Introduction: Lymphatic malformations (LM) are dilated lymphatic vessels that are disconnected from the rest of the lymphatic system. The underlying etiologies of LMs are still largely unknown. This has hindered the development of more advanced and possibly less invasive approaches to managing this disease. Utilizing at whole exome sequencing and characterization of the miRNA expression profiles of lymphatic malformations compared to normal human lymphatic endothelial cells, this thesis seeks to better understand the disease.

Materials and Methods: Eight lymphangioma samples with matching blood underwent whole-exome sequencing (WES) to identify somatic driver mutations. The results of this discovery cohort were validated in an independent group of 30 samples using molecular-inversion probe sequencing. RNA was then extracted from 12 human lymphatic malformation specimens and control human lymphatic endothelial cells for microarray analysis. The microarray data from LM and control endothelial cells was pooled and student's t test was used to compare the data. miRNA bioinformatics databases were used to predict the miRNA regulators of a given gene. Pathway analysis was performed using Qiagen Ingenuity Pathway Analysis (Redwood City, California).

Results: WES identified recurrent alterations to the COSMIC reported gene PIK3CA in three of eight samples. PI3k is an intracellular signaling pathway important for regulating cell cycle and cell proliferation. High-coverage targeted re-sequencing confirmed the variants in these three samples, and identified ten additional samples with PIK3CA mutations. Out of thirty examined 23% had a mutation amino acid mutation changing Glutamic acid (E) for lysine at 542 and another 23% at 545. The majority was of the mutations (54%) occurred at 1047 resulting in a Histidine being switched for an Arginine.

Of the ten most up-regulated and down-regulated miRNA's compared to normal lymphatic endothelial cells only two of these---miR-181b-5p and miR-551b-3p---are predicted to regulate Prox1 and GATA, genes known to be involved with LMs and lymphangiogenesis, respectively. Other miRNA transcripts that were not found in the top ten, but aberrantly expressed, were found to regulate GATA2, Prox1 and VEGFR3, genes also associated with lymphangiogenesis.

Pathway analysis of the miRNA data showed that the CD36 pathway is inhibited by these miRNA's. The CD36 pathway is the thrombospondin-1 receptor that functions to inhibit lymphangiogenesis through the inhibition of VEGF-c and VEGF-d expression in lymphatic channels.