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Germline mutations in the NF2 gene on chromosome 22q12 cause the tumor predisposition syndrome neurofibromatosis-2 (NF2), characterized by development of intracranial and intraspinal tumors - most commonly acoustic schwannomas, meningiomas, and ependymomas. NF2 gene mutations are also found in the sporadic counterparts of these tumors, as well as in malignant mesothelioma, a tumor of mesodermal origin. NF2 mutations in melanoma have been described, although the data in melanoma is conflicting. Merlin, the protein product of the NF2 gene, functions as a tumor suppressor by decreasing cell proliferation and impairing cell motility, adhesion and spreading. In the present study, we have characterized merlin expression and NF2 cDNA sequence in a panel of nine frozen malignant melanomas (2 primary, 7 metastatic) to determine the potential involvement of the NF2 gene in the pathogenesis of malignant melanoma. Merlin was immunoprecipitated from frozen tissue protein lysates derived from the nine melanomas using NF2 antibody (A-19). The immunoprecipitates were Western blotted, incubated with NF2 antibody (C-18) and detected by chemiluminescence. Diminished levels of merlin were detected in 4/9 melanomas analyzed. Direct sequencing using cDNA templates was performed on 6/9 melanomas. Missense mutations were identified in 2/6 tumors. In the first tumor, a T>C transition was identified in exon 2 (L64P) that resulted in substitution of proline (nonpolar) for lysine (basic). The L64P mutation is reported as a cause of the clinical syndrome NF2, and has previously been shown to result in functionally inactive merlin. In the second tumor, two distinct mutations were identified. The first was an A>G transition in exon 2 (D45G) resulting in substitution of glycine (nonpolar) for aspartate (acidic) and the second was an A>G transition in exon 9 (K279E) resulting in substitution of glutamate (acidic) for lysine (basic). All mutations were confirmed by sequencing in both forward and reverse directions. Additionally, sequencing of DNA from non-tumor tissues obtained from the patients with NF2-mutant tumors showed wild-type sequence in all cases, confirming that the identified changes are somatic mutations. None of the three identified mutations have been previously described in melanoma. Correlation of Western blot and sequencing data show that 1/2 mutant tumors displayed diminished merlin expression. For the 4 tumors without mutations, 3/4 displayed diminished merlin expression. These data identify mutations in the NF2 gene and diminished expression of merlin in subsets of malignant melanoma, thus supporting a potential role for the NF2 gene locus in melanomagenesis. Finally, the lack of correlation between mutation status and merlin expression invokes additional mechanisms for regulating merlin expression.

[Proc Amer Assoc Cancer Res, Volume 46, 2005]