Thyroid nodules in xeroderma pigmentosum patients: a feature of premature aging.

PURPOSE: Xeroderma pigmentosum (XP) is an autosomal recessive disease with defective DNA repair, a markedly increased risk of skin cancer, and premature aging. Reports from North Africa have described thyroid nodules in XP patients, but thyroid nodule prevalence has never been determined in XP patients enrolled in our natural history study at the National Institutes of Health (NIH). METHODS: We performed thyroid ultrasound examinations on all 29 XP patients examined from 2011 to 2019 and assessed nodule malignancy using the Thyroid Imaging Reporting and Data System. Thyroid nodule prevalence was also obtained from comparison cohorts. DNA sequencing was performed on thyroid tissue from XP patients who had surgery for thyroid cancer. RESULTS: Thyroid nodules were identified in 18/29 XP patients (62%). The median age of patients with thyroid nodules in our XP cohort (20 years) was younger than that of three comparison groups: 36 years (California study-208 subjects), 48 years (Korean study-24,757 subjects), and 52 years (NIH-682 research subjects). Multiple (2-4) thyroid nodules were found in 12/18 (67%) of the patients with nodules. Autopsy examination revealed follicular adenomas in 4/8 (50%) additional XP patients. DNA sequencing revealed rare mutations in two other XP patients with papillary thyroid cancer. CONCLUSIONS: XP patients have an increased incidence of thyroid nodules at an early age in comparison to the general population. These finding confirm another premature aging feature of XP.

Antiproliferative activity of ecteinascidin 743 is dependent upon transcription-coupled nucleotide-excision repair.

While investigating the novel anticancer drug ecteinascidin 743 (Et743), a natural marine product isolated from the Caribbean sea squirt, we discovered a new cell-killing mechanism mediated by DNA nucleotide excision repair (NER). A cancer cell line selected for resistance to Et743 had chromosome alterations in a region that included the gene implicated in the hereditary disease xeroderma pigmentosum (XPG, also known as Ercc5). Complementation with wild-type XPG restored the drug sensitivity. Xeroderma pigmentosum cells deficient in the NER genes XPG, XPA, XPD or XPF were resistant to Et743, and sensitivity was restored by complementation with wild-type genes. Moreover, studies of cells deficient in XPC or in the genes implicated in Cockayne syndrome (CSA and CSB) indicated that the drug sensitivity is specifically dependent on the transcription-coupled pathway of NER. We found that Et743 interacts with the transcription-coupled NER machinery to induce lethal DNA strand breaks.

Adverse effects of trichothiodystrophy DNA repair and transcription gene disorder on human fetal development.

The effects of DNA repair and transcription gene abnormalities in human pre-natal life have never been studied. Trichothiodystrophy (TTD) is a rare (affected frequency of 10(-6)) recessive disorder caused by mutations in genes involved in nucleotide excision repair (NER) pathway and in transcription. Based on our novel clinical observations, we conducted a genetic epidemiologic study to investigate gestational outcomes associated with TTD. We compared pregnancies resulting in TTD-affected offspring (n = 24) with respect to abnormalities during their antenatal and neonatal periods to pregnancies resulting in their unaffected siblings (n = 18), accounting for correlation, and to population reference values. Significantly higher incidence of several severe gestational complications was noted in TTD-affected pregnancies. Small for gestational age (SGA) <10th percentile [Relative risk (RR ) = 9.3, 95% CI = 1.4-60.5, p = 0.02], SGA <3rd percentile (RR = 7.2, 95% CI = 1.1-48.1, p = 0.04), and neonatal intensive care unit (NICU) hospitalization (RR = 6.4, 95% CI = 1.4-29.5, p = 0.02) occurred more frequently among TTD-affected neonates compared with their unaffected siblings. Compared with reference values from general obstetrical population, pregnancies that resulted in TTD-affected infants were significantly more likely to be complicated by hemolysis, elevated liver enzymes and low platelets (HELLP) syndrome (RR = 35.7, 95% CI = 7.6-92.5, p = 0.0002), elevated mid-trimester maternal serum human chorionic gonadotropin (hCG) levels (RR = 14.3, 95% CI = 7.0-16.6, p < 0.0001), SGA <3rd percentile (RR = 13.9, 95% CI = 7.4-21.1, p < 0.0001), pre-term delivery (<32 weeks) (RR = 12.0, 95% CI = 4.9-21.6, p < 0.0001), pre-eclampsia (RR = 4.0, 95% CI = 1.6-7.4, p = 0.006), and decreased fetal movement (RR = 3.3, 95% CI = 1.6-5.2, p = 0.0018). Abnormal placental development is an underlying mechanism that may explain the constellation of observed complications in our study. Thus, we hypothesize that TTD DNA repair and transcription genes play an important role in normal human placental development.

Trichothiodystrophy: a systematic review of 112 published cases characterises a wide spectrum of clinical manifestations.

Trichothiodystrophy (TTD) is a rare, autosomal recessive disease, characterised by brittle, sulfur deficient hair and multisystem abnormalities. A systematic literature review identified 112 patients ranging from 12 weeks to 47 years of age (median 6 years). In addition to hair abnormalities, common features reported were developmental delay/intellectual impairment (86%), short stature (73%), ichthyosis (65%), abnormal characteristics at birth (55%), ocular abnormalities (51%), infections (46%), photosensitivity (42%), maternal pregnancy complications (28%) and defective DNA repair (37%). There was high mortality, with 19 deaths under the age of 10 years (13 infection related), which is 20-fold higher compared to the US population. The spectrum of clinical features varied from mild disease with only hair involvement to severe disease with profound developmental defects, recurrent infections and a high mortality at a young age. Abnormal characteristics at birth and pregnancy complications, unrecognised but common features of TTD, suggest a role for DNA repair genes in normal fetal development.

Impaired interferon production and natural killer cell activation in patients with the skin cancer-prone disorder, xeroderma pigmentosum.

Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder with sun sensitivity, markedly increased skin cancer susceptibility, and defective DNA repair without consistently identified symptoms of immune deficiency. We examined natural killer (NK) cell activity and interferon production in peripheral blood lymphocytes (PBL) of eight XP patients who had multiple primary skin cancers. The XP patients had normal numbers of T cells and NK cells, as well as normal lymphokine-activated killer cell activity and normal tumor necrosis factor-alpha production. Unstimulated NK cell function was 40% of normal controls in five XP patients, but was normal in three other XP patients. However, PBL from all the XP patients tested showed no enhancement of NK activity by the interferon inducer, polyinosinic acid:polycytidilic acid (polyIC) but enhancement by interferon-alpha was normal, suggesting an impairment in interferon production. Parallel studies in non-XP skin cancer patients revealed that both unstimulated and polyIC-enhanced NK activity were normal. Further investigation using PBL from XP patients revealed that the production of interferon-gamma after stimulation with interferon inducers (polyIC, interleukin 2, or K562 tumor cells) was 13-43% of normals. These data indicate that XP lymphocytes have a defect in production of interferons and suggest that defective interferon production, as well as DNA repair defects, may play an important role in the susceptibility of XP patients to skin cancer.

Abnormal ultraviolet mutagenic spectrum in plasmid DNA replicated in cultured fibroblasts from a patient with the skin cancer-prone disease, xeroderma pigmentosum.

A shuttle vector plasmid, pZ189, was utilized to assess the types of mutations that cells from a patient with xeroderma pigmentosum, complementation group D, introduce into ultraviolet (UV) damaged, replicating DNA. Patients with xeroderma pigmentosum have clinical and cellular UV hypersensitivity, increased frequency of sun-induced skin cancer, and deficient DNA repair. In comparison to UV-treated pZ189 replicated in DNA repair-proficient cells, there were fewer surviving plasmids, a higher frequency of plasmids with mutations, fewer plasmids with two or more mutations in the marker gene, and a new mutagenic hotspot. The major type of base substitution mutation was the G:C to A:T transition with both cell lines. These results, together with similar findings published earlier with cells from a xeroderma pigmentosum patient in complementation group A, suggest that isolated G:C to A:T somatic mutations may be particularly important in generation of human skin cancer by UV radiation.

Retinoid protection against x-ray-induced chromatid damage in human peripheral blood lymphocytes.

Oral administration of isotretinoin (13-cis retinoic acid) was shown previously (Kraemer, K. H., J. J. DiGiovanna, A. N. Moshell, R. E. Tarone, and G. L. Peck. 1988. N. Engl. J. Med. 318:1633-1637) to reduce the frequency of skin cancers in xeroderma pigmentosum (XP) patients. The mechanism of protection was unclear. In the present study, x-ray-induced chromatid damage in PHA-stimulated blood lymphocytes from five XP patients receiving isotretinoin was approximately half that in blood samples from the same patients before or subsequent to treatment. The x-ray-induced chromatid damage in blood lymphocytes from a normal control was reduced significantly by cocultivation with blood or plasma from an XP patient receiving isotretinoin or by addition of 10(-6) M isotretinoin to cultures 1 h before x-irradiation. A similar reduction in x-ray-induced chromatid damage was reported previously by adding to the culture medium, mannitol, a scavenger of the free hydroxyl radical, or catalase, which decomposes hydrogen peroxide; both of these products are generated during ionizing radiation. The present observations suggest that isotretinoin acts as a scavenger of such radiation products, thereby providing protection against x-ray-induced chromatid damage.

Carrier detection in xeroderma pigmentosum.

We were able to detect clinically normal carriers of xeroderma pigmentosum (XP) genes with coded samples of either peripheral blood lymphocytes or skin fibroblasts, using a cytogenetic assay shown previously to detect individuals with cancer-prone genetic disorders. Metaphase cells of phytohemagglutinin-stimulated T-lymphocytes from eight individuals who are obligate heterozygotes for XP were compared with those from nine normal controls at 1.3, 2.3, and 3.3 h after x-irradiation (58 R) during the G2 phase of the cell cycle. Lymphocytes from the XP heterozygotes had twofold higher frequencies of chromatid breaks or chromatid gaps than normal (P less than 10(-5)) when fixed at 2.3 or 3.3 h after irradiation. Lymphocytes from six XP homozygotes had frequencies of breaks and gaps threefold higher than normal. Skin fibroblasts from an additional obligate XP heterozygote, when fixed approximately 2 h after x-irradiation (68 R), had a twofold higher frequency of chromatid breaks and a fourfold higher frequency of gaps than fibroblasts from a normal control. This frequency of aberrations in cells from the XP heterozygote was approximately half that observed in the XP homozygote. The elevated frequencies of chromatid breaks and gaps after G2 phase x-irradiation may provide the basis of a test for identifying carriers of the XP gene(s) within known XP families.

Xeroderma pigmentosum, trichothiodystrophy and Cockayne syndrome: a complex genotype-phenotype relationship.

Patients with the rare genetic disorders, xeroderma pigmentosum (XP), trichothiodystrophy (TTD) and Cockayne syndrome (CS) have defects in DNA nucleotide excision repair (NER). The NER pathway involves at least 28 genes. Three NER genes are also part of the basal transcription factor, TFIIH. Mutations in 11 NER genes have been associated with clinical diseases with at least eight overlapping phenotypes. The clinical features of these patients have some similarities but also have marked differences. NER is involved in protection against sunlight-induced DNA damage. While XP patients have 1000-fold increase in susceptibility to skin cancer, TTD and CS patients have normal skin cancer risk. Several of the genes involved in NER also affect somatic growth and development. Some patients have short stature and immature sexual development. TTD patients have sulfur deficient brittle hair. Progressive sensorineural deafness is an early feature of XP and CS. Many of these clinical diseases are associated with developmental delay and progressive neurological degeneration. The main neuropathology of XP is a primary neuronal degeneration. In contrast, CS and TTD patients have reduced myelination of the brain. These complex neurological abnormalities are not related to sunlight exposure but may be caused by developmental defects as well as faulty repair of DNA damage to neuronal cells induced by oxidative metabolism or other endogenous processes.

UV light-induced cyclobutane pyrimidine dimers are mutagenic in mammalian cells.

We used a simian virus 40-based shuttle vector plasmid, pZ189, to determine the role of pyrimidine cyclobutane dimers in UV light-induced mutagenesis in monkey cells. The vector DNA was UV irradiated and then introduced into monkey cells by transfection. After replication, vector DNA was recovered from the cells and tested for mutations in its supF suppressor tRNA marker gene by transformation of Escherichia coli carrying a nonsense mutation in the beta-galactosidase gene. When the irradiated vector was treated with E. coli photolyase prior to transfection, pyrimidine cyclobutane dimers were removed selectively. Removal of approximately 90% of the pyrimidine cyclobutane dimers increased the biological activity of the vector by 75% and reduced its mutation frequency by 80%. Sequence analysis of 72 mutants recovered indicated that there were significantly fewer tandem double-base changes and G X C----A X T transitions (particularly at CC sites) after photoreactivation of the DNA. UV-induced photoproducts remained (although at greatly reduced levels) at all pyr-pyr sites after photoreactivation, but there was a relative increase in photoproducts at CC and TC sites and a relative decrease at TT and CT sites, presumably due to a persistence of (6-4) photoproducts at some CC and TC sites. These observations are consistent with the fact that mutations were found after photoreactivation at many sites at which only cyclobutane dimers would be expected to occur. From these results we conclude that UV-induced pyrimidine cyclobutane dimers are mutagenic in DNA replicated in monkey cells.

Quantification of expression of linked cloned genes in a simian virus 40-transformed xeroderma pigmentosum cell line.

We wished to determine whether simian virus 40 (SV40)-transformed xeroderma pigmentosum cells, despite their defective DNA repair, were suitable for DNA-mediated gene transfer experiments with linked genes. Expression of a nonselectable gene (cat, coding for chloramphenicol acetyltransferase [CAT]) linked to a selectable gene (gpt, coding for xanthine-guanine phosphoribosyltransferase [XPRT]) in the plasmid pSV2catSVgpt was quantified after transfection of SV40-transformed xeroderma pigmentosum [XP20s(SV40)] and normal human [GM0637(SV40)] fibroblast cell lines. A novel autoradiographic assay with [3H]xanthine incorporation showed 0.5 to 0.7% phenotypic expression of XPRT in both cell lines. Without selection, transient CAT activity was 20 times greater in the GM0637(SV40) than in the XP20s(SV40) cells, and transient XPRT activity was 5 times greater. Both of these transient activities were increased and equalized in both cell lines by transfection with pRSVcat or pRSVgpt. Genotypic transformation to gpt+ occurred at a frequency of 2 X 10(-4) to 4 X 10(-4) in both cell lines with pSV2catSVgpt. After 2 to 3 months in selective medium, stable expression of the (nonselected) cat gene was found in 11 (92%) of 12 gpt-containing clones derived from GM0637(SV40) cells and in 13 (81%) of 16 gpt-containing clones from XP20s(SV40) cells. However, the levels of CAT activity did not correlate with those of XPRT activity, and both of these activities varied more than 100-fold among different clones. Copies (1 to 4) of the gpt gene were integrated in four clones of the GM0637(SV40) cells having an XPRT activity of 1 to 5 nmol/min per mg, but 5 to 80 copies were integrated in four XP20s(SV40) clones with an XPRT activity of 0.8 to 1.8 nmol/min per mg. This study shows that XP20s(SV40) is as suitable for gene transfer experiments as the normal human line GM0637(SV40).

The human XPG gene: gene architecture, alternative splicing and single nucleotide polymorphisms.

Defects in the XPG DNA repair endonuclease gene can result in the cancer-prone disorders xeroderma pigmentosum (XP) or the XP-Cockayne syndrome complex. While the XPG cDNA sequence was known, determination of the genomic sequence was required to understand its different functions. In cells from normal donors, we found that the genomic sequence of the human XPG gene spans 30 kb, contains 15 exons that range from 61 to 1074 bp and 14 introns that range from 250 to 5763 bp. Analysis of the splice donor and acceptor sites using an information theory-based approach revealed three splice sites with low information content, which are components of the minor (U12) spliceosome. We identified six alternatively spliced XPG mRNA isoforms in cells from normal donors and from XPG patients: partial deletion of exon 8, partial retention of intron 8, two with alternative exons (in introns 1 and 6) and two that retained complete introns (introns 3 and 9). The amount of alternatively spliced XPG mRNA isoforms varied in different tissues. Most alternative splice donor and acceptor sites had a relatively high information content, but one has the U12 spliceosome sequence. A single nucleotide polymorphism has allele frequencies of 0.74 for 3507G and 0.26 for 3507C in 91 donors. The human XPG gene contains multiple splice sites with low information content in association with multiple alternatively spliced isoforms of XPG mRNA.

In vitro assay of the effects of psoralens plus ultraviolet radiation on human lymphoid cells.

An in vitro assay system that mimics some of the features of in vivo oral methoxsalen photochemotherapy (PUVA) in relation to human lymphoid cells was developed. In this assay, suspension cultures of human lymphoid cells were subjected to therapeutic concentrations of 8-MOP and to a UVA radiation spectrum modified to approximate the dermal exposure. The assay permitted correlation and quantitation of multiple 8-MOP plus UVA-induced biological and physical alterations in the same assay system. Thus were demonstrated inhibition of DNA synthesis, reduction of cell survival, production of DNA cross-links, and loss of mixed leukocyte reactivity induced by combinations of 8-MOP and UVA in or near the presumed therapeutic range. This assay may be useful for predicting lymphoid cellular toxicity of other photoactive agents as well as for examining the molecular effects of these agents.

Analysis of point mutations in an ultraviolet-irradiated shuttle vector plasmid propagated in cells from Japanese xeroderma pigmentosum patients in complementation groups A and F.

To assess the contribution to mutagenesis by human DNA repair defects, a UV-treated shuttle vector plasmid, pZ189, was passed through fibroblasts derived from Japanese xeroderma pigmentosum (XP) patients in two different DNA repair complementation groups (A and F). Patients with XP have clinical and cellular UV hypersensitivity, increased frequency of skin cancer, and defects in DNA repair. The XP DNA repair defects represented by complementation groups A (XP-A) and F (XP-F) are more common in Japan than in Europe or the United States. In comparison to results with DNA repair-proficient human cells (W138-VA13), UV-treated pZ189 passed through the XP-A [XP2OS(SV)] or XP-F [XP2YO(SV)] cells showed fewer surviving plasmids (XP-A less than XP-F) and a higher frequency of mutated plasmids (XP-A greater than XP-F). Base sequence analysis of more than 200 mutated plasmids showed the major type of base substitution mutation to be the G:C----A:T transition with all three cell lines. The XP-A and XP-F cells revealed a higher frequency of G:C----A:T transitions and a lower frequency of transversions among plasmids with single or tandem mutations and a lower frequency of plasmids with multiple point mutations compared to the normal line. The spectrum of mutations in pZ189 with the XP-A cells was similar to that with the XP-F cells. Seventy-six to 91% of the single base substitution mutations occurred at G:C base pairs in which the 5'-neighboring base of the cytosine was thymine or cytosine. These studies indicate that the DNA repair defects in Japanese XP patients in complementation groups A and F result in different frequencies of plasmid survival and mutagenesis but in similar types of mutagenic abnormalities despite marked differences in clinical features. These results, together with comparable studies from United States patients in XP complementation groups A and D, suggest that G:C----A:T somatic mutations may be important in the generation of human skin cancer by UV radiation.

Detection of ataxia telangiectasia heterozygous cell lines by postirradiation cumulative labeling index: measurements with coded samples.

Coded skin fibroblast cell strains from ataxia telangiectasia (AT) families and apparently normal individuals were obtained from two different sources. AT homozygous strains were clearly identified on the basis of marked hypersensitivity to cell killing by X-irradiation. AT heterozygotes were intermediate in their cytotoxic response between AT homozygotes and five normal reference cell strains. When density-inhibited cultures were X-irradiated and immediately subcultured to low density, a large fraction of AT heterozygous cells were irreversibly blocked in G1 as determined by cumulative labeling indices following incubation with [3H]thymidine. No such block occurred in four reference normal or AT homozygous strains. Three coded cell strains from apparently normal individuals resembled AT heterozygotes in their response; two of these strains were heterozygous for lysosomal storage disease. Thus, although the phenotype associated with the cellular response of AT heterozygous cells to X-irradiation is not specific to this disorder, the cumulative labeling indices assay may be a useful method for the detection of AT heterozygotes in kindreds with known AT.

Ultraviolet mutagenesis in a plasmid vector replicated in lymphoid cells from patient with the melanoma-prone disorder dysplastic nevus syndrome.

The hereditary dysplastic nevus syndrome (DNS) is an autosomal dominant disorder in which affected individuals have increased numbers of dysplastic (premalignant) nevi and a greater than 100-fold increased risk of developing cutaneous melanoma. Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with hereditary DNS have been shown to be hypermutable to UV radiation (M.I.R. Perera et al., Cancer Res., 46: 1005-1009, 1986). To examine the mechanism involved in this UV hypermutability, we used a shuttle vector plasmid, pZ189, which carries a 160-base pair marker gene, supF, and can replicate in human cells. pZ189 was treated with UV radiation and transfected into DNS6BE, a lymphoblastoid cell line from a patient with hereditary DNS. Plasmid survival after UV was similar with the DNS6BE line and with a lymphoblastoid cell line from a normal donor. Plasmid mutation frequency was greater with the DNS line in accord with the DNS cellular hypermutability. Base sequence analysis was performed on 69 mutated plasmids recovered from the DNS line. There were significantly more plasmids with single base substitution mutations (P less than 0.01) in comparison to UV-treated plasmids passed through normal fibroblasts. pZ189 hypermutability and an increased frequency of single base substitutions was previously found with a cell line from a melanoma-prone xeroderma pigmentosum patient. These differences may be related to the increased melanoma susceptibility in both DNS and xeroderma pigmentosum.

Hereditary dysplastic nevus syndrome: lymphoid cell ultraviolet hypermutability in association with increased melanoma susceptibility.

The hereditary dysplastic nevus syndrome (DNS) is a well-characterized disorder in which affected individuals have increased numbers of premalignant (dysplastic) nevi and a markedly increased risk of developing cutaneous melanoma. Seeking evidence of a systemic disorder in DNS, we examined the effect of ultraviolet radiation on cultured lymphoid cells. Epstein-Barr virus-transformed lymphoblastoid cell lines from patients with hereditary DNS had similar survival values following treatment with 2.3 to 9.0 J of 254-nm ultraviolet radiation per m2 as did lines from control individuals. Mutagenesis at the hypoxanthineguanine phosphoribosyltransferase locus was assessed by measuring the induction of resistance to thioguanine using a microtiter well assay. Three lymphoblastoid cell lines from patients with hereditary DNS and melanoma had a 2- to 3-fold greater frequency of induced mutants per clonable cell than three normal lines following exposure to 4.5 to 9.0 J of ultraviolet radiation per m2. Expanded clones of mutated DNS lymphoblastoid cell lines had less than 6% of normal hypoxanthine-guanine phosphoribosyltransferase activity. Inhibition and recovery of DNA synthesis following ultraviolet exposure were similar in 2 DNS and 2 normal lines. Repair by DNS lines of ultraviolet-induced DNA damage was in the normal range as measured by alkaline elution. Thus, hereditary DNS exhibits in vitro hypermutability which may reflect increased susceptibility to ultraviolet-induced somatic mutations in vivo. This abnormality may be related to the increased melanoma susceptibility of patients with hereditary DNS.

Sequence specificity of aflatoxin B1-induced mutations in a plasmid replicated in xeroderma pigmentosum and DNA repair proficient human cells.

The mutagenic spectrum induced by aflatoxin-DNA lesions in DNA repair deficient and repair proficient human cells was investigated. The reactive metabolite aflatoxin B1-8,9-epoxide was synthesized and reacted in vitro with the shuttle vector plasmid pS189. Plasmids were transfected into human fibroblasts and allowed to replicate, and the recovered plasmids were screened in indicator bacteria for plasmid survival and mutations in the supF marker gene. Sequence data were obtained from 71 independently arising mutants recovered from DNA repair deficient xeroderma pigmentosum (XP) cells [XP12BE(SV40)] and 60 mutants recovered from a DNA repair proficient cell line (GM0637). Plasmid survival was lower and mutation frequency higher with the XP cells, and the mutation hotspots differed substantially for the 2 cell lines. Most mutations (> 90%) were base substitutions at G:C pairs, only about one-half of which were G:C-->T:A transversions, the expected predominant mutation. One-third of the mutations at GG sites and none of those at isolated Gs were G:C-->A:T transitions. Tandem base substitutions also occurred only at GG sites and were found only with XP cells. The location of mutation hotspots with either cell line did not correlate with the level of modification within the sequence as assessed by a DNA polymerase stop assay. These results suggest that the DNA repair deficiency associated with XP can influence not only the overall frequency of mutations but also the distribution of mutations within a gene. The finding of transition mutations exclusively at GG sites may be of predictive value in attempts to link dietary aflatoxin exposure to cancers associated with specific mutations in the c-ras oncogene and the p53 tumor suppressor gene.