Neurofibromatosis 1 (NF1) is a common genetic disorder characterized by abnormalities of tissues derived from the neural crest. To define germ-line mutations in the NF1 gene, we studied 20 patients with familial or sporadic cases of NF1 diagnosed clinically and one patient with only café-au-lait spots and no other diagnostic criteria. A protein truncation assay identified abnormal polypeptides synthesized in vitro from five RT-PCR products that represented the entire NF1 coding region. Truncated polypeptides were observed in 14 individuals. The mutations responsible for the generation of abnormal polypeptides were characterized by DNA sequencing. Thirteen previously unpublished mutations were characterized in the 14 individuals. The mutation 2027insC was observed in two unrelated individuals; the other 12 mutations were unique. The sequence changes included seven nonsense and four frameshift mutations that created premature translation termination signals, and two large in-frame deletions that led to the synthesis of truncated polypeptides. One of the mutations was found in the child with a single clinical diagnostic criterion, providing her with a presumptive diagnosis of NF1. Our results confirm that truncating mutations are frequent in both familial and sporadic NF1 cases. The identification of mutations in 14 of 21 individuals studied (67%) suggests that the use of protein truncation assays will rapidly accelerate the rate of identification of NF1 mutations. Because we scanned the entire NF1 coding region in each individual, the distribution of NF1 truncating mutations was discerned for the first time. The mutations were relatively evenly distributed throughout the coding region with no evidence for clustering.

Evolutionary studies have suggested that mutation rates vary significantly at different positions in the eukaryotic genome. The mechanism that is responsible for this context-dependence of mutation rates is not understood. We demonstrate experimentally that frameshift mutation rates in yeast microsatellites depend on the genomic context and that this variation primarily reflects the context-dependence of the efficiency of DNA mismatch repair. We measured the stability of a 16.5-repeat polyGT tract by using a reporter gene (URA3-GT) in which the microsatellite was inserted in-frame into the yeast URA3 gene. We constructed 10 isogenic yeast strains with the reporter gene at different locations in the genome. Rates of frameshift mutations that abolished the correct reading frame of this gene were determined by fluctuation analysis. A 16-fold difference was found among these strains. We made mismatch-repair-deficient (msh2) derivatives of six of the strains. Mutation rates were elevated for all of these strains, but the differences in rates among the strains were substantially reduced. The simplest interpretation of this result is that the efficiency of DNA mismatch repair varies in different regions of the genome, perhaps reflecting some aspect of chromosome structure.genetic instability ͉ microsatellite ͉ mutation rate ͉ Saccharomyces cerevisiae C omparisons of amino acid or base sequences of orthologous genes indicate that different genes evolve at different rates (1). Differences in the rates of accumulation of amino acid changes or nonsynonymous base substitutions are influenced by selective constraints (1). For highly expressed genes, the rate of synonymous base substitutions is affected by GC content and codon bias (2, 3). In Saccharomyces cerevisiae and mammalian cells, the rates of synonymous substitutions also vary by a factor of Ϸ10, depending on the position of the gene in the genome (4-6). The interpretation of these observations is unclear. It is possible that the misincorporation rates of the replicative DNA polymerases are different at different positions in the genome. Alternatively, the fidelity of DNA polymerases may be invariant, but the detection and repair of misincorporation events may be context-specific.Microsatellites are regions of DNA in which a single base or a small number of bases is repeated in tandem. The polyGT sequence is a particularly common microsatellite in many eukaryotes (7). We have developed (8, 9) methods of measuring the rate of microsatellite alterations. In this study, we used this assay to measure the mutation rates of the same polyGT microsatellite placed in 10 different chromosomal contexts in the yeast genome. We show that the microsatellite mutation rates vary by more than an order of magnitude among different genomic positions in yeast strains that have wild-type DNA mismatch repair. We have demonstrated (8) that the mutation rates of microsatellites are greatly elevated in yeast strains with deficient mismatch repair. In this study, we find that microsatellite in...

We have measured the mutation rates of G(17) and A(17) repeat sequences in cultured mammalian cells with and without mismatch repair and have compared these rates to those of a (CA)(17) repeat sequence. Plasmids containing microsatellites that disrupt the reading frame of a downstream neomycin-resistance gene were introduced into the cells by transfection and revertants were selected using the neomycin analog G418. Comparison of mutation rates within cell lines showed that the mutation rates of A(17) and (CA)(17) sequences were similar in the mismatch repair proficient cells, but the mutation rate of G(17) was significantly higher than that of either A(17) or (CA)(17). In the mismatch repair deficient cells, the G(17) and (CA)(17) mutation rates were similar and were significantly higher than the A(17) rate. PCR analysis of the mutants showed that 1 bp insertions predominated in both mononucleotide repeats in the mismatch repair proficient cells; in mismatch repair deficient cells, 2 bp deletions were the most common mutation in the A(17) sequence, but 1 bp insertions and 2 bp deletions were equally represented in the G(17) sequence. These results indicate that a G(17) repeat is less stable than an A(17) repeat in both mismatch repair proficient and mismatch repair deficient mammalian cells. This observation implies that the replication fidelity is lower in G(17) repeats.

Purpose: Microsatellite instability (MSI) is found in 10%