PMC2684809_01
Xeroderma pigmentosum patients with and without neurological disease. A. Patient XP21BE, at age 5 years, had extensive lentiginous pigmentation on sun exposed portions of her face, chest and shoulders. B. Patient XP329BE, at age 19 months, had extensive freckle-like pigmentation on the sun exposed portions of his face.
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By age 13 years patient XP329BE had developed 36 histologically confirmed basal cell carcinomas (Table 1 and Figure 1B).
Serial audiograms from the right ear of patient XP21BE. Audiograms from 250 Hz to 8000 Hz were performed at age 5 yr, 7 yr, 10 yr, 12 yr, 22 yr and 25 yr. There was a mild-to-profound sensorineural hearing loss across the entire frequency range at age 5 yr that progressed to a severe-to-profound sensorineural hearing loss at age 25 yr.
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Progression to a severe-to-profound sensorineural hearing loss was documented from age 5 years to 25 years (Figure 2).
Post UV cell survival and photoproduct removal in XP cells. A. Post-UV viability of normal (open circles), XP329BE (closed triangles) and XP21BE (closed circles) fibroblasts measured by MTS assay. Mean +- SEM of quadruplicate cultures. B. Removal of 6-4 photoproducts from normal (open circles), XP21BE (closed circles) and XP329BE (closed triangles) cells following 10 J/m2 UV exposure measured by ELISA assay. Mean +- SD of duplicate experiments are shown. C. Removal of CPD from normal (open circles), XP21BE (closed circles) and XP329BE (closed triangles) cells following 10 J/m2 UV exposure measured by ELISA assay. Mean +- SD of duplicate experiments are shown.
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We examined the UV sensitivity cells from both patients and a normal control using an MTS assay (Figure 3A). An ELISA assay indicated that normal fibroblasts had a rapid post-UV removal of 6-4 PP, with 5% remaining by 3h and 2% at 6h (Figure 3B). CPD photoproducts was slower in normal cells with 56% remaining at 6h and 29% remaining at 24h (Figure 3C). CPD removal was delayed in both XP-C cells with 75-80% remaining at 6h and 45-60% at 24h.
XPC Initiation codon mutation in XP21BE and XP329BE cells. A. Sequence of XPC gene in genomic DNA from normal, XP21BE and XP329BE cells. The T base of the ATG initiation codon is substituted by a G in the XP cells (arrows). B. RFLP detection of c.2T>G mutation in genomic DNA and cDNA in cells from a normal donor, XP21BE, XP329BE and his father and mother. Upper gel: N1aIII digestion of a 106 bp fragment of XPC genomic DNA results in products of 31 bp and 75 bp in the normal sequence containing a T (arrows in lanes 3, 12 and 15). BanII digestion of the XPC genomic DNA fragment containing a G results in products of 34 bp and 72 bp (arrows in lanes 5, 8, 11, and 14). Undigested bands are indicated by *. Patients XP21BE and XP329BE are homozygous for the c.2T>G mutation and the parents of XP329BE are heterozygous for the mutation. Lower gel: N1aIII digestion of XPC cDNA results in products of 31 bp and 75 bp in the normal sequence containing a T (lanes 3, 9 and 12) from the normal donor and both parents. BanII digestion of the XPC cDNA fragment containing the T>G mutation results in products of 34 bp and 72 bp (lane 5) in cDNA from XP329BE. There is no detectable BanII digested cDNA in the cells from the parents indicating that only the normal allele is expressed. C. Western blot of protein extracted from cells from normal, XP24BE, XP21BE, XP25BE and XP329BE cells using XPC (upper row) and actin (lower row) antibodies. There was no detectable XPC protein in the XP cells. D. Absence of activity of XPC cDNA containing the c.2T>G mutation in a host cell reactivation assay. XP4PA-SV-EB XPC cells were co-transfected with a UV treated CAT plasmid (1000 J/m2) and a plasmid with either wild type XPC cDNA (pXPC-HAN; n=9), XPC cDNA with the c.2T>G mutation (pXPC-HAN-2T>G; n=6), or the empty vector (pEBS7; n=9). CAT activity was measured after 48 hr. The wild type XPC cDNA showed increased CAT activity (p=0.0002) while CAT activity with the XPC cDNA with the c.2T>G mutation was not significantly different from the empty vector. Mean +- SEM shown.
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This mutation was determined both in the genomic DNA (Figure 4A) and cDNA (data not shown) from the patients' cells. G transition created a new BanII restriction site in XPC exon 1 and abolished an N1aIII restriction site (Figure 4B). Genomic DNA from the XP-C patients and their parents was digested in order to determine whether the mutations were homozygous or hemizygous. Restriction enzyme digestion of cDNA from patient XP329BE revealed digestion with BanII but not N1aIII indicating that all of the cDNA contained the c.2T>G mutation (Figure 4B lower gel - lanes 5 and 6). In contrast cDNA from both parents was digested by N1aIII and not BanII indicating that there was no detectable mutated cDNA expressed in these cells (Figure 4B lower gel - lanes 8,9, 11 and 12). High levels of XPC protein were observed in the cells from the normal donors (Figure 4C). In contrast, there was no detectable XPC protein in the cells from the XP-C patients (Figure 4C). This was similar to the previously reported undectable levels of XPC protein in cells from patients XP24BE [Compound heterozygote: Intron 5.1 A to G at -2; and c.463C>T, p.Arg155*] and XP25BE [Homozygous: Intron 11 Del -1. -2 AG with Insertion of CC between -6 and -7] (Figure 4C and). XP21BE or XP329BE cells XPC protein was not detected (Figure 5 - top row, left panels), in agreement with the Western blots (Figure 4C). XPG protein was detected in the un-irradiated normal and XP cells (Figure 5 - top row, right panels).
Lack of recruitment of XPC and other NER proteins to localized DNA damage in XP21BE and XP329BE cells following UV irradiation. Normal cells (AG13145) were labeled with 0.8 mum latex beads and XP21BE and XP329BE cells were labeled with 2 mum latex beads. In the absence of UV, XPC and XPG protein was stained in the normal cells (yellow *). XPG protein was present in the XP cells (green *) but XPC protein was not detected. Following 100 J/m2 UV irradiation delivered through a 5 mum filter, cells were fixed at 5 min or 30 min post-irradiation and immunostained with pairs of antibodies to simultaneously assess the location of DNA damage and NER proteins. The arrows indicate sites of localized immunostaining: normal - yellow arrows, patient - green arrows. While CPD photoproducts were detected in the XP and normal cells at 5 min and 30 min after UV exposure, the NER proteins (XPC, XPG, XPB, XPD, XPA and XPF) were only localized in the normal cells (yellow arrows). Symbols below each image indicate localization (+) or non-localization (-) of NER proteins or photoproducts in patient cells/normal cells.
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Restriction enzyme digestion of cDNA from patient XP329BE revealed digestion with BanII but not N1aIII indicating that all of the cDNA contained the c.2T>G mutation (Figure 4B lower gel - lanes 5 and 6). In contrast cDNA from both parents was digested by N1aIII and not BanII indicating that there was no detectable mutated cDNA expressed in these cells (Figure 4B lower gel - lanes 8,9, 11 and 12). The cells were UV-irradiated through 5-mum diameter pores of a polycarbonate isopore membrane filter to follow localization of NER proteins to sites of UV-induced DNA damage in vivo as a measure of their DNA repair activity (Figure 5). XPC protein was detected in the un-irradiated normal cells (Figure 5 - top row, left panels). XP21BE or XP329BE cells XPC protein was not detected (Figure 5 - top row, left panels), in agreement with the Western blots (Figure 4C). XPG protein was detected in the un-irradiated normal and XP cells (Figure 5 - top row, right panels). Assay within 5 min of UV irradiation produced fluorescent CPD foci in the nuclei of the normal and both the XP-C patients' cells (Figure 5 - second row, right panels). Localization of the XPC protein (green) to the DNA damaged site was observed in the normal cells (yellow arrows) but not in the cells from the XP-C patients (Figure 5 - second row, left panels). By 30 min after UV treatment CPD were still detectable in the normal and XP-C patients' cells (Figure 5 - third row, right panels).