Abstract Lymphangioleiomyomatosis is a low grade neoplasm that causes progressive lung destruction, lung cyst formation, and respiratory failure. Bi-allelic mutations in TSC2 (or much less commonly TSC1) have been known as the main genetic driver of LAM in both individuals with TSC as well as sporadic LAM. It has also been thought that the distinction between TSC-LAM and sporadic LAM was well-defined. However, recent studies by PI Kwiatkowski and co-investigator Darling have shown that mosaicism for TSC1/TSC2 is common in adults with TSC-LAM, and associated with a milder clinical phenotype that may be missed in some apparent sporadic LAM patients. In addition, detailed analyses of LAM lung lesions have been able to identify TSC1 or TSC2 mutations in only a fraction of sporadic LAM patients, suggesting the involvement of other genes. Furthermore, a LAM GWAS led by the PI has identified SNPs on chromosome 15 near the transcription factor NR2F2 as having alleles that show association with sporadic LAM. In this proposal, we examine all three of these issues in greater detail. Two of the Aims will use massively parallel sequencing (MPS) and a novel technology we have developed, Multiplex High-sensitivity PCR Assay (MHPA), that is capable of highly sensitive variant detection in TSC2, down to an allele frequency of 0.05%, 10-fold lower than our previous targeted capture assay. In Aim 1, we will determine whether mutations in other mTOR pathway genes and/or MITF family member translocation or amplification cause sporadic LAM in a set of 100 LAM patients. In Aim 2, we will determine whether the presence of TSC2 mutations in cell free (cf) DNA is a biomarker of LAM; and examine the frequency of genetic mosaicism in selected subsets of apparent sporadic LAM patients; simultaneously, also in 100 LAM patients. We will enrich the patients studied for those with singleton TSC lesions, such as hypomelanotic macule (HMM) or facial angiofibroma, or bilateral angiomyolipoma. We will use our new MHPA assay for this analysis. In Aim 3, we will examine the role of NR2F2 in LAM development, by examining allelic imbalance in the H3K27ac ChIP-Seq data, performing NR2F2 ChIP-Seq, using Binding and expression target analysis to infer the genes most likely to have their expression driven by NR2F2, determine if NR2F2 is part of the Core transcription Regulatory Circuitry (CRC) in angiomyolipoma, and assess effects of NR2F2 expression modulation, and treatment with activators and inhibitors in the human angiomyolipoma cell line 621-101.