Characterisation of likely pathogenic variants in a gene suspected to be responsible for digenic inheritance of primary lymphedema

Details

Supervisors

Vikkula, Miikka ; Brouillard, Pascal

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

enPrimary lymphedema (PL) is a rare genetic disorder characterized by developmental or functional defects of the lymphatic vasculature. A key enzyme in heparan sulphate biosynthesis localised in the cis-Golgi has recently emerged as a candidate gene that may contribute to PL pathogenesis through a digenic mechanism, according to our patient database. This project aimed to functionally characterize rare variants in the gene encoding this enzyme identified in individuals with PL. Using the Highlander software, which integrates next-generation sequencing data from over 1000 PL patients, 13 rare variants were identified in 23 individuals. Bioinformatic tools, including gnomAD, deCAF, RGC, AlphaMissense, Mutation Taster, Consurf, SpliceAI and VENUS were used to predict pathogenicity. A digenic analysis revealed 134 individuals with variants in at least two lymphedema-associated genes of the ANGPT/TIE or the VEGFC/VEGFR3 pathway including our gene of interest. In vitro mutagenesis was used to generate plasmids containing the variants in the gene coupled to a FLAG. HEK293T cells were transiently transfected, and protein expression was assessed by Western blot using antibodies for FLAG, ß-actin, as a loading control, and NeomycinR for transfection efficiency. Due to heterogeneous and weak FLAG signals, stable HEK293T cell lines were generated for each variant through geneticin selection, followed by clonal isolation. However, expression remained faint, prompting the evaluation of an antibody directed to our protein of interest to improve detection. Enzymatic activity of our protein was also evaluated, using an ELISA-based assay on HEK293T lysates. In parallel, immunocytofluorescence was performed on transiently transfected HEK293T cells to evaluate the co-localization with the Golgi marker GM130. Overall, our results indicate that three of the variants affect enzymatic activity, but further investigations are required to fully elucidate the functional impact of the variants and their potential role in digenic PL.