, 2002; Balmain et al., 2003). Mouse models of cancer have been extensively used for the analysis of the genetic basis of tumor susceptibility, and have led to the mapping of multiple loci that confer, either alone or in specific combinations, increased cancer risk (Dietrich et al., 1993; Fijneman et al., 1996; Ruivenkamp et al., 2002; Demant, 2003; Dragani, 2003; Mao and Balmain, 2003). Using a two-stage skin carcinogenesis model involving dimethylbenz(a)anthracene (DMBA) initiation and subsequent promotion with 12-O-tetradecanoyl-phorbol-13-acetate (TPA), we previously identified 15 skin tumor susceptibility loci in an interspecific F1 backcross ((NIH/Ola × Mus spretus) × NIH/Ola) (NSP) study by quantitative trait locus (QTL) analysis (Nagase et al., 1995, 1999; Ewart-Toland et al., 2003). Skts1, the strongest papilloma resistance locus, was mapped to a broad interval of at least 20 cM on proximal chromosome 7 (Nagase et al., 1995). Approaches to the fine resolution mapping of susceptibility variants include derivation of congenic mice by multiple rounds of breeding to transfer regions containing the variant from the susceptible to the resistant strain, or vice versa (Vogel et al., 1994). Haplotype segregation may also be used in combination with linkage analysis to fine map QTLs if at least one of the parental strains in the crosses is outbred. The latter approach was recently used to identify Aurora-A as a candidate tumor susceptibility gene (Ewart-Toland et al., 2003), a conclusion that has been supported by several association studies using human population-based cases and controls (Ewart-Toland et al., 2005).
An additional approach seeks to exploit, by analogy with loss of the wild-type allele in tumors of patients carrying germline mutations in high-penetrance tumor susceptibility genes such as Rb (Knudson, 1971; Friend et al., 1986), the effects of polymorphisms in low-penetrance susceptibility genes on patterns and allele specificity of somatic alterations in tumors (Nagase et al., 2003). The feasibility of this approach is demonstrated by recent data showing that polymorphisms in specific low-penetrance tumor susceptibility genes affect allele-specific amplification or mutation events in human and mouse tumors (Ewart-Toland et al., 2003; Hienonen et al., 2006; To et al., 2006). Here, we have used a new panel of congenic lines, together with analysis of somatic chromosomal imbalances in tumors, to reduce the large QTL containing Skts1 to a relatively small interval. Although the congenic mapping alone reduced the interval to approximately 0.9 cM, the physical distance remained large (15 Mb), presumably owing to a low rate of meiotic recombination that limits the usefulness of this approach. The region could however, be further refined using tumor analysis to identify the markers showing the highest frequency of preferential allelic imbalance. We conclude that analysis of preferential allelic imbalance in mouse tumors can complement standard genetic approaches to the identification of germline variants that influence cancer risk, and suggest that similar strategies may also help to define genomic regions containing human low-penetrance susceptibility genes.
Results and discussion
Mouse chromosome 7 contains at least two independent skin tumor susceptibility loci (Skts1 and Skts2) as shown by the presence of two broad LOD score peaks detected by standard QTL analysis (Nagase et al., 1995). By further analysis of the original F1 backcross (N2), we demonstrated two clear peaks on proximal chromosome 7, that is, one at 26.7 cM in agreement with earlier mapping of Skts1 and one at 9.6 cM (now called Skts14; Mao et al., 2006). These findings indicate that, as seen with other tumor susceptibility QTLs (Cormier et al., 2000), the locus is complex and may contain two or more overlapping susceptibility regions.
To confirm the presence of low-penetrance susceptibility genes at the identified regions, multiple congenic lines of proximal chromosome 7 were generated. Firstly, we selected several N4 mice, homozygous for Mus musculus alleles on distal chromosome 7 containing Skts2 but heterozygous for proximal chromosome 7 containing Skts1 and Skts14, for further backcrossing with NIH/Ola mice to generate N9 mice. Although N9 mice only have a small fraction of their genome from the resistant M. spretus parent, based on random segregation of alleles, significant linkage of proximal chromosome 7 was detected with papilloma incidence (P=1.0 × 10−3), roughly at the location of Skts1, indicating that M. spretus alleles on proximal chromosome 7 are strong enough, even in the heterozygous state, to confer statistically significant resistance to tumorigenesis. We concluded that these mice constitute a good starting point for the generation of single congenic lines. In contrast to N2 and N4 mice, the LOD score plot of N9 mice did not show a peak at 9.6 cM (data not shown) suggesting that interacting genes necessary for the phenotype of Skts14 were probably lost during breeding (Legare and Frankel, 2000). Subsequently, N9 mice with different recombinations on proximal chromosome 7 were selected to generate 25–30 N10 mice per founder by backcrossing with NIH/Ola mice. Of the seven congenic N10 lines tested, four showed significant linkage with papilloma incidence whereas three did not (comparing heterozygous NS with homozygous NN mice; Table 1, ‘Backcross’ columns, and Figure 1a and b). By intercrossing mice heterozygous for the indicated regions on chromosome 7, the effect of homozygosity of M. spretus alleles on tumorigenesis was determined. Importantly, the same four congenic lines showed highly significant linkage with papilloma incidence (comparing homozygous SS with homozygous NN mice; ‘Intercross’ columns, Table 1, and Figure 1c–e). An additional experiment was carried out with line 1230, which contains the smallest congenic region (Figure 2a), to show that the heterozygotes for this line had intermediate papilloma numbers compared to the two groups of reciprocal homozygous mice (Figure 1e). The effect in the intercross analysis was even more pronounced than in the backcross analysis (Table 1 and Figure 1a–e). Six additional congenic lines were only tested by intercross analysis, of which three showed strong linkage with papilloma incidence (Table 1). By determining the minimum-overlapping region within the seven positive congenic lines and by excluding the regions of the six lines that did not show an effect, we were able to narrow down Skts1 to an interval of 0.9 cM, that is from 20.9 to 21.8 cM (Figure 2a). Importantly, the congenic line 1230, that merely contains M. spretus alleles for exactly this minimum-overlapping region from 20.9 to 21.8 cM, showed strong linkage analysis with papilloma incidence (Table 1, Figure 1e) providing further strong support for the location of Skts1 within this interval. Moreover, these congenic studies allowed us to exclude a number of interesting candidates for the Skts1 gene. Dragani and co-workers have proposed that the gene responsible for the albino phenotype, Tyr encoding the enzyme tyrosinase, is in fact the Skts1 gene (Saran et al., 2004). This gene clearly, however, lies outside the main congenic region and can be excluded as a candidate for Skts1.
During the preparation of this panel of congenic lines, the complete sequence of the mouse genome was determined, allowing us to estimate the physical size of the minimal congenic region containing Skts1 (Waterston et al., 2002). Surprisingly, although the genetic size of the interval corresponded to 0.9 cM (as determined empirically from our linkage analysis of >1000 meioses in interspecific backcross animals) and the average relationship between genetic and physical size in the mouse is approximately 1 cM=2 Mb (Yu et al., 2001), the physical distance between markers D7Mit193 and D7Mit248 that define the minimal congenic region was about 15 Mb (Figure 2a; http://www.ensembl.org). This suggests that there is an unusually low meiotic recombination rate at this location on proximal chromosome 7, leading to this discrepancy between the genetic and physical maps of the region. In order to test the possibility that the M. spretus genome may carry a large inversion on chromosome 7, which could lead to recombination suppression (Silver, 1995) we carried out fluorescence in situ hybridization (FISH) mapping of bacterial artificial chromosome (BACs) (RPCI23-5O6, RPCI23-93H2) located between the markers delimiting the minimal congenic region (Figure 2a). Mouse embryonic fibroblasts were cultured from the congenic line 1230. FISH analysis of BACs from this region demonstrated that the relative orientation with respect to the centromere was the same in NIH/Ola and M. spretus (Figure 2b and c). The higher magnification images shown below panels b and c show that the BAC RPC123–506, located at 56.2 Mb (labeled in red), is further from the centromere (more densely staining region at the top of the image) than BAC RPC123-93H2, located at 43.7 Mb (labeled in green). The same configuration was found in mouse embryonic fibroblasts (MEFs) from the specific congenic lines used for tumor analysis, thus excluding the possibility that a large inversion had occurred during the selection or breeding of the congenic lines. The reasons for the low recombination frequency in this part of chromosome 7 remain unclear, but the results indicated that the application of the congenic method in isolation would not lead to identification of the Skts1 gene.
We, therefore, carried out a detailed investigation of allelic imbalance on mouse chromosome 7 to determine whether somatic changes would allow more refined localization of Skts1. Mutations of Hras induced by DMBA treatment provide the driving force for trisomy of chromosome 7 during skin tumor development (Kemp et al., 1993). In 23 of 26 carcinomas derived from NSP mice, we demonstrated specific activating mutations in the M. musculus allele of Hras (Nagase et al., 2003). Importantly, almost all tumors showed allele-specific imbalances of chromosome 7 that favored the chromosome carrying the mutant Hras allele (Nagase et al., 2003). However, the imbalances of alleles in favor of the M. musculus chromosome could be due to preferential mutation or selection of the Hras gene from the NIH/Ola parent, to the influence of Skts2 located close to Hras on distal chromosome 7, or even to the selection of M. musculus alleles at Skts1 in the proximal part of the chromosome. To resolve this question, we first analysed allele-specific imbalance in papillomas from mice of the N2 or N4 generations. Chromosomal imbalances on proximal chromosome 7 were detected in approximately 70% of papillomas derived from N2 mice (Figure 3b) confirming our earlier findings with carcinomas from these mice (Nagase et al., 2003). Approximately two-thirds of papillomas showed allele-specific imbalances in favor of the susceptible M. musculus alleles. Papillomas from N4 mice also showed imbalances on proximal chromosome 7 with an even larger percentage in favor of M. musculus alleles (Figure 3a and b), in spite of the fact that both parental chromosomes carried the same NIH/Ola Hras gene as well as other alleles on distal chromosome 7 such as Skts2. This clearly shows that one or more low-penetrance tumor susceptibility genes near Skts1 on proximal chromosome 7 can influence somatic genetic changes in tumors, independently from the previously described effects of Hras or the Skts2 locus. Papillomas derived from N9 mice also showed preferential allelic imbalances on proximal chromosome 7, including Skts1, with 95% of imbalances in favor of M. musculus alleles (Figure 3b) which is also significantly higher than the 63% observed in N2 mice (P=0.02, χ2 test). A summary of the results obtained for different markers on proximal chromosome 7 is shown in Figure 3c and d. Most papillomas showed gains of musculus alleles across the whole region (34 papillomas) but others exhibited regional gains involving smaller chromosome fragments (Figure 3c). Maximal allele-specific imbalance was detected using marker D7Mit278 located at approximately 52.7 Mb, which is within the minimum-overlapping region of 0.9 cM identified with the congenic mice. The somatic mapping data allowed us to refine the interval containing Skts1 to a size of 1.5 Mb (between markers D7Mit211 and D7Mit198, Figure 2a), centered at D7Mit278. This interval still contains a relatively large number of genes (more than 12 transcripts), some of which (e.g. Atp10a, Ube3a) now become interesting candidates for further analysis.
These data demonstrate that low-penetrance susceptibility genes, even when present in the heterozygous state in congenic mice, can influence somatic genetic changes in tumors and that these alterations can be exploited to rapidly fine map putative susceptibility loci. This strategy may be particularly useful for refining susceptibility genes within regions of the genome that are recombination cold spots (Yu et al., 2001), and therefore difficult to fine map by conventional linkage or association studies. When combined with novel tools to analyse genome-wide allele-specific alterations in copy number and expression, this approach could greatly accelerate discovery of cancer susceptibility QTLs, helping us in the eventual identification of a subset of these genes.
Materials and methods
Animals and tumor induction
In a large F1 backcross study using NIH/Ola and M. spretus mice (i.e. NSP backcross, (NIH/Ola × M. spretus) × NIH/Ola), the Skts1 locus on chromosome 7 was identified by QTL analysis as a skin tumor susceptibility locus (Nagase et al., 1995). A resistant F1 backcross mouse was selected for further phenotype-assisted backcrossing over at least 10 generations to NIH/Ola mice, ultimately leading to multiple congenic lines containing different overlapping regions of M. spretus proximal chromosome 7 on the NIH/Ola background. Mice from all generations were treated following the same skin tumor induction protocol, as reported previously (Nagase et al., 1995). In short, mice (8–12 weeks old) received a single dose of DMBA (25 μg per mouse in 200 μl acetone) and, starting 1 week after initiation, animals were promoted with TPA (200 μl of 0.1 mM solution in acetone) twice weekly for 20 weeks. Papilloma numbers were counted every other week until 20 weeks after tumor initiation.
DNA preparation, genotyping and allelotyping using microsatellite markers
DNAs were prepared from papillomas and corresponding normal tail tips and microsatellite markers were amplified by standard methods. Each marker's order and distance were estimated from progeny generated during these (NIH/Ola × M. spretus) × NIH/Ola backcross animals (n>1000). Negative binomial regression analysis was used to identify QTLs that control skin tumor susceptibility, as reported previously (Nagase et al., 1995, 1999). To determine susceptibility in the congenic lines, the Wilcoxon rank sum test was used. Allelic imbalances in papillomas of N2, N4 and N9 mice were examined by quantitative polymerase chain reaction (PCR) using fluorescent dye-labeled oligomers designed to amplify SSLP markers (microsatellites). Differences of 50% or more in the intensity ratios of the two alleles in papilloma DNAs compared to DNAs from corresponding tails as normal controls were scored as allelic imbalances, as previously described (Nagase et al., 2003). At least three independent microsatellite markers were analysed in duplicate per papilloma. Percentages of allelic imbalances in different crosses were compared using the χ2 test.
Fibroblast cell lines
The matings of the intercrossed congenic line 1230 were timed and the embryos were dissected at E 13.5. Yolk sacs were isolated and genomic DNAs were prepared from the head of each embryo and genotyped by PCR. Fibroblasts were passaged repeatedly according to the standard 3T3 protocol. Metaphase chromosomes were prepared and used for FISH.
FISH mapping of chromosome 7 BACs
BAC clones (RPCI23-5O6, RPCI23-93H2) were purchased from Oakland Children's Hospital. RPCI23-5O6 was labeled with cy3 by nick translation. RPCI23-93H2 was labeled with fluorescein isothiocyanate (FITC). Hybridization, washing and probe detection were performed according to the manufacturer's instructions (Bioprime DNA labeling system, Invitrogen, Carlsbad, CA, USA).
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We thank R del Rosario and R Contreras for assistance with animal husbandry. This work was supported by an NCI Mouse Models of Human Cancer Consortium Grant (U01 CA84244). The early development of the congenic lines was funded by Cancer Research UK at the Beatson Institute (Glasgow, Scotland). JPdK was supported by a research fellowship through the Dutch Cancer Society. JHM is the recipient of a Leukemia & Lymphoma Society Fellowship. AB acknowledges support of the Barbara Bass Bakar Endowed Chair of Cancer Genetics.
Competing interests statement
The authors declare that they have no competing financial interests.
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de Koning, J., Wakabayashi, Y., Nagase, H. et al. Convergence of congenic mapping and allele-specific alterations in tumors for the resolution of the Skts1 skin tumor susceptibility locus. Oncogene 26, 4171–4178 (2007). https://doi.org/10.1038/sj.onc.1210206
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