Establishing functional assessment-based and long-range PCR-based analytic methods to molecularly characterize genetic defects in TSC patients

• Main Authors: Jung Yu Liao, 廖容幼
• Other Authors: D. C. Chu
• Format: Others
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/09363049315346085646

碩士 === 長庚大學 === 醫學生物技術暨檢驗學系 === 100 === The mutations in TSC1 or TSC2 genes are main causes of tuberous sclerosis complex. TSC1 and TSC2 mutations result in a consequence that their corresponding proteins, hamartin and tuberin, respectively, cannot form a functional protein complex. Due to dysfunction of hamartin or tuberin, hamartomas arise in multiple organs. Previously in our laboratory, 37 tuberous sclerosis cases have been screened for DNA mutation by HRMA and dHPLC followed by DNA sequencing. The analytical results revealed that no sequence variant was identified in four subjects and variants of undetermined significance (VUS) in TSC2 gene, TSC2 c.1565A→C, TSC2 c.4400C→G and TSC2 c.4436C→T, were identified in three subjects. In this study, long-range PCR (LR-PCR) was conducted to elucidate whether the 4 patients without identified TSC gene mutations are large genomic deletion or not. On the other hand, functional assessment was conducted to determine whether the identified VUS are pathogenic mutation or not. The LR-PCR results revealed that no large-scale genomic deletion was detected in the four cases without identified mutation. On the other hand, functional assessment results revealed that TSC2 c.1565A→C and TSC2 c.4400C→G are single nucleotide polymorphisms (SNPs) and TSC2 c.4436C→T is a pathogenic mutation. In conclusion, we have successfully established a model to functionally analyze tuberin translated from TSC2 variants. By using LR-PCR and functional assessment, the TSC cases without identified mutation could be further analyzed following mutation screening.