Mutations in the tsc2 Gene Do Not Always Play a Role in the Pathogenesis of Sporadic Lymphangioleimyomatosis
L Gao, WQ Ling, DJ Kwiatkowski, L Schuger. University of Chicago, Chicago, IL; Harvard Medical School, Boston, MA
Background: Lymphangioleiomyomatosis (LAM) is a rare and fatal lung disease affecting women predominantly of child-bearing age. Mutation of one tsc2 (tuberin) allele, preceded or followed by LOH (loss of herterozygosity) of the other allele, is currently considered to cause sporadic LAM. Therefore it is proposed that the lack of functional tuberin, leading to phosphorylation and activation of the mTOR (molecular target of rapamycin)/S6K1 signaling pathway, is essential to the pathogenesis of LAM disease. The propose of this study is to assess the roles of tsc2 mutations or LOH and mTOR/S6K1 signaling pathway in the pathogenesis of sporadic LAM.
Design: DNA samples prepared from LAM lesions laser-microdissected from frozen lung tissue of 10 patients with sporadic LAM were studied for LOH by MLPA (multiplex ligation-dependent probe assay) and 7 of them for mutations in tsc2. These 7 samples were subject to whole genome amplification, and analyzed by deep sequencing of all tsc2 exons using the Roche-454 platform. In addition, serial sections of FFPE lung tissue sampled from 3 of the sequenced cases were immunostained for LAM markers SM actin and HMB 45, tuberin C-terminus (missed in mutated/truncated tuberin), phosphorylated mTOR and S6K1.
Results: None of the 10 cases showed LOH. Five of the 7 samples sequenced showed one tsc2 mutation. One sample had two different mutations. The mutation frequency was below 10% in all but one case, in which it was 46%. One of the cases without tsc2 mutations (0M), a case with one tsc2 mutation (1M) and the case with two different tsc2 mutations (2M) were selected for IHC studies. Case 0M showed diffuse immunoreactivity for tuberin in LAM lesion, which is similar to that of normal alveoli, reactive epithelial and mesothelial cells (+++), but no immunoreactivity for phosphorylated mTOR and S6K1. Case 1M showed diffuse LAM lesion immunoreactivity for tuberin (+++), while in case 2M positivity for tuberin was focal (++). M1 and M2 cases showed focal immunoreactivity for phosphorylated mTOR and S6K1, which was (+) compared to (++++) in reactive epithelial and mesothelial cells and (-) in normal alveoli. In all cases vascular SM were negative (-) for tuberin, phospho-mTOR and phospho-S6K1.
Conclusions: Sporadic LAM does not require tsc2 LOH to develop. Furthermore, some LAM cases have no abnormalities in either of the two tsc2 alleles. Activation of the mTOR/S6K1 pathway does not always play a role in the pathogenesis of sporadic LAM.
Tuesday, March 23, 2010 1:00 PM
Poster Session IV # 260, Tuesday Afternoon