NF1 mutation analysis using a combined heteroduplex/SSCP approach

Neurofibromatosis type 1 (NF1) is a common autosomal dominant disorder characterized predominantly by neurofibromas, café-au-lait spots, and Lisch nodules. The disease is caused by disruptive mutations of the large NF1 gene, with half of cases caused by new mutation. Less than 100 constitutional mutations have thus far been published, ranging from very large deletions to point mutations. We have pursued NF1 mutation analysis by heteroduplex analysis (HDA) and single-strand conformational polymorphism analysis (SSCP) of individual exons. We streamlined these techniques to eliminate the use of radioactivity, to apply both methods to the same PCR product, and to multiplex samples in gels. Applied simultaneously to a set of 67 unrelated NF1 patients, HDA and SSCP have thus far identified 26 mutations and/or variants in 45 of the 59 exons tested. Disease-causing mutations were found in 19% (13/67) of cases studied. Both techniques detected a variety of mutations including splice mutations, insertions, deletions, and point changes, with some overlap in the ability of each method to detect variants.