Case Report

XP79KO, a 6-y-old boy, had presented with severe photosensitivity since 6 mo old and was tentatively diagnosed as XP at the age of 1 y. He developed freckles on his face at the age of 2 y. He has never shown neurological symptoms. No skin cancer had developed under strict sun avoidance. There was no consanguinity in his parents. His photo irradiation test revealed that MED was 200 J per m² and the peak time causing erythema delayed up to 72 h after ultraviolet (UV)B exposure. He and his family gave their consent for investigating inherited variation of XP genes. The Medical Ethics Committee of Kobe University approved this investigation, which was conducted in accordance with the Declaration of Helsinki principles.

Mutations in the XPA Gene in XP79KO

Genomic DNA was extracted from normal and XP79KO-cultured fibroblasts according to a standard phenol/chroloform protocol. All exons and exon–intron boundaries of the XPA gene were PCR amplified. At first, we examined the frequently mutated sites in exons 3, 4, 6 in Japanese patients with XPA that can be recognized by restriction enzymes AlwNI, MseI, and HphI, respectively, as described (^{Nishigori et al, 1993}). The PCR-RFLP analysis showed that XP79KO is heterozygous for the AlwNI mutation (data not shown). Direct sequence analysis showed that the other undetermined mutation was T to G transversion at the second nucleotide in intron 1 (Figure 1a). This mutation was not found in genomic DNA from 30 non-affected unrelated Japanese individuals or the SNP database network in Japan (http://www.snpnet.jst.go.jp/top_e.html). The father of the patient is heterozygous for the AlwNI site and the mother is heterozygous for this intron 1 mutation.

Figure 1.

Point mutation in intron 1 in XP79KO causes abberant splicing, resulting in frameshift in the coding region and stop site downstream. (a) Sequence of the region around exon 1–intron boundary of XP79KO and a normal individual. T to G transversion in the second base of intron 1 in XP79KO is schematically shown (star). (b) RT-PCR products from xeroderma pigmentosum group A (XPA) mRNA in normal human and XP79KO. Kb ladder DNA was used as size markers. (c) Base sequences of the cloned cDNA corresponding to normal and XP79KO mRNAs with codon numbers (boxed). Underlined codons mean frameshift. Two arrowheads indicate abnormal splice sites. A part of exon 1 in normal sequence is not shown. (d) A scheme of the aberrant splicing of pre-mRNA from the AlwNI and intron 1 mutation alleles in the XPA gene in XP79KO cells.

Full figure and legend (49K)

Aberrant Splicing of the XPA Pre-mRNA in XP79KO Cells

Since this intron 1 mutation disrupts the normal 5' splice donor site of intron 1, we examined the abnormal splicing in XP79KO cells by RT-PCR. Total RNA extracted from XP79KO and normal cells was reverse transcribed and amplified using primers (RT-E1F: 5'-GAGCTAGGTCCTCGGAGTGG-3', RT-E6R: 5'-TGAAAAGGACCAATCTAAATTTCC-3') encompassing the entire coding region of XPA mRNA (Figure 1b). These DNA fragments were cloned and sequenced. Band I had the normal XPA cDNA sequence. Band II for XP79KO contained three PCR products. One was identified as a DNA fragment lacking exon 3, which was designated as def I I (^{Satokata et al, 1992}), and the other two were found to be lacking the latter parts of exon 1, which were named def E1A and def E1B (Figure 1c). The sizes of the RT-PCR products from def I I, def E1A, and def E1B were 811, 816, and 822 bp, respectively, and these products consisted of band II (Figure 1d). Both def E1A and def E1B caused frameshifts that result in a stop codon at the fifth triplet of exon 2 (Figure 1c).

No XPA Protein was Detected in XP79KO Cells

Cell extract was prepared from normal, XP79KO, and XP101KO cells, which were confirmed to have the homozygous AlwNI mutation, and separated by SDS-PAGE (^{Maniatis et al, 1989}). Western blot analysis using two kinds of anti-XPA protein antibodies showed that two bands of 40 and 38 kDa were detected in normal cells (Figure 2, arrows). The 38 kDa protein is tentatively considered to be a degradation product of the 40 kDa protein (^{Miura et al, 1991}). No XPA protein, however, was detected in XP79KO and XP101KO cells (Figure 2).

Figure 2.

XPA protein was not detected in XP79KO. Western blot analysis of normal human, XP79KO and XP101KO cells with mouse monoclonal (BD Pharmingen, San Diego, California: left panel) and rabbit polyclonal (Santa Cruz Biotechnology, Santa Cruz, California: right panel) anti-human xeroderma pigmentosum group A (XPA) antibodies. Horseradish peroxidase-conjugated secondary antibodies were visualized using an enzyme chemiluminescence system. Two arrows show the two bands of human XPA protein. Background (non-specific bands) indicates that the amount of the loaded extract was approximately equal in the lanes.

Full figure and legend (106K)

Cellular UV Sensitivity and UV-Induced Unscheduled DNA Synthesis (UDS)

The ability to repair the UV-induced DNA damage was determined by measuring cellular sensitivity to UV by means of the colony formation assay and the UDS as described (^{Nishigori et al, 1994}). D₀ value of both XP79KO and XP101KO cells was 0.8 J per m² whereas that of normal cells was 5.0 J per m². UV-induced UDS in XP79KO cells was 4.75% of the normal level. Both the post-UV colony-forming ability and UV-induced UDS of the cells from XP79KO's mother showed a normal level.

Discussion

Eighteen mutation sites in the XPA gene in the world have been identified so far (^{Tanaka et al, 1993;States et al, 1998}). We found the nineteenth mutation that is the first mutation around exon 1 of the XPA gene.

The severity of neurological abnormalities in Japanese patients with XPA correlates with the locations of the mutations. XPA patients with the homozygous AlwNI mutation (skipping the zinc-finger motif in exon 3) manifest the hearing impairment, which is one of the earliest neurological symptoms, around the age of 5 y. Around the age of 10 y, they show weak tendon reflexes and gait disturbance. An XPA patient with the homozygous HphI mutation (codon 228 nonsense mutation in exon 6) had less severe neurological manifestations compared with those with the homozygous AlwNI mutation. This difference can be explained by the fact that the mutated XPA protein produced from the homozygous HphI mutation remains zinc-finger domain (exon 3) intact and is more stable than that from the homozygous AlwNI mutation (^{Sato et al, 1987;Miura et al, 1991}). At first, we thought that the other undetermined mutation of XP79KO should be located near the 3' end of the XPA gene that would result in mild clinical manifestations because he had shown no neurological symptoms at the age of 6 y. This patient, however, has the novel intron 1 mutation that results in aberrant mRNA splicing and no XPA protein production. These in vitro data coincide with cellular repair data such as UV sensitivity test and UV-induced UDS that were similar to that of XPA patients with the homozygous AlwNI mutation.

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Acknowledgments

We would like to thank the patients and families who contributed to this study. We are deeply indebted to Ms Horie for her excellent technical assistance. This work was supported by grants from the Ministry of Education, Science, Sports, and Culture of Japan.