Physical evidence of Mcs5, a QTL controlling mammary carcinoma susceptibility, in congenic rats

David J. Samuelson, Jill D. Haag, Hong Lan, Dinelli M. Monson, Millicent A. Shultz, Bradley D. Kolman and Michael N. Gould1

McArdle Laboratory for Cancer Research, University of Wisconsin-Madison, 1400 University Avenue, Madison, WI 53706-1599, USA

1 To whom correspondence should be addressed Email: gould{at}oncology.wisc.edu


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Genetic susceptibility to breast cancer is influenced by high- and low-penetrance genes. The low-penetrance genes contributing to increased and decreased risk likely exist at appreciable frequencies in the human population. To identify high-frequency, low-penetrance modifier genes, we are using a rat genetic model. Eight quantitative trait loci, named mammary carcinoma susceptibility (Mcs) loci, have been genetically identified in two rat strains, Wistar-Kyoto (WKy) and Copenhagen. These strains are resistant to developing mammary cancer compared with susceptible Wistar-Furth (WF) female rats. Here we provide physical evidence of the existence of Mcs5 in the resistant WKy rat and further narrow the candidate region defining the QTL. Two congenic rat lines (C and D) containing large segments of the WKy Mcs5 QTL on chromosome 5 were generated on a WF background. The minimal WKy interval from markers D5Wox7 and D5Uwm37 (line C) conferred resistance to developing 7,12-dimethylbenz- [a]anthracene (DMBA)-induced mammary carcinomas. Line C females that were homozygous for the WKy allele at this interval averaged 1.1±0.3 carcinomas/rat compared with 6.9±0.4 average carcinomas/rat for WF control females (P<0.01). Line D females containing the minimal WKy interval from D5Rat26 to D5Uwm42, were as susceptible to developing mammary carcinomas as WF controls (5.7±0.6 versus 6.9±0.4, respectively). The WKy region in common to these lines from D5Rat26 to D5Uwm37 is thus not expected to contain Mcs5-associated genes. Based on results presented here, the Mcs5 locus has been physically located within a congenic interval on rat chromosome 5 between markers D5Uwm8 and D5Rat26.

Abbreviations: COP, Copenhagen strain; DMBA, 7,12-dimethyl- benz[a]anthracene; Mcs, mammary carcinoma susceptibility loci; WF, Wistar- Furth strains; WKy, Wistar-Kyoto strains


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Breast cancer is a multigenic disease (1). The genetic component of risk for breast cancer is at least 30% (2) and likely greater (3). The genetic etiology of breast cancer consists both of high-penetrance, low-frequency genes such as Brca1 and Brca2, and low-penetrance, high-frequency modifier genes. Given that the large effort to find additional high-penetrance breast cancer genes such as the Brca genes has thus far failed, it has been suggested to be unlikely that additional genes in this class will be discovered (4). In addition, the rate of discovery of low-penetrance modifier genes has been disappointing (5). Much of the discovery effort for these genes has used association studies in case-control populations of breast cancer patients and controls. These studies, for the most part, focused on candidate lists that were mainly comprised of known genes involved in DNA repair (4) or xenobiotic metabolism (6). Unfortunately, while several potential modifier genes were preliminarily identified, most were subsequently rejected based on larger case-control studies or meta-analysis of several similar studies (6). Together these findings suggest the need for alternative approaches to identifying high-frequency, low-penetrance modifier alleles that either increase or decrease breast cancer risk. It is projected that if 50% of these breast cancer modifier genes could be identified it would be possible to assign 80% of the total breast cancer risk to 50% of the population (1). This ‘at risk’ population would then be targeted for increased surveillance and intervention with chemoprevention agents.

In the future, it is likely that one will be able to screen the entire human genome for regions that are associated with modified breast cancer risk. This would require an experimental design in which polymorphic genomic markers such as single nucleotide polymorphisms (SNP) or restriction fragment length polymorphisms covering the entire genome could be used together with a dense haplotype-block map for association studies in very large, case-control or population-based studies. The technology needed for such studies may optimistically become available in the next decade. However, given current technologies for assaying SNPs, it is only feasible to perform such an analysis on small targeted portions of the genome. This requires strategies to prioritize several megabase-sized genomic regions for study.

We have approached this problem through the use of comparative genomics and have chosen the rat for modeling genetic risk to breast cancer. The rat was chosen as a model over the mouse because rat mammary cancer more closely resembles human breast cancer in both hormonal responsiveness and histopathology. Our initial studies showed that the Wistar-Furth (WF) rat strain, which is more sensitive to 7,12-dimethylbenz[a]anthracene (DMBA)-induced mammary carcinogenesis than is the Fischer (F344), possesses dominantly acting genes/alleles for increased sensitivity since (WF x F344)F1 rats were as sensitive to tumor induction as the WF parent (7). Current studies focus on the genetics of two rat strains that are resistant to mammary carcinogenesis: the Copenhagen (COP) and Wistar-Kyoto (WKy) strains. In the WKy strain, four quantitative trait loci (QTLs) that control the susceptibility to DMBA-induced mammary carcinoma multiplicity were identified by genetic linkage analysis. In these experiments no differences were found in the histopathology of the induced mammary tumors between each strain. The locus with the highest LOD score (LOD = 13) and the greatest statistically predicted degree of effect on mammary cancer susceptibility is the mammary carcinoma susceptibility (Mcs)5 locus (8). The genetic identification (8) of Mcs5 was an important first step in identifying this locus. However, it is necessary to physically confirm the existence of Mcs5s as an independently acting resistance allele. Here we produce Mcs5 congenic rats and provide physical evidence for the existence of an independently acting resistance locus within the predicted Mcs5 interval. Using these congenic rats we also measure the degree of resistance conferred by this WKy allele in both its homozygous and heterozygous state.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Animals
WF and WKy rats were obtained from Harlan Sprague-Dawley (Indianapolis, IN). They were bred together to yield (WKy x WF)F1 progeny. Male F1 rats and subsequent backcross male carriers were then bred to female WF rats to the N8 or N9 generations. All potential breeders were genotyped at each generation to maintain the desired Mcs5 interval. Rats were fed Teklad lab blox chow and acidified water ad libitum. They were maintained in a 12-h light/dark cycle. Rats were maintained in an AALACC-accredited facility and all protocols were approved through the University of Wisconsin Medical School Animal Research Committee.

Genotyping
A small tail tip section from each rat was removed at 1–4 weeks of age. To isolate DNA, the tail samples were digested overnight at 55°C in 500 µl of genomic lysis buffer consisting of 20 mM Tris HCl pH 8.0, 150 mM NaCl, 100 mM EDTA and 1% SDS. Two hundred microliters of Protein Precipitation Solution (Gentra Systems, Minneapolis, MN) was added to the lysate solution. DNA in the clear supernatant was precipitated with isopropanol, washed and resuspended in water. DNA was PCR-amplified using polymorphic microsatellite markers (ResGen/Invitrogen, Carlsbad, CA) (Table I). PCR reactions were performed in 96-well plates, combining 2.5 ml of diluted DNA (~50–100 ng) with 2.5 µl PCR master mix, consisting of 10 mM Tris–HCl, pH 8.3, 1.5 mM MgCl2, 50 mM KCl, 0.001% gelatin, 250 µM dNTPs, ~200 nM each primer and 0.2 U AmpliTaq polymerase. Each reaction underwent an initial denaturation of 94°C for 3 min followed by 40 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 30 s and a final extension of 72°C for 7 min. PCR products were resolved on 3% GenPure HiRes agarose gels (ISC BioExpress, Kaysville, UT), stained with SYBR gold (Molecular Probes, Eugene, OR), and imaged using a Typhoon Imager and ImageQuant software (Amersham Biosciences, Piscataway, NJ).


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Table I. Known mammary carcinoma susceptibility (Mcs) loci and markers used to screen lines C and D for congenicity

 
At each backcross generation we selected male carriers for each respective congenic line that retained much of the Mcs5 interval (>70 cM) from D5Wox7 to D5Uwm42. In addition, the carriers were genotyped using markers on other chromosomes, as listed in Table I, to assure that they did not carry WKy alleles at any other known Mcs loci or other chromosomes besides chromosome 5. WF.WKy-D5Wox7/D5Uwm37 (line C) originated at the first backcross generation (N2). WF.WKy-D5Rat26/D5Uwm42 (line D) was derived from an N4 recombinant male whose lineage contained the distal portion of WKy chromosome 5.

Phenotyping
For phenotype analysis, heterozygous male and female congenic carriers were bred to produce female congenic rats of three genotypes: WKy-homozygous, WKy/WF-heterozygous and WF-homozygous. At generation N8 or N9, rats were phenotyped to determine the degree of mammary carcinoma susceptibility. At 50–55 days of age, female N8 or N9 rats that were WKy-homozygous, WKy/WF-heterozygous or WF-homozygous at the Mcs5 congenic intervals were administered a single dose (65 mg/kg body mass) of DMBA (ACROS Organics; Fisher Scientific, Pittsburgh) in sesame oil by gastric intubation. Mammary carcinomas >3 x 3 mm were counted at 15 weeks post-carcinogen administration. At this time, spleen tissue was removed for DNA extraction and confirmation of each animal's genotype. Tumor multiplicity data were analyzed using ANOVA with Dunnett and Sheffe's multiple comparison procedures and the unpaired t-test function of StatView (SAS Institute Inc., Cary, NC).

Comparative genomics
The rat and human genomes were compared within the Mcs5 region using the synteny view of the Ensembl genome browser (9,10). The interval of rat chromosome 5 compared with human was from bases 14 801 800 to 129 629 500, which was defined by the Mcs5 genetic markers D5Uwm8 and D5Rat26. The base-pair positions of these markers on rat chromosome 5 were derived using the BLAT function (11) from the UCSC genome browser (12) and the sequence of the genetic markers D5Uwm8 and D5Rat26 (13). D5Uwm8 starts at base 14 801 808 and D5Rat26 starts at base 129 629 135. The UCSC genome browser used the January 2003 rat genome and the Ensembl browser used the rat genome version 13.2.1 (April 1, 2003) and the human genome version 13.31.1 (May 6, 2003).


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Congenic production
The region predicted to contain Mcs5 from the original QTL scan covered ~90 cM of WKy chromosome 5 (8). The production of a single line covering 90 cM was not feasible due to high recombination rates over such a large interval. Therefore, two lines (C and D) were generated that contained large, contiguous, overlapping WKy regions of chromosome 5 from D5Wox7 to D5Uwm42 (Figure 1) on a WF (susceptible strain) recipient genome. Rats from each backcross generation were screened for the presence of the WKy allele using markers spaced every 10 cM or less within the Mcs5 target region for each line. At generations N4, N5, N7 and N8, carriers were also screened for the absence of WKy alleles at other known Mcs loci and all rat chromosomes, excluding Y, with the markers listed in Table I. The N8 generation yielded carriers for each line that were WF-homozygous at all markers tested outside the Mcs5 target region.



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Fig. 1. Genetic map, QTL scan and congenic line intervals for the Mcs5 QTL region on Wistar-Kyoto (WKy) chromosome 5. Congenic lines that contain specific WKy alleles spanning the region of Mcs5 are designated by the dark vertical bars. Regions of recombination at the ends of the lines are designated by open vertical bars. Rat chromosome 5 markers and genetic map distances are designated on the vertical axis. The horizontal axis is the LOD score associated with the corresponding marker intervals.

 
Mammary carcinogenesis phenotyping assay
To determine the mammary carcinoma susceptibility phenotype conferred by the large Mcs5 WKy allelic intervals, line C and D females that were homozygous for the WKy allele at the N9F1 (line C only), N8F1, N8F2 and N8F3 generations were compared with susceptible WF control females (Figure 2). Data were pooled within lines since no statistically significant differences were detected in phenotypes between generations. The Mcs5 region contained by line C (Figure 1) conferred an 87% reduction in tumor multiplicity, compared with WF-homozygous littermates, when two WKy alleles were present. Line C females homozygous for the WKy locus from D5Wox7 to D5Uwm37 (n = 13) were resistant, averaging 1.1±0.3 carcinomas/rat compared with 6.9±0.4 average carcinomas/rat for WF control females (n = 50; P<0.01). Mammary carcinoma rates of line D females (n = 29) homozygous for the WKy interval from D5Rat26 to D5Uwm42 (Figure 1) were not statistically different than susceptible WF females (5.7±0.6 versus 6.9±0.4, respectively), minimizing the possibility that the line D region contains a WKy allele controlling mammary carcinoma susceptibility.



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Fig. 2. Mammary carcinoma susceptibility phenotypes of WKy chromosome 5 congenic lines. WF-homozygous (open bars), WKy-homozygous (solid bars) and susceptible control WF (hatched bar) females were given DMBA (65 mg/kg body mass) at 50–55 days of age by gastric intubation. Data from N8F1, N8F2, N8F3 and N9F1 females of each line (C and D) were pooled after determining no differences in susceptibility phenotype existed between generations. Plotted are the mean numbers of mammary carcinomas (3 x 3 mm or greater) per rat ± SEM developing at 15 weeks post-carcinogen. WKy-homozygous line C females (mean = 1.1 ± 0.3, n = 13) were resistant (P < 0.01), while line D females (mean = 5.7 ± 0.6, n = 29) were as susceptible as WF (mean = 6.9 ± 0.4, n = 50) females. For both lines, littermate WF-homozygous females were not different from WF control females.

 
Line C females (n = 15) that were WF-homozygous from D5Wox7 to D5Uwm37 averaged 8.3±1.2 carcinomas/rat and line D WF-homozygous females (n = 12) averaged 7.3±1.0 carcinomas/rat (Figure 2). For both lines C and D, the WF-homozygous female littermates were not significantly different than the susceptible WF controls (n = 50) that averaged 6.9±0.4 mammary carcinomas/rat, indicating that the mammary carcinoma susceptibility phenotype observed in each congenic line was not influenced by the presence of random WKy alleles at other loci. Importantly, when these results were considered collectively with the strong resistance phenotype exhibited by line C WKy-homozygous females but not by those of line D, the region suspected to contain Mcs5 was reduced to the interval from markers D5Uwm8 to D5Rat26 (~60 cM). This interval spans approximately 14.8–129.6 Mb or ~115 Mb of rat chromosome 5. As shown in Table II the region of interest contains sequence intervals that have homologies to regions on human chromosomes 1, 6, 8 and 9. Also shown in Table II are examples of genes found in these regions that are related to cancer development and/or play a role in transcription/translation, signal transduction, DNA repair or the cell cycle. To determine if the Mcs5 Wky allele in line C is dominant, heterozygous females from this line were compared with their homozygous littermates. Line C WKy/WF-heterozygous females tended to have slightly higher carcinoma numbers (mean = 2.2±0.4, n = 22) than line C WKy-homozygous females (mean = 1.1±0.3, n = 13); however, a t-test revealed these groups were not statistically different (P = 0.07), indicating that the Mcs5 QTL gene(s) acts in a dominant manner (Figure 3). However, a weak statistical claim of partial-dominance should also be considered. Line C WKy-homozygous and WKy/WF-heterozygous females were both statistically different from the susceptible WF-homozygous littermates (mean = 8.3±1.2, n = 15) (P<0.0001) as they were from the WF controls (mean = 6.9±0.4, n = 50) (P<0.01).


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Table II. Genomic comparison of Mcs5 to human

 


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Fig. 3. Mammary carcinoma susceptibility phenotypes of WKy chromosome 5 congenic line C. WF-homozygous (open bar), WKy-homozygous (solid bar), WKy/WF heterozygous (diagonal bar) and susceptible control WF (hatched bar) females were given DMBA (65 mg/kg body mass) at 50–55 days of age. Plotted are the mean numbers of mammary carcinomas (3 x 3 mm or greater) per rat ± SEM at 15 weeks post-carcinogen. Congenic mammary carcinoma multiplicity data from N8F1, N8F2, N8F3 and N9F1 line C congenic females were pooled after determining that no differences in susceptibility existed between generations. WKy-homozygous (mean = 1.1 ± 0.3, n = 13) congenic line C females tended to have fewer mammary carcinomas than WKy/WF-heterozygous females (mean = 2.2 ± 0.4, n = 22) but the groups were not statistically different from each other. Susceptible WF-homozygous littermates (mean = 8.3 ± 1.2, n = 15) and WF control females (mean = 6.9 ± 0.4, n = 50) were not statistically different.

 

    Discussion
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
We previously used a linkage mapping approach to genetically identify the QTLs that underlie the genetics of susceptibility in the WKy rat, a strain that is resistant to developing chemically induced mammary cancer. The genetic identification of these QTLs was based on statistical analysis and modeling. Four QTLs were identified, three associated with resistance and one associated with increased susceptibility (8). This observation supported the complex multigenic hypothesis of the genetic etiology of human breast cancer. Here we report that one of these statistically predicted (8) WKy loci, Mcs5, have physically been shown to carry gene(s) that confer resistance independently of the other genetically identified QTLs (Mcs6, 7, 8). This observation is in agreement with our negative binomial model of the linkage data (8). The Mcs5 line C WKy/WF-heterozygous congenic rats exhibited a 73% reduction in tumor multiplicity relative to WF-homozygous littermates, similar to the 64% reduction predicted by our model (8) for a single WKy Mcs5 allele. Homozygous rats for the WKy Mcs5 allele had an 87% reduction in carcinomas when compared with those homozygous for the WF Mcs5 allele.

The physical analysis was accomplished using N8 and N9 congenic rats produced from carriers that were selectively screened and bred to remove the WKy alleles at QTLs of Mcs6, 7, 8 and Mcs modifier 1 (Mcsm1). This ‘speed’ strategy gave confidence for phenotyping N8 and N9 rats instead of waiting for the traditional N10 generation. While it was observed that both WKy-homozygous and WKy/WF- heterozygous Mcs5 rats have resistance to DMBA-induced mammary carcinomas, the two groups were not statistically different from each other. This suggests that the Mcs5 WKy allele has a complete-dominant effect on tumor multiplicity. However, this should be considered a preliminary interpretation for two possible reasons. The first is that perhaps the number of rats analyzed provided insufficient statistical power to precisely estimate the mean difference between the WKy-homozygous and WKy/WF-heterozygous rats, suggesting instead that Mcs5 might follow a partial-dominance model. A second possibility is that there may be more than one modifier gene in the interval, with each having varying degrees of dominance. Such a situation might confound our current interpretation.

Besides physically confirming the Mcs5 WKy resistance allele(s), this study defined the Mcs5 region to ~60 cM. While this is still a large interval it is a significant improvement over the results of the linkage analysis that covered most of chromosome 5 (~90 cM). It provides us with a solid starting point to fine-map this region to locate the one or more genes that contribute to the mammary carcinogenesis susceptibility phenotype. While it is premature to speculate on candidate genes at this point, browsing the human homologous regions to Mcs5 reveals there are several cancer-related genes that reside within the Mcs5 interval. We now plan to collect and phenotype recombinant congenic rats within the interval defined by D5Uwm8-D5Rat26 to determine how many subloci (genes) exist with the ability to modify cancer susceptibility as well as to determine their genomic locations. Furthermore, once one or more subloci are defined to exist in a defined region below 2 cM and shown to maintain a quantifiable effect on mammary cancer susceptibility, we will be able to move forward in identifying potential candidate genes. Identifying such genes within the eight QTLs thus far genetically identified in the mammary cancer resistant rat strains COP and WKy will allow us to evaluate alleles of their human homologs for their effect on breast cancer risk using human breast cancer case-control and population studies.


    Acknowledgments
 
We thank Dr Laurie A.Shepel for providing her editorial skills and suggestions for improving the manuscript, and Anand Patani for providing excellent technical assistance. This study was supported by NIH grant CA77494.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received March 17, 2003; revised May 19, 2003; accepted June 21, 2003.