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Elias Bou Farhat, MD







Pulmonary and Clinical Care Medicine


Elias Bou Fahrat*, Victoria-Elisabeth Gruber, Katarzyna Klonowska, Barbara Ogórek, Zachary T. Herbert, Suzanne Miller, Simon R. Johnson, Elzbieta Radzikowska, Renata Langfort, Miquel Àngel Pujana, Yoichiro Mitsuishi, Kuniaki Seyama, Jason L. Hornick, Elizabeth P. Henske, David J. Kwiatkowski

Principal Investigator

David J. Kwiatkowski


Comprehensive Genetic Analysis of Patients with Sporadic Lymphangioleiomyomatosis


Lymphangioleiomyomatosis (LAM) is a rare and progressive cystic lung disease occurring predominantly in woman, with more rapid progression pre-menopausal and during pregnancy. Atypical proliferation of smooth muscle cells that express melanosomal markers is present in the lungs of woman with LAM. These cells are thought to be the cause of the disease. LAM occurs sporadically (sporadic LAM) or in association with tuberous sclerosis complex (TSC-LAM). Previous studies have shown that LAM smooth muscle-like cells possess somatic mutations in TSC2. We are pursuing analysis of formalin-fixed paraffin-embedded (FFPE) lung biopsies from sporadic LAM patients with the goal of defining a causative mutation in every patient. 133 FFPE sporadic LAM blocks are available. We screened H/E slides to exclude patients with < 1% LAM cell content. We extracted DNA from these blocks and employed two methods of analysis: 1) multiplex high-sensitivity PCR assay (MHPA) covering 75% of TSC2; 2) hybrid capture of TSC1/TSC2, forty other mTOR pathway genes, and MITF, TFE3, TFEB. 51 samples were excluded due to low LAM cell content, insufficient or highly fragmented DNA, and poor performance in the MHPA and/or low read depth (< 1000 per amplicon). Mutations were identified using our custom computational pipeline. 63 samples were subject to sequencing and one or two TSC2 mutations were identified in 41 patients (65%); no TSC1 mutations were identified. 25 mutations were insertions or deletions (indels), 8 splice, 7 missense, 14 nonsense, one is a deletion with two nucleotide change, at allele frequencies of 0.09-20.58% (median 2.54%). Eleven mutations have been validated by secondary analysis, and this is in process for all others. 13 of 41 samples had two mutations in TSC2 consistent with bi-allelic inactivation of TSC2. We suspect that the remaining cases had a single mutation accompanied by copy neutral loss of heterozygosity, to cause bi-allelic inactivation.

Clinical Implications

Sporadic LAM is a rare disease, with 200 to 300 newly diagnosed cases in the US each year. Genetic analysis is not commonly done for LAM, due to the low frequency (<10%) of LAM cells in the lung in most patients and limited amounts of tissue for analysis. Sirolimus, an mTORC1 inhibitor, is an FDA approved therapy for LAM. It is possible that mutation of other mTOR pathway genes and/or fusions involving the MITF/TFE genes cause LAM. Identification of such mutations is important for our understanding of the pathogenesis of LAM and could have therapeutic implications.