Mutations in human lymphocytes studied by an HLA selection system

Mutation Research, 198 (1988) 221-226 Elsevier 221 MTR 04520 Mutations in h u m a n lymphocytes studied by an H L A selection system M. Janatipour, K.J. Trainor, R. Kutlaca, G. Bennett 1, j. Hay 1, D.R. Turner and A.A. Morley Department of Haematology, Flinders Medical Centre, Bedford Park, SA 5042, and I Tissue Typing Laboratory, Red Cross Blood Transfusion Service, Pirie Street, Adelaide, SA 5000 (Australia) (Received 28 April 1987) (Revision received 4 August 1987) (Accepted 10 August 1987) Keywords: Human mutagenesis; Autosomal mutation; Mutation frequency; Gene deletion; Human lymphocytes; HLA locus. Summary Human lymphocytes mutated at the HLA-A2 or HLA-A3 alleles were enumerated and studied by primary selection using antibody and complement, followed by limiting dilution cloning and secondary selection using immunofluorescence or antibody and complement. The geometric mean frequency of in vivo mutant lymphocytes was 3.08 × 10 -5 for the HLA-A2 allele and 4.68 x 10 -6 for the HLA-A3 allele. Mutagenesis by X-radiation or mitomycin produced a dose-related increase in mutant frequency. HLA-B phenotyping and Southern Analysis of the HLA-A gene suggested that mutation was frequently due to gene deletion, which was often substantial. In recent years cloning techniques have been developed for studying in vivo and in vitro mutations in human somatic cells, using the lymphocyte as the model nucleated cell. The genetic locus used to date, at least for in vivo studies, has been the hypoxanthine phosphoribosyl transferase (HPRT) locus (Albertini et al., 1982; Morley et al., 1983b). The HPRT locus has proved very informative but it does have some potential limitations. Thus it is not known whether it is representative of other loci in the genome, particularly since, being X-linked, large mutations may be underrepresented owing to involvement of adjacent Correspondence: Dr. A.A. Morley, Department of Haematology, Fiinders Medical Centre, Bedford Park, SA 5042 (Australia). functionally hemizygotic gene(s) essential for cell survival; cells bearing HPRT mutations may be selected against in vivo and the measured mutant frequency may not truly reflect the number of mutational events which have occurred in the past; and since any X-linked gene locus cannot detect mutations involving mitotic recombination (at least in males), the HPRT locus may completely miss a class of mutations which seem to be of great importance in carcinogenesis (Murphree and Benedict, 1984; Frearon et al., 1984; Koufos et al., 1984; Orkin et al., 1984). For these reasons we have developed a mutation assay using the HLA-A locus to detect and study mutations in human T lymphocytes. This locus was chosen since it is autosomal, being on chromosome 6, the alleles are codominant, and past studies (Pious et al., 1973; Kavathas et al., 1980) have shown that it is feasible to use comple- 0027-5107/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division) 222 ment-mediated cytotoxicity to detect HLA-A loss mutants in human lymphoblastoid cell lines. Materials and methods Individuals who were heterozygous at the HLA-A locus and who were either HLA-A2 or HLA-A3 were studied. The two antibodies used were BB7.2 which is a complement fixing IgG 2 antibody directed against HLA-A2 and GAP A3 which is a complement fixing IgM antibody directed against HLA-A3. They were obtained from the American Type Culture Collection (Rockville, MD, U.S.A.). Lymphocytes were isolated by Ficoll-Hypaque, centrifuged, and washed 3 times in McCoy's medium containing 8% foetal calf serum. Selection was performed either immediately, or after stimulation with phytohaemagglutinin (PHA, Wellcome reagent grade, 1/~l/ml) and culture for 3-10 days. For selection the cell suspension was centrifuged, the supernatant removed and the cells resuspended in 100 /,1 of a 1 : 5 dilution of antibody in Hanks' solution. After incubation at 4 ° C for 30 min, the suspension was centrifuged, the supernatant removed, the cells resuspended in 200 /,1 of rabbit complement (Commonwealth Serum Laboratories, Parkville, Victoria, Australia) and held at room temperature for 60 min. Control lymphocytes were exposed to antibody alone, complement alone or medium only. The cells were then mixed with McCoy's medium containing PHA, 104 irradiated lymphocytes and 10% conditioned medium (as source of interleukin-2), and cloned as previously described (Morley et al., 1983a). Based on the original counts before cytotoxicity, wells were set up to contain 2 or 5 cells/well for control lymphocytes and 2 × 104 lymphocytes for cells exposed to antibody and complement. Plates were scored after 16-20 days. The cells in wells showing growth were scored for A2 or A3 positivity using the appropriate antibody, the binding of which was detected either by imunofluorescence using fluorescein-labelled rabbit or goat a n t i - m o u s e - i m m u n o g l o b u l i n a n t i b o d y (Nordic Immunology) or by complement-mediated cytotoxicity. Only wells which were negative were regarded as being presumptively mutant clones. The mutation frequency was calculated from the cloning efficiency without cytotoxicity and from the cloning efficiency of HLA-A lymphocytes. Wells containing cells which were presumptively mutant cells were expanded as previously described (Morley et al., 1985) to obtain 20-50 x 10 6 lymphocytes. At this stage, HLA phenotyping of the A and B loci was performed with polyclonal antisera by the standard N I H technique (Mittal et al., 1968) and genomic DNA was obtained for subsequent Southern blotting. The DNA probe used was a 490-base pair genomic probe from the 3'-untranslated segment of the HLA-A2 gene which at high stringency hybridization has been shown to be specific for HLA-A genotypes (Koller et al., 1984). DNA probing was done only for HLA-A2 mutant clones. Mutagenesis studies were performed essentially as previously described for the H P R T locus (Morley et al., 1985). Freshly isolated lymphocytes were mutagenized either with various doses of 100 kV X-radiation or by exposure to various concentrations of mitomycin. The mitomycin remained in contact with the cells for the first 3 days of culture. After mutagenesis the lymphocytes were cultured for a total of 7-10 days to allow expression and then selection was performed to enumerate and isolate mutant clones. Induced mutation was measured only at the HLA-A2 locus. In one experiment the recovery of H L A - A 3 cells was studied by mixing cells from an HLA-A3 individual with cells from an HLA-A3 + individual in a ratio of 1 : 104. After antibody-mediated cytotoxicity the cells were cloned and the recovery of the H L A - A 2 - cells determined. Results Absolute selection against wild-type cells was not achieved since in all experiments a proportion (5-100%) of the wells showing growth still scored as H L A - A 2 or HLA-A3 positive by immunofluorescence or complement-mediated cytotoxicity. In the majority of the experiments (68%) the number of wells showing growth was sufficiently low for the second round of selection by immunofluorescence or complement-mediated cytotoxicity to be practicable and for results to be obtained. However, some early experiments showed that when all wells showed growth, all or 223 10-4 FREQUENCY OF lO.5 MUTANT CELLS 25, 125. 20. 100, 15, 75, 10, 50, 5, 25' 16o 260 10-6 A3- A2- 400 X-RAY (rad) 0.6250.65 0.0750.:10 0.;125 MITOMYCIN (ug/ml) INDUCED MUTANT FREQUENCY xlO-5 Fig. 1. Frequency of mutant cells in 13 heterozygous individuals as measured by selection against HLA-A2 or HLA-A3. virtually all of these wells would contain wild-type cells. It was therefore subsequently felt that, when all wells in an experiment were found to be positive, second-round selection was not practicable and such experiments were abandoned. The reason for the variable killing by antibody and complement was not established although a number of technical variables were investigated and continue to be investigated. HLA typing showed that 188 of 198 clones studied had lost the index HLA-A2 or HLA-A3 allele; the 10 clones which were HLA-A2 ÷ or HLA-A3 ÷ presumably arose from wild-type cells which had escaped the two rounds of selection. These clones were excluded from the study. The frequencies of presumptively mutant HLAA2 or HLA-A3 cells for 13 individuals are shown in Fig. 1. The geometric mean frequency ( + 1 S.E.) was 3.08 (3.71-12.55)X 10 -5 for H L A - A 2 mutants and 4.68 (5.81-3.76)x 10 -6 for HLAA 3 - mutants. Mutation assays were performed twice in 4 individuals. The mean values of each duplicate are shown in Fig. 1 and the co-efficient of variation for these 4 individuals was 33%. Mutagenesis was produced by X-radiation in lymphocytes from 6 subjects and by mitomycin in lymphocytes from 7 subjects and these data are shown in Fig. 2. Data from all subjects at all doses were not obtained owing either to inadequate cytotoxicity or to different doses of mutagen being used in some experiments. In the mixing experiment, the cloning efficiencies were: H L A - A 3 - cells, 0.25; HLA-A3 ÷ cells without selection, 0.22; HLA-A3 ÷ cells with selec- Fig. 2. Frequency of HLA-A2- mutant cells produced by X-radiation or mitomycin. The results are shown as the mean _+1 S.D. tion 5.6 x 10-6; mixed H L A - A 3 - and HLA-A3 ÷ cells (1 : 104) with selection, 1.6 x 10 -5. From these results it was calculated that 42% of the H L A - A 3 - cells added to the mixture were recovered after selection. The results of phenotyping at the HLA-B locus and of molecular probing of the HLA-A gene are summarized in Table 1 and an example of the results of molecular probing is shown in Fig. 3. Loss of one HLA-B allele was seen in both spontaneous and induced mutants and, for any one individual, always involved the same HLA-B allele, suggesting that the lost allele was on the same chromosome as the index HLA-A locus. Mutants induced by mitomycin showed a significantly greater frequency of loss of the presumptively linked HLA-B allele (P:0.05, X2 test) suggesting that this agent produces particularly large genomic TABLE 1 RESULTS OF HLA PHENOTYPING AND MOLECULAR PROBING OF MUTANT CLONES Mutants Deletion involving HLA-A gene Loss of HLA-B allele Spontaneous mutants in vivo after 3-10 days in culture 13/43 20/40 19/56 26/72 X-Ray-induced 21/27 4/29 Mitomycin-induced 14/18 23/21 224 b b~ Fig. 3. Southern analysis of a control clone (second track from left) and spontaneously mutant clones isolated from one individual by selection against HLA-A2. Genomic DNA was digested with the enzyme Hind III and the arrows show the band attributable to the HLA-A2 allele (5.25 kb). The other HLA-A allele (in this individual, A 11) yields a band at 4.75 kb, immediately below the A2 fragment. loss. Conversely the mutants induced by X-radiation showed a significantly reduced frequency of loss of the presumptively linked HLA-B allele ( P < 0.02, X 2 test) suggesting that this agent produces deletions small with respect to those which arise spontaneously in vivo or in culture. H L A phenotyping and D N A analysis were performed jointly in a total of 119 clones. Gene deletion was observed in 70 of these clones and simultaneous loss of an HLA-B allele was observed in 35 of the 70. Gene deletion was not observed in 49 clones and both HLA-B alleles were detected in 47 of these 49. Discussion The evidence is strong that clones finally scored as H L A - A 2 - or HLA-A3 were actually mutant cells. The H L A - A loss phenotype persisted during the divisions involved in clonal expansion, in 38% of clones was associated with loss of an HLA-B phenotype and in 53% of clones was associated with gene deletion at the H L A - A locus. The results of H L A typing and D N A analysis were in good agreement in that loss of an HLA-B allele was commonly seen when gene deletion was present but was seen in only 2 or 49 clones when gene deletion was not detected. The failure of D N A analysis to detect an abnormality in these two clones may be explicable by the occurrence of a deletion which did not affect the probed restriction fragment which is 3' to the gene but which nonetheless produced phenotypic loss of HLA-A2. Comparison of the results obtained with selection against the two H L A - A alleles was of interest as the spontaneous in vivo mutant frequency measured with the anti-HLA-A2 antibody was almost an order of magnitude higher than that measured with the anti-HLA-A3 antibody. It is possible but inherently unlikely that the spontaneous mutation frequency for the two alleles was different or that the intensity of any in vivo selection differed between the two types of mutants. The most likely explanation for the difference between the results with these two antibodies is a difference in the stringency of the binding conditions for the two antibodies. If the anti-HLA-A2 antibody had very stringent requirements then it would not bind to mutants showing only slight conformational 225 changes of the HLA-A2 epitope; conversely if the HLA-A3 antibody had very relaxed requirements then it would eliminate such mutants. The results obtained with the HLA system showed some similarities to and some differences from those previously obtained with the HPRT system (Morley et al., 1983b; Trainor et al., 1984), although in comparing quantitative results from the two systems, it should be borne in mind that the HPRT system as usually performed involves stringent thioguanine selection and probably underestimates mutation frequency. The frequency of spontaneous mutants detected with the antiHLA-A3 antibody ranged from 1.7 x 10 -6 to 8.5 x 10 -6, which is similar to the range of 1 x 10-6-2 x 10 -5 observed for H P R T mutant frequency (Morley et al., 1983b; Trainor et al., 1984). However, as seen in Fig. 1, the frequency of mutants observed using the anti-HLA-A2 antibody was almost an order of magnitude higher, ranging from 1.5 x 10 -5 to 7.0 x 10 -5. Study of mutation induction by X-radiation or mitomycin was performed only using the antiHLA-A2 antibody. The mean frequency of induced mutant cells was 23.7 x .~9-5 after 400 rad which is somewhat greater than previous results with the HPRT system which showed a frequency of 9.2× 10 -5 after 400 rad (Sanderson et al., 1984). Waldren et al. (1986) studied X-ray mutagenesis at a locus on human chromosome 11 in a CHO hybrid containing that chromosome, and obtained a mutant yield 2 orders of magnitude greater than that obtained by others using the HPRT locus and 20-fold greater than the results obtained in the present experiments using the HLA-A locus. They attributed the difference between their results and those of others to the fact that mutagenesis was carried out at low doses of radiation, 25-150 rad, at which there was little killing, but this cannot account for the difference between their results and our own at both the HPRT and HLA-A loci, since our experiments were carried out in the same dose range and since lymphocytes have virtually the same radiation sensitivity as CHO cells (Kutlaca et al., 1982a, b). It may be that a foreign chromosome in a cell hybrid sustains more damage or is repaired less accurately than an endogenous chromosome in that cell. Molecular analysis of the HLA-A gene was performed only for HLA-A2 mutants and showed that mutation frequently involved deletion, at least of the 3' portion of the gene being probed. Deletion was observed both in spontaneous and induced mutants and was of the same order of frequency as that previously observed using the HPRT system for both spontaneous (Albertini et al., 1985; Turner et al., 1985) and induced mutants (Thacker and Cox, 1984; Vrieling et al., 1985; Skulimowski et al., 1986; Gibbs et al., 1987). The results were also broadly similar to those obtained by Yandell et al. (1986) for spontaneous and induced mutations at the thymidine kinase locus in a lymphoblastoid cell line although they observed a somewhat higher frequency of 71% for gene loss in spontaneous mutations. The results of HLA phenotyping, which showed that loss of an HLA-B allele was common when molecular evidence of gene deletion was present in spontaneous or mitomycin-induced mutants, indicate that many of these deletions must have been quite large, as the HLA-B locus is approximately 1 cM or 1000 kb distant from the HLA-A locus. Deletions at the HPRT locus, which is approximately 34 kb in size, seem to be smaller, for we have never seen complete deletion of this gene in spontaneous mutant lymphocytes, although other workers have (personal communication from R. Albertini). Mutants induced by X-rays predominantly arise from deletion of the HLA-A2 gene. However, in agreement with previous results, these deletions are small in genomic terms and this is reflected in the low frequency (14%) of associated loss of HLA-B phenotype in these mutants. The results from the HLA and HPRT system are therefore in broad agreement. They suggest that neither region of the genome is likely to represent a genetic "hot-spot" and that these genetic systems are likely to provide valid information on the number and types of mutations which affect autosomal and X-linked loci on the human genome. 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