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.

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.

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.

Enhancement of antioxidant and phase II enzymes by oral feeding of green tea polyphenols in drinking water to SKH-1 hairless mice: possible role in cancer chemoprevention.

Following the oral feeding of a polyphenolic fraction isolated from green tea (GTP) in drinking water, an increase in the activities of antioxidant and phase II enzymes in skin, small bowel, liver, and lung of female SKH-1 hairless mice was observed. GTP feeding (0.2%, w/v) to mice for 30 days significantly increased the activities of glutathione peroxidase, catalase, and quinone reductase in small bowel, liver, and lungs, and glutathione S-transferase in small bowel and liver. GTP feeding to mice also resulted in considerable enhancement of glutathione reductase activity in liver. In general, the increase in antioxidant and phase II enzyme activities was more pronounced in lung and small bowel as compared to liver and skin. The significance of these results can be implicated in relation to the cancer chemopreventive effects of GTP against the induction of tumors in various target organs.

An intronic poly (AT) polymorphism of the DNA repair gene XPC and risk of squamous cell carcinoma of the head and neck: a case-control study.

Inherited polymorphisms of DNA repair genes may contribute to variations in DNA repair capacity and genetic susceptibility to cancer. In a hospital-based case-control study of 287 non-Hispanic white patients with newly diagnosed SCCHN and 311 control subjects matched on age, sex, ethnicity, and smoking status, we investigated the role of a newly identified variant allele of XPC, XPC-PAT+. We found that the frequency of the XPC-PAT+ allele was higher in the cases (0.409) than in the controls (0.333; P = 0.007). Fifty cases (17.4%) and 37 controls (11.9%) were XPC-PAT+/+, and 135 (47.0%) cases and 133 controls (42.8%) were XPC-PAT+/-. XPC-PAT+/- and XPC-PAT+/+ subjects were at significantly increased risk for SCCHN [adjusted odds ratios = 1.44 and 1.85, respectively (95% confidence intervals, 1.01-2.05 and 1.12-3.05, respectively; trend test, P = 0.007)]. We did not find ethnic difference in the frequency of XPC-PAT+ allele among four groups aged between 19 and 75 years: non-Hispanic whites, 294; African-Americans, 178; Hispanic-Americans, 103; and native Chinese, 119 (0.333, 0.281, 0.296, and 0.353, respectively). The case-control findings support the hypothesis that the XPC-PAT+ allele may contribute to the risk of developing SCCHN.

Ras p21 farnesylation in ultraviolet B radiation-induced tumors in the skin of SKH-1 hairless mice.

Cutaneous exposure to solar ultraviolet B (UVB) radiation is well recognized as the major cause of skin cancer in humans; however, the precise molecular mechanisms whereby UVB mediates carcinogenesis remains unclear. The involvement of activated ras oncogenes has been demonstrated extensively in both animal and human skin cancers. Activated ras oncogenes encode mutated ras p21 that exist in the guanosine triphosphate-bound active state and, following localization to the inner side of the plasma membrane, cause cellular transformation. This membrane association requires three post-translational modifications occurring at the C-terminus of the ras p21. The farnesylation of p21 by a cytosolic enzyme known as farnesyltransferase (FTase) is the critical step that triggers biologic functions of the ras p21. In this study, FTase activity was found to be substantially higher (approximately threefold) in UVB radiation-induced tumors in SKH-1 hairless mice compared to epidermis from controls. Western blot analysis showed significantly higher levels of Ha-ras p21 in both cytosolic and membrane fractions prepared from tumors compared to epidermis. Pan ras antibody against mutated p21 at codon 12 showed very strong reactivity for ras val-12p21 in tumors but not in normal epidermis, suggesting a gly to val substitution at 12th position in ras p21 in UVB-induced tumors. Our data indicate that enhanced FTase activity and the processing of overexpressed p21 in UVB-induced tumors are correlated, and predict the role of point mutation at the 12th codon of the ras oncogene during photocarcinogenesis in mice.

Clinical, cellular, and molecular features of an Israeli xeroderma pigmentosum family with a frameshift mutation in the XPC gene: sun protection prolongs life.

An Ashkenazi Jewish Israeli family with two children affected with severe xeroderma pigmentosum was investigated. A son, XP12TA, developed skin cancer at 2 y and died at 10 y. A daughter, XP25TA, now 24 y old, was sun protected and began developing skin cancers at 10 y. Their cultured skin fibroblasts showed reductions in post-ultraviolet survival (11% of normal), unscheduled DNA synthesis (10% of normal), global genome DNA repair (15% of normal), and plasmid host cell reactivation (5% of normal). Transcription-coupled DNA repair was normal, however. Northern blot analysis revealed greatly reduced xeroderma pigmentosum complementation group C mRNA. A plasmid host cell reactivation assay assigned the cells to xeroderma pigmentosum complementation group C. Cells from both parents and an unaffected child exhibited normal post-ultraviolet-C survival and normal DNA repair. Sequencing the xeroderma pigmentosum complementation group C cDNA of XP12TA and XP25TA revealed a homozygous deletion of two bases (del AT 669-670) in exon 5 with a new termination site 10 codons downstream that is expected to encode a truncated xeroderma pigmentosum complementation group C protein. Sequence analysis of the xeroderma pigmentosum complementation group C cDNA in cells from the parents found identical heterozygous mutations: one allele carries both the exon 5 frameshift and an exon 15 polymorphism and the other allele carries neither alteration. Cells from the unaffected brother had two normal xeroderma pigmentosum complementation group C alleles. This frameshift mutation in the xeroderma pigmentosum complementation group C gene led to reduced DNA repair with multiple skin cancers and early death. Sun protection delayed the onset of skin cancer and prolonged life in a sibling with the same mutation.

Xeroderma pigmentosum group C splice mutation associated with autism and hypoglycinemia.

A 4 y old boy of Korean ancestry had xeroderma pigmentosum (XP) with sun sensitivity, multiple cutaneous neoplasms, and inability to speak. Neurologic examination revealed hyperactivity and autistic features without typical XP neurologic abnormalities. Cultured skin fibroblasts (XP22BE) showed decreased post-UV survival, reduced post-UV plasmid host cell reactivation and defective DNA repair (16% of normal unscheduled DNA synthesis in intact cells and undetectable excision repair in a cell free extract). In vitro and in vivo complementation assigned XP22BE to XP group C (XPC) and a markedly reduced level of XPC mRNA was found. Two XPC cDNA bands were identified. One band had a deletion of 161 bases comprising the entire exon 9, which resulted in premature termination of the mutant XPC mRNA. The larger band also had the same deletion of exon 9 but, in addition, had an insertion of 155 bases in its place (exon 9a), resulting in an in-frame XPC mRNA. Genomic DNA analysis revealed a T-->G mutation at the splice donor site of XPC exon 9, which markedly reduced its information content. The 155 base pair XPC exon 9a insertion was located in intron 9 and was flanked by strong splice donor and acceptor sequences. Analysis of the patient's blood showed persistently low levels of glycine (68 microM; NL, 125-318 microM). Normal glycine levels were maintained with oral glycine supplements and his hyperactivity diminished. These data provide evidence of an association of an XPC splice site mutation with autistic neurologic features and hypoglycinemia.

A stop codon in xeroderma pigmentosum group C families in Turkey and Italy: molecular genetic evidence for a common ancestor.

Xeroderma pigmentosum family G from Van, Turkey had two severely affected children: a son with multiple skin cancers who died at age 10 (XP67TMA), and an 8 y old daughter who began developing skin cancer before 3 y of age (XP68TMA). XP67TMA and XP68TMA cells were hypersensitive to killing by ultraviolet and the post-ultraviolet DNA repair level was 12-16% of normal. Host cell reactivation of an ultraviolet-treated reporter plasmid cotransfected with a vector expressing wild-type XPC cDNA assigned XP67TMA to xeroderma pigmentosum complementation group C. The XPC mRNA level was markedly reduced. Sequencing of the 3.5 kb XPC cDNA from XP67TMA showed a C-T mutation in XPC exon 8 at base pair 1840. This mutation converts the CGA codon of arginine at amino acid 579 to a UGA stop codon resulting in marked truncation of the 940 amino acid xeroderma pigmentosum C protein. Restriction fragment length polymorphism analysis of XPC exon 8 DNA in XP67TMA and XP68TMA showed that both affected children had a homozygous mutation and that both parents had heterozygous normal and mutated sequences at the same position consistent with a history of consanguinity in the family. The mutated allele also contained two XPC single nucleotide polymorphisms. The same mutated XPC allele was reported in an Italian family. Studies of 19 microsatellite markers flanking the XPC gene on chromosome 3 suggest that the XPC allele passed between Italy and Turkey approximately 300-500 y ago. This XPC allele containing a nonsense mutation is associated with severe clinical disease with multiple skin cancers and early death.

Effect of coexposure to asbestos and kerosene soot on pulmonary drug-metabolizing enzyme system.

This article reports the effect of coexposure to Indian chrysotile asbestos (5 mg/rat) and kerosene soot (5 mg/rat) on the pulmonary phase I and phase II drug-metabolizing enzymes 1, 4, 8, 16, 30, 90, and 150 days after a single intratracheal inoculation. Exposure to soot resulted in a significant induction of the pulmonary microsomal cytochrome P450 and the activity of dependent monooxygenase, benzo(a)pyrene (B[a]P) hydroxylase, and epoxide hydrase at all time intervals. On the other hand, the cytosolic glutathione S-transferase (GST) activity was induced at days 1, 4, 8, 16, and 30 after exposure, followed by inhibition in the enzyme activity. In contrast, chrysotile exposure depleted cytochrome P450, B[a]P hydroxylase, epoxide hydrase, and GST at initial stages, while all these parameters except GST were induced at later stages. However, coexposure to chrysotile and soot led to a significant inhibition in the cytochrome P450 levels, activities of B[a]P hydroxylase, epoxide hydrase, and GST at initial stages of exposure. At advanced stages, however, an additional increase in cytochrome P450, B[a]P hydroxylase, and epoxide hydrase but a decrease in GST was observed. These results clearly show that the intratracheal coexposure to high levels of asbestos and kerosene soot alters the metabolic activity of the lung, which is turn may retain toxins in the system for a longer period, resulting in adverse pathological disorders.

Mechanism of asbestos-mediated DNA damage: role of heme and heme proteins.

Several observations, including studies from this laboratory, demonstrate that asbestos generates free radicals in the biological system that may play a role in the manifestation of asbestos-related cytotoxicity and carcinogenicity. It has also been demonstrated that iron associated with asbestos plays an important role in the asbestos-mediated generation of reactive oxygen species. Exposure to asbestos leads to degradation of heme proteins such as cytochrome P450-releasing heme in cytosol. Our simulation experiments in the presence of heme show that such asbestos-released heme may increase lipid peroxidation and can cause DNA damage. Further, heme and horseradish peroxidase (HRP) can cause extensive DNA damage in the presence of asbestos and hydrogen peroxide/organic peroxide/hydroperoxides. HRP catalyzes oxidation reactions in a manner similar to that of prostaglandin H synthetase. Iron released from asbestos is only partially responsible for DNA damage. However, our studies indicate that DNA damage mediated by asbestos in vivo may be caused by a combination of effects such as the release and participation of iron, heme, and heme moiety of prostaglandin H synthetase in free radical generation from peroxides and hydroperoxides.

Subjective and objective visual acuity testing techniques.

The Catford apparatus for determining the objective visual acuity was elevated with 20 normal (20 eyes) and 40 abnormal (75 diseased eyes) patients. The vision of the normal individuals was fogged with neutral-density filters and convex lenses. Eyes with normal or near normal vision showed good correlation between optokinetic response and visual acuity, but no correlation was observed in eyes with poor vision. These findings, which vary from those of Catford, indicate that objective methods of visual acuity testing using a nystagmoid response do not appear useful for general clinical purposes.

Intravitreal hemorrhage associated with rapid increase in intracranial pressure (Terson's syndrome).

Four patients manifested Terson's syndrome in association with increased intracranial pressure. Three patients had subarachnoid hemorrhages, while the fourth suffered strangulation. Vitreous hemorrhage probably is related to the rapid increase in intracranial pressure with compression of the central retinal vein and its choroidal anastamotic channels.

Protection against ultraviolet B radiation-induced effects in the skin of SKH-1 hairless mice by a polyphenolic fraction isolated from green tea.

In prior studies we and others have shown that oral feeding of a polyphenolic fraction isolated from green tea (GTP) or water extract of green tea affords protection against ultraviolet B (UVB) radiation-induced carcinogenesis in SKH-1 hairless mice (Wang et al., Carcinogenesis 12, 1527-1530, 1991). It is known that exposure of murine skin to UVB radiation results in cutaneous edema, depletion of the antioxidant-defense system and induction of ornithine decarboxylase (ODC) and cyclooxygenase activities. In this study we assessed the protective effect of GTP on these UVB radiation-caused changes in murine skin. Oral feeding of 0.2% GTP (wt/vol) as the sole source of drinking water for 30 days to SKH-1 hairless mice followed by irradiation with UVB (900 mJ/cm2) resulted in significant protection against UVB radiation-caused cutaneous edema (P < 0.0005) and depletion of the antioxidant-defense system in epidermis (P < 0.01-0.02). The oral feeding of GTP also resulted in significant protection against UVB radiation-caused induction of epidermal ODC (P < 0.005-0.01) and cyclooxygenase activities (P < 0.0001) in a time-dependent manner. Our data indicate that the inhibition of UVB radiation-caused changes in these markers of tumor promotion in murine skin by GTP may be one of the possible mechanisms of chemopreventive effects associated with green tea against UVB-induced tumorigenesis. The results of this study suggest that green tea, specifically polyphenols present therein, may be useful against inflammatory responses associated with the exposure of skin to solar radiation.

Effect of chrysotile asbestos on cytochrome P-450-dependent monooxygenase and glutathione-S-transferase activities in rat lung.

The in vitro and in vivo effect of a carcinogenic variety of asbestos, chrysotile, both on xenobiotic metabolizing enzymes such as benzo[a]pyrene hydroxylase, epoxide hydrolase as well as glutathione-S-transferase activities and microsomal lipid peroxidation in rat lung were examined. The in vitro incubation of chrysotile with microsomes significantly adsorbed heme proteins, cytochrome P-450 and P-448 with the concomitant decrease in the dependent monooxygenase activities. The prolonged incubation of this mineral fibre with microsomes also resulted in the release of heme. It also led to the depletion in the activities of epoxide hydrolase and glutathione-S-transferase. However, it induced lipid peroxidation. When these in vitro effects were validated in vivo, the exposure to early stages produced similar alterations as observed in in vitro studies. However, reverse pattern in the alterations was observed after 90 days of exposure except in the case of lipid peroxidation which remained induced.

Strand breakage in DNA by silicic acid.

The alkaline unwinding assay has been used to demonstrate the formation of single-strand breaks in DNA on treatment with silicic acid. Double-stranded DNA, containing no single-strand breaks, when incubated with increasing concentrations of silicic acid, showed the formation of an increasing number of strand breaks per molecule. Experiments on reduction of silicic acid-treated DNA with NaBH4 suggested the possibility of creation of apurinic or apyrimidinic sites. The significance of silicic acid interaction with cellular DNA during asbestos exposure is discussed.

A rapid and convenient filter-binding assay for ras p21 processing enzyme farnesyltransferase.

Because it is the target for the development of anti-cancer agents, the mammalian cytosolic enzyme farnesyltransferase (FTase) has received significant attention in recent years. FTase catalyzes the transfer of a farnesyl group from farnesylpyrophosphate (FPP) to cysteine 185/186 at the carboxyl terminal end of ras proteins (ras p21), a reaction essential for the localization of ras p21 to the plasma membrane for their cellular functions including cell transformation in case of oncogenic ras p21. Here, we report the development of a rapid and convenient assay procedure for FTase using phosphocellulose paper which has a binding affinity for proteins. The FTase is assayed as the transfer of [3H]farnesyl group from [3H]FPP to the ras p21 at pH 7.4 and 37 degrees C in the presence of rat brain cytosol followed by the binding of radioactive farnesylated ras p21 to the phosphocellulose paper. The radioactivity associated with ras p21 bound to the phosphocellulose paper was determined by scintillation counting after soaking the paper in trichloroacetic acid and washing with distilled water. Utilizing [3H]FPP and recombinant Ha-ras p21 as substrates in the reaction, the FTase followed Michaelis-Menten kinetics with Km values of 1.0 and 7.69 microM for respectively [3H]FPP and recombinant Ha-ras p21. The method reported here has the advantages over the other published assay procedures of being rapid, convenient and economical, and can be successfully used for the basic assaying of FTase in different organs and distinct species and for the screening of novel inhibitors of FTase.