Genetic Mapping of the Thymoma Susceptible Locus, Tsr1, in BUF/Mna Rats
Authors: A. Oyabu, K. Higo, C. Ye, H. Amo, M. Saito, S. Yagyu, H. Morita, K. Maeda, T. Serikawa, M. Takahashi, M. Matsuyama
Journal: JNCI Journal of the National Cancer Institute (1999)

BUF/Mna (BUF) rats have been reported to develop spontaneous thymomas in virtually 100% of animals (1–4). The thymomas in BUF rats have been defined as epithelial cell tumors, having varying proportions of lymphocytic components that are non-neoplastic in nature (5,6). These thymomas start to develop in rats at the age of 9 months and they reach their maximum growth by the time the animals are 18 months of age (7). It was found that thymoma development in this strain was regulated by an autosomal dominant gene, Tsr1 (8). However, the chromosomal location of the gene is not yet determined. The aim of this study is to determine the location of Tsr1, using microsatellite markers and backcross of rats between the thymoma-susceptible BUF strain and thymoma nonsusceptible ACI/NMs (ACI) strain. A highly inbred rat strain, BUF, was established and has been maintained by brother–sister mating for 21 years as a thymoma-prone strain in our laboratory (2,3,8). ACI rats have also been maintained, as described earlier (8). Backcross rats were generated by mating female ACI rats with a male (BUF × ACI)F1 rat. The use of these animals in the experimental protocols described was approved by the Nagoya University Animal Care Committee. The rats were killed at the age of 18 months, and complete autopsies were performed. Thymoma was diagnosed macroscopically and microscopically. Genomic DNAs were extracted from ear tissues of the rats by a standard phenol/proteinase K technique. Microsatellite sites in each genomic DNA (100 ng) sample were amplified by use of a standard polymerase chain reaction (PCR) method (9) employing a DNA thermocycler (PJ 2000; The Perkin-Elmer Corp., Foster City, CA) and a reaction mixture containing 10 mM Tris–HCl, pH 8.3; 50 mM KCl; 1.5 mM MgCl2, 200 mM each of deoxyadenosine triphosphate, deoxycytidine triphosphate, deoxyguanosine triphosphate, and deoxythymidine triphosphate; 1 mM each of sense and antisense primers; and 0.6 U of Taq DNA polymerase (Takara Shuzo, Shiga, Japan) in a final volume of 25 mL. Programming of the temperature and time cycles was as follows: 1) 7 minutes at 94 °C; 2) 35 cycles of 1 minute at 94 °C, 1 minute at 55 °C, and 1 minute at 72 °C; and 3) an elongation step of 7 minutes at 72 °C. PCR products were resolved by electrophoresis in 8% polyacrylamide gels and stained with ethidium bromide. Four hundred forty microsatellite markers were selected to see length polymorphisms between BUF and ACI rats; primers for 94 markers were prepared by use of a DNA synthesizer (Model 392; Applied Biosystems, Foster City, CA) as described previously (9– 16), and the rest of the primers, except those for D7Cep2, were purchased from Research Genetics, Huntsville, AL. D7Cep2 is a random, cloned, sequencetagged microsatellite site, rich in AC repeats (Serikawa T: unpublished observations). The backcross rats were genotyped into two groups, BUF/ACI heterozygote and ACI/ACI homozygote, for each microsatellite marker according to the bands obtained by PCR and electrophoresis. The Fisher’s exact test (two-tailed) was used for analysis of associations between the development of thymoma and genotypes of microsatellite markers. All of the backcross rats were phenotypically classified into either the thymoma group or the nonthymoma group. Associations were judged to be statistically significant when the P values of the Fisher’s exact test were <.01. All data were analyzed by use of MAPMAKER/EXP and MAPMAKER/ QTL computer programs (17,18), and map order, map distance, and the location of genetic loci associated with thymoma development were determined, with reference to the genetic map of the rat (19). One hundred thirty-seven backcross rats were generated. Thirteen of them died of various neoplasms—malignant lymphomas, mammary carcinomas, glioma, interstitial cell tumors of the testis, and leiomyosarcoma of the small intestine. The remaining 124 rats that survived for 18 months were killed and thymomas were found in 17 of them. All of these thymomas were of lymphocytepredominant type. Of 440 microsatellite markers tested, 138 markers located on all of the chromosomes, except for the Y chromosome, showed length variation of the PCR products between BUF and ACI rats (Table 1). For chromosome 7, the D7Rat17 , D7Ra t100 , D7Ra t21 , D7Rat112, and D7Rat10 markers showed the heterozygous BUF/ACI type in 16 (94%) of 17 rats in the thymoma group, in contrast to the heterozygosity in 50–53 (47%–50%) of 107 rats of the nonthymoma group, and the associated P values from the Fisher’s exact test were all <.001. Therefore, these loci were thought to show close linkage to Tsr1. The D7Rat69, D7Cep2, D7Rat24, D7Mit5, D7Rat86, D7Rat53, D7Mit24, and D7Rat4 markers on this chromosome also yielded low P values, which were .008, .009, .004, .002, .001, .002, .002, and .003, respectively. The other 15 markers on this chromosome and the 110 markers on the remaining chromosomes yielded larger P values, showing no statistically significant linkage with thymoma development. All data were analyzed by MAPMAKER/EXP and MAPMAKER/QTL computer programs to identify the ge-