Neurofibromatosis type 1 (NF1), a common autosomal dominant disorder caused by mutations of the NF1 gene, is characterized by multiple neurofibromas, pigmentation anomalies, and a variety of other possible complications, including an increased risk of malignant neoplasias. Tumorigenesis in NF1 is believed to follow the two-hit hypothesis postulated for tumor-suppressor genes. Loss of heterozygosity (LOH) has been shown to occur in NF1-associated malignancies and in benign neurofibromas, but only few of the latter yielded a positive result. Here we describe a systematic approach of searching for somatic inactivation of the NF1 gene in neurofibromas. In the course of these studies, two new intragenic polymorphisms of the NF1 gene, a tetranucleotide repeat and a 21-bp duplication, could be identified. Three tumor-specific point mutations and two LOH events were detected among seven neurofibromas from four different NF1 patients. Our results suggest that small subtle mutations occur with similar frequency to that of LOH in benign neurofibromas and that somatic inactivation of the NF1 gene is a general event in these tumors. The spectrum of somatic mutations occurring in various tumors from individual NF1 patients may contribute to the understanding of variable expressivity of the NF1 phenotype.

A reciprocal t(17;22)(q11.2;q11.2) was found in a female patient with neurofibromatosis type 1 (NF1) and in her affected daughter. Sequence analysis of cloned junction fragments traversing the breakpoints allowed the identification of the structures involved in the rearrangement. Aberrant bands in Southern hybridizations of restriction enzyme-digested DNA of the patient pointed to the disruption of the NF1 gene in intron 31. Semispecific polymerase chain reaction analysis of the genomic DNA of the patient with the specific primer anchored at NF1 exon 31 was used to obtain the breakpoint-spanning fragment of the derivative chromosome 17. The intron 31 sequence turned out to be interrupted within a large irregular (AT) repeat. The chromosome 22-derived sequence of the der(17) junction fragment allowed us to identify cosmids of the corresponding region from a chromosome 22 specific cosmid library. With the support of the breakpoint-spanning cosmids, the chromosome 22 region upstream of the fragment carried by the der(17) was characterized. Primers deduced from the sequence of this upstream region were used in combination with a primer in NF1 intron 31 distal to the breakpoint on chromosome 17 to amplify the der(22) junction fragment. The structure of the junction sequences suggested that the translocation had arisen by unequal homologous recombination between (AT)-rich repeats on chromosome 22 and on chromosome 17 in intron 31 of the NF1 gene. However, our data support the assumption of additional rearrangements prior to, or in the course of, the recombination event, leading to a loss of the sequences between the involved (AT) repeats on chromosome 22. In the direct vicinity of these (AT) repeats, two members of a previously undescribed low-copy repetitive sequence have been found, copies of which are also present on human chromosome 13.

Stop mutations are known to disrupt gene function in different ways. They both give rise to truncated polypeptides because of the premature-termination codons (PTCs) and frequently affect the metabolism of the corresponding mRNAs. The analysis of neurofibromin transcripts from different neurofibromatosis type 1 (NF1) patients revealed the skipping of exons containing PTCs. The phenomenon of exon skipping induced by nonsense mutations has been described for other disease genes, including the CFTR (cystic fibrosis transmembrance conductance regulator) gene and the fibrillin gene. We characterized several stop mutations localized within a few base pairs in exons 7 and 37 and noticed complete skipping of either exon in some cases. Because skipping of exon 7 and of exon 37 does not lead to a frameshift, PTCs are avoided in that way. Nuclear-scanning mechanisms for PTCs have been postulated to trigger the removal of the affected exons from the transcript. However, other stop mutations that we found in either NF1 exon did not lead to a skip, although they were localized within the same region. Calculations of minimum-free-energy structures of the respective regions suggest that both changes in the secondary structure of the mRNA and creation or disruption of exonic sequences relevant for the splicing process might in fact cause these different splice phenomena observed in the NF1 gene.

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In healthy humans, insulin is secreted in an oscillatory manner. While the underlying mechanisms generating these oscillations are not fully established, increasing evidence suggests a central role for phosphofructo-1-kinase/muscle subtype (PFK1-M), which also serves as the predominantly active PFK1 subtype in the pancreatic beta-cell. The fact that normal oscillatory secretion is impaired in subjects with impaired glucose tolerance and healthy relatives of patients with type 2 diabetes suggests that this defect may be involved in the secretory dysfunction. To evaluate a possible link between inherited PFK1-M deficiency in humans (Tarui's disease or glycogenosis type VII) and altered insulin oscillations, in vivo studies were performed. We determined basal insulin oscillations during 2 h of frequent plasma sampling in two related teen-aged individuals with homozygous and heterozygous PFK1-M deficiency compared with nondeficient, unrelated control subjects. As predicted by the underlying hypothesis, normal oscillations in insulin secretion were completely abolished in the individual with homozygous deficiency of PFK1-M and significantly impaired in the heterozygous individual, as shown by spectral density and autocorrelation analyses. Thus, deficiency of PFK1-M subtype in humans appears to be associated with an impaired oscillatory insulin secretion pattern and may contribute to the commonly observed secretion defects occurring in type 2 diabetes.