Adult-onset xeroderma pigmentosum neurological disease--observations in an autopsy case.

Xeroderma pigmentosum (XP) is an inherited disease with defective DNA repair. Patients develop skin cancer because of unrepaired DNA damage produced by the ultraviolet radiation (UV) in sunlight. Many XP children also develop XP neurological disease (ND), consisting of sensorineural hearing loss (SNHL) and a primary neuronal degeneration of the central and peripheral nervous systems. Since the harmful UV in sunlight cannot reach the nervous system, the cause of the death of XP neurons has been hypothesized to result from the inability to repair their DNA that has been damaged by endogenous metabolites. Progressive XP ND originating in an adult has been identified in only a single case. Although clinically asymptomatic at the age of 47 years, the patient had audiometric evidence of a developing mild SNHL together with elicited signs and electrophysiologic evidence of a peripheral neuropathy. She died of metastatic endocervical adenocarcinoma at 49 years of age. We describe here the neuropathological findings in this patient, including examination of the inner ear. Despite clinical evidence of SNHL, there were no anatomic abnormalities of the inner ear. However, the dorsal root ganglia (DRG) showed ongoing neuronal loss. Our findings indicate that XP ND originating in this adult is, like XP ND in children, a primary neuronal degeneration that manifests first in the peripheral nervous system.

A 32P-postlabeling assay for the oxidative DNA lesion 8,5'-cyclo-2'-deoxyadenosine in mammalian tissues: evidence that four type II I-compounds are dinucleotides containing the lesion in the 3' nucleotide.

8,5'-Cyclopurine-2'-deoxynucleotides, which are strong blocks to mammalian DNA and RNA polymerases, represent a novel class of oxidative DNA lesion in that they are specifically repaired by nucleotide excision repair but not by base excision repair or direct enzymatic reversion. Previous studies using thin layer chromatography of (32)P-postlabeled DNA digests have detected several bulky oxidative lesions of unknown structure, called I-compounds, in DNA from normal mammalian organs. We investigated whether any of these type II I-compounds contained 8,5'-cyclo-2'-deoxyadenosine (cA). Two previously detected type II I-compounds were found to be dinucleotides of the sequence pAp-cAp and pCp-cAp. Furthermore, a modification of the technique resulted in detection of two additional I-compounds, pTp-cAp and pGp-cAp. Each I-compound isolated from neonatal rat liver DNA matched authentic (32)P-labeled cA-containing chromatographic standards under nine different chromatographic conditions. Their levels increased significantly after normal birth. The (32)P-postlabeling technique used here is capable of detecting 1-5 lesions/diploid mammalian cell. Thus, it should now be possible to detect changes of cA levels resulting from low level ionizing radiation and other conditions associated with oxidative stress, and to assess cA levels in tissues from patients with the genetic disease xeroderma pigmentosum who are unable to carry out nucleotide excision repair.

The oxidative DNA lesion 8,5'-(S)-cyclo-2'-deoxyadenosine is repaired by the nucleotide excision repair pathway and blocks gene expression in mammalian cells.

Xeroderma pigmentosum (XP) patients with inherited defects in nucleotide excision repair (NER) are unable to excise from their DNA bulky photoproducts induced by UV radiation and therefore develop accelerated actinic damage, including cancer, on sun-exposed tissue. Some XP patients also develop a characteristic neurodegeneration believed to result from their inability to repair neuronal DNA damaged by endogenous metabolites since the harmful UV radiation in sunlight does not reach neurons. Free radicals, which are abundant in neurons, induce DNA lesions that, if unrepaired, might cause the XP neurodegeneration. Searching for such a lesion, we developed a synthesis for 8,5'-(S)-cyclo-2'-deoxyadenosine (cyclo-dA), a free radical-induced bulky lesion, and incorporated it into DNA to test its repair in mammalian cell extracts and living cells. Using extracts of normal and mutant Chinese hamster ovary (CHO) cells to test for NER and adult rat brain extracts to test for base excision repair, we found that cyclo-dA is repaired by NER and not by base excision repair. We measured host cell reactivation, which reflects a cell's capacity for NER, by transfecting CHO and XP cells with DNA constructs containing a single cyclo-dA or a cyclobutane thymine dimer at a specific site on the transcribed strand of a luciferase reporter gene. We found that, like the cyclobutane thymine dimer, cyclo-dA is a strong block to gene expression in CHO and human cells. Cyclo-dA was repaired extremely poorly in NER-deficient CHO cells and in cells from patients in XP complementation group A with neurodegeneration. Based on these findings, we propose that cyclo-dA is a candidate for an endogenous DNA lesion that might contribute to neurodegeneration in XP.

Cockayne syndrome and xeroderma pigmentosum.

OBJECTIVES: To review genetic variants of Cockayne syndrome (CS) and xeroderma pigmentosum (XP), autosomal recessive disorders of DNA repair that affect the nervous system, and to illustrate them by the first case of xeroderma pigmentosum-Cockayne syndrome (XP-CS) complex to undergo neuropathologic examination. METHODS: Published reports of clinical, pathologic, and molecular studies of CS, XP neurologic disease, and the XP-CS complex were reviewed, and a ninth case of XP-CS is summarized. RESULTS: CS is a multisystem disorder that causes both profound growth failure of the soma and brain and progressive cachexia, retinal, cochlear, and neurologic degeneration, with a leukodystrophy and demyelinating neuropathy without an increase in cancer. XP presents as extreme photosensitivity of the skin and eyes with a 1000-fold increased frequency of cutaneous basal and squamous cell carcinomas and melanomas and a small increase in nervous system neoplasms. Some 20% of patients with XP incur progressive degeneration of previously normally developed neurons resulting in cortical, basal ganglia, cerebellar, and spinal atrophy, cochlear degeneration, and a mixed distal axonal neuropathy. Cultured cells from patients with CS or XP are hypersensitive to killing by ultraviolet (UV) radiation. Both CS and most XP cells have defective DNA nucleotide excision repair of actively transcribing genes; in addition, XP cells have defective repair of the global genome. There are two complementation groups in CS and seven in XP. Patients with the XP-CS complex fall into three XP complementation groups. Despite their XP genotype, six of nine individuals with the XP-CS complex, including the boy we followed up to his death at age 6, had the typical clinically and pathologically severe CS phenotype. Cultured skin and blood cells had extreme sensitivity to killing by UV radiation, DNA repair was severely deficient, post-UV unscheduled DNA synthesis was reduced to less than 5%, and post-UV plasmid mutation frequency was increased. CONCLUSIONS: The paradoxical lack of parallelism of phenotype to genotype is unexplained in these disorders. Perhaps diverse mutations responsible for UV sensitivity and deficient DNA repair may also produce profound failure of brain and somatic growth, progressive cachexia and premature aging, and tissue-selective neurologic deterioration by their roles in regulation of transcription and repair of endogenous oxidative DNA damage.

DNA repair and ultraviolet mutagenesis in cells from a new patient with xeroderma pigmentosum group G and cockayne syndrome resemble xeroderma pigmentosum cells.

Xeroderma pigmentosum (XP)/Cockayne syndrome (CS) complex is a combination of clinical features of two rare genetic disorders in one individual. A sun-sensitive boy (XP20BE) who had severe symptoms of CS, with dwarfism, microcephaly, retinal degeneration, and mental impairment, had XP-type pigmentation and died at 6 y with marked cachexia (weight 14.5 lb) without skin cancers. We evaluated his cultured cells for characteristic CS or XP DNA-repair abnormalities. The level of ultraviolet (UV)-induced unscheduled DNA synthesis was less than 5% of normal, characteristic of the excision-repair defect of XP. Cell fusion studies indicated that his cells were in XP complementation group G. His cells were hypersensitive to killing by UV, and their post-UV recovery of RNA synthesis was abnormally low, features of both CS and XP. Post-UV survival of plasmid pSP189 in his cells was markedly reduced, and post-UV plasmid mutation frequency was higher than with normal cells, as in both CS and XP. Sequence analysis of the mutated plasmid marker gene showed normal frequency of plasmids with multiple base substitutions, as in CS, and an abnormally increased frequency of G:C-->A:T mutations, a feature of XP. Transfection of UV-treated pRSVcat with or without photoreactivation revealed that his cells, like XP cells, could not repair either cyclobutane pyrimidine dimers or non-dimer photoproducts. These results indicate that the DNA-repair features of the XP20BE (XP-G/CS) cells are phenotypically more like XP cells than CS cells, whereas clinically the CS phenotype is more prominent than XP.

Fluorescent light-induced chromatid breaks distinguish Alzheimer disease cells from normal cells in tissue culture.

The neurodegeneration and amyloid deposition of sporadic Alzheimer disease (AD) also occur in familial AD and in all trisomy-21 Down syndrome (DS) patients, suggesting a common pathogenetic mechanism. We investigated whether defective processing of damaged DNA might be that mechanism, as postulated for the neurodegeneration in xeroderma pigmentosum, a disease with defective repair not only of UV radiation-induced, but also of some oxygen free radical-induced, DNA lesions. We irradiated AD and DS skin fibroblasts or blood lymphocytes with fluorescent light, which is known to cause free radical-induced DNA damage. The cells were then treated with either beta-cytosine arabinoside (araC) or caffeine, and chromatid breaks were quantified. At least 28 of 31 normal donors and 10 of 11 donors with nonamyloid neurodegenerations gave normal test results. All 12 DS, 11 sporadic AD, and 16 familial AD patients tested had abnormal araC and caffeine tests, as did XP-A cells. In one of our four AD families, an abnormal caffeine test was found in all 10 afflicted individuals (including 3 asymptomatic when their skin biopsies were obtained) and in 8 of 11 offspring at a 50% risk for AD. Our tests could prove useful in predicting inheritance of familial AD and in supporting, or rendering unlikely, the diagnosis of sporadic AD in patients suspected of having the disease.

Gene specific DNA repair of damage induced in familial Alzheimer disease cells by ultraviolet irradiation or by nitrogen mustard.

We have measured gene specific DNA repair in a normal human fibroblast cell line, and in fibroblast lines from two patients with familial Alzheimer disease (AD). Cells were treated with either ultraviolet radiation (UV) or the chemotherapeutic alkylating agent, nitrogen mustard (HN2). DNA damage formation and repair were studied in the active dihydrofolate reductase (DHFR) gene for the main lesions introduced by each of these two types of DNA damaging agents. The gene specific repair of UV induced cyclobutane pyrimidine dimers in the human DHFR gene was 86% complete in the AD cells after 24 h of repair incubation. This repair efficiency was similar to what we and others have found in normal human fibroblasts. After treatment of the AD cells with HN2, we found the frequency of HN2 induced lesions in the DHFR gene to be similar to the frequency in the transcriptionally inactive delta-globin gene. The gene specific repair of HN2 induced lesions in the DHFR gene was completed within 8-24 h in the normal fibroblast line and in the familial AD line, and the repair kinetics were similar for both cell lines. These results indicate that familial AD fibroblasts have normal gene specific repair of both UV induced and HN2 induced DNA damage in active genes.

Gene-specific DNA repair in xeroderma pigmentosum complementation groups A, C, D, and F. Relation to cellular survival and clinical features.

We have examined the gene- and strand-specific DNA repair of UV-induced cyclobutane pyrimidine dimers in fibroblasts from normal individuals and from patients with the DNA repair-deficient disorder xeroderma pigmentosum (XP). Cells were studied from XP complementation groups A, C, D, and F. DNA repair was assessed in the essential, active gene, dihydrofolate reductase (DHFR), in the active c-myc protooncogene, and in the transcriptionally inactive delta-globin gene. In addition, repair was studied in the individual strands of the DHFR gene in normal and group C cells. In the two strains of group C cells, we find preferential DNA repair of the DHFR gene and a strand bias of the repair with more repair in the transcribed strand. This is in general accordance with previously published reports (Venema, J., van Hoffen, A., Natarajan, A.T., van Zeeland, A.A., and Mullenders, L.H.F. (1990) Nucleic Acids Res. 18, 443-448; Venema, J., van Hoffen, A., and Mullenders, L.H.F. (1991) Mol. Cell. Biol. 11, 4128-4134), but we now find that there is more repair in the nontranscribed strand and less in the transcribed strand than what has been observed previously. In XP group A and D strains, we find little or no gene-specific DNA repair. In cells from an individual in XP complementation group F, we find less repair of dimers in the active gene than what has been observed for the overall genome. We have also measured the colony-forming ability of the strains after treatment with UV and find that this measure of survival does not correlate with the level of gene-specific repair of dimers. Thus, XP group F represents a novel repair phenotype with little or no gene-specific repair of dimers, but with relatively high UV resistance. We also evaluate the XP patients' clinical features in relation to gene-specific repair of dimers.

Clinically asymptomatic xeroderma pigmentosum neurological disease in an adult: evidence for a neurodegeneration in later life caused by defective DNA repair.

Xeroderma pigmentosum is a genetically heterogeneous disease caused by DNA repair defects resulting in skin cancer and, in some patients, a primary neuronal degeneration which has in all previous reports become symptomatic prior to age 21 years. A 47-year-old xeroderma pigmentosum patient is described who, although neurologically asymptomatic, has sensorineural hearing loss together with clinical signs and electrophysiologic evidence of a developing peripheral neuropathy. This case suggests that defective DNA repair may cause neurodegeneration in adults as well as in children.

Skin cancer and chromosomal aberrations induced by ultraviolet radiation. Evidence for lack of correlation in xeroderma pigmentosum variant and group E patients.

Ultraviolet radiation (UV) in sunlight induces an abnormally high incidence of skin cancer in patients with xeroderma pigmentosum (XP), an autosomal recessive disease with defects in the repair of damaged DNA. We determined the frequency of UV-induced chromosomal aberrations in cultured lymphoblast lines from a patient with the variant form of XP, from a patient with the complementation group E form, and from two patients with the complementation group C form. In contrast to results with patients having other forms of XP, the group E and variant patients showed no abnormal increase in UV-induced chromosomal aberrations. Even in the presence of caffeine, which exacerbates the postreplication repair defect of UV-irradiated XP variant cells, there was still no abnormally elevated frequency of UV-induced chromosomal aberrations in the variant cells. These results, indicating that the level of UV-induced chromosomal aberrations is not correlated with these patients' marked susceptibility to skin cancer, suggests that some mechanism other than genetic transposition is causatively related to these XP patients' high incidence of sunlight-induced skin cancer.

Evidence for defective repair of cyclobutane pyrimidine dimers with normal repair of other DNA photoproducts in a transcriptionally active gene transfected into Cockayne syndrome cells.

Cockayne syndrome (CS) and xeroderma pigmentosum (XP), autosomal recessive diseases with clinical and cellular hypersensitivity to UV radiation, differ in ability to repair UV DNA photoproducts in their overall genome: normal repair in CS, defective repair in XP. In order to characterize a DNA repair defect in an active gene in CS, we measured the capacity of cells from patients with CS and XP to reactivate 2 major types of UV-induced DNA damage, photoreactivatable (i.e., cyclobutane pyrimidine dimers) and non-photoreactivatable (primarily pyrimidine-(6-4)pyrimidone photoproducts), in the actively transcribing chloramphenicol acetyltransferase (cat) gene of the plasmid expression vector pRSV-cat. Epstein-Barr virus-transformed lymphoblast lines from 4 normal persons and from 3 patients with CS and from two with XP were transiently transfected with the plasmid, and the cat activity in cell extracts was determined. When the cells were transfected with UV-irradiated plasmid, expression was abnormally decreased in both the CS and XP cells. When the cyclobutane pyrimidine dimers in the UV-irradiated plasmid were removed by photoreactivation prior to transfection, cat expression in the CS, but not in the XP, lines reached normal levels. These data imply that both the XP and CS cells are unable to repair normally the cyclobutane pyrimidine dimer photoproducts which block transcription of cat. However, the CS, but not XP, cells can repair normally the other UV-induced photoproducts which block transcription. The ability of CS, but not XP, cells to repair these non-dimer photoproducts indicates that the active gene repair mechanism treats the cyclobutane pyrimidine dimer differently from the non-dimer photoproducts.

Neurological disease in xeroderma pigmentosum. Documentation of a late onset type of the juvenile onset form.

Xeroderma pigmentosum (XP) is an autosomal recessive, neurocutaneous disorder characterized by sunlight-induced skin cancers and defective DNA repair. Many XP children develop a primary neuronal degeneration. We describe 2 unusual XP patients who had a delayed onset of XP neurological disease. Somatic cell genetic studies indicated that they have the same defective DNA repair gene and are both in XP complementation group A. These 2 patients, together with a group A patient previously reported from London, establish as a distinct clinical entity the late onset type of the juvenile onset form of XP neurological disease. The functional capacity of these patients' cultured fibroblast strains to survive after treatment with ultraviolet radiation indicates that their DNA repair defect is less severe than that of typical group A patients who have a more severe neurodegeneration with an earlier symptomatic onset. The premature death of nerve cells in XP patients (which is presumably due to their inherited defects in DNA repair mechanisms) suggests that normal repair of damaged DNA in neurons is required to maintain integrity of the human nervous system.

Induction by ionizing radiation of the gadd45 gene in cultured human cells: lack of mediation by protein kinase C.

The effect of ionizing radiation on the expression of two DNA-damage-inducible genes, designated gadd45 and gadd153, was examined in cultured human cells. These genes have previously been shown to be strongly and coordinately induced by UV radiation and alkylating agents in human and hamster cells. We found that the gadd45 but not the gadd153 gene is strongly induced by X rays in human cells. The level of gadd45 mRNA increased rapidly after X rays at doses as low as 2 Gy. After 20 Gy of X rays, gadd45 induction, as measured by increased amounts of mRNA, was similar to that produced by the most effective dose of the alkylating agent methyl methanesulfonate. No induction was seen after treatment of either human or hamster cells with 12-O-tetradecanoylphorbol-13-acetate, a known activator of protein kinase C (PKC). Therefore, gadd45 represents the only known mammalian X-ray-responsive gene whose induction is not mediated by PKC. However, induction was blocked by the protein kinase inhibitor H7, indicating that induction is mediated by some other kinase(s). Sequence analysis of human and hamster cDNA clones demonstrated that this gene has been highly conserved and encodes a novel 165-amino-acid polypeptide which is 96% identical in the two species. This gene was localized to the short arm of human chromosome 1 between p12 and p34. When induction in lymphoblast lines from four normal individuals was compared with that in lines from four patients with ataxia telangiectasia, induction by X rays of gadd45 mRNA was less in the cell lines from this cancer-prone radiosensitive disorder. Our results provide evidence for the existence of an X-ray stress response in human cells which is independent of PKC and which is abnormal in taxia telangiectasia.

Radiosensitive Down syndrome lymphoblastoid lines have normal ionizing-radiation-induced inhibition of DNA synthesis.

The extent of X-ray-induced inhibition of DNA synthesis was determined in radiosensitive lymphoblastoid lines from 3 patients with Down syndrome and 3 patients with ataxia telangiectasia (AT). Compared to 6 normal control lines, the 3 AT lines were abnormally resistant to X-ray-induced inhibition of DNA synthesis, while the 3 Down syndrome lines had normal inhibition. These results demonstrate that radiosensitive human cells can have normal X-ray-induced inhibition of DNA synthesis and provide new evidence for the dissociation of radiosensitivity from radioresistant DNA synthesis.

Ultraviolet light-induced chromosomal aberrations in cultured cells from Cockayne syndrome and complementation group C xeroderma pigmentosum patients: lack of correlation with cancer susceptibility.

Both Cockayne syndrome (CS) and xeroderma pigmentosum (XP) are inherited diseases with defective repair of damage induced in DNA by UV. Patients with XP, but not those with CS, have an increased susceptibility to formation of sunlight-induced skin tumors. We determined the frequency of UV-induced chromosomal aberrations in cultured lymphoblastoid cell lines from five CS patients and three complementation-group-C XP patients to determine whether such aberrations were abnormally increased only in the XP cells. We found that CS cells had the same abnormally increased number of induced aberrations as the XP cells, indicating that the number of UV-induced aberrations in XP group C cells does not account for the susceptibility of these XP patients to sunlight-induced skin cancer.

Alzheimer's disease fibroblasts have normal repair of N-methyl-N'-nitro-N-nitrosoguanidine-induced DNA damage determined by the alkaline elution technique.

Cultured fibroblast strains from two normal persons and from two patients with the neurodegeneration of Alzheimer's disease were exposed to the alkylating chemical N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Immediately after exposure and also after a 24-h repair incubation period the single-strand breaks in the cells' DNA were quantified by the alkaline elution technique. In contrast to a report by others using alkaline elution, MNNG, and these same strains, we found no evidence of deficient repair of MNNG-induced DNA damage in the Alzheimer's disease cells. The putative DNA repair defect in Alzheimer's disease should be investigated by methods other than the alkaline elution technique which measures only a small fraction of the damage induced by an alkylating chemical such as MNNG.