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Human Molecular Genetics, 2004, Vol. 13, No. 3 343-352
DOI: 10.1093/hmg/ddh026

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

Sikandar G. Khan1, Ahmet Metin2, Engin Gozukara3, Hiroki Inui1, Tala Shahlavi1, Vanessa Muniz-Medina4, Carl C. Baker4, Takahiro Ueda1, Juliet R. Aiken5, Thomas D. Schneider5 and Kenneth H. Kraemer1,*

1Basic Research Laboratory, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA, 2Department of Dermatology, Yüzüncü Yil University Medical School, Van, Turkey, 3Department of Biochemistry, Inönü University Medical School, Malatya, Turkey, 4Laboratory of Cellular Oncology, Center for Cancer Research, National Cancer Institute, Bethesda, MD, USA and 5Laboratory of Experimental and Computational Biology, National Cancer Institute, Frederick, MD, USA

Received September 24, 2003; Revised November 15, 2003; Accepted November 23, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The lariat branch point sequence (BPS) is crucial for splicing of human nuclear pre-mRNA yet BPS mutations have infrequently been reported to cause human disease. Using an inverse RT–PCR technique we mapped two BPS to the adenosine residues at positions –4 and –24 in intron 3 of the human XPC DNA repair gene. We identified homozygous mutations in each of these BPS in two newly diagnosed Turkish families with the autosomal recessive disorder xeroderma pigmentosum (XP). Cells from two severely affected children in family A harbor a homozygous point mutation in XPC intron 3 (–9 T to A), located within the downstream BPS. Using a real-time quantitative reverse transcriptase–polymerase chain reaction (QRT–PCR) assay, these cells expressed no detectable (<0.1%) normal XPC message. Instead they expressed an XPC mRNA isoform with deletion of exon 4 that has no DNA repair activity in a host cell reactivation (HCR) assay. In contrast, in cells from three mildly affected siblings in family B, the BPS adenosine located at the –24 position in XPC intron 3 is mutated to a G. Real-time QRT–PCR revealed 3–5% of normal XPC message. These cells from family B had a higher level of HCR than cells from the severely affected siblings in family A, who had multiple skin cancers. Mutations identified in two BPS of the XPC intron 3 resulted in alternative splicing that impaired DNA repair function, thus implicating both of these BPS as essential for normal pre-mRNA splicing. However, a small amount of normal XPC mRNA can provide partial protection against skin cancers.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The expression of eukaryotic genes requires the accurate removal of introns and joining of the flanking exons by RNA splicing (1–3). This occurs by a two-step transesterification mechanism: in the first step, the intron 5' splice site (donor) is cleaved to generate a 5'exon and a lariat intermediate, and in the second step, the intron 3' splice site (acceptor) is cleaved to generate ligated exons (spliced product) and an excised lariat intron. RNA splicing is carried out by spliceosomes assembled on the pre-mRNA. Both 5' and 3' splice site sequences have critical roles in the early steps of spliceosome assembly (1,2). The 3' end of an intron contains three sequence elements critical for the splicing as follows: the splice lariat branch point sequence (BPS), the polypyrimidine (Py) tract, and the conserved dinucleotide AG at the 3' splice site. In recent years many mutations at splice sites have been reported which have been shown to affect exon definition and the splicing of pre-mRNA (4), but few mutations in BPS have been identified that result in human genetic diseases (5). Those mutations have all been in dominant or X-linked disorders (6–18), but not in a recessive disorder such as xeroderma pigmentosum (XP). The relationship of mutations in BPS of introns to severity of clinical symptoms has not been clearly established.

XP is a rare recessive disease characterized by extreme sun sensitivity, abnormal pigmentation, and a greater than 1000-fold increased incidence of skin cancers that occur at an average age of <10 years in association with defective DNA repair (19,20). The xeroderma pigmentosum group C (XPC; MIM 278720) DNA repair gene encodes a 940 amino acid protein involved in global genome nucleotide excision repair. We previously determined the genomic structure of the XPC gene to facilitate the detection of mutations in introns and exons of this gene (21). Earlier studies indicated that XP-C patients have mutations within exons or at intron–exon junctions and generally have similarly severe clinical features (22–26). Here we report splicing defects in the XPC gene in five cell lines from two Turkish XP-C families as a consequence of mutations within two different functional BPS in the same intron. We found a relationship between expression of normal and alternatively spliced XPC mRNA isoforms, DNA repair status and severity of clinical disease.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Clinical findings
Turkish siblings from family A, XP100TMA, a 20-year-old male, and XP101TMA, his 16-year-old sister, were both severely affected with xeroderma pigmentosum (Fig. 1A and B). They began developing skin lesions at 3 years of age. Both had cutaneous atrophy, telangiectasia, actinic keratoses and multiple skin cancers including squamous cell carcinomas, basal cell carcinomas and melanomas. Their parents are distant cousins, as shown in the pedigree (Fig. 1C).



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  		Figure 1. Severely affected XP-C siblings from Turkey in family A. (A) The affected proband, XP100TMA, a 20-year-old male and (B) his 16-year-old sister, XP101TMA, began developing skin lesions at 3 years of age. They had cutaneous atrophy, telangiectasia, actinic keratoses and multiple skin cancers including squamous cell carcinomas, basal cell carcinomas and melanomas. (C) Pedigree of family of XP100TMA. Five generations of this consanguineous family are shown. The proband, XP100TMA (solid square), and his affected sister, XP101TMA (solid circle), have an unaffected brother and two sisters (open symbols). The mother (XPH102TMA) is an obligate heterozygote (half solid symbol).

 
In contrast, the Turkish sisters from family B, XP72TMA, 20 years old, XP73TMA, 18 years old and XP123TMA, 11 years old, were mildly affected with xeroderma pigmentosum (Fig. 2A–C). Skin lesions began at age 3–5 years. They had freckling but no atrophy, telangiectasia or actinic keratoses. XP73TMA had a squamous cell carcinoma excised from her face at age 12 years. The other sisters did not have skin cancer. Their parents are first cousins (Fig. 2D).



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  		Figure 2. Mildly affected XP-C siblings from Turkey in family B. (A) The affected proband, XP72TMA, a 20-year-old girl, (B) XP73TMA, an 18-year-old girl, and (C) their 11-year-old sister, XP123TMA, had freckling but not atrophy, telangiectasia or actinic keratoses. XP73TMA developed a squamous cell carcinoma on her face. Skin lesions began at age 3–5 years. (D) Pedigree of family of XP72TMA. Four generations of this consanguineous family are shown. The proband, XP72TMA (solid circle), has two affected sisters, XP73TMA and XP123TMA (solid circles), and three unaffected brothers (open symbols). Their parents are half cousins (half solid symbols).

 
The families lived in rural villages about 200 km apart in Van province in eastern Turkey near the border with Iran. This is a very sunny region about 2000 m above sea level. The children in both families were not protected from the sun before diagnosis in 1996 when XP72TMA and XP100TMA were 13 and 12 years old, respectively. They were then instructed to practice sun protection. The severely affected siblings in family A strictly avoid sunlight and remain in a dark room all day. In contrast, the mildly affected sisters in family B wear a headscarf to cover their hair and neck but do not strictly avoid sun exposure.

Relationship of DNA repair status to clinical phenotypes
DNA repair status of the cell lines studied here was determined employing a post-UV host cell reactivation (HCR) assay that measures the ability of cells to repair DNA damage in a UV-treated plasmid (which contained a luciferase reporter gene) 2 days after transfection in vivo. The normal cells had higher post-UV relative luciferase activity than that of the cell lines from all of the XP patients (Fig. 3A). The post-UV relative luciferase activity in cells from severely affected XP patients (XP100TMA and XP101TMA) was lower than that in the mildly affected patients' cells (XP72TMA, XP73TMA and XP123TMA). The cells from clinically normal mother of XP100TMA (XPH102TMA), an obligate XP heterozygote, had close to normal activity. These results indicate a relationship between DNA repair status of the cells and the clinical phenotypes in the XP patients.



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  		Figure 3. Post-UV host cell reactivation (HCR) studies. HCR was employed to measure DNA repair status of cell lines studied, to determine the complementation group and to assess the function of abnormally spliced XPC mRNA. (A) The relative luciferase activity was considerably higher in the lymphoblastoid cell lines from mildly affected siblings (XP72TMA, XP73TMA and XP123TMA) than in the cells from severely affected siblings (XP100TMA and XP101TMA). The XP heterozygote (XPH102TMA) had post-UV relative luciferase activity close to the normal cells. (B and C) Assignment to XP-C. UV-treated (1000 J/m2) reporter gene plasmid (pCMVLuc) was cotransfected with an XP cDNA containing plasmid [pXPA (circle), pXPC (triangle) or pXPD (diamond)] into triplicate cultures of primary patients fibroblasts. Each symbol represents the relative reporter gene activity in an independent transfection experiment 48 h after transfection compared with the corresponding unirradiated control reporter gene plasmid. The correction was achieved only by cotransfection with pXPC, indicating that these cells are in complementation group C: (B) XP100TMA, (C) XP72TMA. (D and E) Deletion of XPC exon 4 abolishes DNA repair function. Co-transfection of UV-treated pCMVLuc plasmid into XPC cells [XP4PA (SV40)] (D) or normal cells (GM00637) (E) along with full-length XPC cDNA expression vector (XPC/pcDNA3.1A) (solid circle) with the XPC cDNA expression vector with deletion of exon 4 (XPC-del Ex4/pcDNA3.1A; solid triangle) or with the empty vector (pcDNA3.1A; diamond). Each symbol represents the relative luciferase activity in triplicate independent transfection experiments measured 48 h after transfection compared with the corresponding unirradiated control pCMVLuc plasmid. In the XPC cells increased luciferase activity was seen after co-transfection with the full length XPC cDNA but not with the cDNA with deletion of exon 4. Overexpression of the XPC plasmid with deletion of exon 4 did not positively or negatively influence the normal cells' ability to repair UV-damaged plasmid DNA.

 
Assignment of XP cells to complementation group C
We performed complementation group assignment for probands XP72TMA and XP100TMA cells by use of HCR. The UV irradiated luciferase reporter gene plasmid was transfected into XP100TMA and XP72TMA fibroblasts along with plasmids expressing cloned XP group A, C or D cDNA. Only co-transfection with a plasmid containing the XPC cDNA led to a markedly increased post-UV HCR in the XP100TMA and XP72TMA cells thus assigning these cells to the XP complementation group C (Fig. 3B and C).

Mutations in the XPC gene
In comparison to wild-type cells (Fig. 4A), the cells from the severely affected XP patients in family A (XP100TMA and XP101TMA) had a point mutation (–9 T to A) in XPC intron 3 (Fig. 4B). RT–PCR of mRNA from these cells (using primer pair A1 and Exon5R) resulted in a short band (171 bp) and no detectible band of the normal size (295 bp; Fig. 5B, lanes 1 and 2). Sequence analysis of this cDNA band revealed an XPC mRNA isoform with a 124 bp deletion comprising the entire exon 4 (Fig. 5C) and no normally spliced XPC mRNA. Thus, the T to A mutation at the –9 position disrupted the normal splicing of intron 3. The cells from their mother (XPH102TMA) were heterozygous for the XPC intron 3 –9 T to A mutation (data not shown) and showed both normal and rapidly migrating XPC mRNA isoforms (Fig. 5B, lane 3).



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  		Figure 4. Mutational analysis of XPC gene in XP100TMA and XP72TMA cells. The sequence of a portion of the genomic DNA of XPC intron 3–exon 4 junctions from (A) wild-type (GenBank accession: AF261895 bases 323–356), (B) XP100TMA and (C) XP72TMA was determined by use of an ABI automated sequencer. The homozygous mutations in the XPC intron 3 are indicated by arrows. XP100TMA has a point mutation (T to A) at –9 position (AF261895 344 T to A) and XP72TMA contains a point mutation at –24 position (A to G); (AF261895 329 A to G). (The mutated A and G are enlarged.) The branch point adenosines at –4 and –24 positions are enlarged with yellow background and the branch point sequences in XPC intron 3 are indicated by red lines. The 5' end of exon 4 is indicated by a black line.

 


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  		Figure 5. Analysis of XPC cDNA exon 4 skipping in the XP cells. (A) Schematic diagram of XPC isoforms containing exon 4 (295 bp) and skipping exon 4 (171 bp) using primers A1 and 5R. (B) RT–PCR assay comparing the profiles of XPC mRNA species. Total RNA was subjected to RT–PCR as described in Materials and Methods, and the products were displayed on a 2% Nu-sieve agarose gel stained with ethidium bromide. The PCR products representing XPC mRNA species with (295 bp) and without (171 bp) exon 4 is indicated alongside the gel by an arrow. Lane 1, RNA from XP100TMA cells; lane 2, XP101TMA; lane 3, XPH102TMA; lane 4, XP72TMA; lane 5, XP73TMA; lane 6, XP123TMA; lane 7, normal lymphoblastoid (KR06057); lane 8, marker, 100 bp DNA ladder (MBI Fermentas). (C) Sequence analysis of the faster migrating RT–PCR products (171 bp) from the XP patient's cells. The bands were agarose gel purified and sequenced employing reverse primer Exon5R by use of an ABI automated sequencer. The faster migrating band showed deletion of the entire 124 bases of exon 4. The strand complementary to the RNA is shown.

 
The cells from the mildly affected siblings in family B (XP72TMA, XP73TMA and XP123TMA) had a point mutation in XPC intron 3 (–24 A to G; Fig. 4C). RT–PCR of mRNA from these cells using a primer pair flanking XPC exon 4 (A1 and Exon5R) resulted in two bands: one of normal size and a second of shorter size (Fig. 5B, lanes 4–6). Sequencing of the shorter band revealed a deletion of 124 bases comprising the entire exon 4 (data not shown). The normal size band in these patients and in the normal cells (Fig. 5B, lane 7) had inclusion of exon 4. Thus, these mildly affected XP-C patients in family B express two XPC mRNA isoforms: (i) normal full-length and (ii) XPC mRNA isoform with deletion of exon 4 (Fig. 5A).

Lariat formation occurs at conserved sequences within the intron
We were surprised that the mutations that we found in the XP patients were relatively far away from the intron–exon junction, raising the possibility that they involved the splice lariat intermediate. We thus decided to map the location of the BPS for the splice lariat intermediates of XPC intron 3 by use of RT–PCR with gene-specific sense and anti-sense primers. The first-strand cDNA synthesis was initiated using an anti-sense primer at the 5' end of the XPC intron 3 (S75), which was expected to produce a product that can be extended beyond the BPS (Fig. 6A and B). Mapping of the BPS was achieved by use of Superscript II because of its ability to read through the 2'–5' phosphodiester bond present in lariat splicing intermediates, converting the lariat circle into linear cDNA that was amplified by inverse PCR and subsequently sequenced (Fig. 6) (27). The RNA from four normal fibroblasts yielded two bands with the different primer pairs: 238 and 218 bp (Fig. 6C, lanes 3–6, using primers S18 and S76) or 177 and 157 bp (using primers S18 and S30, data not shown). Neither of these bands was seen in RNA from the severely affected XP patient's cells (XP100TMA; Fig. 6C, lane 1). However, in the cells from mildly affected XP-C patient (XP72TMA), a faint band corresponding to the BPS adenosine at the –4 position was observed (Fig. 6C, lane 2). The Bioanalyzer electropherogram data for lanes 1–3 are shown in Figure 6D. The upper and lower bands in Figure 6C lane 3 correspond to the 238 and 218 bp peaks seen for the normal fibroblast sample in Figure 6D. A minor non-specific peak of 230 bp is also seen for this sample. XP72TMA (Fig. 6C, lane 2) shows only the specific 238 bp peak and the non-specific peak at 230 bp. This data correlates well with the expected elimination of the shorter lariat intermediate due to mutation of the upstream BP. Both specific peaks are absent for XP100TMA (Fig. 6C, lane 1) which shows only the non-specific 230 bp peak. This result is consistent with the complete elimination of exon 4 inclusion by mutation of the downstream BPS.



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  		Figure 6. BPS Mapping. Mapping of the two XPC intron 3 BP adenosines at 4 and 24 bases upstream of the intron 3–exon 4 junction by inverse RT–PCR and sequencing. (A) Diagram of an intron-containing splicing intermediate in a lariat configuration in which the G at the 5' end of the intron is linked by a 2'–5' phosphodiester bond (RNA branch) to a single adenosine residue near the 3' end of the intron. The cDNA synthesis initiating at an anti-sense primer (S75) in the 5' end of XPC intron 3 by Superscript II was predicted to stop at the branching nucleotide, but instead continued and yielded a molecule that extended into the 3' end of the intron. The blue line represents antisense synthesis at the 5' end of the intron leading through the lariat and proceeding in an antisense direction (red line). (B) Diagram of a primer pair (S76 and S18) designed for inverse PCR and sequencing. The red line indicates the BPS at the 3' end of intron 3 and the blue line indicates the 5' end of intron 3. (C) RT employing a gene-specific primer (S75) followed by PCR with inverse primers S18 and S76 generated two lariat-specific bands of 238 and 218 bp with RNA from four different normal fibroblasts (lanes 3, AG13145; lane 4, AG04659; lane 5, AG13354; and lane 6, AG13129) which were separated using an Agilent 2100 Bioanalyzer. Lane 1, XP100TMA showed no lariat-specific bands. Lane 2, XP72TMA showed only the upper band. (D) The Agilent 2100 Bioanalyzer electropherogram data for lanes 1–3 that were used to create the image in Figure 6C. The upper and lower bands in panel C lane 3 correspond to the 238 and 218 bp peaks seen for the normal fibroblast sample (lane 3). A minor non-specific peak of 230 bp is also seen for this sample. XP72TMA (lane 2) shows only the specific 238 bp peak and the nonspecific peak at 230 bp. Both specific peaks are absent for XP100TMA (lane 1) which shows only the non-specific 230 bp peak. (E and F) Sequence of the PCR products obtained using S18 primer. (E) The BPS including the A at –4 position (underlined in red) is extended to the 5' end of the intron 3 sequence (underlined in blue). (Note an extra A has been introduced by the polymerase.) (F) The BPS near the A at the –24 position (underlined in red) is extended to the 5' end of the intron 3 sequence (underlined in blue). (Note the A has been skipped by the polymerase.)

 
Sequence analysis of multiple clones placed the first BPS adenosine 4 nucleotides upstream and the second BPS adenosine 24 nucleotides upstream of the intron 3–exon 4 junction (Fig. 6E and F). These sequences demonstrate that the G residue at the 5' end of intron 3 is linked to the BPS adenosine. The 20 bp difference (Fig. 6C and D) was the size expected for two lariat-derived products extended from the primer past two different BPS into the upstream XPC intron 3 sequence. The BPS that we mapped within XPC intron 3 in the normal cells are 5'-TGTTGAT-3' (BP adenosine at the –4 position) and 5'-TACTGAT-3' (BP adenosine at the –24 position; Fig. 6E and F and Fig. 4, red bars). The detection of two bands corresponding to both BP adenosines in the XPC intron 3 at the –4 and –24 positions in the four normal fibroblasts indicates that the normal splicing utilizes BP formation at both –4 and –24 positions (Fig. 6C, lanes 3–6).

DNA repair function of XPC mRNA isoform with deletion of exon 4
The alternatively spliced XPC mRNA isoform with deletion of exon 4 found in XP100TMA and XP72TMA cells (Fig. 5) resulted in a frameshift and truncation that was expected to have altered DNA repair function. We constructed an expression vector containing XPC cDNA with a deletion of exon 4 and assessed its function by employing a transient post-UV HCR complementation assay. Transfection of plasmid containing XPC cDNA skipping exon 4 (pcDNA3.1/V5-His/A-XPC-deletedexon4) resulted in no increase in luciferase activity in the XPC cells in comparison to the empty vector control (Fig. 3D). Thus, XPC cDNA skipping exon 4 is not functional in this DNA repair assay. To test for dominant negative function we transfected normal cells with XPC cDNA skipping exon 4. This did not alter its ability to repair the UV-damaged plasmid (Fig. 3E).

Association of XPC message levels with mild to severe clinical phenotypes
We used allele-specific primers to measure the levels of wild-type and alternatively spliced XPC mRNA in cells from the XP patients and normal controls using real time QRT–PCR (21). Exon 7 inclusion and exon 12 inclusion assays were used to assess the levels of total XPC mRNA while the exon 4 inclusion assay measured full-length XPC mRNA (Table 1). The normal cells (KR06057) had 240 fg total message (exon 12 inclusion), which was in the range that was previously reported for normal cells (21). The cells from the XP patients all had substantially lower levels of total message (45.2–81.5 fg). Similar differences between the XP and normal cells were seen for exon 7 inclusion. This low level of message is consistent with nonsense-mediated message decay, resulting from the frameshift changes leading to premature stop codons (28).


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  		Table 1. Full-length and alternatively spliced XPC mRNA isoforms in cells from XP-C patients
 
The normal cells had a measurable level of exon 4 skipping (4.3 fg) that was similar to that reported previously (21). In contrast the cells from the 5 XP patients all had elevated levels of alternatively spliced XPC message with skipping of exon 4 (24.7–49.7 fg) as expected from a mutation in the 3' portion of intron 3 that affects splicing. The level of message with exon 4 skipping was similar in both severely affected XP patients (XP100TMA and XP101TMA), and in one of the mildly affected patients (XP123TMA; 49.7, 47.9 and 46.8 fg, respectively). In addition one mildly affected XP patient (XP72TMA) had a similar level of message with exon 4 skipping as the clinically normal heterozygous mother of the severe patients (XPH102TMA; 24.7 and 24.8 fg, respectively). Thus the amount of alternatively spliced XPC mRNA with skipping of exon 4 did not correlate with the clinical symptoms, a finding that is consistent with the lack of a dominant negative function in the HCR assay (Fig. 3E). In contrast (Table 1, column 2), there was about 3–5% of normal level of XPC mRNA that included exon 4 in the clinically milder patients (XP72TMA, XP73TMA and XP123TMA; 5.5–8.2 fg) and no measurable level (<0.1%) of XPC mRNA including exon 4 in the cells from the severely affected patients (XP100TMA and XP101TMA). The cells from the clinically normal heterozygous mother of the severely affected patients (XPH102TMA) had a level of full-length XPC mRNA (22%) that was intermediate between that of the normal cells and that of the XP patients (Table 1).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Splice lariat BPS mutations in human disease
Two different homozygous mutations in two BPS of intron 3 in the XPC gene provide the molecular basis of severe or mild clinical symptoms in five patients with the autosomal recessive disorder XP from different families with the same ethnic heritage. These mutations influenced the splicing efficiency of XPC pre-mRNA to different degrees and correlated with DNA repair levels. Splice lariat BPS mutations have previously been associated only with dominant and X-linked disorders and not in recessive diseases such as XP: fish-eye disease (lecithin : cholesterol acetyltransferase (MIM 606967) mutation) (12), hepatic lipase deficiency (MIM 151670) (6), congenital contractual arachnodactyly [FBN2 (MIM 121050) mutation] (14), Sandhoff disease [beta-hexosaminidase beta-subunit (MIM 606873) mutations] (8), Ehlers–Danlos syndrome type II (MIM 1300010) (7), neurofibromatosis 2 [NF2 (MIM 607379) mutation] (17), extrapyramidal movement disorder [Segawa's syndrome; tyrosine hydroxylase (MIM 191290) mutation] (10), nail patella syndrome [LMX1B (MIM 602575) mutation] (9), X-linked hydrocephalus [neural cell adhesion molecule L1 (MIM 308840) mutation] (15), X-linked Reifenstein syndrome [androgen receptor (MIM 313700) mutation] (18), and X-linked hyper-IgM syndrome [CD40 ligand (MIM 300386) mutation] (16). In contrast to these clinical observations, laboratory studies suggested that a mutated BPS is easily replaced by a cryptic BPS with only moderately reduced splicing efficiency (29–32).

Information theory, lariat mutations and alternative splicing
Information theory is an important tool to rank normal and mutant splice junctions (33). An information content of ~2.4 bits is the apparent minimal functional value for normal splicing (4). The very low information content (–0.1 bits) of the normal XPC acceptor at exon 4 would suggest that this might be a very poor splice site and should result in even greater levels of exon 4 skipping in the normal cell lines than we found (Table 1) (21). The information contents for the XPC exon 4 splice acceptor and three other junctions (all in the CYP gene family) are near or below zero bits and yet these junctions supported accurate and efficient pre-mRNA splicing (4). Almost all mammalian introns contain a cis-acting element, the Py tract, which is usually located between the BPS and the 3'-splice site. Early in spliceosome assembly, the specific binding of U2AF65 to the polypyrimidine tract promotes the base pairing of U2 snRNA with the BPS (34,35). However, the only uniform Py tract in the XPC intron 3 (a run of five uridines) is not between the BP adenosine at –4 position and the 3' splice junction but instead lies adjacent to the second BP adenosine at –24 position, further upstream of the 3' splice junction (Fig. 4), thereby explaining the very low information content of the XPC exon 4 splice acceptor.

The BPS establishes base pairing interactions with a specific sequence of U2 snRNA, bulging out the BP nucleotide, adenosine (i.e. having no base opposite it), that forms a 2'–5' phosphodiester bond (RNA branch) with the 5' end of the introns (Fig. 6A and B) (35,36). Mutations in the BPS can weaken the binding of U2 snRNP with the BPS thus destabilizing the U1 snRNP and U2AF interactions with the pre-mRNA (37). If these interactions are destabilized, the exon boundaries will not be defined. This may be a mechanism for the exon skipping that we observed in the XPC gene as a consequence of mutation in the BPS in the cells from the XP patients.

The two BPS we mapped in XPC intron 3 (Fig. 6) are similar to previously recognized BPS for which the average information content of the BPS site is 5.4±3.1 bits (T.D. Schneider and J.R. Aiken, manuscript in preparation). In the cells from the severely affected siblings in family A, a homozygous point mutation (–9 T to A) in the downstream BPS in XPC intron 3 completely abolished normal splicing of the XPC gene (Table 1). The severity of the splicing defect probably reflects the fact that the downstream BPS overlaps the polypyrimidine tract and mutations in this region therefore affect both elements. Thus this mutation reduced the information content of the natural splice acceptor of exon 4 from –0.1 to –2.3 bits. Simultaneously the information content of the downstream BPS dropped from a low 0.4 to –1.4 bits. Splice site elements with information values below zero should not be functional (33).

In contrast, in cell lines from mildly affected siblings in family B, a homozygous point mutation (–24 A to G) in the second BPS of the same XPC intron 3 only 15 bases further upstream only slightly decreased the information content of the XPC exon 4 acceptor from –0.1 to –0.4 bits. This mutation reduced the information content of the BPS located at this same –24 adenosine position, from 9.3 to an estimated –2.6 bits. The experimental data (Fig. 6C and D, lane 2) indicate that the mutated BPS is not used, and this is consistent with the information analysis. Instead, the small amount of normally spliced XPC mRNA is the result of splicing that uses the downstream BPS. It is interesting that the upstream BPS, which has high information content (9.3 bits) and is immediately followed by a pyrimidine tract (UUUUU) that should strongly bind U2AF65, is insufficient to support splicing in the presence of the downstream BPS mutation. These data suggest that the two BPSs are not redundant, since both are essential for efficient inclusion of exon 4. The mutation in the upstream BPS has a dramatic effect on the downstream BPS and vice versa. These results provide evidence that two BPS in the same intron can be required for accurate and efficient splicing of human nuclear pre-mRNA and that multiple factors can influence splice site selection.

XPC mRNA levels and clinical disease
Since consanguinity was present in both families (Figs 1C and 2D) the cells contained the same mutation on both alleles. This permits us to assign a molecular function to each homozygous mutation. The level of the XPC mRNA isoform with deletion of exon 4 (having no DNA repair function; Fig. 3D) was similar in the mild and the severely affected patients (Table 1); thus this isoform was not responsible for the different clinical features. This is consistent with the absence of a dominant negative function as seen in the HCR assay (Fig. 3E). In contrast, there was a low but measurable (3–5%) amount of normal XPC mRNA containing exon 4 in the mildly affected XP-C patients and no measurable (<0.1%) level of XPC mRNA with exon 4 in the cell lines from the severely affected patients (Table 1). Therefore the reduction or absence of the exon 4 containing transcript leads to clinical disease. The DNA repair status of cells from the mildly affected patients was higher than in the cells from the severely affected XP-C patients (Fig. 3A), suggesting that even low levels of XPC mRNA (Table 1) and DNA repair provide some protection against UV induced skin cancer. XPC knockout mice have markedly increased post-UV skin cancer (38). Further, increased skin cancer incidence was also observed in XPC heterozygous mice with different genetic backgrounds (38,39), indicating that XPC gene products may be a rate-limiting factor in the removal of UV-induced DNA lesions. Low levels of XPC protein increased the amount of unrepaired photoproducts in the DNA (40).

Interestingly, cells from the clinically normal heterozygous mother of the severely affected patients (XPH102TMA) had an intermediate level of normal XPC message (including exon 4) and XPC mRNA with skipping of exon 4 was higher than with the normal cells (Table 1 and Fig. 5B, lane 3). This is apparently due to the combination of both nonsense-mediated message decay in the mutated allele (28) and a gene dosage effect, and has been reported previously in only one other XP-C heterozygote (25). This suggests a mechanism for the observation that XP heterozygotes may be at a higher risk for the occurrence of sun-induced skin cancers (41). Our data indicates that the amount of normal XPC message in the patient's cells can be a major determinant of clinical phenotypes.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Cell lines, culture conditions and DNA/RNA isolation
The patients were studied under a protocol (99-C-0099) approved by the Institutional Review Boards of the National Cancer Institute (Bethesda, MD, USA) and Yuzuncu Yil University Medical School, Van, Turkey. Fibroblast and lymphoblastoid cell cultures from Turkish family A—XP100TMA (GM15709, GM15710), XP101TMA (GM15715, GM15716) and XPH102TMA (GM15717, GM15718); family B—XP72TMA (GM14877, GM14876A), XP73TMA (GM14879, GM14878A), XP123TMA (GM15723), and normal primary fibroblasts (AG13145, AG04659, AG13354 and AG13129), normal SV40-transformed fibroblasts (GM00637) and lymphoblastoid (KR06057) cells were obtained from the Human Genetic Mutant Cell Repository (Camden, NJ, USA). SV40-transformed XP-C (XP4PA-SV-EB) cells were a gift from Dr R. Legerski (MD Anderson Hospital, Houston, TX). These cell lines were cultured as described (42). The separation of RNA and DNA was performed as described (21).

Mutation detection by PCR amplification, sub-cloning and nucleotide sequencing
The 16 XPC gene exons including splice donor and acceptor sites, BPS and Py tracts were PCR amplified using intronic primers flanking these sequences and sequenced as described previously (21).

Analysis of XPC exon 4 skipping
The first strand cDNA was synthesized as described (21) and was utilized to amplify the XPC exon 4 region using primer pair A1 and Exon5R and Advantage cDNA PCR kit (Clontech, CA, USA) as per the vendor's protocol following annealing/extension at 66°C. The PCR products were resolved on a 2% Nu-sieve agarose (BMA) gel stained with ethidium bromide. The fast and slow migrating PCR bands were agarose gel purified and sequenced using primer Exon5R as described (21).

Host cell reactivation and functional analysis of XPC mRNA isoform
The pCMVLuc reporter gene plasmid (43) (a generous gift from M. Hedayati and L. Grossman, Johns Hopkins University, Baltimore, MD, USA) was used to measure HCR in order to assess DNA repair ability and to assign XP cells to a specific complementation group as described previously (23,24,26,42).

We used a transient plasmid HCR assay to measure functional correction of the XPC DNA repair defects by the full-length wild-type and XPC mRNA isoform skipping exon 4 as described previously (42). In brief, for construction of an expression vector with deletion of exon 4, the XPC full-length cDNA containing the entire human XPC cDNA as well as 5'- and 3'-untranslated sequence was first released from a plasmid pBS.XPC.SK (a generous gift from Dr Fumio Hanaoka) and then subcloned at the NotI-site of the vector pcDNA3.1/V5-His/A (Invitrogen). The XPC exon 4 region was RT–PCR amplified with primer pair G1, A2 and RNA from XP100TMA cells (product size 416 bp) and subcloned into the pCR2.1-TOPO vector. Bsu36I and NheI (New England Biolabs) digestion of this construct released an XPC cDNA fragment with deleted exon 4 which replaced a similarly released wild-type XPC cDNA fragment with exon 4 in plasmid pcDNA3.1/V5-His/A-XPC that contains full-length XPC cDNA (pcDNA3.1/V5-His/A-XPC-deletedexon4).

DNA sequence information analysis
Sequences were scanned with the donor and acceptor individual information weight matrices and the identified sites were displayed as described previously (33,44). These analyses can be performed on a web server: www.lecb.ncifcrf.gov/~toms/delilaserver.html.

Mapping of precise lariat intermediates via an inverse RT-PCR and nucleotide sequencing
The methodology for mapping of the BPS in XPC intron 3 was adopted as previously published (27) with minor modifications. The cDNA was synthesized by Superscript II reverse transcriptase (Invitrogen) as per the manufacturer's protocol using an anti-sense primer (S75) in the 5' end of the XPC intron 3 (GenBank number AF261894) and RNA from normal fibroblasts (AG13145, AG04659, AG13354 and AG13129) and XP-C fibroblasts (XP100TMA and XP72TMA). The lariat intermediate was amplified by PCR using the primers as indicated in Table 2. The cDNA was amplified by PCR with a sense (S18) in the 3' region of the XPC intron 3 (GenBank number AF261895) and an anti-sense primer in the 5' end of the XPC intron 3 (either S76 or S30) using the Advantage cDNA PCR kit (Clontech, CA, USA) under the conditions described by the manufacturer. The PCR steps were conducted as follows: 94°C for 1 min, then 35 cycles of amplification (94°C for 30 s and 66°C for 3 min), ending with 66°C for 3 min. The PCR products (2 µl) were analyzed using DNA 1000 LabChip kit on the ‘Agilent 2100 Bioanalyzer’ as per the manufacturer's protocol (Agilent Technologies, Wilmington, DE, USA). The PCR products were gel purified, subcloned into the pCR 2.1-TOPO vector (TOPO TA cloning kit, Invitrogen) and sequenced.


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  		Table 2. Human XPC gene-specific primers for mapping lariat intermediate, construction of vector skipping XPC exon 4, RT–PCR for exon 4 skipping and real-time QRT–PCR assays
 
Real time quantitative reverse transcriptase-polymerase chain reaction (QRT–PCR) for XPC mRNA isoforms
XPC mRNA isoforms were quantitated using isoform-specific primer pairs (Table 2) employing real-time QRT–PCR as described (21). Real-time QRT–PCR assays were carried out on a Bio-Rad iCycler iQ system (Bio-Rad, Hercules, CA, USA) using intercalation of SYBR Green as the fluorescence reporter.

Electronic Database Information.
Online Mendelian Inheritance in Man (OMIM): www3.ncbi.nlm.nih.gov/Omim/; GenBank: www.ncbi.nlm.nih.gov:80/.


   FOOTNOTES
 
* To whom correspondence should be addressed at: DNA Repair Section, Basic Research Laboratory, National Cancer Institute, Building 37 Room 4002 MSC 4258, Bethesda, MD 20892-4258, USA. Tel: +13014969033; Fax: +3015943409; Email: kraemerk{at}nih.gov


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 MATERIALS AND METHODS
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