Molecular dissection of isolated disease features in mosaic neurofibromatosis type 1.

Elucidation of the biological framework underlying the development of neurofibromatosis type 1 (NF1)-related symptoms has proved to be difficult. Complicating factors include the large size of the NF1 gene, the presence of several NF1 pseudogenes, the complex interactions between cell types, and the NF1-haploinsufficient state of all cells in the body. Here, we investigate three patients with distinct NF1-associated clinical manifestations (neurofibromas only, pigmentary changes only, and association of both symptoms). For each patient, various tissues and cell types were tested with comprehensive and quantitative assays capable of detecting low-percentage NF1 mutations. This approach confirmed the biallelic NF1 inactivation in Schwann cells in neurofibromas and, for the first time, demonstrated biallelic NF1 inactivation in melanocytes in NF1-related cafe-au-lait macules. Interestingly, both disease features arise even within a background of predominantly NF1 wild-type cells. Together, the data provide molecular evidence that (1) the distinct clinical picture of the patients is due to mosaicism for the NF1 mutation and (2) the mosaic phenotype reflects the embryonic timing and, accordingly, the neural crest-derived cell type involved in the somatic NF1 mutation. The study of the affected cell types provides important insight into developmental concepts underlying particular NF1-related disease features and opens avenues for improved diagnosis and genetic counseling of individuals with mosaic NF1.

Conservation of hotspots for recombination in low-copy repeats associated with the NF1 microdeletion.

Several large-scale studies of human genetic variation have provided insights into processes such as recombination that have shaped human diversity. However, regions such as low-copy repeats (LCRs) have proven difficult to characterize, hindering efforts to understand the processes operating in these regions. We present a detailed study of genetic variation and underlying recombination processes in two copies of an LCR (NF1REPa and NF1REPc) on chromosome 17 involved in the generation of NF1 microdeletions and in a third copy (REP19) on chromosome 19 from which the others originated over 6.7 million years ago. We find evidence for shared hotspots of recombination among the LCRs. REP19 seems to contain hotspots in the same place as the nonallelic recombination hotspots in NF1REPa and NF1REPc. This apparent conservation of patterns of recombination hotspots in moderately diverged paralogous regions contrasts with recent evidence that these patterns are not conserved in less-diverged orthologous regions of chimpanzees.

Comprehensive NF1 screening on cultured Schwann cells from neurofibromas.

Neurofibromatosis type 1 (NF1) is mainly characterized by the occurrence of benign peripheral nerve sheath tumors or neurofibromas. Thorough investigation of the somatic mutation spectrum has thus far been hampered by the large size of the NF1 gene and the considerable proportion of NF1 heterozygous cells within the tumors. We developed an improved somatic mutation detection strategy on cultured Schwann cells derived from neurofibromas and investigated 38 tumors from nine NF1 patients. Twenty-nine somatic NF1 lesions were detected which represents the highest NF1 somatic mutation detection rate described so far (76%). Furthermore, our data strongly suggest that the acquired second hit underlies reduced NF1 expression in Schwann cell cultures. Together, these data clearly illustrate that two inactivating NF1 mutations, in a subpopulation of the Schwann cells, are required for neurofibroma formation in NF1 tumorigenesis. The observed somatic mutation spectrum shows that intragenic NF1 mutations (26/29) are most prevalent, particularly frameshift mutations (12/29, 41%). We hypothesize that this mutation signature might reflect slightly reduced DNA repair efficiency as a trigger for NF1 somatic inactivation preceding tumorigenesis. Joint analysis of the current and previously published NF1 mutation data revealed a significant difference in the somatic mutation spectrum in patients with a NF1 microdeletion vs. non-microdeletion patients with respect to the prevalence of loss of heterozygosity events (0/15 vs. 41/81). Differences in somatic inactivation mechanism might therefore exist between NF1 microdeletion patients and the general NF1 population.

Somatic loss of wild type NF1 allele in neurofibromas: Comparison of NF1 microdeletion and non-microdeletion patients.

Neurofibromatosis type I (NF1) is an autosomal dominant familial tumor syndrome characterized by the presence of multiple benign neurofibromas. In 95% of NF1 individuals, a mutation is found in the NF1 gene, and in 5% of the patients, the germline mutation consists of a microdeletion that includes the NF1 gene and several flanking genes. We studied the frequency of loss of heterozygosity (LOH) in the NF1 region as a mechanism of somatic NF1 inactivation in neurofibromas from NF1 patients with and without a microdeletion. There was a statistically significant difference between these two patient groups in the proportion of neurofibromas with LOH. None of the 40 neurofibromas from six different NF1 microdeletion patients showed LOH, whereas LOH was observed in 6/28 neurofibromas from five patients with an intragenic NF1 mutation (P = 0.0034, Fisher's exact). LOH of the NF1 microdeletion region in NF1 microdeletion patients would de facto lead to a nullizygous state of the genes located in the deletion region and might be lethal. The mechanisms leading to LOH were further analyzed in six neurofibromas. In two out of six neurofibromas, a chromosomal microdeletion was found; in three, a mitotic recombination was responsible for the observed LOH; and in one, a chromosome loss with reduplication was present. These data show an important difference in the mechanisms of second hit formation in the 2 NF1 patient groups. We conclude that NF1 is a familial tumor syndrome in which the type of germline mutation influences the type of second hit in the tumors.