Analysis of the NF1 gene by temperature gradient gel electrophoresis reveals a high incidence of mutations in exon 4b

A total of 196 unrelated patients with neurofibromatosis type 1 (NF1) was s creened for mutations in exons 4a-c of the NF1 gene by temperature gradient gel electrophoresis (TGGE) of polymerase chain reaction (PCR)-amplified ge nomic DNA fragments using intron-based primers. DNA samples with abnormal T GGE band patterns were subjected to sequence analysis. Sequence alterations were identified in ten patients (5.1%). 496delGT (1), 499delTGTT (4), T528 A=D176E (2), T539A=L180X (1), 540insA (1), C574T=R192X (1). Thus, a total o f six different mutations was identified in exon 4b but none in exons 4a an d 4c. Only the missense mutation D176E, which we assume to be a nonpathogen ic polymorphism, and the 4-base pair (bp) deletion 499delTGTT have been des cribed before. The reason for the high incidence of mutations in exon 4b is obviously a tetranucleotide tandem repeat comprising nucleotides 495-502 ( TGTTTGTT) that may give rise to slipped mispairing and subsequent deletion of one repeat unit during replication. Additionally, the recurrent 4 bp del etion was found as a second hit in a malignant schwannoma of a further NF1 patient, suggesting that micro-lesions may be as frequent among somatic as among germline mutations. This is the first report of a systematic study of NF1 exons 4a-c in a large group of NF1 patients.