DNA POLYMORPHISMS AND CONDITIONS FOR SSCP ANALYSIS OF THE 20 EXONS OFTHE RET PROTOONCOGENE

Recently identified mutations affecting different domains of the RET p roto-oncogene are associated with Multiple Endocrine Neoplasia type 2A (MEN 2A) and type 2B (MEN 2B), familial and sporadic Medullary Thyroi d Carcinomas (MTC) and Hirschsprung disease (HSCR). In order to facili tate the screening for RET mutations, and to study possible genotype-p henotype correlations, we established exon-intron junctions and extend ed the intronic sequences flanking the 20 exons of this gene. This mad e it possible to design primers and to develop PCR conditions useful f or SSCP analysis of the whole RET coding sequence. Nine conformational variants were observed which after sequencing turned out to be 8 sile nt mutations and a conservative amino acid substitution. Restriction a nalysis performed on DNA samples from unrelated controls confirmed the polymorphic nature of six of these nucleotide changes and made it pos sible to estimate the frequency of the corresponding alleles.