A FUNCTIONAL ASSAY FOR HETEROZYGOUS MUTATIONS IN THE GTPASE-ACTIVATING PROTEIN RELATED DOMAIN OF THE NEUROFIBROMATOSIS TYPE-1 GENE

The GTPase-activating protein related domain of the human neurofibroma tosis type 1 protein (NF1GRD) can down-regulate RAS in Saccharomyces c erevisiae, Using a technique termed the FASAY method, for Functional A nalysis of Separated Alleles in Yeast, we designed a rapid method for detection of heterozygous NF1GRD loss-of-function mutations, In our me thod, PCR amplified NF1GRD cDNA is directly cloned into a centromeric vector by homologous recombination in a cdc25 temperature-sensitive mu tant strain expressing human Ha-ras. This strain is dependent on the H a-uas for growth, allowing a simple growth assay for NF1GRD loss-of-fu nction mutations, In a test of our method, two alternatively spliced N F1GRD cDNAs (type I and II) inhibited yeast growth whereas four mutant s with amino acid substitutions at highly conserved residues did not, This simple method thus permits the rapid screening for heterozygous g ermline or somatic NF1GRD mutations, In an initial application of this method, no mutations disrupting NF1GRD function were detected in lymp hoblasts from 11 previously untested neurofibromatosis type 1 patients .