Genetic discrimination in health insurance: an overview and analysis of the issues.

The problem of genetic discrimination in health insurance will increase as genetic knowledge expands and the number of genetic tests proliferates. Unless appropriate legislative protections are developed and enforced, a consequence of the genetic revolution may be that more people are put at risk for losing their health insurance. The current situation requires people to make difficult choices about taking tests that could save or prolong their lives. Unless these people believe that they and their families will be adequately protected from discrimination and from the possibility of losing or being denied health insurance, many will choose not to be tested for genetic conditions or predisposition to disease. Solutions to this problem require continuing research and debate and the creation of new policies and laws that protect the people while maintaining the economic viability of insurance companies. This article explores the problem of genetic discrimination as it relates to health insurance in the United States. The goal of this article is to assist nurses and other health care professionals to better understand the important and complex issues and concepts related to genetics, genetic testing, and genetic discrimination in health insurance.

Cloning of a candidate gene for ataxia-telangiectasia group D.

Transfection, with a human cosmid clone library, of an ataxia-telangiectasia (AT) cell line (AT5BIVA) from complementation group D previously resulted in the isolation of a cell line (1B3) with partially restored resistance to ionizing radiation. We rescued the integrated cosmid sequences within 1B3 and obtained two cosmid clones that contained overlapping DNA from chromosomal region 11q23, previously shown to be the region containing the AT gene(s) from three complementation groups. Isolation of an apparently full-length 3.0-kb cDNA from a HeLa cell library demonstrated a previously unidentified gene (ATDC) within these cosmid clones. The transfected copy of the ATDC gene in 1B3 is truncated at the 3' end but is a complete transcription unit, because of the presence of SV40 termination sequences within the adjacent cosmid DNA. After further screening of cosmid clones from a chromosome 11 library, we identified contiguous DNA that contained the missing portion of the gene. Southern blot analysis indicated that the ATDC gene is present in a single copy in the human genome; however, RNA blot analysis revealed mRNA of several sizes (1.8, 2.6, 3.0, 4.7, and 5.7 kb) that varied among different cell lines. Because no large rearrangements were detected in AT5BIVA cells by Southern or RNA blot analysis, any alteration in the ATDC gene in this cell line would involve a point mutation or a small rearrangement. Transfection of the AT5BIVA cell line with one of the cosmids partially restored radioresistance. Analysis of 100 X-radiation hybrid cell lines containing various fragments from the chromosomal region 11q23 showed that the ATDC gene is closely linked to THY1. The ATDC gene therefore lies outside the linkage region predicted to contain the AT gene(s) for complementation groups A and C, indicating a separate locus for the AT complementation group D gene.

Replication intermediates as substrates for DNA rearrangements.

A model for the formation of DNA rearrangements in somatic cells is presented. Two double-strand breaks at the junctions between unreplicated DNA and newly replicated DNA generate four double-stranded DNA molecules that can recombine to form tandem duplications, inversions, deletions and extrachromosomal DNA circles.

Modification of deoxyribose-phosphate residues by extracts of ataxia telangiectasia cells.

The release of DNA 5'-terminal deoxyribose-phosphate residues from enzymatically incised apurinic/apyrimidinic sites by human cell extracts has been under investigation. During the course of these studies, we observed that ataxia telangiectasia cell extracts modify deoxyribose-phosphate (dRp) residues by converting them to an altered form, dRp-X, which shows altered chromatographic properties on HPLC analysis. The chemical nature of the adduct is as yet unknown, but dRp-X is stable to both heat and acid. The modification requires an enzymatic activity and a low-molecular weight co-factor. Extracts of normal cells contain a dialyzable inhibitor that suppresses the reaction occurring with ataxia telangiectasia cell extracts. Formation of dRp-X has been observed in 7 out of 7 ataxia telangiectasia lymphoblastoid lines which represent at least 3 genetic complementation groups. Similar modification of dRp did not occur with extracts of cells of normal origin, nor those representing Fanconi's anaemia, xeroderma pigmentosum, Bloom's syndrome, Werner's syndrome or Friedreich's ataxia.

Stable radioresistance in ataxia-telangiectasia cells containing DNA from normal human cells.

SV40-transformed ataxia-telangiectasia (AT) cells were transfected with a cosmid that contains a normal human DNA library and a selectable marker, the neo gene, which endows successfully transformed mammalian cells with resistance to the antibiotic G418. After a three-part selection protocol for G418 resistance and radioresistance, a cell line stably resistant to ionizing radiation was recovered. Cells from this line were irradiated with 50 Gy of X-rays and fused with non-transfected AT cells. Among the G418-resistant colonies recovered was one that was stably resistant to radiation. Resistance to ionizing radiation of both the primary transfectant line and its fusion derivative was intermediate between that of AT cells and normal cells, as assayed by colony-forming ability and measurement of radiation-induced G2 chromatid aberrations; both cell lines retained AT-like radioresistant DNA synthesis. These results suggest that, because radioresistance in the transfected cells was not as great as that in normal human cells, the two hallmarks of AT, radiosensitivity and radioresistant DNA synthesis, may still be the result of a single defective AT gene.

Radioresistant DNA synthesis and human genetic diseases.

Sixty-eight human fibroblast cell strains were assayed for radioresistant DNA synthesis (RDS), which is defined here as the absence of a steep component of inhibition of DNA synthesis in a dose-response curve when rate of DNA synthesis is plotted against radiation doses from 0 to 20 Gy or more. Twenty-seven strains from patients who were previously diagnosed to have ataxia-telangiectasia (AT) were positive for this feature. Among the cell strains that did not show RDS were two from AT obligate heterozygotes (i.e., the parents of AT patients), two from patients with Alzheimer disease, two from patients with Friedreich ataxia, one from a patient with Bloom syndrome, one from a patient with Down syndrome, and six from patients with various immunodeficiencies. Four strains demonstrated RDS that was less pronounced than in most AT cells: one was from a patient with Nijmegen breakage syndrome, one was from a patient without ataxia but with choreiform movement disorder, telangiectasia, and elevated concentrations of alpha-fetoprotein in the blood, and two were from AT patients. RDS therefore is not a necessary trait of human genetic diseases that involve radiosensitivity or immunodeficiency. Although recent reports suggest that some AT patients do not exhibit RDS, we found RDS in all the AT cells we tested.

Ataxia-telangiectasia as a model system for studies of radiation protection mechanisms.

Patients with ataxia-telangiectasia (AT), a human autosomal recessive genetic disease, are uniformly hypersensitive to ionizing radiation as measured by colony-forming ability and by chromosomal aberrations. Obligate heterozygotes, i.e., parents of AT patients, are slightly more radiosensitive than normal humans in terms of both colony-forming ability and chromosomal aberrations formed in G2. Thus, this system not only furnishes a model system to study factors that are responsible for radioresistance in normal human beings, but is also a unique tool for determining the role of gene dosage on radiation-induced cell killing. Because AT cells seem to be hypomutable to ionizing radiation, they also can be used to study the relationship between radiosensitivity and mutability and, therefore, carcinogenesis. Isolation of the defective gene that causes hypersensitivity in AT cells and its counterpart in normal cells should lead to a breakthrough in our understanding of radiation effects and how they can be prevented in human beings.

A Chinese hamster ovary cell line hypersensitive to ionizing radiation and deficient in repair replication.

An X-ray-sensitive Chinese hamster ovary cell line was isolated by means of a semi-automated procedure in which mutagenized cells formed colonies on top of agar, were X-irradiated, and were photographed at two later times. We compared the photographs to identify colonies that displayed significant growth arrest. One of the colonies identified in this manner produced a stable line (irs1SF) that is hypersensitive to ionizing radiation. The X-ray dose at which 10% of the population survives (D10) is 2.25 Gy for irs1SF and 5.45 Gy for the parental line. The new mutant is also moderately sensitive to ethyl methanesulfonate. irs1SF performs only half as much X-ray-induced repair replication as the parental line, indicating a defect in excision repair. This defect is believed to be the primary cause of the line's radiosensitivity. Although irs1SF repairs DNA double-strand breaks at a normal rate, it repairs single-strand breaks more slowly than normal. irs1SF has an elevated number of spontaneous chromatid aberrations and produces significantly higher numbers of X-ray-induced chromatid aberrations after exposure during the G1 phase of the cell cycle. The line is hypomutable, with X-ray exposure inducing only one-third as many 6-thioguanine-resistant colonies as the parental line.

Absence of DNA overreplication in Chinese hamster cells incubated with inhibitors of DNA synthesis.

Previous reports have suggested that transient inhibition of DNA synthesis by chemicals or ultraviolet light causes some of the DNA to replicate more than once in one cell cycle, i.e., that it induces overreplication of DNA. The data that led to this suggestion were obtained from cesium chloride equilibrium density gradient analyses, in which cells were incubated with bromodeoxyuridine so that the DNA synthesized after incubation with the inhibitor could be densitometrically separated from the DNA that had been radioactively labeled before incubation with the inhibitor. An unresolved problem with these analyses was that the data also suggested that overreplication must have occurred in control cells, i.e., those not incubated with an inhibitor of DNA synthesis. We show here that the latter result is probably due to an artifact of cesium chloride equilibrium density gradient analysis, probably because of nonspecific trapping of DNA in regions of the gradients where there are large amounts of DNA. We also used another protocol that avoids this artifact; with this protocol any overreplicated DNA would be found where heavy-heavy DNA bands and nonspecific trapping cannot occur. When this protocol was used there was no evidence that transient inhibition of DNA synthesis induces overreplication of DNA.

The role of acentric chromosome fragments in gene amplification.

We assessed the role of acentric chromosome fragments in gene amplification by using cell fusion techniques to introduce the fragmented chromosomes of a donor Chinese hamster ovary (CHO) cell line that contained the dihydrofolate reductase (dhfr) gene(s) into a CHO cell line deficient for dhfr. Chromosome fragments were successfully integrated into cells at a frequency of approximately 3%. Methotrexate-resistant variants arose much more frequently in two cell lines derived from these successful cell fusions than in wild-type CHO cells. The hybrid cell lines also amplified their dhfr genes more readily than did the CHO cell line used as dhfr donor.

DNA synthesis in irradiated mammalian cells.

One of the first responses observed in S phase mammalian cells that have suffered DNA damage is the inhibition of initiation of DNA replicons. In cells exposed to ionizing radiation, a single-strand break appears to be the stimulus for this effect, whereby the initiation of many adjacent replicons (a replicon cluster) is blocked by a single-strand break in any one of them. In cells exposed to ultraviolet light (u.v.), replicon initiation is blocked at fluences that induce about one pyrimidine dimer per replicon. The inhibition of replicon initiation by u.v. in Chinese hamster cells that are incapable of excising pyrimidine dimers from their DNA is virtually the same as in cells that are proficient in dimer excision. Therefore, a single-strand break formed during excision repair of pyrimidine dimers is not the stimulus for inhibition of replicon initiation in u.v.-irradiated cells. Considering this fact, as well as the comparative insensitivity of human ataxia telangiectasia cells to u.v.-induced inhibition of replicon initiation, we propose that a relatively rare lesion is the stimulus for u.v.-induced inhibition of replicon initiation.

Establishment and characterization of two human cell lines with amplified dihydrofolate reductase genes.

Two SV40-transformed human cell lines, GM637, derived from a normal human subject, and GM5849, derived from a patient with ataxia-telangiectasia (A-T), were grown in increasing concentrations of the cytotoxic agent methotrexate (MTX). The GM637 line was naturally more resistant to methotrexate than was GM5849 and, over a 5-month period, became resistant even to very high concentrations (up to 100 microM). The GM5849 line became resistant to 500 nM methotrexate during the same period. However, dot blot and Southern blot analyses showed that both cell lines had amplified their dihydrofolate reductase (dhfr) genes to about the same extent, approx. 50-fold. Using the GM5849 line with amplified dhfr, we attempted to determine if interruption of DNA synthesis by hydroxyurea would cause DNA to be replicated twice within a single cell cycle, as has been reported for Chinese hamster ovary cells. No evidence for such a phenomenon was obtained.

Chromosomal changes without DNA overproduction in hydroxyurea-treated mammalian cells: implications for gene amplification.

It has been reported that a 6-h incubation of early S-phase Chinese hamster cells with hydroxyurea promotes DNA overproduction, i.e., replication of DNA a second time within a single cell cycle, and that this could be the basis for gene amplification in drug-treated mammalian cells. When we incubated methotrexate-resistant Chinese hamster cells that were approximately 2 h into the S phase with hydroxyurea for 6 h, DNA that had been replicated before the incubation with hydroxyurea (early S-phase DNA) was replicated again within 11 h after the hydroxyurea treatment. However, incubation with colchicine or Colcemid after hydroxyurea treatment virtually abolished this overreplication, as well as that of the amplified dihydrofolate reductase genes in these cells, indicating that the second replication had occurred in a second cell cycle. Cells collected in the first mitosis after incubation with hydroxyurea never contained overreplicated DNA but did contain abundant chromosome aberrations. Early S-phase DNA replicated again on schedule during the first few hours after mitosis. Asymmetric segregation of chromosome fragments or unequal sister chromatid exchange may be the actual basis for gene amplification in drug-treated mammalian cells.

Inhibition of mammalian cell DNA synthesis by ionizing radiation.

A semi-log plot of the inhibitory effect of ionizing radiation on the rate of DNA synthesis in normal mammalian cells yields a two-component curve. The steep component, at low doses, has a D0 of about 5 Gy and is the result of blocks to initiation of DNA replicons. The shallow component, at high doses, has a D0 of greater than or equal to 100 Gy and is the result of blocks to DNA chain elongation. The target size for the inhibition of DNA replicon initiation is about 1000 kb, and the target size for inhibition of DNA chain elongation is about 50 kb. There is evidence that the target for both components is DNA alone. Therefore, the target size for inhibition of DNA chain elongation is consistent with the idea that an effective radiation-induced lesion in front of the DNA growing point somehow blocks its advance. The target size for inhibition of DNA replicon initiation is so large that it must include many replicons, which is consistent with the concept that a single lesion anywhere within a large group (cluster) of replicons is sufficient to block the initiation of replication of all replicons within that cluster. Studies with radiosensitive human cell mutants suggest that there is an intermediary factor whose normal function is necessary for radiation-induced lesions to cause the inhibition of replicon initiation in clusters and to block chain elongation; this factor is not related to poly(ADP-ribose) synthesis. Studies with radiosensitive Chinese hamster cell mutants suggest that double-strand breaks and their repair are important in regulating the duration of radiation-induced inhibition of replicon initiation but have little to do with effects on chain elongation. There is no simple correlation between inhibition of DNA synthesis and cell killing by ionizing radiation.

A cytogenetic investigation of DNA rereplication after hydroxyurea treatment: implications for gene amplification.

Although the mechanisms leading to gene amplification are poorly understood, it has recently been proposed that the initial event of amplification is the rereplication of a variable, but relatively large, amount of the genome within a single cell cycle. We sought evidence for rereplication of DNA as a basis for gene amplification through two cytogenetic techniques: differential staining for sister-chromatid exchange analysis and premature chromosome condensation. Synchronized Chinese hamster ovary cells were incubated continuously with bromodeoxyuridine and treated with hydroxyurea (HU) when cells were approximately 2 h into the S phase. After 6 h exposure to HU, the drug was removed and at 3 h intervals thereafter metaphase cells were collected and the chromosomes were stained by the fluorescence-plus-Giemsa procedure. No staining patterns consistent with rereplication of DNA were observed. Since HU causes cytogenetic damage, the premature chromosome condensation technique was used to determine the kinetics of chromosome damage after removal of HU. Extensive G2 chromosome damage within 1 h after removal of HU from the medium was found, although cesium chloride gradient analysis showed that there was no rereplication of DNA during this time. Contrary to a previous report, these results provide no evidence that incubation of cells with HU during S phase induces rereplication of DNA within a single cell cycle. The results observed are consistent with the hypothesis that drug-induced aberrations and the subsequent abnormal segregation of chromosomal fragments are the first steps in the process that leads to gene amplification in drug-treated mammalian cells.

3-Aminobenzamide does not affect radiation-induced inhibition of DNA synthesis in human cells.

3-Aminobenzamide (3AB), a potent inhibitor of poly(ADP-ribose) synthesis, does not affect the dose response for ionizing radiation-induced inhibition of DNA synthesis in human fibroblasts. If the radioresistant DNA synthesis observed in fibroblasts from patients with ataxia-telangiectasia (A-T) were due to reduced poly(ADP-ribose) synthesis after irradiation, as has been proposed, the response in normal cells incubated with 3AB would have been similar to that observed in A-T cells. Therefore, altered poly(ADP-ribose) synthesis in A-T cells is not solely responsible for their radioresistant DNA synthesis.

Establishment and characterization of a permanent pSV ori--transformed ataxia-telangiectasia cell line.

A permanent ataxia-telangiectasia (A-T) cell line has been established from the fibroblast strain AT2SF after transfection with the bacterial plasmid pSV ori-, which contains replication origin-defective SV40 sequences. The original transfection frequency, as measured by transformed foci, was markedly reduced in two A-T strains when compared with either normal or xeroderma pigmentosum fibroblasts. As with SV40 virion-transformed fibroblasts, pSV ori--transformed cells entered a crisis phase, from which about one-fourth of the original clones from A-T and normal fibroblasts recovered. Both the pSV ori--transformed TAT2SF cell line and an SV40 virion-transformed AT5BI (GM5489) cell line retained their characteristic sensitivity to the lethal effects of ionizing radiation, as well as their X ray-resistant DNA synthesis. Southern blot analysis of cellular SV40 sequences demonstrated a single major integration site of pSV ori- in the AT2SF cells. In contrast, AT5BI cells transformed with SV40 virions demonstrated a high degree of heterogeneity of integrated viral sequences. Neither the TAT2SF nor the GM5489 transformed cell line contains any detectable freely replicating SV40 viral sequences, which are seen in many other semipermissive SV40-transformed cells.

Inhibition and recovery of DNA synthesis in human cells after exposure to ultraviolet light.

The inhibition of DNA synthesis in normal human cells by UV is a complex function of fluence because it has several causes. At low fluences, inhibition of replicon initiation is most important. This is made clear by the fact that it occurs to a lesser degree in cells from patients with ataxia telangiectasia (AT). Assuming that only leading strand synthesis is blocked by UV-induced lesions, single lesions between replicons in parental strands for leading strand synthesis inhibit DNA synthesis by acting as temporary blocks until they are replicated by extension of the lagging strand of the adjacent replicon. A more severe inhibition occurs when two lesions are induced between adjacent growing replicons, because one in four possible configurations may result in a long-lived unreplicated region (LLUR). In the absence of excision repair, these may eventually be replicated by activation of an otherwise unused origin within the LLUR. The frequency of LLURs increases steeply with fluence. Activation of normally unused origins to replicate LLURs may facilitate recovery from inhibition of DNA synthesis, but repair of lesions is probably more important. In excision-repair-defective cells, an LLUR without an origin to initiate its replication may be a lethal lesion.