We describe a novel in vitro assay system that detects the generation of suppressor T cells after exposure of human lymphocytes to class I alloantigens. We have used this system to study immune functions in a group of seven patients with ataxia-telangiectasia (AT). Normal T lymphocytes exposed to cells differing at the A and B locus histocompatibility locus antigens (HLA) become activated for suppression of Epstein-Barr virus (EBV)-induced immunoglobulin (Ig) production. In contrast to the normal, T cells from patients with AT demonstrate no inhibitory effect after allostimulation. These data indicate that patients with AT have a profound defect involving responses to class I antigens of the major histocompatibility complex (MHC).
The AT cell fails to pause sufficiently after X-ray or similar radiomimetic insults to repair damage. Rather, it launches with undue speed into DNA replication. It may incorporate errors into DNA that lead to the chromatid and chromosome breaks. Breakpoints have been noted at 7p13, 7q33-35, 14q11-12, and 14q32. The regions at 7q33-35, and 14q11-12 are specific to T cells and include T cell receptor genes. The region at 14q11-12 is involved in T-cell malignancies. The region at 14q32 contains immunoglobulin heavy-chain genes and is involved in B-cell malignancies.
By in situ hybridization of cloned human alpha-fetoprotein cDNA to human mitotic chromosome preparations, the alpha-fetoprotein gene was localized within the q11-22 region on the long arm of human chromosome 4. In addition, the human alpha-fetoprotein gene was isolated from a genomic phage library. The gene is split into 15 exons and 14 introns, and the entire structure is contained within two large (9.5 and 9.0) and two small (0.3 and 0.25 kb) EcoRI fragments of contiguous chromosomal DNA. The structure of alpha-fetoprotein and its gene is very similar to the corresponding structures of serum albumin, indicating a common evolutionary origin of these two serum proteins. However, the two genes are differentially expressed during normal development and under certain pathological conditions such as hepatomas, germ-cell tumors, or ataxia-telangiectasia. The molecular basis of this differential gene expression remains to be understood.
Retrospective studies of cancer incidence in AT families suggest that the major detrimental impact of the AT gene on the health of the general population stems from its disease-predisposing potential in heterozygotes. The absence of a definitive marker for routine identification of such carriers, however, has previously precluded direct validation of this predicted effect of AT heterozygosity. We demonstrate that chronic gamma-ray exposure, because it expands the difference in radiation cytotoxicity between normal and heterozygotic strains compared to acute dose delivery, may point the way to development of a reliable laboratory diagnostic procedure for identification of carriers of a defective AT gene.
We have examined several AT-related lesions in lymphoblastoid cell lines (LCLs) derived from AT patients. Diminished sensitivity to gamma-irradiation was found in six of seven AT-LCLs. A seventh line, from a patient with apparently normal T-cell immunity, responded normally following radiation. Constitutive proteins from exponentially growing AT-LCLs were assessed by SDS-PAGE analysis and did not differ significantly from normals. IgM synthesis was also normal except for one AT-LCL that contained native IgM molecules of different sizes, corresponding to the presence of pentamers and oligomers. Analysis under reducing conditions showed normal-sized secretory mu-chains. Finally, we examined mRNAs corresponding to two oncogenes, c-myc and c-myb, in AT and normal LCLs and found marked overproduction of c-myc in one AT-LCL (ie,, ATL6). The latter findings suggest that AT cells might be prone to aberrantly express cellular oncogenes as a result of chromosomal instability and consequent transposition of oncogenes.
In a number of laboratories genetic analysis of ataxia-telangiectasia (AT) has been performed by studying the expression of the AT phenotype in fused somatic cells or mixtures of cell-free extracts from different patients. Complementation of the defective response to ionizing radiation was observed frequently, considering four different parameters for radiosensitivity in AT. The combined results from studies on cultured fibroblasts or lymphoblastoid cells from 17 unrelated families revealed the presence of at least four and possibly nine complementation groups. These findings suggest that there is an extensive genetic heterogeneity in AT. More extensive studies are needed for an integration of these data and to provide a set of genetically characterized cell strains for future research of the AT genetic defect.
Gm marker studies were performed on seven families with ataxia-telangiectasia in order to determine genetic linkage. For statistical analysis, the disorder was assumed to be monogenic. Using LOD scores computed by the program LIPED, tight linkage was excluded at a recombination fraction of less than or equal to 2 cM. This distance (equivalent to approximately two million nucleotides) includes most or all of the 14q32 chromosomal region.
The nature of the soluble mediators of immune regulation, the lymphokines, is now being determined and this allows consideration of new approaches to treatment of disorders involving immune dysfunction, such as ataxia-telangiectasia. The immunomodulatory effects of interleukin-2, interferons, and known chemotherapeutic agents such as cyclophosphamide are considered in relation to the immune disorders of ataxia-telangiectasia. The possible etiology of the disease and its treatment are considered in relation to the immunopathologic effects that are observed.
Four common sites of chromosome breakage have been observed in patients with ataxia-telangiectasia (AT): 7p14, 7q35, 14q11.2, and 14q32. These sites appear to coincide with the location of genes for the T-cell receptor subunits (alpha, beta, and gamma) and IGH. Each of these genes involves rearrangements of DNA for its expression, suggesting that an abnormal DNA processing enzyme or family of enzymes underlies this propensity for chromosomal breakage in AT patients. Such a defect could also explain the radiation hypersensitivity of AT fibroblasts. In view of these findings, it is perhaps surprising that AT patients do not manifest more severe immunological defects although they would explain the lack of uniformity of these defects from one patient to the next. Two other genes utilize DNA rearrangement, IGK (on chromosome 2p12) and IGL (on chromosome 22q11), and have not been noted previously to be involved in translocations in these patients. We report here a 2:14 translocation (p14:q32) in a phytohemagglutinin-stimulated lymphocyte from a patient with AT.
The ability of lymphocytes from 11 patients with ataxia-telangiectasia to produce specific antiinfluenza virus antibody in vitro was evaluated. Lymphocytes from these patients produced markedly less antibody than lymphocytes from normal controls when stimulated with type A influenza viruses. Additional studies were undertaken to evaluate the function of the B cells, T cells, and adherent cells of these patients in specific antibody production. B cells from the AT patients produced one-third to one-half as much antiinfluenza virus antibody as did B cells from normals when stimulated with the polyclonal activator Epstein-Barr virus or, in the two cases studied, when stimulated with influenza virus in the presence of normal HLA-identical T-cells, suggesting that a partial B-cell defect contributed to the deficient antibody response in these patients. Helper T-cell function of T-cells from two patients was evaluated in coculture with their HLA-identical sibling's B cells; these studies revealed that the patients' T-cells could provide less help than normals' T-cells but that this help was not entirely deficient. Furthermore, T-cells from AT patients could provide allostimulated helper T-cell function in coculture with allogeneic normal B cells. Taken together, these results suggest that partial defects of B- and T-cell function both contribute to the decreased antiinfluenza virus antibody production by patients with AT.
According to the Hardy-Weinberg principle, the frequency in the general population of heterozygous carriers of a gene causing an autosomal recessive syndrome in homozygotes is likely to be between 0.1% and 5%. It is thus important to know whether carriers of the AT gene have a risk of cancer or diabetes greater than comparable noncarriers. A retrospective study of blood relatives in 26 AT families, and follow-up of the obligatory heterozygotes in those families, demonstrated an excess of diabetes, deaths from cancer, and deaths from ischemic heart disease among obligatory or probable AT heterozygotes. Hypotheses about the disease-predisposing effects of the AT gene in the heterozygote are now being reexamined, retrospectively and prospectively, in almost 150 newly identified AT families. Specific tests for the AT gene will permit even more rigorous tests of these hypotheses.
DNA synthesis in cells from ataxia-telangiectasia (AT) patients differs from that in normal cells in two principal ways. In unirradiated cultures, AT cells have a lower inherent rate of DNA synthesis (and, therefore, a longer S phase) than normal cells and, in cultures exposed to ionizing radiation, DNA synthesis is not inhibited as it is in normal cells; this radioresistant DNA synthesis is due to completely resistant DNA chain elongation and partially resistant DNA replicon initiation. It is probable that the defects in DNA synthesis in irradiated and unirradiated cells are related and are at least partially involved in the pathogenesis of this syndrome.
The unique neuropathology of AT is discussed, focusing on changes in the cerebellum--site of the most consistent and severe findings. In the cerebellum, despite marked Purkinje cell and granule cell loss and thinning of the molecular layer, the basket cells are relatively preserved, as demonstrated by the Bielschowsky staining. To document that basket cells do, indeed, represent a "footprint" of where Purkinje cells once existed, we examined the cerebellum of patients with chronic alcohol abuse. We again found normal numbers of "empty" basket cells. This suggests that AT is a degenerative condition in which the Purkinje cell layer forms, perhaps abnormally, but then undergoes neuronal depletion.
Heterozygotes of ataxia-telangiectasia (AT) can, in certain parts of the world, represent a significant proportion of the population. Epidemiological studies suggest that they are more cancer prone than normal individuals. Fibroblasts of five AT heterozygotes are significantly more sensitive to gamma irradiation (mean D0 = 1.18 Gy) than five normals (mean D0 = 1.49 Gy) although some overlap in response is observed. Experiments designed to maximize differences in survival by allowing a period for the repair of potentially lethal damage (PLD) showed that only one out of five AT heterozygotes was defective in the repair of PLD. This technique does not, therefore, permit an improved discrimination of AT heterozygotes. Two AT heterozygotes were tested for their ability to repair lesions that give rise to micronuclei. Both, like the homozygote, were seen to be defective in this capacity. Defects in the repair of chromosome damage may permit a cellular discrimination of the heterozygotes.
Studies of the in vitro phenotype of a series of AT strains established in Israel revealed the following features: premature senescence and increased demands for growth factors, normal sensitivity to the cytotoxic effect of alkylating agents, hypersensitivity to agents that damage the deoxyribose moiety of DNA via a "targeted" free radical attack (this hypersensitivity is coupled with reduced inhibition of DNA synthesis compared to normal cells), varying degrees of intermediate hypersensitivity to the same agents in AT heterozygous cells, lack of potentially lethal damage repair and sublethal damage repair in AT homozygous cells following treatment with free radical-producing agents. We conclude that AT involves a DNA repair defect and that the AT DNA lesion is probably a gap with the 3'-phosphate or 3'-phosphoglycolate end left in the DNA following sugar destruction.
An attempt was made to relate expression of neuronal abnormalities in ataxia-telangiectasia (AT) to the sequence of normal cellular events in the developing human cerebellum. Previous light and electron microscopic analyses indicate that the cerebellar cortex in humans develops during a protracted period that spans 8 fetal and 12 postnatal months. However, the Purkinje cells that comprise the most obvious lesion in the AT disorder are all generated before the end of the fourth fetal month. Correlative Golgi studies in human and [3H]thymidine labeling of DNA in dividing cerebellar cells in rhesus monkey demonstrate that after the last mitotic division Purkinje cells migrate to the cortical plate where they form a well-defined stratum below the embryonic molecular layer. Only thereafter do they begin to differentiate and develop their large dendritic tree. The dendrites grow in coordination and simultaneously with the genesis of parallel fibers in the molecular layer. The parallel fibers--the horizontal portion of granule cell axons--form between the fourth fetal and twelfth postnatal month in a well-defined inside-outside order, the earliest generated fibers being situated near the Purkinje cell layer and the last fibers generated lying closer to the pial surface. The four cases of AT examined in this study showed the usual neuropathological changes, which include a variable degree of Purkinje and granule cell loss. However, we emphasize here an abnormality of dendritic arborization and the presence of displaced Purkinje cells, which are situated in the middle and superficial strata of the molecular layer. Based on the sequence of histogenetic events, we argue that neither abnormal arborization nor aberrant position could be attained after parallel fibers of the deeper strata have been laid down and after Purkinje cells have formed their dendritic tree. Therefore, we suggest that the AT disorder in these cases must affect Purkinje cell differentiation or the interaction of these cells with parallel fibers during the first half of gestation, which is considerably earlier than any other recognized expression of the disorder. The subsequent degeneration of Purkinje cells is apparently not related to the aberrant position of the somas, since many Purkinje cells situated in normal position also die and there is no evidence that displaced cells degenerate at a slower or more rapid rate. The early expression of AT in the central nervous system provides new insight into possible pathogenesis and opens new avenues for research.
