A recurrent nonsense mutation occurring as a de novo event in a patient with recessive dystrophic epidermolysis bullosa.

glossia, excessive caries, premature loss of teeth and microstomia ( fig. 1 e). The patient also complained of ocular pain, which was in agreement with the findings of blisters, erosions and scarring of the cornea on ophthalmic examination ( fig. 1 f). She presented other physical deformities, such as joint contractures, and exhibited esophageal strictures, chronic anemia and growth retardation. Since the age of 33 years, she had undergone repeated surgical interventions for the excision of primary and recurrent squamous cell carcinomas (SCC) of both hands/arms ( fig. 1 g) and of the left foot and leg. In all cases, histopathological examination showed well to moderately differentiated SCC ( fig. 1 h, i), as usually described in RDEB patients. At the time of the last surgery, complete staging investigations – including ultrasound, CT and PET scans – were negative. Following written informed consent, skin and blood samples were obtained from the patient. Routine histological examination of a skin biopsy showed subepidermal blisters and indirect immunofluorescence, using the monoclonal antibody LH7.2, complete absence of VII collagen protein ( fig. 1 j, k) in accordance with the clinical presentation. Genomic DNA was extracted from peripheral blood lymphocytes of the patient and her parents by standard methods, and mutations were identified and characterized by PCR amplification and direct sequencing as described previously [3] . The patient was a heterozygous carrier for the R1763X mutation located in exon 60 and the c.6266delCCCC mutation (also designated as c.6266_6269del) located in exon 75 ( fig. 2 a) and predicted to result in a frameshift and a stop codon in exon 82. Thus, the patient bore two premature-termination-codon-causing mutations in her COL7A1. Further mutation analysis in the patient’s parents revealed that the mother was a carrier of the c.6266delCCCC mutation, this deletion has already been reported by our group in this patient [patient No. 25 in reference 3 ]; however, the second disease-causing mutation (the de novo mutation) had not been disclosed at that time. In contrast, the father did not carry either mutation, suggesting that the p.R1763X mutation occurred de novo on the paternal allele. Paternity was therefore confirmed by human leukocyte antigen (HLA) haplotype analysis ( fig. 2 b) and intragenic SNP genotyping ( fig. 2 c) [4] . The nonsense mutation p.R1763X, previously identified in a DEB patient from Central Europe [2] is due to a C ] T transition that affects a CpG dinucleotide. CpG dinucleotides are frequent sites of mutations due to hypermutability of 5-methyl-cytosine to thymine [5] . The occurrence of the same mutation in DEB patients of a different geographical origin suggests that the residue 1763 of the COL7A1 represents a mutation ‘hotspot’, probably due to the vulnerability of the nucleotide sequence.