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.