dna repair 8 ( 2 0 0 9 ) 114–125
available at www.sciencedirect.com
journal homepage: www.elsevier.com/locate/dnarepair
XPC initiation codon mutation in xeroderma pigmentosum
patients with and without neurological symptoms
Sikandar G. Khana, Kyu-Seon Oha, Steffen Emmerta,1, Kyoko Imotoa, Deborah Tamuraa,
John J. DiGiovannaa,c, Tala Shahlavia, Najealicka Armstronga, Carl C. Bakerb,
Marcy Neuburgd, Chris Zalewskie, Carmen Brewere, Edythe Wiggsf,
Raphael Schiffmannf, Kenneth H. Kraemera,∗
aBasic Research Laboratory, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States
bLaboratory of Clinical Oncology, National Cancer Institute, National Institutes of Health, Bethesda, MD, United States
cDivision of Dermatopharmacology, Department of Dermatology, The Warren Alpert Medical School of Brown University,
Providence, RI, United States
dDepartment of Dermatology, Medical College of Wisconsin, Milwaukee, WI, United States
eOtolaryngology Branch, National Institute on Deafness and Other Communication Disorders, National Institutes of Health,
Bethesda, MD, United States
fDevelopmental and Metabolic Neurology Branch, National Institute of Neurological Diseases and Stroke,
National Institutes of Health, Bethesda, MD, United States
a r t i c l ei n f o
Received 25 July 2008
Received in revised form
3 September 2008
Accepted 17 September 2008
Published on line 14 November 2008
Sensorineural hearing loss
a b s t r a c t
Two unrelated xeroderma pigmentosum (XP) patients, with and without neurological abnor-
malities, respectively, had identical defects in the XPC DNA nucleotide excision repair (NER)
gene. Patient XP21BE, a 27-year-old woman, had developmental delay and early onset of
sensorineural hearing loss. In contrast, patient XP329BE, a 13-year-old boy, had a normal
neurological examination. Both patients had marked lentiginous hyperpigmentation and
multiple skin cancers at an early age. Their cultured fibroblasts showed similar hypersensi-
a homozygous c.2T>G mutation in the XPC gene which changed the ATG initiation codon
to arginine (AGG). Both had low levels of XPC message and no detectable XPC protein on
Western blotting. There was no functional XPC activity in both as revealed by the failure
of localization of XPC and other NER proteins at the sites of UV-induced DNA damage in a
tion was functionally inactive in a post-UV host cell reactivation (HCR) assay. Microsatellite
markers flanking the XPC gene showed only a small region of identity (∼30kBP), indicating
that the patients were not closely related. Thus, the initiation codon mutation resulted in
DNA repair deficiency in cells from both patients and greatly increased cancer susceptibility.
The neurological abnormalities in patient XP21BE may be related to close consanguinity and
simultaneous inheritance of other recessive genes or other gene modifying effects rather
than the influence of XPC gene itself.
Published by Elsevier B.V.
∗Corresponding author at: DNA Repair Section Basic Research Laboratory, CCR, NCI, Building 37 Room 4002 MSC 4258, Bethesda, MD 20892,
United States. Tel.: +1 301 496 9033; fax: +1 301 594 3409.
E-mail address: email@example.com (K.H. Kraemer).
1Present address: Department of Dermatology, University of Goettingen, Germany.
1568-7864/$ – see front matter. Published by Elsevier B.V.
dna repair 8 ( 2 0 0 9 ) 114–125
Xeroderma pigmentosum (XP) is a rare autosomal reces-
sive disorder caused by a defect in the nucleotide excision
repair (NER) pathway [1–4] which removes a wide spectrum
of structurally unrelated DNA lesions including cyclobutane
pyrimidine dimers (CPD) and 6-4 photoproducts induced by
ultraviolet radiation (UV) from sunlight. Cells from XP patients
fall into seven genetic complementation groups XP-A through
XP-G, corresponding to seven of the gene products involved
in NER and a variant form with a defect in trans-lesion
polymerase eta. XP patients have increased freckle-like pig-
mentation in response to sun exposure and a greater than
1000-fold increased incidence of UV-induced skin cancers at
an early age [4,5].
XP complementation group C (XP-C) is one of the more
common forms in the United States . Cells from XP-C
sion repair (TCR) but defective global genome nucleotide
excision repair (GGR) of damaged DNA while cells from XP
complementation groups A, B, D, F and G are defective in both
GGR and TCR . The XPC DNA repair gene encodes a 940
amino acid protein that forms an in vivo stable heterotrimeric
complex with one of the two human orthologs of Saccha-
romyces cerevisiae Rad23p (RAD23A or RAD23B) and centrin 2,
a component of the centrosome, and functions as a DNA-
damage sensor and repair recruitment factor in GGR [3,7,8].
About 20% of XP patients exhibit progressive neurodegen-
eration [4,9]. However, neurological symptoms are rarely seen
in XP-C patients. Most of the XP patients with neurological
symptoms are in XP complementation groups XP-A, XP-B, XP-
D or XP-G [2,6]. Since the development of neurologic involve-
ment has grave clinical prognostic implications, understand-
ing the relationship between complementation group and
neurologic degeneration is extremely important. We report
here two XP patients (XP21BE and XP329BE) with the same
homozygous initiation codon mutation in theXPCgene. While
both patients have multiple skin cancers, XP21BE has devel-
opmental delay and sensorineural hearing loss while XP329BE
has no neurological abnormalities. The neurological abnor-
malities in XP21BE may not be related to the XPC gene defect.
2.Materials and methods
After obtaining informed consent, the XP patients were stud-
ied at the Clinical Center, NIH under protocols approved by
the NCI Institutional Review Board. Both patients had thor-
ough skin examinations and biopsy of lesions suspicious for
skin cancer. Examinations included detailed ophthalmology,
neurology, audiology, and other assessments as medically
Cell lines, culture conditions and DNA/RNA
XP-C families were studied: Family A: XP21BE (GM09943,
and lymphoblastoidcell culturesfromtwo
GM09942); Family B: XP329BE (2851839, JA1356), XPH395BE
his father (JA1357) and XPH396BE his mother (JA1358).
Normal SV40-transformed fibroblast (GM00637), normal pri-
mary skin fibroblast (AG13145) and normal lymphoblastoid
(KR06057) cells and XP24BE (GM11638) and XP25BE (KR04489)
fibroblasts  were obtained from the Human Genetic
Mutant Cell Repository (Camden, NJ). SV40-transformed
XP-C (XP4PA-SV-EB) cells [8,11] were a gift from Dr. R.
Legerski (M. D. Anderson Hospital, Houston, TX). Cell cul-
ture and separation of RNA and DNA was performed as
2.3. Measurement of UV sensitivity
H-tetrazolium (MTS) assay (Promega) as described .
genomic DNA by ELISA
Measurement of UV-induced photoproducts in
Cultured cells were washed with Dulbecco’s phosphate-
buffered saline, UV-irradiated and incubated for various times
to allow repair. Cells were harvested and genomic DNA
was isolated using QIAamp Blood Kit (Qiagen, Hilden, Ger-
many). 6-4 PP and CPD were quantified by an enzyme-linked
immunosorbent assay (ELISA) using 64M-2 and TDM-2 mono-
clonal antibodies as described [13–15].
assignment and functional analysis of mutation
Post-UV HCR assay for complementation group
The post-UV host cell reactivation (HCR) assay was used to
assign XP cells to XP complementation group C as described
previously . A plasmid with XPC cDNA containing the
c.2T>G, initiation codon mutation was assessed for func-
tion in the post-UV HCR assay. For the construction of an
expression vector with the initiation codon mutation (pXPC-
HAN-2T>G), the expression vector pXPC-HAN that contains
full-length XPC cDNA was subjected to site-directed muta-
genesis using the QuikChange Site-Directed Mutagenesis Kit
(Stratagene, LaJolla, CA) as per vendor’s protocol and for-
ward primer 5?CAGACAAGCAACAGGGCTCGGAAACGC3?and
reverse primer 5?GCGTTTCCGAGCCCTGTTGCTTGTCTG3?. The
expression vector pXPC-HAN was constructed by insert-
ing the full-length wild-type XPC cDNA fragment obtained
from pcDNA3/HA-XPC (a generous gift from Dr. Fumio
Hanaoka, Osaka University, Osaka, Japan) into the empty
expression vector pEBS7 (a generous gift from Dr. Randy
Legerski, University of Texas, M.D. Anderson Cancer Center,
Mutation detection by PCR amplification and
The 16 XPC gene exons including splice donor and acceptor
sites were PCR amplified using intronic primers flanking these
sequences and sequenced as described previously .
dna repair 8 ( 2 0 0 9 ) 114–125
Real time quantitative RT-PCR (QRT-PCR) for XPC
XPC mRNA was quantified using gene specific primer
pairs employing real time QRT-PCR as described . The
allele-specific primer pairs, oVMM-21/oVMM-22 and oCCB-
331/oCCB-337 were used to measure the amount of XPC mRNA
including exon 4 and exon 12, respectively. All real-time QRT-
PCR data is expressed as fg of a full-length wild type XPC cDNA
2.8.XPC protein detection by Western blotting
Western blotting for the XPC protein was performed as
Inc., Cambridge, MA) and anti-?-Actin polyclonal antibodies
following localized UV irradiation
Immunocytochemistry of NER protein localization
Fibroblasts were labeled with 0.8?m (normal donors) or 2?m
(patients) latex beads (Polybead carboxylate microspheres;
Polysciences, Warrington, PA) and cultured until confluent
. Cells were combined, overlaid with 5?m Millipore fil-
ters, irradiated with 100J/m2UV-C and double stained for
immunofluorescent localization of NER proteins and photo-
products as described [13,17–19]. Cells were visualized with a
LSM 510 confocal microscope (Carl Zeiss).
2.10.Microsatellite marker analysis
Microsatellite markers flanking the XPC gene on chromosome
3 were genotyped using fluorescently labeled oligonu-
cleotide probes as previously described . PCR products
were separated on ABI Prism 3100 Genetic analyzer, and
were analyzed with the GeneScan, version 3.1.2, software
Both patient XP21BE (Fig. 1A) and patient XP329BE (Fig. 1B) 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 (Table 1). Both patients
began to develop skin cancers by age 3 years.
33 squamous cell carcinomas. By age 27 years she had a total
of 7 basal cell carcinomas, 62 squamous cell carcinomas, and
confirmation of 16 keratoacanthomas and multiple angioker-
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. Academ-
ically she was considered learning disabled by the time she
was 8 years old. At the age of 10 she developed simple par-
tial seizures and was treated with carbamazepine which was
Fig. 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 freckle-like pigmentation on the sun exposed portions of his face.
dna repair 8 ( 2 0 0 9 ) 114–125
Table 1 – Clinical findings in two unrelated patients with identical XPC gene mutations compared to clinical features of
XP neurological disease and XP/CS complex.
XP21BEXP329BEXP neurological diseaseXP/CS complex
Age at last observation
Onset of lentiginous
sun exposed skin
Age of first skin cancer
Basal cell carcinoma
By age 10 months
By age 8 months
frequency hearing loss
Audiology Sensorineural hearing
loss across entire freq
Normal – gave birth to
DNA repair defect
XPA, XPD, XPG
XPB, XPD, XPG
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 through-
out her schooling. Medication for the treatment of attention
deficit/hyperactivity disorder was discontinued after a tic dis-
order developed. As a high school student in a work-study
program she was trained to monitor inventory and order sup-
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
part time as a clerk while raising her 4-year-old child.
An audiogram at age 5 years showed bilateral, symmetri-
across the entire test frequency range (250–8000Hz). Progres-
sion to a severe-to-profound sensorineural hearing loss was
documented from age 5 years to 25 years (Fig. 2).
On examination at age 24 years she was cheerful and coop-
erative. Her speech was clear without frank dysarthria. Muscle
toes on plantar stimulation. Fine motor movements were nor-
mal 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
77.4kg, height 152.8cm) with head circumference 53.3cm (17
percentile). CT and MRI of the brain were normal at age 25
years and 27 years, respectively.
Patient XP21BE had serial neuropsychological evaluations
beginning at age 7 years using the appropriate Wechsler scale
for age (Table 2). 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 years and 12 years were disparate from those obtained
as a younger child and as an adult. This is coincident with
Table 2 – Stability of IQ of patient XP21BE.
aSeizure disorder treated with carbamazepine followed by valproic
acid. Seizures resolved and medications discontinued.
bAttributed to significant decline on most tests that are timed.
dna repair 8 ( 2 0 0 9 ) 114–125
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 Pic-
ture Arrangement and a 5–6 point raw score drop on Object
Assembly that contributed to the 25 points 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 disor-
der. 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 consis-
tent, with the exception of the 12th year performance. Word
recognition reading and spelling were better than math and
full scale IQ. Overall, there was no evidence of progressive
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 histolog-
ically confirmed basal cell carcinomas (Table 1 and Fig. 1B).
He had normal neurological examination and normal audio-
gram. 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
We examined the UV sensitivity cells from both patients and
a normal control using an MTS assay (Fig. 3A). The cells from
both patients showed a higher sensitivity to UV than normal
cells. After exposure to 14J/m2UV the XP cells had 35–45% via-
bility 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.
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 (Fig. 3B). This is similar to previous reports
for normal cells [13,14,21]. 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 .
As previously described [13,14,21,22] post-UV removal of CPD
photoproducts was slower in normal cells with 56% remain-
ing at 6h and 29% remaining at 24h (Fig. 3C). 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 .
Thus, the cells from the XP-C patient with neurological abnor-
malities (XP21BE) and with normal neurological examinations
(XP329BE) showed similar levels of reduction in the repair of
Assignment of XP cells to XP complementation
The post-UV host cell reactivation 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
Fig. 2 – Serial audiograms from the right ear of patient XP21BE. Audiograms from 250Hz to 8000Hz were performed at age 5
years, 7 years, 10 years, 12 years, 22 years and 25 years. There was a mild-to-profound sensorineural hearing loss across
the entire frequency range at age 5 years that progressed to a severe-to-profound sensorineural hearing loss at age 25 years.
dna repair 8 ( 2 0 0 9 ) 114–125
Fig. 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±S.E.M. of quadruplicate cultures.
(B) Removal of 6-4 photoproducts from normal (open circles), XP21BE (closed circles) and XP329BE (closed triangles) cells
following 10J/m2UV exposure measured by ELISA assay. Mean±S.D. of duplicate experiments are shown. (C) Removal of
CPD from normal (open circles), XP21BE (closed circles) and XP329BE (closed triangles) cells following 10J/m2UV exposure
measured by ELISA assay. Mean±S.D. of duplicate experiments are shown.
A, C or D cDNA. Only co-transfection with a plasmid contain-
ing the wild-type XPC cDNA led to a markedly increased post-
cells to the XP complementation group C (data not shown).
3.6.Initiation codon mutation in the XPC gene
We characterized the causative mutations in these cells by
determined both in the genomic DNA (Fig. 4A) and cDNA (data
not shown) from the patients’ cells.
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 restric-
tion site (Fig. 4B). 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 prod-
ucts from XP21BE and XP329BE were cut only by BanII and the
restriction endonucleases indicating that both the parents of
XP329BE were heterozygous for the same mutation in that one
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
3.7. XPC mRNA levels in the cells from XP-C patients
A real-time quantitative reverse transcriptase-PCR (QRT-PCR)
assay  with allele-specific primers that detect XPC mRNAs
Table 3 – XPC mRNA levels IN XP-C cells.
Cell lineXPC exon 4 inclusion XPC exon 12 inclusion
Avg (fg) Avg (fg)
containing either exon 4 or exon 12 (Table 3) was used to mea-
sure 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 24–42% in
cells from XP21BE and XP329BE. Interestingly, the levels of
XPC mRNA in these patients with an initiation codon muta-
tion were slightly higher compared to cells from patients with
premature termination codons (PTC) . 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 muta-
tion (Fig. 4B lower gel—lanes 5 and 6). In contrast cDNA from
both parents was digested by N1aIII and not BanII indicating
cells (Fig. 4B lower gel—lanes 8,9, 11 and 12).
Reduced XPC protein levels in the cells from XP-C
To measure the XPC protein levels in the cells from XP-
C patients and their parents, Western blot analysis was
dna repair 8 ( 2 0 0 9 ) 114–125
Fig. 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 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 106bp fragment of XPC genomic DNA results in products of 31bp and 75bp 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 34bp and 72bp (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 31bp and 75bp 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 34bp and 72bp (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 (1000J/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 48h. 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±S.E.M. shown.
performed on total cellular extracts using XPC specific mono-
clonal and anti-?-actin polyclonal antibodies . High levels
of XPC protein were observed in the cells from the normal
donors (Fig. 4C). In contrast, there was no detectable XPC pro-
tein in the cells from the XP-C patients (Fig. 4C). This was
tein 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] (Fig. 4C and ). 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 .
Reduced DNA repair function of initiation codon
with c.2T>G mutation (pXPC-HAN-2T>G) and assessed its
function employing a transient post-UV HCR assay (Fig. 4D)
The recovery of chloramphenicol acetyl transferase (CAT)
activity reflects the ability of the transfected cells to repair
the UV-induced plasmid DNA damage . 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 com-
plemented. Transfection of pXPC-HAN-2T>G plasmid resulted
in a markedly reduced CAT activity in XP4PA-SV-EB XP-
dna repair 8 ( 2 0 0 9 ) 114–125
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 func-
tional 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).
3.10.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-?m diameter pores
of a polycarbonate isopore membrane filter to follow local-
ization of NER proteins to sites of UV-induced DNA damage
in vivo as a measure of their DNA repair activity (Fig. 5). The
NER proteins were detected by immunofluorescence and con-
focal microscopy. Their localization to the site of DNA damage
reflected their DNA repair activity. XPC protein was detected
in the un-irradiated normal cells (Fig. 5, top row, left panels).
In contrast, in un-irradiated XP21BE or XP329BE cells XPC pro-
with the Western blots (Fig. 4C). XPG protein was detected in
the un-irradiated normal and XP cells (Fig. 5, top row, right
panels). Assay within 5min of UV irradiation produced fluo-
rescent CPD foci in the nuclei of the normal and both the XP-C
patients’ cells (Fig. 5, second row, right panels). Localization of
in the normal cells (yellow arrows) but not in the cells from the
XP-C patients (Fig. 5, second row, left panels). By 30min after
UV treatment CPD were still detectable in the normal and XP-
C patients’ cells (Fig. 5, 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 (yel-
low arrows) indicating that the NER was functionally active
(Fig. 5, 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 (Fig. 5,
rows 3, 4, 5 and 6). These findings indicate that there was no
detectible functional XPC protein activity in the cells of either
in other XP-C and normal cells .
3.11.Genetic marker analysis
XP21BE and XP329BE were homozygous for the same XPC
initiation codon mutation (c.2T>G), suggesting the possibil-
ity that they might have a common ancestor. This can be
tested by analyzing microsatellite or single nucleotide poly-
morphism (SNP) markers near the XPC gene on chromosome 3
used arranged along chromosome 3 as indicated by the maps
of the human genome http://www.ncbi.nlm.nih.gov/ Exami-
nation of DNA from each of the XP patients, both of whom had
gous over a region of 670kBP (Table 4). While both of these
patients have the same homozygous initiation codon muta-
tion, their alleles were different for 11 out of 12 microsatellite
markers (Table 4 and data not shown). There was a com-
mon region of about 30kBP within the XPC gene (bold type
in Table 4). Thus these XP-C patients were not closely related.
In addition, the markers from XP21BE were heterozygous out-
side of the 670kBP homozygous region, indicating that these
cells were not hemizygous.
XPC initiation codon mutation, DNA repair and
patients create premature termination codons [10,20,23–28].
PTCs can reduce the levels of XPC message and XPC pro-
tein in cells by means of nonsense-mediated mRNA decay
been reported. These are Pro334His  and Trp690Ser ,
which alters the stability of the encoded mutant protein .
Splice site mutations may result in severe or mild symptoms
depending on the level of XPC mRNA. Our previous studies
demonstrated that undetectable levels of normal XPC mRNA
were associated with severe clinical symptoms in 2 siblings
including multiple skin cancers at an early age . In con-
trast, 3–5% of normal levels of full-length XPC mRNA resulted
in mild symptoms in 3 affected siblings . The differential
expressions of XPC message in these cells were associated
with different mutations in two functional lariat branch point
sequences (BPS) in same XPC intron 3.
The patients in this report had a c.2T>G mutation in the
in mammals play a role in specifying which initiation site is
likely to be used during the protein translation. The sequence
near the first initiation codon in the XPC gene (AGCAACaugG)
matches well with the known optimal recognition sequence
with an A in position −3 and G in position +4. The initiation
sites are reached via a scanning mechanism. The ribosome
AUG and usually initiates translation at this first AUG [32,33].
In the cells from both XP-C patients, XPC mRNA with mutated
first AUG may not be recognized by most of the ribosomes
recruited at the 5?cap-structure on the XPC mRNA thus result-
ing in an undectable level of XPC protein. The next in-frame
unmutated AUG is located at codon 118, however, the sur-
with the known optimal recognition sequence. The A in posi-
tion −3 is similar to the optimal recognition sequence but A in
position +4 is distinct. There are many out of frame AUGs in
between the mutated initiation codon and the AUG at codon
118 which would inhibit ribosome scanning . The use of
this AUG would delete the NH2-terminal 117 amino acids in
the XPC protein. This truncated protein has been shown to
have functional activity [8,22].
While XPC mRNA levels from XP21BE and XP329BE were
slightly higher (Table 2) than in patients with XPC nonsense
mutations , all of the mRNA contained the mutation which
rendered the allele completely inactive (Fig. 4D). There was
no detectable normal size XPC protein on the Western blots
(Fig. 4C) and no functional protein present as judged by the
failure to recruit NER proteins to sites of UV damage (Fig. 5).
The failure to detect localization of XPC protein to UV dam-
dna repair 8 ( 2 0 0 9 ) 114–125
Fig. 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?m latex beads and XP21BE and XP329BE cells were labeled
with 2?m 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 100J/m2UV irradiation delivered through a
5?m filter, cells were fixed at 5min or 30min 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 5min and 30min 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.
aged DNA is in agreement with other studies of XP cells and
in vitro assays of NER protein interactions [18,19,35]. Thus, in
the absence of XPC protein, the other core NER proteins (XPA,
XPB, XPD, XPG, and XPF) did not localize to the damage site.
This sensitive assay is able to detect functional activity of the
3–5% of normal full-length XPC mRNA in cells from patients
with XPC splice mutations (Khan, Oh, et al. unpublished
dna repair 8 ( 2 0 0 9 ) 114–125
Table 4 – Analysis of genomic DNA from XP21BE and XP329BE.
Location CHR3 (BP)XP329BE maternalXP329BE paternal XP21BE Allele IXP21BE
UniSTS or SNP name
between XP-C patients
Genetic markers detecting the relationship
Genetic analysis of the patients by use of microsatellites
and SNP’s can reveal common ancestry. We previously used
microsatellite markers to provide evidence of a relationship
300–500 years ago between XPC families in Turkey and in
Italy . Individuals that are closely related will show larger
regions of identity than people who are more distant relatives.
In our patients the common region was only about 30kBP
within the XPC gene (bold type in Table 4). Thus these XP-C
patients were not closely related.
The presence of homozygosity of this region surrounding
XPC also suggests that these patients may be homozygous for
genetic changes in other regions that may be critical for their
different clinical symptoms.
XPC gene mutations and neurological
XP is characterized by sun sensitivity and early onset of lentig-
inous pigmentation and skin cancer in sun exposed parts
of the body. About 20% XP patients have a distinct group of
neurological symptoms including microcephaly, progressive
neurological degeneration with sensorineural hearing loss
beginning at high frequencies, loss of intellectual functions,
ataxia, loss of coordination and ability to walk, and cerebral
atrophy with dilated ventricles in association with primary
neuronal degeneration (Table 1 and [9,36,37]). These patients
have defects in XP complementation groups XP-A, XP-D, or
XP-G. A correlation between the neurological status of the XP
patients and the post-UV colony-forming ability (CFA) of their
cells was reported . Some XP patients have been identified
with a second clinical entity: the XP/Cockayne syndrome (CS)
complex (Table 1). These patients exhibit cutaneous abnor-
malities of XP and CS type of neurological changes including
myelination of the brain, and calcification of basal ganglia
in association with progressive cachectic dwarfism [9,36,37].
These patients have been described in XP complementa-
tion groups XP-B, XP-D and XP-G. In contrast, the neurologic
involvement in patient XP21BE differs from typical XP neu-
rologic involvement by having normal reflexes, sensorineural
deafness at many frequencies, normal CT and MRI of her brain
and normal eye exam. Although she had learning disabilities,
she also had good coordination and was able to win medals
and compete in gymnastic events in the Special Olympics for
disabled individuals. The post-UV cell survival of her cells
was similar to that of patient XP329BE who had no neurologi-
cal abnormalities. Similarly, her normal retina, normal height,
head circumference, and normal fertility, and normal CT and
MRI as well as her agility and increased body mass index dis-
(Table 1). Thus, her neurological and cellular abnormalities
were not typical of XP neurological disease or of the XP/CS
reported in only two other XP-C patients [24,39,40]. Patient
XP1MI was a 17-year-old black woman with multiple basal cell
carcinomas, scleralization of the cornea and XP associated
neurological and developmental abnormalities including
microcephaly (<1%ile), mental retardation (IQ 39-48), growth
retardation, and delayed sexual development (primary amen-
orrhea). However, unlike typical XP neurological disease, she
had normal hearing and reflexes and did not have ataxia
[39,41]. In addition to XP she had systemic lupus erythe-
matosus with joint involvement and positive serology. There
was no consanguinity reported. The cells from XP1MI had
a homo- or hemizygous missense mutation (c.1106C>A,
p.Pro334His) in the XPC gene . XP22BE was a 4-year-old
boy of Korean ancestry with multiple skin cancers who had
neurological abnormalities not usually found with either
XP or CS, including autistic features hyperactivity, normal
hearing, hyperactivity, normal reflexes, and normal MRI with-
out dilated ventricles or microcephaly along with persistent
hypoglycinemia [24,40]. He was born with a cleft palate and
duplex right kidney. This patient was the product of close
consanguinity. He had a homozygous XPC splice mutation
(c.IVS10 +2T>G) . Until additional patients are found either
with the same XPC mutation or with XP and hypoglycinemia
we cannot ascribe his symptoms to the defect in the XPC
dna repair 8 ( 2 0 0 9 ) 114–125
The hearing loss seen in XP patients is usually high fre-
quency loss that is progressive and occurs gradually over
many years [37,41]. Clinically asymptomatic high frequency
hearing loss  was present in one adult patient (XP1BE)
with a c.1292 1293delAA, p.Lys431Argfs*6 defect in the XPC
gene . In contrast, the hearing loss in XP21BE was iden-
tified at an early age and spanned the entire frequency range.
Patient XP21BE was a child of a close consanguineous mat-
ing with second-degree relationship. In this close mating
the proportion of genes shared is 25% (identical by descent),
leading to the chance of homozygosity by descent of 1/8
. Thus close consanguinity confers an increased probabil-
ity of simultaneous occurrence of other recessive disorders.
Nonsyndromic hearing loss has been reported to occur at
increased frequency in association with parental consan-
guinity [44,45]. We therefore looked for other genetic causes
of sensorineural hearing loss. Approximately 50% of non-
syndromic sensorineural hearing loss is due to mutations in
GJB2 which encodes connexin 26  but we were unable to
is the result of a distinct genetic abnormality. The other neu-
rological abnormalities in patient XP21BE, as well as in patient
XP22BE may be related to simultaneous inheritance of other
recessive genes or other gene modifying effects rather than
the influence of XPC gene itself.
Conflict of interest
This research was supported by the Intramural Research Pro-
gram of the NIH, National Cancer Institute, Center for Cancer
Research. We are grateful to the patients and their families for
their cooperation in this research. S.E. was supported in part
by a grant from the Deutsche Forschungsgemeinschaft DFG
 K.H. Kraemer, T.M. Ruenger, Genome instability, DNA repair
and cancer, in: K. Wolff, L.A. Goldsmith, S.I. Katz, B.A.
Gilchrest, A.S. Paller, D.J. Leffell (Eds.), Fitzpatrick’s
Dermatology in General Medicine, McGraw Hill, New York,
2008, pp. 977–986.
 T.M. Ruenger, J.J. DiGiovanna, K.H. Kraemer, Hereditary
diseases of genome instability and DNA repair, in: K. Wolff,
L.A. Goldsmith, S.I. Katz, B.A. Gilchrest, A.S. Paller, D.J. Leffell
(Eds.), Fitzpatrick’s Dermatology in General Medicine,
McGraw Hill, New York, 2008, pp. 1311–1325.
 E.C. Friedberg, G.C. Walker, W. Siede, R.D. Wood, R.A.
Schultz, T. Ellenberger, DNA Repair and Mutagenesis, ASM
Press, Washington, DC, 2006.
 K.H. Kraemer, M.M. Lee, J. Scotto, Xeroderma pigmentosum.
Cutaneous, ocular, and neurologic abnormalities in 830
published cases, Arch. Dermatol. 123 (1987)
 K.H. Kraemer, M.-M. Lee, A.D. Andrews, W.C. Lambert, The
role of sunlight and DNA repair in melanoma and
nonmelanoma skin cancer: the xeroderma pigmentosum
paradigm, Arch. Dermatol. 130 (1994) 1018–1021.
 S. Moriwaki, K.H. Kraemer, Xeroderma
pigmentosum—bridging a gap between clinic and laboratory,
Photodermatol. Photoimmunol. Photomed. 17 (2001) 47–54.
 S.G. Khan, V. Muniz-Medina, T. Shahlavi, C.C. Baker, H. Inui,
T. Ueda, S. Emmert, T.D. Schneider, K.H. Kraemer, The
human XPC DNA repair gene: arrangement, splice site
information content and influence of a single nucleotide
polymorphism in a splice acceptor site on alternative
splicing and function, Nucleic Acids Res. 30 (2002) 3624–3631.
 R. Legerski, C. Peterson, Expression cloning of a human DNA
repair gene involved in xeroderma pigmentosum group C,
Nature 359 (1992) 70–73 [published erratum appears in 1992
December 10;360 (6404):610].
 K.H. Kraemer, N.J. Patronas, R. Schiffmann, B.P. Brooks, D.
Tamura, J.J. DiGiovanna, Xeroderma pigmentosum,
trichothiodystrophy and Cockayne syndrome: a complex
genotype-phenotype relationship, Neuroscience 145 (2007)
 S.G. Khan, K.S. Oh, T. Shahlavi, T. Ueda, D.B. Busch, H. Inui, S.
Emmert, K. Imoto, V. Muniz-Medina, C.C. Baker, J.J.
DiGiovanna, D. Schmidt, A. Khadavi, A. Metin, E. Gozukara,
H. Slor, A. Sarasin, K.H. Kraemer, Reduced XPC DNA repair
gene mRNA levels in clinically normal parents of xeroderma
pigmentosum patients, Carcinogenesis 27 (2006) 84–94.
 L. Daya-Grosjean, M.R. James, C. Drougard, A. Sarasin, An
immortalized xeroderma pigmentosum, group C, cell line
which replicates SV40 shuttle vectors, Mutat. Res. 183 (1987)
 S. Emmert, H. Slor, D.B. Busch, S. Batko, R.B. Albert, D.
Coleman, S.G. Khan, B. Abu-Libdeh, J.J. DiGiovanna, B.B.
Cunningham, M.M. Lee, J. Crollick, H. Inui, T. Ueda, M.
Hedayati, L. Grossman, T. Shahlavi, J.E. Cleaver, K.H.
Kraemer, Relationship of neurologic degeneration to
genotype in three xeroderma pigmentosum group G
patients, J. Invest. Dermatol. 118 (2002) 972–982.
 K. Imoto, N. Kobayashi, S. Katsumi, Y. Nishiwaki, T.A.
Iwamoto, A. Yamamoto, Y. Yamashina, T. Shirai, S.
Miyagawa, Y. Dohi, S. Sugiura, T. Mori, The total amount of
DNA damage determines ultraviolet-radiation-induced
cytotoxicity after uniformor localized irradiation of human
cells, J. Invest. Dermatol. 119 (2002) 1177–1182.
 Y. Nishiwaki, N. Kobayashi, K. Imoto, T.A. Iwamoto, A.
Yamamoto, S. Katsumi, T. Shirai, S. Sugiura, Y. Nakamura, A.
Sarasin, S. Miyagawa, T. Mori, Trichothiodystrophy
fibroblasts are deficient in the repair of ultraviolet-induced
cyclobutane pyrimidine dimers and (6-4) photoproducts, J.
Invest. Dermatol. 122 (2004) 526–532.
 A. Nakagawa, N. Kobayashi, T. Muramatsu, Y. Yamashina, T.
Shirai, M.W. Hashimoto, M. Ikenaga, T. Mori,
Three-dimensional visualization of ultraviolet-induced DNA
damage and its repair in human cell nuclei, J. Invest.
Dermatol. 110 (1998) 143–148.
 N.G. Jaspers, D. Bootsma, Genetic heterogeneity in
ataxia-telangiectasia studied by cell fusion, Proc. Natl. Acad.
Sci. U.S.A. 79 (1982) 2641–2644.
 N. Kobayashi, S. Katsumi, K. Imoto, A. Nakagawa, S.
Miyagawa, M. Furumura, T. Mori, Quantitation and
visualization of ultraviolet-induced DNA damage using
specific antibodies: application to pigment cell biology,
Pigment Cell Res. 14 (2001) 94–102.
 K.S. Oh, K. Imoto, J. Boyle, S.G. Khan, K.H. Kraemer, Influence
of XPB helicase on recruitment and redistribution of
nucleotide excision repair proteins at sites of UV-induced
DNA damage, DNA Repair (Amst.) 6 (2007) 1359–1370.
 M. Volker, M.J. Mone, P. Karmakar, A. van Hoffen, W. Schul,
W. Vermeulen, J.H. Hoeijmakers, R. van Driel, A.A. van
Zeeland, L.H. Mullenders, Sequential assembly of the
dna repair 8 ( 2 0 0 9 ) 114–125
nucleotide excision repair factors in vivo, Mol. Cell. 8 (2001)
 E.M. Gozukara, S.G. Khan, A. Metin, S. Emmert, D.B. Busch, T.
Shahlavi, D.M. Coleman, M. Miller, N. Chinsomboon, M.
Stefanini, K.H. Kraemer, A stop codon in xeroderma
pigmentosum group C families in Turkey and Italy:
molecular genetic evidence for a common ancestor, J. Invest.
Dermatol. 117 (2001) 197–204.
 L. Riou, E. Eveno, A. van Hoffen, A.A. van Zeeland, A.
Sarasin, L.H. Mullenders, Differential repair of the two major
UV-induced photolesions in trichothiodystrophy fibroblasts,
Cancer Res. 64 (2004) 889–894.
 S. Emmert, N. Kobayashi, S.G. Khan, K.H. Kraemer, The
xeroderma pigmentosum group C gene leads to selective
repair of cyclobutane pyrimidine dimers rather than 6-4
photoproducts, Proc. Natl. Acad. Sci. U.S.A. 97 (2000)
 F. Chavanne, B.C. Broughton, D. Pietra, T. Nardo, A. Browitt,
A.R. Lehmann, M. Stefanini, Mutations in the XPC gene in
families with xeroderma pigmentosum and consequences
at the cell, protein, and transcript levels, Cancer Res. 60
 S.G. Khan, H.L. Levy, R. Legerski, E. Quackenbush, J.T.
Reardon, S. Emmert, A. Sancar, L. Li, T.D. Schneider, J.E.
Cleaver, K.H. Kraemer, Xeroderma pigmentosum group C
splice mutation associated with autism and
hypoglycinemia, J. Invest. Dermatol. 111 (1998) 791–796.
 L. Li, E.S. Bales, C.A. Peterson, R.J. Legerski, Characterization
of molecular defects in xeroderma pigmentosum group C,
Nat. Genet. 5 (1993) 413–417.
 A.J. Ridley, J. Colley, D. Wynford-Thomas, C.J. Jones,
Characterisation of novel mutations in Cockayne syndrome
type A and xeroderma pigmentosum group C subjects, J.
Hum. Genet. 50 (2005) 151–154.
 A. Rivera-Begeman, L.D. McDaniel, R.A. Schultz, E.C.
Friedberg, A novel XPC pathogenic variant detected in
archival material from a patient diagnosed with Xeroderma
Pigmentosum: a case report and review of the genetic
variants reported in XPC, DNA Repair (Amst.) 6 (2007)
 H. Slor, S. Batko, S.G. Khan, T. Sobe, S. Emmert, A. Khadavi,
A. Frumkin, D.B. Busch, R.B. Albert, K.H. Kraemer, Clinical,
cellular, and molecular features of an Israeli Xeroderma
Pigmentosum Family with a Frameshift Mutation in the XPC
gene: sun protection prolongs life, J. Invest. Dermatol. 115
 L.E. Maquat, Nonsense-mediated mRNA decay in mammals,
J. Cell. Sci. 118 (2005) 1773–1776.
 G. Yasuda, R. Nishi, E. Watanabe, T. Mori, S. Iwai, D. Orioli, M.
Stefanini, F. Hanaoka, K. Sugasawa, In vivo destabilization
and functional defects of the xeroderma pigmentosum C
protein caused by a pathogenic missense mutation, Mol.
Cell. Biol. 27 (2007) 6606–6614.
 S.G. Khan, A. Metin, E. Gozukara, H. Inui, T. Shahlavi, V.
Muniz-Medina, C.C. Baker, T. Ueda, J.R. Aiken, T.D.
Schneider, K.H. Kraemer, Two essential splice lariat
branchpoint sequences in one intron in a xeroderma
pigmentosum DNA repair gene: mutations result in reduced
XPC mRNA levels that correlate with cancer risk, Hum. Mol.
Genet. 13 (2004) 343–352.
 M. Kozak, An analysis of vertebrate mRNA sequences:
intimations of translational control, J. Cell. Biol. 115 (1991)
 M. Kozak, Initiation of translation in prokaryotes and
eukaryotes, Gene 234 (1999) 187–208.
 M. Kozak, Some thoughts about translational regulation:
forward and backward glances, J. Cell. Biochem. 102 (2007)
 A. Tapias, J. Auriol, D. Forget, J.H. Enzlin, O.D. Scharer, F.
Coin, B. Coulombe, J.M. Egly, Ordered conformational
changes in damaged DNA induced by nucleotide excision
repair factors, J. Biol. Chem. 279 (2004) 19074–19083.
 I. Rapin, Y. Lindenbaum, D.W. Dickson, K.H. Kraemer, J.H.
Robbins, Cockayne syndrome and xeroderma pigmentosum,
Neurology 55 (2000) 1442–1449.
 J.H. Robbins, K.H. Kraemer, M.A. Lutzner, B.W. Festoff, H.G.
Coon, Xeroderma pigmentosum. An inherited disease with
sun sensitivity, multiple cutaneous neoplasms, and
abnormal DNA repair, Ann. Intern. Med. 80 (1974) 221–248.
 A.D. Andrews, S.F. Barrett, J.H. Robbins, Xeroderma
pigmentosum neurological abnormalities correlate with
colony-forming ability after ultraviolet radiation, Proc. Natl.
Acad. Sci. U.S.A. 75 (1978) 1984–1988.
 J. Hananian, J.E. Cleaver, Xeroderma pigmentosum
exhibiting neurological disorders and systemic lupus
erythematosus, Clin. Genet. 17 (1980) 39–45.
 E.J. Quackenbush, K.H. Kraemer, W.A. Gahl, V. Schirch, D.A.
Whiteman, K. Levine, H.L. Levy, Hypoglycinaemia and
psychomotor delay in a child with xeroderma pigmentosum,
J. Inherit. Metab. Dis. 22 (1999) 915–924.
 J.H. Robbins, R.A. Brumback, M. Mendiones, S.F. Barrett, J.R.
Carl, S. Cho, M.B. Denckla, M.B. Ganges, L.H. Gerber, R.A.
Guthrie, Neurological disease in xeroderma pigmentosum.
Documentation of a late onset type of the juvenile onset
form, Brain 114 (1991) 1335–1361.
 J.H. Robbins, R.A. Brumback, A.N. Moshell, Clinically
asymptomatic xeroderma pigmentosum neurological
disease in an adult: evidence for a neurodegeneration in
later life caused by defective DNA repair, Eur. Neurol. 33
 P.S. Harper, Practical Genetic Counselling,
Butterworth-Heinemann Ltd., Oxford, 1988.
 A.H. Bittles, S.G. Sullivan, L.A. Zhivotovsky, Consanguinity,
caste and deaf-mutism in Punjab, 1921, J. Biosoc. Sci. 36
 M. Sajjad, A.A. Khattak, J.E. Bunn, I. Mackenzie, Causes of
childhood deafness in Pukhtoonkhwa Province of Pakistan
and the role of consanguinity, J. Laryngol. Otol. (2008) 1–7.
 M.B. Petersen, P.J. Willems, Non-syndromic,
autosomal-recessive deafness, Clin. Genet. 69 (2006)