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@#ObjectiveTo design and construct CRISPR/Cas9 gene editing system targeting Tsc1 and Tsc2 genes,and verify the effectiveness of gene editing at cellular level.MethodsThree sgRNA guide sequences were designed for mouse Tsc1 and Tsc2 genes respectively. The sgRNA expression vector was constructed and co-transfected with the Cas9 expression plasmid into mouse N2a cells. After the positive cells were obtained through drug screening,the DNA fragments at the targeting site were amplified by PCR,and the targeting efficiency was verified by TA clone sequencing.ResultsThe five targets of Tsc1-M-sgRNA2 and Tsc1-M-sgRNA3 of Tsc1 gene and Tsc2-M-sgRNA1,Tsc2-M-sgRNA2 and Tsc2-M-sgRNA3 of Tsc2 gene were all edited,and the editing efficiency was 40%,80%,30%,30% and 20%,respectively.ConclusionA CRISPR-Cas9 gene editing system with editing efficiency targeting mouse Tsc1 and Tsc2 genes was successfully constructed.
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Objective To evaluate the value of targeted second-generation sequencing (NGS) in the genetic diagnosis of tuberous sclerosis complex (TSC) associated with renal complications.Methods The clinical data of 43 patients (with 33 patients of definite diagnosis and 10 patients of possible diagnosis)with tuberous sclerosis complex associated with renal complications were analyzed.There were 26 females and 17 males with a mean age of 28 (10 ～ 48) years ranging from 10 to 48 years old.All patients had renal complications,including 39 renal angiomyolipomas,3 renal cysts and 1 renal cell carcinoma.Written informed consents were signed,and 5ml peripheral blood was drawn for DNA extraction.Mutations of TSC1 and TSC2 genes were detected by the NGS technology,and then confirmed by Sanger sequencing.Results TSC1 or TSC2 pathogenetic mutants were identified in 39 patients (90.7％),which were consistent with clinical phenotypes and two non-significant mutations were identified in two individuals,while no mutation was detected in the other two cases.Fourteen novel mutations were reported for the first time,including 8 frameshift mutation,3 deletion mutation,2 nonsense mutation and 1 splicing mutation.Conclusion NGS is an accurate and less time-consuming technology in detecting TSC1 or TSC2 gene mutation,which has great value in genetic diagnosis of tuberous sclerosis complex associated with renal complications.
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OBJECTIVES: Balloon cells and dysplastic neurons are histopathological hallmarks of the cortical tubers of tuberous sclerosis complex (TSC) and focal cortical dysplasia (FCD) of the Taylor type. They are believed to be the epileptogenic substrate and cause therapeutic drug resistant epilepsy in man. P-glycoprotein (P-gp) is the product of multidrug resistance gene (MDR1), and it maintains intracellular drug concentration at a relatively low level. The authors investigated expression of P-gp in balloon cells and dysplastic neurons of cortical tubers in patients with TSC. METHODS: An immunohistochemical study using the primary antibody for P-gp, as an indicative of drug resistance, was performed in the cortical tuber tissues in two patients of surgical resection for epilepsy and six autopsy cases. RESULTS: Balloon cells of each lesion showed different intensity and number in P-gp immunopositivity. P-gp immunopositivity in balloon cells were 28.2%, and dysplastic neurons were 22.7%. These immunoreactivities were more prominent in balloon cells distributed in the subpial region than deeper region of the cortical tubers. Capillary endothelial cells within the cortical tubers also showed P-gp immunopositivity. CONCLUSION: In this study, the drug resistance protein P-glycoprotein in balloon cells and dysplastic neurons might explain medically refractory epilepsy in TSC.