Identification of an insertion and accompanying deletion in exon 31 of the neurofibromatosis type 1 gene

Neurofibromatosis type I (NF1) is a common hereditary disease predisposing to neoplasia, affecting approximately 1 in 3,500 births annually, with no apparent bias for sex or ethnicity (Riccardi and Eichner, 1986; Crowe et al., 1956). Individuals with NF1 display a broad range of clinical features, which include cafe-au-lait spots, Lisch nodules, learning disabilities, skeletal abnormalities, cutaneous and subcutaneous neurofibromas, and malignancies of the central and peripheral nervous systems (Riccardi and Eichner, 1986). The pleiotropic nature of NF1 distinguishes it from other genetic predispositions to neoplasia, such as familial retinoblastoma or Wilms’ tumor. NF1 can occur in both familial and sporadic forms, with approximately 50% of all cases occurring sporadically (Huson et al., 1989). The mutations identified to date have been dispersed throughout the gene with no apparent “hot spot” or hypermutable region identified. The NFI gene includes 59 exons and encodes at least four transcripts (Viskochil et al., 1993; Li et al., 1995). While the function of the NF1 gene product neurofibromin is not fully understood, it contains a region with structural and functional homology to the GTPase activating proteins (GAPs) of mammals and yeast (Ballester et al., 1990; Xu et al., 1990). The GAPs play a pivotal role in the transduction of extracellular growth and differentiation regulatory signals within the cell. Early studies demonstrated a loss of heterozygosity in NF1 malignancies compared to nontumor tissue from the same patients, suggesting that biallelic inactivation of the NFI gene was necessary for tumor formation (Skuse et al., 1989; Skuse, 1990). Those findings are consistent with the finding of GAP homology within the NFI gene, a region termed the GAP-related domain (GRD), and the postulated role ofneurofibromin as a tumor suppressor through its interaction with RAS. In order to identify NF1 mutations, we screened DNA isolated from peripheral blood lymphocytes, obtained from 58 unrelated NFl patients, for singlestrand conformation polymorphisms (SSCPs) (Orita et al., 1989a,b). We amplified a region of the NFI gene, using polymerase chain reaction (PCR) primers (Fig. l), which flank exon 31, so that the PCR product of 193 bases included the entire exon and the splice sites at its borders (Cawthon et al., 1990). We detected an abnormal band in the SSCP analysis of one of the patients screened (NF70) compared to the others (Fig. 2, lane 2). At the time that the sample was obtained, the patient was a 46-year-old man with typical manifestations of NF1, including caf6-au-lait spots, multiple cutaneous neurofibromas, and severe scoliosis. He had no known affected relatives, so his case is most likely sporadic. In order to better understand the abnormality detected, we amplified exon 3 1 from that patient and ran the PCR products on a nondenaturing polyacrylamide gel. The results shown in Figure 2 (lane 1) indicate that the patient has an insertion of approximately 200 base pairs (bp) on one homologue of chromosome 17. The band containing the insertion was eluted from a polyacrylamide gel, ligated to the TA cloning vector PCR2000 (Invitrogen, San Diego, CA), cloned in Escherichia cob, and the sequence flanking the insertion