Development of a flow-cytometric HLA-A locus mutation assay for human peripheral blood lymphocytes

doi:10.1016/0165-1161(92)90005-7

Mutation Research/Environmental Mutagenesis and Related Subjects

Volume 272, Issue 1, August 1992, Pages 17-29

https://doi.org/10.1016/0165-1161(92)90005-7 Get rights and content

Abstract

A flow-cytometric technique was developed to measure the frequency of variant lymphocytes lacking expression of HLA-A2 or A24 allele products among donors heterozygous for HLA-A2 or A24. It was found that the variant frequency of lymphocytes in peripheral blood was of the order of 10⁻⁴ and increased with donor age. Molecular analyses of mutant clones revealed that about one-third were derived from somatic recombinations and that the remaining two-thirds did not show any alterations after Southernblotting analysis. In contrast, mutants obtained after in vitro X-ray mutagenesis study were found to be mostly derived from large chromosomal deletions. A small-scale study on atomic bomb survivors did not show a significant dose effect.

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Cited by (29)

Possible role of natural killer cells in negative selection of mutant lymphocytes that fail to express the human leukocyte antigen-A2 allele
2001, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
Citation Excerpt :
Granulocyte fractions were stained with biotin-labeled anti-HLA-A2 antibody and FITC-labeled anti-CD16 antibody followed by PE-labeled streptavidin. The frequency of HLA-A2-negative cells was measured by flow cytometry as described previously in [8]. An Epstein–Barr virus (EBV)-transformed B cell line (designated MIB) was established from an HLA-A2 (Het) individual (MI) [20].
Increased frequencies of cells carrying mutations at several loci have been found in the blood cells of atomic-bomb (A-bomb) survivors upon testing four or five decades after the bombing. Interestingly, though, we have been unable to demonstrate any radiation-associated increases in the frequencies of mutant blood cells in which human leukocyte antigen (HLA)-A expression has been disrupted; this is true both of preliminary tests on the T cells of a small subset of A-bomb survivors and of the much more extensive study reported here in which we screened a much larger group of survivors for HLA-A2 loss mutations in B cells and granulocytes as well as in T cells. In attempting to explain our inability to detect any increases in HLA-A2-negative cell numbers in HLA-A2 heterozygous individuals exposed to A-bomb irradiation, we decided to test the hypothesis that HLA-A mutant lymphocytes might well have been induced by radiation exposure in much the same way as every other type of mutant we encountered, but may subsequently have been eliminated by the strong negative selection associated with their almost inevitable exposure to autologous natural killer (NK) cells in the bloodstream of each of the individuals concerned. We now report that mutant B lymphocyte cell lines that have lost the ability to express the HLA-A2 antigen do indeed appear to be much more readily eliminated than their parental heterozygous counterparts during co-culture in vitro with autologous NK cells. We make this claim first because we have observed that adding autologous NK cells to in vitro cultures of HLA-A2 heterozygous B or T cell lines appeared to cause a dose-dependent decrease in the numbers of HLA-A2-negative mutants that could be detected over a period of 3 days, and second because when we used peripheral blood HLA-A2 heterozygous lymphocyte cultures from which most of the autologous NK cells had been removed we found that we were able to detect newly-arising HLA-A2 mutant T cells in substantial numbers. Taken together, these results strongly support the hypothesis that autologous NK cells are responsible for eliminating mutant lymphocytes that have lost the ability to express self-HLA class I molecules in vivo, and may well therefore explain why we have been unable to detect increased frequencies of HLA-A2 mutants in samples from any of the 164 A-bomb survivors whose HLA-A2 heterozygote status made their lymphocytes suitable for our tests.
Interindividual variation in mitotic recombination
1999, American Journal of Human Genetics
Mitotic recombination (MR) between homologous chromosomes is a mutational event that results in loss of heterozygosity in half of the segregants at mitosis. Loss of heterozygosity may have important biological consequences. The purpose of this study was to describe human variation in the spontaneous frequency of MR. Using an immunoselection technique for isolating the somatic mutations that result in loss of expression of one of the codominant alleles at the HLA-A locus, we have measured the frequency and molecular basis of somatic mutations in lymphocytes from a population of young adults. Mutations were classified as being the result of intragenic changes, major deletions, or MR. Here we show that the MR mutation frequency in females was significantly greater than that in males but that intragenic mutation frequency showed no association with sex. Individual variation in MR frequency ranged over more than two orders of magnitude and was not normally distributed. Furthermore, the observed number of individuals from whom no mutants resulting from MR were obtained was significantly greater than was expected. The endogenous level of MR may be under genetic control. Given the association of loss of heterozygosity with cancer initiation and progression, low endogenous MR may confer a reduced lifetime risk of cancer, and the converse may apply.
Erythrocyte as a potential model for study of lifelong somatic mutations
1998, Medical Hypotheses
The detection and measurement of somatic cell mutation in vivo is an important subject of research for the assessment of human cancer risk induced by various environmental genotoxic factors. The possible mechanisms which influence the persistence of mutant cells of the hematoimmune system in the peripheral blood are presented. The erythroid system is a system which accumulates mutational lesions and so stably generates red blood cells with various phenotypic changes.
Evolution and the inevitability of human cancer
1998, Seminars in Cancer Biology
Natural selection, which is absolutely dependent on genetic differences between individuals, is the process by which life has evolved on this planet. Genetic variability is ultimately depended on the occurrence of new mutations in the germ-line of species. The rate at which this occurs appears not to be arbitrary or dependent on chance external events. Rather the available evidence suggests that it is highly controlled and determined by endogenous processes. However, the body does not have separate mechanisms for controlling mutation frequency in the germinal and somatic lineages and the selective process described inevitably has also led to somatic cells being subject to mutation accumulation. Indeed, since mutation frequency increases exponentially with time, the human somatic mutation frequency at approximately 80 years of age in epithelial tissues appears to be more than 10-fold higher than in the human germline. This normal but highly elevated somatic mutation frequency is sufficient to account for the complex multi-step process of human tumorigenesis even in the absence of the effects of major external mutagens or rare transitions to even more elevated mutation frequencies. Thus, scrutiny of the apparently disparate biological phenomena of evolution and tumorigenesis leads to the postulate that they are in fact two interdependent manifestations of the same underlying process and that given an evolutionary process dependent on mutation accumulation then cancer in long lived organisms is an inevitable consequence.
Frequency of spontaneous and induced micronuclei in the peripheral blood of aging mice
1997, Mutation Research - Fundamental and Molecular Mechanisms of Mutagenesis
The mouse peripheral blood micronucleus assay, a measure of DNA damage in erythroblastic cells, was used to determine: (1) the incidence of spontaneously occurring micronucleated reticulocytes (MNRETs) as a function of age, and (2) the induction of micronuclei following treatment of young and old animals with mitomycin C. Male C57BL/6 mice, 92 weeks of age, exhibited a significantly higher frequency of spontaneously occurring peripheral blood MNRETs than mice that were 6 or 10 weeks of age. Mice that were 5–6 weeks or 91–92 weeks old were treated with one dose, or two consecutive doses of mitomycin C; this resulted in dose-related increases in the frequency of MNRETs. Mitomycin C, at a single dose of 1 or 2 mg/kg, induced one-third as many MNRETs in the older animals as compared to the younger animals. When treated with a split dose of mitomycin C (total dose 0.5 to 2 mg/kg), older animals displayed on average two-thirds the mutagenic response of the younger animals. However, analysis of variance performed on these data indicated that the age of the animals did not have a significant effect on their mutagenic response to mitomycin C at any dose level. It appears that aging mice may not be more sensitive to the mutagenic effects of chemically-induced DNA damage than younger mice, suggesting that the higher spontaneous mutation frequency in older mice could be the result of an increased load of accumulated DNA damage.
Loss of heterozygosity or: How I learned to stop worrying and love mitotic recombination
1997, American Journal of Human Genetics

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Mutation Research/Environmental Mutagenesis and Related Subjects

Abstract

References (28)

Systematic cloning of human minisatellites from ordered array charomid libraries

Genomics

Increased somatic cell mutant frequency in atomic bomb survivors

Mutation Res.

Measurement of in vivo HGPRT-deficient mutant cell frequency using a modified method for cloning human peripheral blood T-lymphocytes

Mutation Res.

Mutations in human lymphocytes studied by an HLA selection system

Mutation Res.

Frequency of mutant T lymphocytes defective in the expression of the T-cell antigen receptor gene among radiation-exposed people

Mutation Res.

The HLA-A mutation assay: Improved technique and normal results

Mutation Res.

Use of T-cell receptor gene probes to quantify the in vivo hprt mutations in human T-lymphocytes

Mutation Res.

In vitro induction, expression and selection of thioguanine-resistant mutatnts with human T-lymphocytes

Mutation Res.

Mutations in human lymphocytes: effects of X- and UV-radiation

Mutation Res.

Detection of somatic mutants in man: HPRT mutations in lymphocytes and hemoglobin mutations in erthrocytes

Mutation Res.

A novel human DNA polymorphism resulting from transfer of DNA from chromosome 6 to chromosome 16

Genomics

Molecular genetic analysis of recessive mutations at a heterozygous autosomal locus in human cells

Mutation Res.

T-cell cloning to detect the mutant 6-thioguanine-resistant lymphocytes present in human peripheral blood

Identification of a chromosome 18q gene that is altered in colorectal cancers

Science

Cited by (29)

Possible role of natural killer cells in negative selection of mutant lymphocytes that fail to express the human leukocyte antigen-A2 allele

Interindividual variation in mitotic recombination

Erythrocyte as a potential model for study of lifelong somatic mutations

Evolution and the inevitability of human cancer

Frequency of spontaneous and induced micronuclei in the peripheral blood of aging mice

Loss of heterozygosity or: How I learned to stop worrying and love mitotic recombination