The Correlation Between NF2 Gene Alterations And Clinical Behaviors In Sporadic Vestibular Schwannoma

?OBJECTIVE? The present study is aimed at determining the genetic alteration of NF2 gene in correlation with its mRNA/protein expression and phosphorylation alteration in sporadic VS,and finding out clinical association.We also detected the expression of cyclin D1 and apoptosis related proteins to find out the potential mechanism of the influence of NF2 gene alteration on the tumor biological behavior.?METHODS? 1.We reviewed a cohort of 40 patients with sporadic VS,clinical characteristics such as sex,age,clinical symptoms,tumor size,facial nerve function and hearing level were recorded in an electronic medical record system completely;2.PCR and bidirectional sequencing and MLPA were conducted to detect intragenic small mutations and exons deletion on NF2 gene;The clinical behaviors were analyzed between each mutation group;3.Realtime PCR and Western blotting were conducted to evaluate the expression level of NF2 mRNA and merlin in tumors with or without detectable alterations;4.We analyzed the methylation statuses of CpG islands in NF2 gene promoter region by MSP technique.P-merlin S518 expression level was analyzed in tumors without any detectable NF2 mutations to find out potential mechanism of sporadic vestibular schwannomas;5.p53,cyclin D1,BAX,Bcl-2 and cleaved caspase 3 expression levels were analyzed using Western blotting to find out the influences of NF2 mutations on the cell signaling;6.Lentivirus-based shRNAs were used to determine whether silencing of merlin potentiates cell proliferation and cell-cycle progression in HSC and the potential mechanism.?RESULTS? 1.Genetic mutation of NF2 is common event in sporadic VS.Double hits were found in 17 cases(42.5%),a single event was discovered in 10 cases(25.0%)and there were only 13 tumors(32.5%)with no molecular genetic alteration of NF2;2.Tumors with NF2 gene alterations had higher GI;Patients with mutations in exon 2 or exon 3 are younger at diagnosis than patients with mutations in other exons;3.Promoter methylation was rare in VS.Phosphorylation on merlin S518 was only found in proportion of tumors harboring no detectable mutations and was correlated to tumor size;4.Down-regulated merlin protein expression was associated with down-regulated p53 expression and up-regulated cyclin D1 expression in mutational tumors.There was no significant difference between patients with or without mutation of BAX/Bcl-2 and cleaved caspase 3 expression;5.In HSC cells,merlin loss could suppress p53 expression,and hence the dysregulation of cell-cycle regulatory proteins;NF2 gene knockdown promoted cell proliferation;?CONCLUSION? 1.Genetic mutation of NF2 is common event in sporadic VS.Point mutation was the first hit followed by allele deletion as a second hit;Alterations in NF2 gene play a role in the pathogenesis of sporadic VS;2.Phosphorylation of merlin on S518 may be a supplemental mechanism to VS tumorigenesis;NF2 gene alteration may promote the cell proliferation rather than apoptosis;3.Lack of merlin resulted in G1/S cell cycle progression and thereby the uncontrolled cell proliferation mainly via the regulation of p53-mediated pathways.