Compound heterozygosity for the Xeroderma pigmentosum complementation group A gene associated with a mild phenotype

ARTICLE

Xeroderma pigmentosum (XP) is a rare autosomal recessive hereditary disease characterized by hyperphotosensitivity, DNA repair defects and a predisposition to skin cancers. XP is classified into seven genetic complementation groups (A through G type) which have a defect in the nucleotide exchange repair and a variant type that is proficient in excision repair. Some are associated with neurological metabolic problems, others with trichothiodystrophy (TTD). The genes responsible for each complementation group except for group E have been cloned. The most frequently occurring type worldwide, XP group A (XPA) is particularly common in Japan and shows the most severe clinical manifestations, including neurological deterioration. Some XPA patients, however, show mild clinical symptoms and it has been reported that there is a close relationship between the clinical features and the site of gene mutations [1-6]. Tanaka et al. have cloned the XPA complementing (XPAC) gene [7] and more than 20 types of mutation in the XPAC gene have been reported in the world to date [8]. In Japan, 5 types of mutation have been shown in 84 XPA patients [9] and exon 3, intron 3 and exon 6 are hot spots for these mutations, which are easily detected by a combination of the polymerase chain reaction (PCR) and restriction length polymorphism (RFLP) analysis [1-5] (Table I). We examined DNA from the patient focusing on these mutations.

Case report

A 7-year-old boy was born from non-consanguineous phenotypically normal parents. At the age of 5 months, severe photosensitivity was noted and the patient was treated with a sunscreen. The patient visited our department at the age of 5 years since brownish macules were gradually growing on the face. Multiple brownish freckles on the bilateral cheeks, nose and upper lip were noted (Fig. 1). On the preauricular regions and earlobes, mild erythema with pytriatic scales was present. Similar freckles were scattered on the bilateral forearms and thighs. Neither telangiectasias nor malignant skin tumors were observed. Clinically, there were no obvious physical and neurological abnormalities, and no evidence of mental retardation.

Based upon these clinical symptoms, xeroderma pigmentosum was suspected. In order to complete a genetic diagnosis, dermal fibroblasts were expanded for ultraviolet (UV)-induced unscheduled DNA synthesis (UDS), gene complementation and PCR-RFLP assays.

Methods

UV irradiation

Three 10-W germicidal lamps emitting predominantly 254 nm (Toshiba GL10) were used as the source of UV light. Fluence rates were measured with a UV meter (TOPKON, Tokyo).

Measurement of unscheduled DNA synthesis after UV irradiation

UV-induced UDS was measured as described by Moriwaki et al. [1]. Cells (5 x 10⁴) were seeded on a glass cover split in 35-mm dishes. After incubation for 18 hrs, cells were washed with phosphate-buffered saline and were irradiated with UV (0 and 30 J/m²), followed by incubation in a medium containing 10 muCi/ml of methyl-[³H]-thymidine (25 Ci/nmol, Amarsham, UK) for 3 hrs. After labeling, cells were fixed with Carnoy solution and washed with 5% trichloroacetic acid. Slides were dipped in nuclear track emulsion, NTB3 (Eastman Kodack, Rochester, NY), for autoradiography. The number of grains per interphase nucleus was scored for 100 nuclei in each specimen. UDS was determined by the percent of net count when the net count of normal cells was set at 100%. Net count is determined by subtracting the mean grain count of the unirradiated cells from the mean grain count of the UV-irradiated cells.

DNA repair capacity after UV irradiation

Plasmids: pSRVcat, an expression vector of chloramphenicol acetyltransferase (CAT), was a generous gift from D. K.H. Kraemer. An expression vector, pcDNA3 was purchased form Invitrogen and pcDNA3-XPA carrying XPA gene was kindly gifted from Dr. Tanaka.

Assessment of post-UV DNA repair by plasmid host cell reactivation was performed as previously described [10, 11]. Briefly, pSRVcat was diluted to 31 mug/ml in sterile distiled water and was irradiated on ice using a germicidal lamp at a rate of 5.5 J/m²/s. One day before the transfection, 2 x 10⁵ cells were seeded into 60 mm dishes. Transfection was carried out by use of an activated dendrimer (SuperFect Reagent, QIAGEN, Germany) according to the manufacture's protocol. 0.5-2 mug of UV-treated or untreated pRSVcat was introduced into the cells co-transfected with 0.5-2 mug of pcDNA3 or pcDNA3-XPA. Forty-eight hrs after the transfection, the cell extracts were prepared and used for CAT assay by measurement of [³H] acethylchlorampenicol (Amersham, Life Science, England). The DNA repair capacity was calculated based on scintillation counts as the percentage of the residual CAT gene expression after repair of damaged DNA compared with undamaged DNA, which was set as 100%.

PCR-RFLP analysis and DNA sequencing

Genomic DNA was extracted from the fibroblasts of both the patient and a normal subject, and from peripheral blood cells of the parents with informed consent. Genomic DNA spanning exon 4 or exon 6 including splice sites was amplified by PCR using the primer sets as reported before [1-4]. For exon 4, primer 4a; 5'-GGGAATTCTTGCTGGGCTATTTGCAAAC-3' (intron 3) and 4b; 5'-GGGGATCCGCCAAACCAATTATGAC-3' (intron 4), for exon 6, 6a; 5'-GGGAATTCGGATTCACCTGAATAGCACC-3' (intron 5) and 6b; 5'-GGGGATCCACATTGTGCACACAACCAGG-3' (intron 6) were used. The primers 4a and 6a contain an Eco RI site at the 5' end and the primers 4b and 6b bear a Bam HI site at the 5' end for subcloning. PCR was carried out under the following conditions; 30 cycles of 1 min at 94° C, 1.5 min at 55° C, 1 min at 72° C. The amplified DNA fragments were digested with the restriction endonuclease, Alw NI or Hph I for 3 hrs at 37° C and then separated on a 6% polyacrylamide gel. In order to establish DNA sequencing, the fragments were cloned into pBluescript II SK⁺. Five or six clones were sequenced using BigDye Terminater Cycle Sequencing Kit (Applied Biosystems), an ABI PRISM^® 310 Genetic Analyzer (Applied Biosystems) and Gene Scan^® Analysis Software (Applied Biosystems).

Results

The mean number of grains of the patient's cultured fibroblasts after UV-irradiation was 40.26 (Fig. 2). The degree of UDS in the patient's fibroblasts was to 8.7% of the normal fibroblasts. This result strongly suggested that the patient belonged to the XPA group. The DNA repair ability after UV-irradiation of the patient's fibroblasts was complemented by the XPAC gene (data not shown). These results lead to our diagnosis of XPA.

PCR-RFLP analysis revealed two independent mutations (Fig. 3). One of these was a splicing mutation at the splicing acceptor site of intron 3, which was detected using Alw NI restriction enzyme (Alw NI mutation) (Fig. 3 left). Alw NI cleaved the PCR products from the patient (lane 2) and his father (lane 3) into two fragments, 84 and 244 bp in length, while the fragments of his mother (lane 4) and a normal individual (lane 1) were not cleaved. The other mutation was a nonsense mutation in exon 6 detected by Hph I (Hph I mutation) (Fig. 3 right). Hph I cleaved the PCR products from the patient (lane 2) and his mother (lane 4) into three fragments, 79, 312 and 35 bp, while it cleaved the PCR products from his father (lane 3) and a normal individual (lane 1) at one site. The mutations were confirmed using DNA sequencing. In the case of Alw NI mutation, both normal and mutated (IV3 -1G => C) alleles were obtained from the patient's and paternal PCR products (Fig. 4a), while all subclones from maternal PCR products contained a normal sequence. This point mutation causes a frameshift and a premature stop codon (TGA) at the second codon in exon 4. In the case of the Hph I mutation, both normal and mutated (C => T) sequences were obtained from the patient and his mother (Fig. 4b), while all subclones from his father carried a normal allele. The later mutation is a nonsense leading to Arg²²⁸ (CGA) => stop (TGA).

Discussion

XPA is the most frequently occurring type in the world and more than 20 different mutations of the XPAC gene have been demonstrated to date [8]. From Japan, 84 cases have been genetically diagnosed and 5 different mutations have been reported [9] (Table I). Four of them are readily detected using PCR-RFLP analysis. The genetic characterization of these mutations is important, since genotypes are well correlated with clinical manifestations [1-6]. The Alw NI mutation was detected in 78 patients in Japan (Table I) and most of the patients carrying this mutation homozygously develop severe clinical symptoms after the age of 10 years [2, 4]. Neurologic abnormalities such as hearing impairment, gait disturbance and mental retardation as well as skin cancers develop progressively. Compared to this genotype, patients with a compound heterozygous mutation of and Hph I show milder clinical symptoms [2, 4, 6]. For example, two patients at the age of 13 revealed mild skin and ocular symptoms but no hearing impairment. In another case, the patient could walk normally even at the age of 25 years with only mild mental retardation and hearing impairment. It has been reported that the patients with the mutation of Tyr²⁰⁸ => stop show severe phenotype [3].

It is likely that the severity of clinical symptoms depends on the length of the XPA gene products. We have reported a case of sporadic XPA, in a compound heterozygote for the Alw NI and the Hph I mutations. The subject exhibits neither obvious neurologic symptoms nor malignant skin tumors at the present age of 8. Although XP is a rare hereditary disorder, genetic analyses are quite useful both for prediction of prognosis and for genetic counseling.

Article accepted on 29/07/02

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