Intragenic Tsc2 Somatic Mutations as Knudson's Second Hit in Spontaneous and Chemically Induced Renal Carcinomas in the Eker Rat Model

We searched for the rat homologue of the human tuberous sclerosis (TSC2) gene mutations in loss of heterozygosity (LOH)-negative Eker rat renal carcinomas (RCs) by polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) analysis using 45 primer sets covering all 41 coding exons and one leader exon including splicing donor/acceptor sites. We have identified intragenic somatic mutations in 7 of 21 spontaneous RCs, including one cell line (33%), and in 3 of 9 (33%) N-ethyl-N-nitrosourea (ENU)-induced LOH-negative RCs. Interestingly, five mutations in the spontaneous RCs were either deletion or duplication (5/7 = 71%). In contrast, all three in ENU-induced RCs were base substitutions (3/3 = 100%), as expected. Thus, a qualitative difference in the second hit might exist between spontaneous and ENU-induced mutations (e.g., deletion or duplication versus point mutation). By a direct cloning approach utilizing the restriction length difference caused by germline insertional mutation or reverse transcriptase-PCR analysis in two applicable cases, we could clearly show the presence of intragenic somatic mutations in the second copy (wild-type) of the Tsc2 gene. This is the first demonstration at the DNA sequence level of the validity of Knudson's two-hits hypothesis in the Tsc2 gene.