Molecular pathogenesis of Tuberous Sclerosis Complex (TSC) in patients with no mutation identified in TSC1 or TSC2. M. E. Tyburczy1, Y. Chekaluk1, K. Dies2, M. Sahin2, J. Glass3, D. Franz3, S. Camposano4, E. Thiele4, D. Kwiatkowski1 1) Brigham and Women's Hospital, Harvard Medical School, Boston, MA; 2) Childrens Hospital, Boston, MA; 3) Cincinnati Children's Hospital Medical Center, Cincinnati, OH; 4) Massachusetts General Hospital, Boston, MA.

   Tuberous sclerosis complex (TSC) is an autosomal dominant disorder caused by mutations in TSC1 or TSC2. Two-thirds of TSC cases are sporadic, and mosaicism is known to occur at low frequency in TSC. The use of Sanger sequencing and deletion analysis of TSC1 and TSC2 results in 10-15% of TSC patients being diagnosed with no mutation identified (NMI). We hypothesized that NMI occurs due to: mosaic mutations, intronic mutations, technical failure to identify the usual mutations, and possibly the existence of a third, as yet undiscovered, TSC locus. We used a series of molecular genetic techniques, including next-gen sequencing (NGS), to investigate this hypothesis in 51 TSC NMI patients. We performed NGS of the genomic extent of TSC1 and TSC2 including promoter regions, all exons, and most of the introns on DNA prepared from blood cells or saliva from patients with a definite clinical diagnosis of TSC. We found a TSC1 genomic deletion in 1 case and TSC2 genomic deletions in 3 cases by MLPA. NGS was performed on the remaining 47 patients. Variants of known or potential pathogenic significance were identified in 32 (68%) patients: 26 (81%) variants in TSC2 and 6 (19%) in TSC1. All of these variants were absent in DNA samples from unaffected parents, supporting pathogenicity, and all were confirmed by secondary assays. Mutations were mosaic in 17 (53%) patients with mutant allele frequencies ranging from 1% to 34%, and splice site mutations were seen in 14 (44%) cases. In one patient a 740 nt deletion was found in the TSC2 promoter. We also identified 4 heterozygous mutations in TSC1 and TSC2 coding exons that appear to have been missed by previous analyses. We also searched for mutations in skin tumors (angiofibromas) from 3 NMI patients. In these patients, we identified mosaic mutations in angiofibromas from 2 of 3 samples studied, and neither was detected in respective blood samples (< 0.1% allele frequency). In conclusion, our study clearly indicates that NGS is necessary to detect low allelic mutations in TSC1 and TSC2 as these results detected mutations in 68% of TSC NMI patients. Mosaic and splice region mutations were common, and were missed by previous analyses. NGS has the potential for more efficient and sensitive mutation detection in TSC. Furthermore, analysis of accessible TSC skin tumors enables mutation detection, and defines a set of TSC patients with no detectable mutation in blood or saliva.

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