Abstract 24

MAS is characterized for polyostotic fibrous dysplasia, café au lait spots, sexual precocity, and hyperfunctional endocrinophaties. This condition is caused by activating embrionic mutations in the Gsα gene resulting in the substitution of Arg201 with either His or Cys. Because this somatic mutation occurs early during embriogenesis, patients with MAS are mosaic for the mutant gene, and even severe affected patients often have no evidence of mutation in DNA prepared from blood leukocytes, limiting the utility of regular molecular methods. In the present study we applied allele-specific PCR (AS-PCR) method to characterize mutation at codon 201 of Gsα gene. Three patients with clinical features of MAS, presenting gonadotropin-independent precocious puberty, typical skin spots, and bone lesions, were studied DNA samples were prepared from blood and different tissues available at time of corrective surgery (bone, muscle, skin, thyroid, ovary). Sense primers were designed differing by their 3′ terminal nucleotide that coincided with the site of the possible point mutation, recognizing either the wild-type Arg201 codon, or the mutated forms: His201, Cys201. Separated PCR reactions were set for each DNA sample differing in the sense primer, wild or mutated-types. The cycling conditions were adjusted using DNA samples obtained from both normal and Arg201. His mutated in order to produce the following PCR products: only with the wild-type on the normal DNA sample, and with both wild and mutated-type on the Arg201. His mutated DNA sample. All DNA samples were also analyzed by PCR amplification and direct sequencing Arg201. His mutation was evident in 5/13 DNA samples analyzed by sequencing and 9/13 by AS-PCR. AS-PCR, besides to be rapid, economic and non-radioactive, appears as a more sensitive method for screening of point mutations, specially in such cases as MAS where a postnatal somatic mutation results in a mosaicism, and only some of the cells carry the mutation in the Gsα gene.