Tuberous sclerosis complex (TSC [MIM 191092]), a dominantly inherited disease with a prevalence of 1/6,700 (Sampson et al. 1989; Osborne et al. 1991; Ahlsen et al. 1994) is characterized by hamartomas found in almost every organ system and by malignancy in some organ systems. Two genetic loci have been identified: the TSC1 gene, on chromosome 9q34. 3, and TSC2, on chromosome 16p13. 3 (The European Chromosome 16 Tuberous Sclerosis Consortium 1993; van Slegtenhorst et al. 1997). A tumor-suppressor function has been suggested for TSC2 by loss of heterozygosity (LOH) in hamartomas (Green et al. 1994; Henske et al. 1995, 1996; Carbonara et al. 1996; Sepp et al. 1996). Thus far, no reports have demonstrated mutations in both alleles of the TSC2 gene in hamartomas in humans. Further evidence of a tumor-suppressor function for TSC2 comes from the Eker rat, a naturally occurring model of tumorigenesis. The Eker rat has been determined to have a germline mutation in the Tsc2 gene (the homolog of the TSC2 gene in humans)(Yeung et al. 1994; Kobayashi et al. 1995; Kubo et al. 1995). LOH and second somatic hit studies of the Tsc2 gene have been reported in 33% of spontaneous or chemically induced Eker rat renal-cell carcinoma (RCC) lines (Kobayashi et al. 1997). The mechanism for cell transformation in humans is expected to be different from that in rats, because RCCs are detectable in 100% of Eker rats by age 1 year, whereas only a small percentage (2.5%) of humans with TSC develop RCCs (Eker et al. 1981; Bjornsson et al. 1996; Cook et al. 1996; Al-Saleem et al. 1998). We collected a variety of hamartomas—angiomyolipomas (AMLs) from three patients, cortical tubers from two patients, and facial angiofibromas (FAs) from one patient—from patients in whom the germline TSC2 mutation had been identified (Au et al. 1998). These tissues were tested for the second hit by Southern blotting, with the TSC2 gene as a probe, and by LOH studies of the TSC2 gene and surrounding markers. The second hit for the TSC2 gene was detected in AMLs from two independent patients and in FAs from another patient, providing direct evidence that mutations in both alleles are required for tumorigenesis in humans affected by TSC (Knudson 1971).

The patients in this study have been described elsewhere (Au et al. 1998). All were diagnosed with TSC according to standard criteria by Roach et al.(1998). Informed consent approved by the institutional review boards at The University of Texas Medical School–Houston, The University of Texas Southwestern Medical School, and the Scottish Rite Hospital in Dallas was obtained. Tissue samples collected included 6 AMLs from the right kidney and 1 AML from the left kidney of patient HOU23-01, 1 AML from patient HOU23-03, 14 AMLs from patient TS94-104, 3 FAs from patient TS93-41, and 1 cortical tuber each from patients TS93-14 and TS94-53.