Both patient XP21BE () and patient XP329BE () had a history of lentiginous hyperpigmentation in sun exposed areas before the first year of age and did not have the acute photosensitivity with blistering burns after brief sun exposure which is present in some XP patients (). Both patients began to develop skin cancers by age 3 years.
Figure 1 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 (more ...)
Clinical Findings in Two Unrelated Patients With Identical XPC Gene Mutations Compared to Clincal Features of XP Neurological Disease and XP/CS Complex
By age 13 years patient XP21BE had 2 basal cell carcinomas and 33 squamous cell carcinomas. By age 27 years she had a total of 7 basal cell carcinomas, 62 squamous cell carcinomas, and 13 melanomas (). In addition, she had histopathological confirmation of 16 keratoacanthomas and multiple angiokeratomas.
Her developmental milestones were delayed; she walked at 19 months and spoke in sentences by the age of 3 years. Her mother reported hearing difficulties from 6 months of age. Upon school entrance at age 3 years, she was enrolled in regular classes with speech and hearing support. Academically she was considered learning disabled by the time she was 8 years old. At the age of 10 she developed simple partial seizures and was treated with carbamazepine which was changed to valproic acid during a two year period. At the age of 12 years she was placed into classes for the cognitively disabled. Patient XP21BE was reported to be hyperactive, impulsive and distractible as a young child, features that continued throughout her schooling. Medication for the treatment of attention deficit/hyperactivity disorder was discontinued after a tic disorder developed. As a high school student in a work-study program she was trained to monitor inventory and order supplies for the school cafeteria. However, she was physically agile and won medals in the balance beam and other gymnastic events for disabled individuals in high school. She graduated from high school at the age of 20 and took some community college courses. At 23 years of age she gave birth to a clinically normal son. At the time of her last evaluation she was working part time as a clerk while raising her 4 year old child.
An audiogram at age 5 years showed bilateral, symmetrical sensorineural hearing loss, ranging from mild-to-profound across the entire test frequency range (250–8000 Hz). Progression to a severe-to-profound sensorineural hearing loss was documented from age 5 years to 25 years ().
Figure 2 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 (more ...)
On examination at age 24 years she was cheerful and cooperative. Her speech was clear without frank dysarthria. Muscle strength was normal, tendon reflexes +1, in the upper extremities, +3 in the knees and +2 in the ankles with probable upgoing toes on plantar stimulation. Fine motor movements were normal in upper and lower extremities. Sensory examination was normal. Regular gait, hopping, heel and toe walk were normal. Nerve conduction studies performed at age 25 years showed mild sensory axonal peripherally neuropathy with decrease in the SNAP amplitudes compared to a baseline study at age 10 years. At age 26 years she was obese (BMI 33.2, weight 77.4 kg, height 152.8 cm) with head circumference 53.3 cm (17 percentile). CT and MRI of the brain were normal at age 25 and 27 years respectively.
Patient XP21BE had serial neuropsychological evaluations beginning at age 7 years using the appropriate Wechsler scale for age (). A consistent pattern was performance IQ greater than verbal IQ. The difference was always significant and ranged between 12 and 32 points which is typical of an individual with a hearing impairment. Test results at the age of 11 and 12 years were disparate from those obtained as a younger child and as an adult. This is coincident with her treatment for a seizure disorder. Compared to that of her age peers, her vocabulary was exceptionally low during those years (below the 1st percentile). At the age of 12 years there was a precipitous drop of 18–19 raw score points on Picture Arrangement and a 5 to 6 point raw score drop on Object Assembly that contributed to the 25 point deterioration in performance IQ, and by extension, the drop in full scale IQ that year. Neurologic evaluation at that time suggested that this reduction in test results might be due to either the seizure disorder or the medications used to treat the seizure disorder. She was not tested again until the age of 25 when her performance IQ returned to former levels. As an adult her vocabulary improved to the low average range but continued to be a relatively weak area. Verbal abilities were otherwise stable. Nonverbal abilities, always stronger, were also consistent, with the exception of the 12th year performance. Word recognition reading and spelling were better than math and reading comprehension. Test scores were commensurate with full scale IQ. Overall, there was no evidence of progressive intellectual deterioration.
STABILITY OF IQ OF PATIENT XP21BE
Patient XP21BE was reported to be the child of a second degree consanguineous mating. Early onset of sensorineural hearing loss and developmental delay has not been reported in other XP-C patients.
By age 13 years patient XP329BE had developed 36 histologically confirmed basal cell carcinomas ( and ). He had normal neurological examination and normal audiogram. He is an excellent student, plays the piano and is an avid reader. Patient XP329BE was reported to be the child of a fourth degree consanguineous mating.
Elevated UV sensitivity and reduced DNA photoproduct repair
We examined the UV sensitivity cells from both patients and a normal control using an MTS assay (). The cells from both patients showed a higher sensitivity to UV than normal cells. After exposure to 14 J/m2 UV the XP cells had 35–45% viability while the normal cells had 70%. Though the XP patients had marked differences in their neurological status their cells showed no significant differences in UV sensitivity.
Figure 3 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 (more ...)
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 (). This is similar to previous reports for normal cells [13
]. In contrast about 80–90% of the 6–4 PP remained in cells from both XP patients at 3h and 75–80% at 6h. This is similar to other studies for XPC cells [22
]. As previously described [13
] post-UV removal of CPD photoproducts was slower in normal cells with 56% remaining at 6h and 29% remaining at 24h (). CPD removal was delayed in both XP-C cells with 75–80% remaining at 6h and 45–60% at 24h. This is similar to the other XP-C cells [22
]. Thus, the cells from the XP-C patient with neurological abnormalities (XP21BE) and with normal neurological examinations (XP329BE) showed similar levels of reduction in the repair of photoproducts.
Assignment of XP cells to complementation group C
The post-UV host cell reactivation (HCR) assay was used to assign these cells to XP complementation groups. The UV irradiated reporter gene plasmid was transfected into the cells from the patients along with plasmids expressing wild-type, XP group A, C or D cDNA. Only co-transfection with a plasmid containing the wild-type XPC cDNA led to a markedly increased post-UV HCR in the cells from both XP patients thus assigning these cells to the XP complementation group C (data not shown).
Initiation codon mutation in the XPC gene
We characterized the causative mutations in these cells by nucleotide sequencing. We found a homozygous c.2T>G mutation changing the ATG initiation codon to arginine (AGG) in the XPC exon 1 in cells from both XP-C patients. This mutation was determined both in the genomic DNA () and cDNA (data not shown) from the patients’ cells.
Figure 4 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 (more ...)
We developed genomic PCR and RFLP based method to characterize this new initiation codon mutation in patients and their parents. The T to G transition created a new BanII restriction site in XPC exon 1 and abolished an N1aIII restriction site (). Genomic DNA from the XP-C patients and their parents was digested in order to determine whether the mutations were homozygous or hemizygous. The PCR products from XP21BE and XP329BE were cut only by BanII and the PCR product from normal was cut only by N1aIII. The PCR products from parents of XP329BE were cut by both BanII and N1aIII restriction endonucleases indicating that both the parents of XP329BE were heterozygous for the same mutation in that one allele behaved like normal and the other one had the initiation codon mutation like their son. These results indicate that the XP-C patient XP329BE is homozygous for the initiation codon mutation. We do not have access to DNA from the parents of XP21BE.
XPC mRNA levels in the cells from XP-C patients
A real-time quantitative reverse transcriptase-PCR (QRT-PCR) assay [10
] with allele-specific primers that detect XPC mRNAs containing either exon 4 or exon 12 () was used to measure the levels of XPC mRNA in cells from the XP-C patients, their heterozygous parents and normal controls. Relative to the normal control, the XPC mRNA levels were 24 to 42% in cells from XP21BE and XP329BE. Interestingly, the levels of XPC mRNA in these patients with an initiation codon mutation were slightly higher compared to cells from patients with premature termination codons (PTC)[10
]. This suggests that RNA harboring PTC is more likely to be degraded by nonsense mediated message decay pathway. However, the levels of XPC mRNA in the cells from the parents of XP329BE were similar to the normal cells. 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 ( 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 ( lower gel – lanes 8,9, 11 and 12).
XPC mRNA LEVELS IN XP-C CELLS
Reduced XPC protein levels in the cells from XP-C patients
To measure the XPC protein levels in the cells from XP-C patients and their parents, Western blot analysis was performed on total cellular extracts using XPC specific monoclonal and anti-β-actin polyclonal antibodies [10
]. High levels of XPC protein were observed in the cells from the normal donors (). In contrast, there was no detectable XPC protein in the cells from the XP-C patients (). 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] ( and [10
]). The parents of XP329BE, who are obligate heterozygotes, had XPC protein levels similar to that of the normal control (data not shown). These findings were similar to those previously reported in XP-C, normal and parental cells [10
Reduced DNA repair function of initiation codon mutation
We constructed an expression vector containing the XPC cDNA with c.2T>G mutation (pXPC-HAN-2T>G) and assessed its function employing a transient post-UV HCR assay () The recovery of chloramphenicol acetyl transferase (CAT) activity reflects the ability of the transfected cells to repair the UV-induced plasmid DNA damage [12
]. The expression vector with wild type full-length XPC
cDNA (pXPC-HAN) resulted in increased CAT activity (p=0.0002) indicating that the repair defect in XP4PA-SV-EB XP-C cells could be complemented. Transfection of pXPC-HAN-2T>G plasmid resulted in a markedly reduced CAT activity in XP4PA-SV-EB XP-C cells that was not significantly different from the CAT activity with the empty vector control (pEBS7). Thus, XPC cDNA with the initiation codon mutation was not functional in this DNA repair assay. Over-expression of XPC cDNA with the initiation codon mutation in normal GM00637 cells did not alter the normal repair capability of the cells (data not shown).
Absence of functional XPC protein in vivo
XP and normal cells were labeled by uptake of different sized polystyrene beads in their cytoplasm and then co-cultured. The cells were UV-irradiated through 5-μm 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 (). The NER proteins were detected by immunofluorescence and confocal microscopy. Their localization to the site of DNA damage reflected their DNA repair activity. XPC protein was detected in the un-irradiated normal cells ( – top row, left panels). In contrast, in un-irradiated XP21BE or XP329BE cells XPC protein was not detected ( – top row, left panels), in agreement with the Western blots (). XPG protein was detected in the un-irradiated normal and XP cells ( – 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 ( – 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 ( – second row, left panels). By 30 min after UV treatment CPD were still detectable in the normal and XP-C patients’ cells ( – third row, right panels). In the normal cells all the NER components examined (XPC, XPB, XPG, XPA, XPD, and XPF) were localized to the sites of DNA damage (yellow arrows) indicating that the NER was functionally active ( – rows 3, 4, 5 and 6). In the absence of binding of XPC protein to the DNA damaged site, none of these NER proteins examined localized to the damaged site in the XP cells ( – rows 3, 4, 5 and 6). These findings indicate that there was no detectible functional XPC protein activity in the cells of either patient. These findings are similar to those reported previously in other XP-C and normal cells [19
Figure 5 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 μm latex beads and XP21BE and XP329BE cells were labeled with 2 μm (more ...)
Genetic marker analysis
XP21BE and XP329BE were homozygous for the same XPC
initiation codon mutation (c.2T>G), suggesting the possibility that they might have a common ancestor. This can be tested by analyzing microsatellite or single nucleotide polymorphism (SNP) markers near the XPC
gene on chromosome 3 [20
]. shows 9 microsatellite markers and 3 XPC SNP’s we used arranged along chromosome 3 as indicated by the maps of the human genome http://www.ncbi.nlm.nih.gov/
Examination of DNA from each of the XP patients, both of whom had a history of consanguinity, revealed the markers to be homozygous over a region of 670 kBP (). While both of these patients have the same homozygous initiation codon mutation, their alleles were different for 11 out of 12 microsatellite markers ( and data not shown). There was a common region of about 30 kBP within the XPC
gene (bold type in ). Thus these XP-C patients were not closely related. In addition, the markers from XP21BE were heterozygous outside of the 670 kBP homozygous region, indicating that these cells were not hemizygous.
ANALYSIS OF GENOMIC DNA FROM XP21BE AND XP329BE