Study On Gene Mutation And MTOR Pathway Of Renal Vascular Smooth Muscle Lipoma Associated With Nodular Sclerosis

Part I Detection and analysis of TSC1/TSC2 mutations in patients with TSC-RAMLObjective:To identify the TSC1/TSC2 mutations in TSC-RAML patients, and to explore the relationship between genotype and phenotype.Methods:Totally 24 cases enrolled in the study group, including 22 TSC patients and 2 suspected cases. Among the 22 TSC patients,20 cases were diagnosed with TSC-RAML. After informed consents,3ml whole blood was collected from the peripheral vein. Through the microarray chip capture, high throughput sequencing was performed on the Illumina HiSeq 2500 platform, and compared the test results with LOVD database.Results:Among the 22 TSC patients,4 cases with TSC1 mutation,15 with TSC2 mutation, and no mutation was detected in other 3 cases. In the 20 cases of TSC-RAML, the mutation rate of TSC was 85%(17/20), the proportion of TSC1:TSC2 is about 1:4.7 (3 vs 14), the distribution of TSC1/TSC2 mutations are much scattered, and the mutation types are mainly nonsense mutation (41.2%) and frameshift mutation (35.3%). In patients with TSC1 and TSC2 mutations, the minimum age was 14 and 8 years, the maximum diameter of RAML was 10.3 and> 20cm, and the number of cases with diameter greater than 10cm was 1 and 8, respectively. Comparing our results with LOVD database,8 new mutations were detected,1 TSC1 and 7 TSC2 mutations. In the 3 families, the maximum diameter of the RAML of proband was 10,10.3 and 16cm, respectively. However, the maximum diameter of the RAML of the family members was 0,0 and 1cm, respectively.Conclusions:TSC mutations are mainly TSC2 in TSC-RAML patients, and the mutation sites were often scattered, with the majority of mutation types of nonsense mutation and frameshift mutation. Patients with TSC2 mutation are often with the relatively earlier onset age of RAML and more severe clinical manifestations than patients with TSC1 mutation. TSC-RAML are often with large individual differences, but may not be related to the site and type of mutation.Part Ⅱ Research on the level of activation of the mTOR signaling pathway in TSC-RAMLObjective:To study the activation level of the mTOR signal pathway in TSC-RAML tissue.Methods:We collected 5 TSC-RAML specimens as the study group, and collected 10 S-RAML specimens and 10 normal renal tissues with non renal tumor as the control groups. With the immunohistochemical technique, the expression of p-mTOR, p-S6K1 and p--4EBP1 in the 3 groups was detected.Results:The p-mTOR was positive or strong positive expression in TSC-RAML, and weakly positive expression in S-RAML. The expression of p-S6K1 was positive in TSC-RAML and negative or weakly positive in S-RAML. The p-4EBP1 was positive or weakly positive in TSC-RAML, and weakly positive in S-RAML. The p-mTOR, p-S6K1 and p-4EBPl were negative in normal kidney tissues. In the 5 TSC-RAML specimens, the p-mTOR and p-4EBP1 expression level in the 3 specimens of patients with TSC2 mutation was relatively higher than 2 cases of patients with TSC1 mutation. There was no significant difference on the expression of p-S6K1 between the specimens of patients with TSC1 mutation and TSC2 mutation.Conclusions:Compared with the S-RAML and normal renal tissues, the mTOR signaling pathway has a relatively higher degree of activation in the TSC-RAML tissues. And compared with the TSC1 mutation, the activation of the mTOR signaling pathway was relatively higher in RAML tissue of patients with TSC2 mutantion.Part Ⅲ A preliminary study on the efficacy and safety of the mTOR inhibitor in the treatment of TSC-RAMLObjective:To evaluate the efficacy and safety of the mTOR inhibitor everolimus in treating patients with TSC-RAML.Methods:This is a single center, open label, nonrandomized clinical study, and the subjects were clinically diagnosed TSC-RAML patients, who should be in accordance with the inclusion criteria and exclusion criteria. Patients, who were in accordance with the standards through a rigorous screening, were given everolimus 10mg/d orally, and the treatment time was one year. Routine follow-up time was started after 3,6,12 and 24 months later during the treatment and the dosage can be adjusted timely according to the tolerance of patients. A detailed assessment and record of the baseline data and the condition change of patients before treatment and after treatment was performed, as well as the treatment related adverse events.Results:Up to now, there have been 5 patients with TSC-RAML enrolled and administrated everolimus orally for 3 months. Compared with the baseline data, each patient’s RAML were significantly decreased. The mean maximum diameter of RAML decreased 3.8 (1.5～6) cm, and the average percentage of maximum diameter reduction was 28.2%(15.4%-38%). Facial blood vessels fibers in the 5 patients also became thin and flat. 2 patients with psychiatric symptoms have been significantly improved. Stomatitis, rash, cough and the upper respiratory tract infection were the most common adverse events, of which, only 1 case occurred grade 3 adverse event, and the rest cases were all with grade 1-2 adverse events.Conclusions:The mTOR inhibitor everolimus has significantly curative effect in the treatment of TSC-RAML, and the TSC-RAML was obviously reduced in diameter. With the grade 1-2 adverse events, it is good safety and tolerability for patients with TSC-RAML.