Tuberous sclerosis type 1: three novel mutations detected in exon 15 by a combination of HDA and TGGE

Tuberous sclerosis (TSC) is an autosomal dominant disorder which is genetically heterogeneous with two genes, TSC1 and TSC2. TSC1 consists of 23 exons with exon 15 being the largest one comprising 559 bp. Representing 16% of the coding region exon 15 harbors 37% of the already identified point mutations in TSC1. Mutation screening of large DNA fragments as TSC1 exon 15 by SSCP has been a problem because of the low sensitivity of this method without subdivision. Therefore, we simultaneously performed heteroduplex analysis (HDA) and temperature gradient gel electrophoresis (TGGE) which are both more suitable for evaluation of fragments of this size. DNA samples of 159 patients with the clinical diagnosis of TSC were screened and a total of seven different mutations in nine unrelated cases were identified, including the three novel mutations 1754delT, 1836delT and R500Q. Comparing the two methods applied, HDA showed a higher sensitivity in detecting frameshift mutations, while TGGE seemed to be more sensitive for the detection of base exchanges. We conclude that the combination of these two methods is appropriate to reach a high degree of sensitivity for the detection of all types of small mutations in large DNA fragments.