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ICANCER RESEARCH57. 1188-I 193. March 15. 9971 High Frequency in Vivo Loss of Heterozygosity Is Primarily a Consequence of Mitotic Recombination1 Pawan K. Gupta,2Amrik Sahota,SimeonA. Boyadjiev,StevenBye, ChangshunShao, J. PatrickO'Neill, Timothy C. Hunter, Richard J. Albertini, Peter J. Stambrook, and Jay A. Tischfleld3 Department ofMedical and Molecular Genetics. Indiana University School ofMedicine, Indianapolis, Indiana 46202-5251 (P. K. G.. A. S., S. A. B., S. B.. C. S., J. A. TI: Genetics Laboratory. University of Vermont, Burlington, Vermont 05401 /1. P. 0., T. @. H., R. J. A.): and Department of Cell Biology. Neurobiology and Anatomy, College of Medicine, The University of Cincinnati, Cincinnati. Ohio 45267 (P. J. S.] junctional chromosomal loss with or without reduplication as well as gene conversion and point mutations (3â€”5). We have used the adenine phosphoribosyltransferase gene (A.PRT; A number of different assays have been developed to determine the 16q24) to investigate the mechanisms of loss of heterozygosity (LOH) in mechanisms of point mutations and multilocus events in mammalian normal human somatic cells in vivo. APRT-deficient ([email protected], APRT'Â°) cells in vitro (5, 6). Mutagenesis studies using markers that are T lymphocytes from the peripheral blood of four obligate APRT heterozy functionally (e.g., X-linked HPRT) or structurally (e.g., Chinese ham gotes ([email protected]) with characterized germ-line mutations were selected in ster ovary Aprt) hemizygous usually detect intragenic point mutations medium containing 100 @LM 2,6-diaminopurine. A total of 80 2,6-diamin and relatively small deletions only, at least in aneuploid cell lines opurine-resistant T-cell clones from 2 of the heterozygotes were analyzed for this study. The presence or absence of LOll of proximal linked (6â€”8). It is believed that clones with larger deletions may not be microsatellite repeat markers was used to divide the clones into two viable due to the loss of adjacent genes essential for survival (8). The groups: (a) those in which LOH was likely due to localized changes In assumed limitations inherent in studying mutations at hemizygous loci APRT (e.g., point mutations); and (b) those with LOH at additional loci. A have led to the development of assays using autosomal genes such as total of 61 clones (76%) exhibited LOH of linked microsatellite repeat APRT and tk (9). Selection of mutants from cells with heterozygous markers at different locations on 16q, which extended from the smallest autosomal loci has been shown to occur at higher frequency than that measured region (<5.5 cM) to the entire 16q arm. The remaining 19 from cells having a hemizygous target locus (9, 10). These results clones (24%) had point mutations in APRT or other relatively minor have been interpreted as evidence for a relatively closely linked alterations. Ten clones with LOH encompassing different regions of 16q were examined by conventional cytogenetics and by fluorescence in situ gene(s) with an essential function whose loss limits the size of survivable deletions at the hemizygous or X-linked locus. Whereas hybridization using an I4.PRT cosmid probe. All dones exhibited a normal diploid karyotype, and nine exhibited two copies ofAPRT. The one clone deletion of such an essential gene will always be lethal when it is hemizygous, lethality in diploid cells will only be observed when the that was hemizygous for APRT had the smallest observed region of LOH deleted essential gene is heterozygous, and its normal allele is in in clones from that individual. These results indicate that mitotic recom binatlon and, to a much lesser extent, deletion may be the primary cis-configuration with the deleted normal allele of the marker gene mechanisms for the relatively high frequency of in vivo LOH observed in (I 1â€”14). normal human T cells. Became LOH leads to the expression of recessive LOH can only be studied with heterozygous autosomal loci. In the tumor suppressor genes in many cancers, these data have significant present study, we have used APRT as a marker to select normal human implications for the role of LOH in the early stages of tumor development, T cells that have undergone in vivo LOH. APRT catalyzes the syn especially in breast cancer. thesis of AMP from adenine and 5-phosphoribosyl-l-pyrophosphate. A deficiency of this enzyme frequently leads to 2,8-dihydroxyadenine stone formation in the kidney (15). The APRT gene, located at INTRODUCTION l6q24.3, encodes a selectable marker in cell cultures, and the se quences of the genomic and coding regions (about 2.6 and 0.54 kb, Allelic LOH4 plays an important role in carcinogenesis. A number respectively) are known (16). We have analyzed germ-line mutations of inherited cancers have been shown to be the direct consequence of in this gene in more than 33 families (15, l7)[email protected] Germ-line mutations a germ-line mutation in one allele and a subsequent somatic alteration seem to cluster at the intron 4 splice donor site and at or near codon that inactivates the remaining functional allele (1, 2). The majority of 87 (exon 3) and consist entirely of point mutations and small frame these germ-line mutations are intragenic point mutations that are shifts. Analysis of in vivo APRT somatic point mutations in the T cells generally thought to be due to DNA damage or to errors in DNA of three Japanese heterozygotes showed a similar clustering (17). We repair or replication. The subsequent somatic alterations resulting in have selected DAP-resistant T-cell clones arising in vivo in the blood LOH are generally more complex and may include multilocus chro of parents (obligate APRT heterozygotes) from two unrelated APRT mosomal events such as deletion, mitotic recombination, or nondis deficient families and have examined the entire spectrum of in vivo APRT LOH in the clones from two of the parents. Although the Received 9/30/96; accepted 1/19/97. The costs of publication of this article were defrayed in part by the payment of page studies reported here also describe point mutations, the focus is ABSTRACT charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. , Supported by NIH Grants DK38185 (to J. A. T.) States Department of Energy Grant F6028760502 05652 (to J.A.T. and PJ.S.). 2 Present address: Department of Medicine, and CA3O688 (to R. J. A.), whom requests for reprints should Division of Medical Genetics, MATERIALS Thomas be addressed, at Department of Medical and Molecular Genetics, Indiana University School of Medicine, 975 West Walnut Street. 1B130, Indianapolis. IN 46202-5251. Phone: (317) 274-5738: Fax: (317) 274-1069; E-mail: [email protected] 4 The abbreviations used are: LOH, on multilocus chromosomal events as determined by LOH of (to R. J. A.), and NIEHS Grant ES Jefferson University, Philadelphia, PA 19107. 3 To mainly flanking MR markers and by FISH. United loss of heterozygosity; MR. microsatellite repeat; APRT, adenine phosphoribosyltransferase; HPRT, hypoxanthine guanine phosphoribo syltransferase; tk, thymidine kinase; DAP, 2,6-diaminopurine; TG, 6-thioguanine; CE, AND METHODS T.Lymphocyte Isolation and Mutant Selection. An outline of the proto col used for lymphocyte isolation and mutant selection is given in Fig. 1. Human peripheral blood samples were collected by venipuncture from two APRT-deficient cloning efficiency; TCR, T-cell receptor; FISH, fluorescence in situ hybridization. S A. Sahota families, and J. A. one from Tischfield, Belgium unpublished (family 1) and the other observations. 1188 Downloaded from cancerres.aacrjournals.org on May 10, 2016. © 1997 American Association for Cancer Research. from LOSS OF [email protected] AND MITOTICRECOMBINATION clones was digested with Hind!!!, and a Southern blot was hybridized with TCR-y and -f3probes. Clones showing different RFLP patterns were consid ered to be of independent origin and hence were considered to represent Blood from APRT Heterozygote (APRT +â€˜@, HPRT ) I Isolation independent mutations. Any clones showing identical TCR gene patterns and the same APRT mutation were considered to represent a TCR gene-defined mutant set and to represent the progeny of a single mutational event, but they were considered to be of independent origin if the APRT mutations were of T Lymphocytes different. SelectIon of Microsatellite Analysis ofDAP-resistant Clones. A number of MR mark ers and their relative positions on chromosome 16 are shown in Table 2 (21â€”23).All of these markers were first tested for their informativeness Mutant Cells arising In vlvo APRT-'- (heterozygosity) HPRT 2,6-Diaminopurine (DAP) 6-Thiogua Inc (TG) with putative @4S\@ mtragenic point mutations from those with allelic loss. A second informative repeat marker, Dl6S4l3, about 1.1 CMcloser to APRT, was used for subsequent analyses (24). Primers for PCR were either purchased from Growthof DAPt or [email protected] (Calculate using DNAs from the whole blood of the two heterozygotes from family 1. One informative repeat marker, D16S305, which is approxi mately 6.6 CMproximal to the APRT gene, was first used to distinguish clones Colonies Research Genetics (Huntsville, AL) or synthesized in-house. The PCR reaction Mutant Frequency) conditions were as follows: approximately 100 ng of DNA, 100 ng of each primer, I 50 mM KC1, 10 mM Tns-HC1 (pH 8.3), 1.5 nmi MgCI2, 0.2 mM each deoxyribonucleotide, 10 pCi of [a-32PJdC'FP (specific activity, 3000 Ci' mmol), and 0.5 unit of Taq polymerase (Perkin-Elmer Corp., Foster City, CA) in a total volume of 20 @l.Samples were processed through 30 cycles Analysis of T Cell Receptor (TCR)Gene Rearrangement consisting of 45 s at 95Â°C,45 s at 55Â°C,and 30 s at 72Â°C.Aliquots of the amplified DNA were mixed with 2 volumes of loading gel buffer containing 95% formamide, heat-denatured at 98Â°Cfor 5 mm, and electrophoresed on a Molecular Analysis of APRT LOH and HPRT MutatIon 5% Fig. 1. A diagram of T-lymphocyte mutant selection. DAP- and TG-resistant lymphocytes were selected as described in â€œMaterialsand Methods.â€• T denaturing TaqI RFLP Germany (family 2), and from normal controls (Table 1). The mononuclear cell fraction was isolated by sedimentation and washed, and the cells were enumerated. Isolated lymphocytes were immediately plated into culture me dium as described below for the selection of preexisting APRT-deficient (APRr' or [email protected]) T lymphocytes that, presumably, arose spontaneously in vivo as a consequence of LOH (somatic alterations in the normal APRT allele). Cells were incubated at a density of 1 X 106/ml in RPM! 1640 containing 20% nutrient medium HL-1, 5% bovine calf serum, and 1 mg/ml purified phytohemagglutinin for 36â€”40h to promote mitogen stimulation. This time is sufficient for optimal T-lymphocyte stimulation but is not suffi cient for cell division. The cells were again sedimented, washed, and enumer ated. The cells were reincubated at a density of 1â€”2X 104/well in the same medium, this time containing 125 @g/mlphytohemagglutinin, optimal amounts @ of interleukin 2 obtained from cell-free conditioned medium, irradiated (90 Gy) human lymphoblastoid feeder cells (an APRr' cell strain designated C2 that has a single base substitution in the APRTgene) at 1 X [email protected]/well (18), and 100 DAP. Initial experiments with cells from a homozygous APRT deficient individual allowed optimization of the concentration of DAP for selection ofAPRr' cells. As shown in Fig. 1, lymphocytes were also seeded into parallel cultures in similar medium containing 10 @.tM TG to identify TG-resistant clones for analysis ofHPRTgene mutations (19) and in drug-free medium to determine the nonselection cloning efficiency. Mutant Frequency Determinatiam. Isolated lymphocytes were assayed for cloning efficiency by plating 1, 2, 5, and 10 cells/well in the absence of DAP or TG and at 1â€”2 X [email protected]/well in the presence of DAP or TG. The microliter dishes were incubated for 10â€”14 days, and growing colonies were visualized using an inverted phase-contrast microscope. The CE was calculated polyacrylamide gel. The retention of CE in the presenceand absenceof selective agent(DAP or TG), respectively. Mutant and wild-type (unselected) clones from two of the obligate APRr' heterozygotes (the father and mother in family 1) were propagated to obtain a sufficient number of cells for additional analyses. Genomic DNA was isolated @ from 0.5â€”1.5x cells from each of the DAP-resistant clones. This DNA for in intron 2 ofAPRT that can be assayed by PCR (25). The mother was not informative for the TaqI RFLP, but she was informative for a marker (D16S303) about 2 CMdistal to APRT and for one or more polymorphic markers located in the 5' or 3' flanking regions of the gene (26). Thus, the presence or absence of two distinct APRT alleles in clones retaining heterozy gosity for D16S305 or D16S413 could be confirmed. Clones that retained both APRT alleles were analyzed for mutations by PCR amplification and sequenc ing (either directly or after subcloning) of the APRT gene (27). LOH of D16S305 or D16S413 suggested that there might be LOH elsewhere on chromosome 16, and these clones were analyzed with additional MR markers. Chromosome Analysis. Ten DAP-resistant clones from the father (family 1) with allelic loss of various regions of 16q were cultured, and metaphase preparations and GTG-banding were produced by standard techniques. FISH was done with an APRT-containing cosmid probe (a gift from D. F. Callan, Adelaide, Adelaide Children's Hospital, Australia). Cosmids were purified with a Qiagen kit (Chatsworth, CA) and biotin-labeled with the Bionick kit (Life Technologies, Inc., Gaithersburg, MD). Chromosomes were denatured at 70Â°Cin 70% formamide in 2 X SSC for 2 mm, and hybridization was done overnight at 37Â°C.Hybridization signals were detected by applying FITC avidin/anti-avidinlFlTC-avidin sandwich amplification. The presence of two Table 1 [email protected] and HPRT mutant cellfrequencies in human T-lymphocytesâ€• Themutantcellfrequencyis theratioofthe meanCEin selectivemedium([email protected] TG or 100 [email protected] DAP) divided by the mean CE in drug-free medium. CE was calculated by the Poisson distribution, in which CE = [email protected]/N (where N equals the average number of cells/well). The APRTgenotypes are indicated in the table. TheAPRTgerm-line mutations in the families have been described (Refs. 15 and 17). cell frequency ([email protected] by the Poisson distribution in which CE = â€”[email protected]/N(in which N represents the average number of cells/well). The mutant frequency is the ratio of the mean heterozygosity D16S413 and D16S305 was interpreted as suggesting LOH localized to APRT (e.g., intragenic mutation). The heterozygous father is also informative for a DAPr1 Family Subject (genotypes)Mutant Father([email protected]') Mother ([email protected]') 2 Child (APRr'@) Control (APRr'@) Father ([email protected]'@) Mother ([email protected]) Child ([email protected]) Control ([email protected]'@)10.5 was used for TCR gene rearrangement studies, mutational analysis of the APRT gene, and analysis of LOH. <0.27a TCR Gene Rearrangements. Cell clonality and independenceof muta HPRr.b All individuals were tions were determined by RFLP analysis of TCR gene rearrangements using drug-freemedium. Values were approximately two probes, [email protected] and [email protected] (20). Genomic DNA from DAP-resistant I 189 TGr 21.8 7.8 6.8 49.7 4.2 6.2 9.4 1, equal to the colony-forming 153.9 21.3 097b <0.12 24.3 115.1 1.02â€• efficiency in Downloaded from cancerres.aacrjournals.org on May 10, 2016. © 1997 American Association for Cancer Research. LOSS OF HETEROZYGOSITYAND MITOTICRECOMBINATION Table 2 WH in DAP-resistant T-cell clones from APRT heterozygotes LOHof MRmarkerson chromosome16in DAPrcells.Zygositywasassayedby PCRwithlocus-specificprimersfollowedby electrophoresisin 5%denaturingpolyacrylamide gels. The most likely cytogenetic localization (21â€”24)of each repeat is shown above its designation (e.g., D16S283). A filled circle indicates no LOH, whereas an empty space indicates observedLOH. A. Father, family locationl6pl3.3qll.2qll.2 ql3q24.2q24.3q24.3q24.3No. of [email protected] (5398)(8422)(5305)(5413)(APRT)1 No. of independent mutationsâ€• 118 â€¢â€¢â€¢I â€¢6 â€¢â€¢17 â€¢5 ...4 ....15 â€¢â€¢â€¢â€¢â€¢B. 17 1 6 16 5 4 15 Cytogenetic q12.lq12.l (5261)(S3O8) (S283)(S298)(SPN) â€¢â€¢â€¢ Sâ€¢â€¢ â€¢â€¢â€¢ ... ... â€¢Iâ€¢ ql3 (Sl86) â€¢â€¢ .. .. . . â€¢ â€¢S Mother, family Il6pll.2 No. of [email protected] (S303)4 No. of independent [email protected]' ...I â€¢â€¢â€¢â€¢I â€¢â€¢Iâ€¢I â€¢I â€¢â€¢â€¢4 Sâ€¢â€¢â€¢.a (5420)ql2.l 4 I I I I 4 q22.l (5421) (S265)Cytogenetic (S261)ql3 â€¢â€¢â€¢â€¢ â€¢â€¢â€¢â€¢ .... location q24.2 (S402)q24.3 (S305)q24.3 (5413)q24.3(APRT)q24.3 q24.2 (S422) â€¢ . Number of independent mutations by TCR analysis (65 of 67). b Number of independent mutations by TCR analysis (13 of 13). copies of the chromosome Thirteen DAP-resistant 16 centromeric region was confirmed by FISH with DI6Z2, a chromosome 16-specific a-satellite probe (Oncor, Gaithersburg, MD) as described above (28). At least 10 metaphases for each clone were visualized by FISH. RESULTS Comparison of APRT and HPRT Mutant Frequencies. The APRT and HPRT mutant frequencies in two unrelated APRT-deficient families (father, mother, proband, and control) are shown in Table 1. As expected, T cells from both the APRT' children exhibited about the same colony-forming efficiency in DAP medium as in drug-free medium, indicating optimal selection conditions for APRT' clones. The 4 heterozygotes showed frequencies ranging from 21 X l0_6 to 154 X lO_6, a difference of about 7-fold. The frequencies of HPRT clones in the same individuals (except for the father in family 2, who exhibited an unusually high frequency of 50 X lO_6) varied within a 2â€”5-foldrange and are consistent with extensive previous data (19, 20). Thirteen clones from the mother and 67 clones from the father from family I were propagated for LOH and mutation analyses. Analysis of Heterozygosity. LOH of D16S413 was observed in 48 clones from the father, and retention of heterozygosity was observed in the remaining 19 clones. Subsequent analysis of the 19 clones by TaqI RFLP indicated the retention of both APRT alleles in 15 clones and the retention of only 1 allele in 4 clones, suggesting a chromo somal LOH of less than 5.5 CMin the latter group. Therefore, these 4 clones were grouped with the 48 DAP-resistant clones with LOH for at least D16S413 (e.g., multilocus LOH). The 15 clones retaining both APRT alleles were analyzed for intragenic point mutations (see be low). The 52 clones (48 with LOH of D16S413 and 4 with LOH for APRT) were tested with additional MRs. As shown in Table 2A, LOH in these clones encompassed variable lengths of l6q (23). When LOH was observed at a particular locus, the same allele was lost in all instances, consistent with LOH of only those alleles that are physi cally linked to the normal APRT in this particular heterozygote. Analysis of ICR gene RFLPs indicated that 65 of 67 clones from the father are likely to be the result of independent mutations (Table 2A). analyzed as described clones from the mother in family 1 were above. Because D16S303 was informative in this individual, we derived data concerning LOH distal to APRT. One clone did not show LOH for Dl6S4l3 (which is 4 CMproximal to APRT), and four clones did not exhibit LOH for either APRT or D16S3O3. DNA sequencing indicated point mutations in all four clones without APRT LOH (see below). As shown in Table 2B, LOH in the remaining nine clones encompassed variable lengths of l6q (22, 23). When LOH was observed at a particular locus, the same allele was lost in all instances, as was the case with the father. TCR gene analysis indicated that each of the 13 clones is the result of an independent mutation (Table 2B). Chromosome Analysis of Clones with Allelic Loss. GTG banding revealed a normal karyotype (46,XY) in all 10 DAP-resistant clones from the father in family 1 (data not shown). FISH analysis indicated the presence of 2 APRT alleles in 9 clones that exhibited LOH for APRT and D16S413 and the presence of only 1 allele in the remaining clone (29 of 33 metaphases). The clone exhibiting only one APRT allele (clone Fl6) was one of four in which LOH did not include D16S413. Fig. 2 shows a typical FISH metaphase from clone Fl6. Because D16S303, which is distal to APRT, was not informative in this individual, we could not determine the extent of allelic loss in the telomeric region of 16q. However, karyotype analysis of these 10 clones did not suggest terminal deletions. These results, along with the MR LOH data, suggest that interstitial deletion occurred in only 1 of the 10 clones that were cytogenetically analyzed and that multilocus LOH in the other 9 clones was not a consequence of l6q deletion or deletion and translocation. Clones with Point Mutations. As indicated above, 15 clones from the father and 4 clones from the mother in family 1 did not show LOH for APRT or flanking markers. DNA sequencing of the previously wild-type APRT alleles in these clones revealed point mutations in nine clones from the father and in all four clones from the mother. The results are summarized in Table 3. There were four pairs of clones that exhibited the same point mutation, but in each case, TCR gene rearrangement analysis indicated that the mutations were of independ 1190 Downloaded from cancerres.aacrjournals.org on May 10, 2016. © 1997 American Association for Cancer Research. LOSS OF HETEROZYGOSITYAND MITOTICRECOMBINATION 4 Fig. 2. Metaphase from clone Fl6 that exhibits only APRT LOH. Only a single hybridizing region was observed (arrowhead). FISH with an APRT containing cosmid was performed as described in â€œMaterialsand Methods.â€• a ent origin. The entire APRT gene was sequenced in one of the six clones from the father that exhibited no mutation. In the other five clones that exhibited no mutation, only the coding regions and splice junctions were sequenced. Clonal Origin of Mutant Clones. A comparison of TCR gene rearrangement RFLPs and the nature of mutation in DAP-resistant clones revealed that most clones (78 of 80) were the result of inde pendent mutations. Several groups of clones, however, merit com ment. For example, two clones yielding identical TCR gene rearrange ment patterns exhibited two different mutations (point mutation and allelic loss, respectively). Among three clones with identical TCR RFLP patterns, allelic losses of two different sizes and a point muta tion were observed. These five clones (6%) may represent oligo clonality of mutants (two or more mutants with the same TCR gene rearrangement pattern). This estimate of oligoclonality is similar to that reported for HPRT mutations (29). There were two pairs of clones (5%) with identical TCR gene rearrangement RFLPs and identical mutations, and they are likely to represent true sibling clones. markers along the length of chromosome 16. Because these heterozy gotes are the parents of APRT-deficient probands, the germ-line mutation in each is known (15, 17). The focus of this study is in vivo somatic mutations that arise spontaneously in the T cells of these heterozygotes and produce LOH. T lymphocytes are well suited to these studies because they can be easily obtained from peripheral blood and propagated in vitro as clones from a single cell that has previously undergone in vivo somatic mutation. Furthermore, as was shown in this study and previously (6), T lymphocytes grown in vitro maintain a normal karyotype. We have also measured the frequency of mutation locus in the same individuals We have developed an assay for determining the mechanisms of in vivo LOH in normal human T cells based on individuals that are heterozygous for a selectable telomeric marker (APRT) and MR that produce in vivo LOH and thus provide insight mutations in DAP-resistant T-cell LOHofAPRT. clones were analyzed for either an informative Taql restriction fragment polymorphism (25) or flanking polymorphism (26) to determine whether or not there had been fromthe In 13 of 19 clones without LOH ofAPRT, specific point mutations could be identified within APRT. No mutations were observed in six clones. F and M indicate clones father (27).Clone and mother in family 1, respectively. The intron/exon, codon, and nucleotide numbers of the protein coding regions of APRT are as described previously ChangeP2, No. of independent mutationsâ€• Mutation @ StopM5, FlO 2 C @ ThrF33 M14 FrameshiftMl errorM4 1 error?F28, 2 1 1 1 MetF30 F42 @ @ @ errorF63 MetF19, liea F4l T for we have used polymor into molecular mechanisms. Among 80 DAP-resistant T-lymphocyte clones from two heterozy gotes, 61 (76%) were a consequence of allelic loss. Of the remaining 19 clones, 13 were demonstrated to be and 6 are likely to be the result of point mutations in APRT. Despite a 7-fold higher frequency of DISCUSSION Table 3 APRTpoint HPRT to the APRT data. Furthermore, phic MR markers to initially identify DAP-resistant clones with either chromosomally extensive or localized LOH. Subsequently, the clones were further characterized with additional CA repeat markers, FISH analysis, and DNA sequencing ofAPRT. Therefore, this assay could discriminate between most types of mutational changes clonesDAPr at the X-linked comparison Intron/exon no. Codon El 7 G A del CC A @â€˜ C C @â€˜ A E2 E2 E2 13 37 56-57 62 2 G A E3 I 1 2 G 0 C @â€˜ A A T E3 E4 E5 Nucleotide no. 19 CAG @s TAG, Arg 272 331â€”332 349 133 1 GCC ACC, Ala 84 1406 GTG ATG. Val 107 I 26 135 1477 1806 2063 GTG ACC Splice ATG, Val ATC, Thr @s Splice Splice Number of independent mutations by TCR analysis. 1191 Downloaded from cancerres.aacrjournals.org on May 10, 2016. © 1997 American Association for Cancer Research. LOSS OF [email protected] AND MITOTICRECOMBINATION mutant clones in the father as compared to the mother in family 1 (Table 1), the fraction of clones due to large chromosomal events was relatively constant (0.77 and 0.69, respectively) in each. These results are similar to our observations in T cells from three Japanese APRT heterozygotes (17). The frequency of loss of APRT alleles is consist ent with that observed at other loci. LOH of approximately 73% was observed in primary retinoblastomas (30). Spontaneous tk-deficient clones from a heterozygous human TK6 lymphoblastoid cell line exhibited a 71% frequency of LOH (31). As expected, hemizygous Aprt in CHO cells exhibited a lower (43%) spontaneous frequency of allelic loss (32). Although the proportion of mutant cells due to allelic loss may be similar in vitro and in vivo, the relative frequencies of mutants due to particular molecular mechanisms may differ. For example, karyotypic instability, including chromosome loss and trans location, may be a major cause of DAP-resistant clones in [email protected]' human heteroploid MR12-1 fibrosarcoma cells (33)â€¢6 High frequency of APRT LOH has also been reported in human TK6 lymphoblasts, but in 36 of 38 clones it began at 16ql2, and translocations were detected in 20% of clones (34). Linked recessive lethal alleles likely play a key role in limiting the extent of allelic loss in cells. A study of two clonally related cell lines indicated that one had a much higher rate of LOH (96% of clones) than the other (47% of clones). Furthermore, LOH was observed to extend 98 CMin the former but only 4 CMin the latter. These results were explained by postulating a relatively essential heterozygous gene whose normal allele is linked to the normal tk allele in the cells with reduced LOH (12â€”14).The initial observation of no deletions distal to APRT in human 5W620 cells and an open reading frame downstream of APRT that was noted by our group (Genbank accession number, U04709)7 led to the description of a unique heterozygous transcribed gene downstream ofAPRT, in the opposite orientation (1 1). This gene is postulated to provide a function that is essential to cellular survival. Allelic loss extending through all regions of 16q in our T-cell clones would suggest the absence of a heterozygous recessive lethal gene whose normal allele is linked to the functional APRT allele in these two individuals. Also, the similar proportions of clones with point mutations or larger chromosomal events from the mother and father in family 1 suggest that the observed 7-fold difference in mutation frequency is not due to linked recessive breast cancers (36â€”38).LOH for Dl6S4l3 was shown to be 25% for ductal carcinoma in situ, a preinvasive lesion, and 43% for invasive ductal carcinoma, suggesting that LOH of 16q24.3 is an early event in the development of breast cancer (39). There are a number of common fragile sites located on l6q that have been implicated in allelic loss during carcinogenesis. For example, in human hepatocellular carci noma, LOH is common between l6q22.l and 16q22.3â€”q23.4 (40). We speculate that LOH in l6q in T cells is without phenotypic consequence because T cells are inherently apoptotic or because genes on 16q that are involved in breast cancer are not active in T cells. Because LOH encompasses different regions of 16q in our DAP resistant of mitotic recombination that REFERENCES 1. Knudson, A. G., Jr. Hereditary cancer, oncogenes and antioncogenes. Cancer Res., 45: 1437â€”1443,1985. 2. Loeb, L. A. Mutator phenotype may be required for multistage carcinogenesis. Cancer Res.,51:3075â€”3079, 1991. 3. Stanbridge, E. J. Human suppressor genes. Annu. Rev. Genet., 24: 615â€”657, 1990. 4. Levine, A. J. The tumor suppressor gene. Annu. Rev. Biochem., 62: 623â€”651, 1993. 5. Meuth, M. The structure of mutation in mammalian cells. Biochim. Biophys. Acts. 1032: 1â€”17, 1990. 6. Albertini, R. J., Nicklas, J. A., O'Neill, J. P., and Robison, S. H. in vivo somatic mutations in humans: measurement and analysis. Annu. Rev. Genet., 24: 305â€”326, 1990. 7. Phear, G., Armstrong, W., and Meuth, M. Molecular basis of spontaneous mutation at the Aprt locus of hamster cells. J. Mol. Biol., 209: 577â€”582,1989. 8. de Jong, P. J., Grosovsky, A. J., and Glickman, B. W. Spectrum of spontaneous mutation at the APRT locus of Chinese hamster ovary cells: an analysis at the DNA sequence level. Proc. Natl. Acad. Sci. USA, 85: 3499â€”3503,1988. 9. Yandell, D. W., Dryja, T. P., and Little, J. B. Somatic mutations at a heterozygous lethal genes. Mitotic recombination seems to be the major pathway for in vivo LOH. All 10 DAP-resistant clones with variable allelic loss, encom passing primarily APRT and the distal portion of 16q up through most of l6q, showed normal chromosomes, and nine clones showed two APRT alleles by FISH. The only plausible explanation for these data is mitotic recombination because gene conversion is not known to affect such large regions. At least one maternal clone, however, is likely due to whole-chromosome loss or translocation because it also exhibited LOH for a marker on l6p (Table 2B). Mitotic recombination also seems to produce about 30% of lymphocytes that have lost the expression of one codominant HLA-A allele on chromosome 6 (35). Furthermore, we have observed that mouse primary fibroblasts exhibit a high frequency of Aprt LOH that is mostly due to mitotic recom bination.8 Thus, the phenomenon is not restricted to T lymphocytes. The observation of high-frequency mitotic recombination in normal cells has significant implications for the etiology of cancer. It suggests that LOH caused by mitotic recombination may trigger carcinogenesis or at least be an early event in this process. Several groups have reported LOH for at least two regions of 16q in about 50% of primary T-cell clones as a consequence seems to begin within different chromosomal regions, a set of such clones could be used to establish the order ofpolymorphic loci on l6q. With a sufficient number of loci, a map of the distances between loci, based on mitotic recombination, could be produced and compared to the meiotic map. Our T-cell data suggest an order for polymorphic CA repeats that is consistent with their known chromosomal locations, as indicated in the top row of Table 2, A and B (22). Whether or not events other than mitotic recombination skew the data is not known. It would be of interest to examine LOH due to mitotic recombination in individuals with different heterozygosity for flanking markers and in different tissues. autosomal locus in human cells occur more frequently by allele loss than intragemc structural alteration. Somatic Cell Mol. Genet., 12: 255â€”263,1986. 10. Amundson, S. 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USA, 87: 6791â€”6794,1990. 1193 Downloaded from cancerres.aacrjournals.org on May 10, 2016. © 1997 American Association for Cancer Research. High Frequency in Vivo Loss of Heterozygosity Is Primarily a Consequence of Mitotic Recombination Pawan K. Gupta, Amrik Sahota, Simeon A. Boyadjiev, et al. Cancer Res 1997;57:1188-1193. Updated version E-mail alerts Reprints and Subscriptions Permissions Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/57/6/1188 Sign up to receive free email-alerts related to this article or journal. To order reprints of this article or to subscribe to the journal, contact the AACR Publications Department at [email protected] To request permission to re-use all or part of this article, contact the AACR Publications Department at [email protected] Downloaded from cancerres.aacrjournals.org on May 10, 2016. © 1997 American Association for Cancer Research.