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Loss of Heterozygosity and Point Mutation at Aprt Locus in T Cells and ®Broblasts of Pms27/7 Mice

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Loss of Heterozygosity and Point Mutation at Aprt Locus in T Cells and ®Broblasts of Pms27/7 Mice Oncogene (2002) 21, 2840 ± 2845 ã 2002 Nature Publishing Group All rights reserved 0950 ± 9232/02 $25.00 www.nature.com/onc Loss of heterozygosity and point mutation at Aprt locus in T cells and ®broblasts of Pms27/7 mice Changshun Shao1, Moying Yin2, Li Deng1, Peter J Stambrook3, Thomas Doetschman2 and Jay A Tisch®eld*,1 1Department of Genetics, Rutgers, the State University of New Jersey, Piscataway, New Jersey, NJ 08854-8082, USA; 2Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati College of Medicine, Cincinnati, Ohio, OH 45267-0521, USA; 3Department of Cell Biology, Neurobiology, and Anatomy, University of Cincinnati College of Medicine, Cincinnati, Ohio, OH 45267-0521, USA Mice null for the Pms2 mismatch repair (MMR) gene tion (Harfe and Jinks-Robertson, 2000). Third, MMR exhibit a predisposition to lymphoma, microsatellite plays a role in the regulation of apoptosis (Gong et al., repeat instability, and failure of spermatogenesis. To 1999; Toft et al., 1999; Zhang et al., 1999; Zeng et al., study the role of Pms2 in the maintenance of in vivo 2000). In the absence of MMR, apoptosis is genomic integrity in somatic cells, we characterized Aprt compromised in cells with DNA damage, and the mutations in T cells and ®broblasts of 1296C3H increased chance of survival and proliferation of those Pms27/7Aprt+/7 mice. The spontaneous frequency of cells may allow further accumulation of mutations. DAP-resistant T lymphocytes, as a consequence of Organisms and cells that are MMR-de®cient typi- APRT-de®ciency, was increased threefold. Point muta- cally exhibit an increased level of genetic instability, or tion, which accounted for less than 20% of the DAPr a mutator phenotype. The mutator phenotype includes mutant clones in Pms2+/+ mice, was predominant in the instability of microsatellite repeats, elevation of point mutant T cell clones from Pms27/7 mice. These point mutation and increase of recombination between mutations were predominantly TA to CG transitions. divergent sequences (homeologous recombination). In Fibroblasts of Pms27/7 mice exhibited only a modest humans, MMR-de®ciency can lead to several types of increase in the frequency of clones with point mutations, cancer. For example, inherited mutations in a subset of such that mitotic recombination was still the primary MMR genes, including MSH2, MLH1 and PMS2, are cause of APRT de®ciency. Thus, the mutator phenotype responsible for hereditary non-polyposis colorectal as a consequence of PMS2 de®ciency is tissue-dependent, cancer (HNPCC). which may be related to the tissue-speci®c tumor To elucidate the biological functions of the MMR proneness of Pms27/7 mice. genes in vivo, mouse mutants of various MMR homologs Oncogene (2002) 21, 2840 ± 2845. DOI: 10.1038/sj/ have been generated through targeted gene disruption in onc/1205358 embryonic stem cells. Depending on the MMR gene, the homozygous mutant mice displayed one, two or all of Keywords: mismatch repair; PMS2; APRT; mouse the following three phenotypes (Buermeyer et al., 1999): model; somatic mutation; loss of heterozygosity (i) tumor predisposition, (ii) mutator phenotype, and (iii) meiotic defect(s). For example, Mlh17/7 mice exhibit all three phenotypes, whereas Pms17/7 mice only show a Introduction mild mutator phenotype. Most studies of the mutator phenotype in MMR- DNA mismatch repair (MMR) proteins contribute to de®cient mice were based on observations of micro- the maintenance of genomic integrity in multiple satellite repeat markers (Yao et al., 1999) or bacterial pathways (Modrich and Lahue, 1996, Kolodner and transgenes, such as supF (Narayanan et al., 1997), lacI Marsischky, 1999; Buermeyer et al., 1999, Harfe and and cII (Andrew et al., 1997, 2000; Baross-Francis et Jinks-Robertson, 2000). First, MMR corrects mis- al., 2001). However, microsatellite repeat markers only paired or extrahelical nucleotides that are infrequently re¯ect frameshift changes in sequences containing introduced during DNA replication, by mis-incorpora- mono- or dinucleotide repeats. While bacterial trans- tion or by slippage. Second, MMR-mediated suppres- genes can show base substitutions, frameshifts and sion of recombination is believed to reduce the other intragenic alterations, there are concerns as to incidence of both translocation and mitotic recombina- whether or not their behavior mimics that of endogenous genes since they usually reside as multiple copies in a host cell, are hypermethylated and transcriptionally inactive (Mirsalis et al., 1995). We *Correspondence: JA Tisch®eld; E-mail: [email protected] Received 3 December 2001; revised 21 January 2002; accepted 22 have documented the use of an endogenous, and February 2002 ubiquitously expressed, APRT (adenine phosphoribo- Somatic mutations in Pms27/7 mice C Shao et al 2841 syltransferase) gene as a reporter for loss of hetero- The shift of the mutational spectrum between the two zygosity (LOH) in vivo (Shao et al., 1999, 2000, 2001). groups is highly signi®cant (P50.0001). Analysis of Because Aprt is autosomal, it can detect most genetic ¯anking SSLP showed that almost all of the class I alterations at the chromosome level, such as mitotic variants in either Pms2+/+ or Pms27/7 mice were recombination and multi-loci deletion, in addition to derived from mitotic recombination (Table 2). The intragenic mutations and small deletions. In this study, frequency of such clones, as calculated by multiplying we characterized somatic mutations at Aprt in the median frequency of DAPr clones with the fraction normal T lymphocytes and ®broblasts of 1296C3H of mitotic recombination in each group, was Pms27/7Aprt+/7 mice. 19.261076 and 25.761076 in Pms2+/+ and Pms27/7 mice, respectively. This indicates that mitotic recombi- nation in T cells are not aected by the loss of PMS2. The median frequency of class II T cells, on the Results other hand, diers dramatically between Pms2+/+ and Pms27/7 mice, 2.961076 vs 47.661076, a 16-fold T cells of Pms27/7 mice exhibit increased point mutation dierence. Class II variants can either be caused by at the Aprt and Hprt loci point mutation or by epigenetic silencing of the wild- Though the frequency of DAP-resistant T cells varied type (untargeted) Aprt allele (Shao et al., 1999, Rose et greatly within each group, Pms27/7 mice exhibited a al., 2000). We sequenced all ®ve exons, part of the higher frequency than Pms2+/+ mice (P=0.017, promoter region and three introns of the Aprt gene in Mann ± Whitney U-test). The median frequency was 63 clones recovered from Pms27/7 mice and detected increased threefold in Pms27/7 mice, from 22.661076 point mutations in 51. This indicates that the increase to 73.361076 (Figure 1). in the frequency of DAPr T cells was primarily the We divided the DAPr clones into two classes consequence of point mutation. Strikingly, more than according to the absence or retention of the untargeted half of the mutations in Pms27/7 mice were T to C Aprt allele (Shao et al., 1999). Class I variants, those transitions (Table 3). This predominance of TA to CG exhibiting loss of the untargeted Aprt, were predomi- transitions is consistent with the mutational spectrum nant in Pms2+/+ mice (40/46, 87%), which is of Hprt in Pms27/7 mice (Shaddock et al., 2001), but consistent with our previous study (Shao et al., 2000). is in contrast to the mutational spectrum observed in However, class I clones, those retaining the untargeted lacIofPms27/7 mice (Andrew et al., 1997, Baross- allele, replaced class I as the primary cause of APRT- Francis et al., 2001), in which CG to TA transitions de®ciency in Pms27/7 mice (110/170, 65%) (Table 1). predominate. In addition to base substitution, eight frameshift mutations at di- or mononucleotide runs were detected in class II clones recovered from Pms7/7 mice (Table 3, Figure 2). Four of the 51 (8%) clones that were con®rmed to have point mutation were putative sib clones (having the same mutation), indicating that most of mutants had originated independently. Thus, the higher fre- quency of T cell variants in Pms27/7 mice is caused by an increased mutation rate. We also estimated the frequency of 6-TG resistant (HPRT-de®cient) T cells in Pms27/7 and Pms2+/+ mice. Since Hprt is X-linked, HPRT de®ciency is presumably caused only by intragenic mutations. Consistent with the observations with Aprt, Pms27/7 mice also exhibited a dramatic increase in the frequency of the 6-TGr T cells, 2.3+ 1.461076 (s.e.m., n=9) in Pms2+/+ mice vs 109.8+16.361076 (n=12) in Pms27/7 mice. In comparison to the 16-fold increase in the median frequency of class II DAPr variants, which are primarily caused by point muta- tion, in Pms27/7 mice, the magnitude of increase for 6- TGr variants is much greater (48-fold). The large size Table 1 Class II variants DAPr T cells in Pms27/7 mice No. mice Class I Class II Total % of class II Figure 1 Frequency of DAPr T cells in individual mouse +/+ spleens. Each spot represents one spleen. Bars represent median Pms2 9 40 6 46 13 7/7 values Pms2 15 60 110 170 65 Oncogene Somatic mutations in Pms27/7 mice C Shao et al 2842 Table 2 Mutational spectrum of class I variant clones ®broblasts in Pms27/7 mice (35/54), though the Mitotic Deletion or Total no. fraction of class I was lower in Pms27/7 mice (65%), Cell type Genotype recombination gene conversion of clones than in Pms2+/+ mice (83%) (Table 4). T cells Pms2+/+ 35 1 36 As with the T cells, most of the class I ®broblast Pms27/7 43 0 43 clones were caused by mitotic recombination in Fibroblasts Pms2+/+ 14 1 15 ®broblasts.
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