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UBC Theses and Dissertations

Polycomb repressive complex 2 related syndromes : functional studies of human gene variants In Drosophila Cyrus, Sharri Sonjene


The Polycomb Repressive complex 2 is an epigenetic regulator responsible for the mono-, di- and trimethylation of histone 3 lysine 27. Pathogenic mutations in the core members of the complex, namely EZH2, EED and SUZ12 cause the childhood-onset diseases Weaver syndrome, Cohen Gibson syndrome and SUZ12-related overgrowth respectively. Additionally, somatic mutations in these genes are seen in a variety of solid tumours and blood malignancies, and individuals with Weaver syndrome have a 5% risk of cancer development in the first 25-30 years of life. As exome and genome studies become more routine in clinical practice, classifying and reporting rare variants in genes associated with diverse clinical outcomes will become more complex and varied. Thus, detailed phenotyping of individuals with variants in these genes paired with functional studies is essential to truly characterise the effect of PRC2 variants in human populations. In this work we use now-published clinical data to generate a phenotypic summary of Weaver syndrome, Cohen Gibson syndrome and SUZ12-related overgrowth. We show that these syndromes are typically characterised by neurodevelopmental delay and overgrowth, with the variable presence of abnormalities in other organ systems. As SUZ12-related overgrowth was the most recently described syndrome, we describe an additional 10 individuals, further expanding the clinical phenotype and increasing the reported case count from 3 to 13. To bridge genotype to function, we use Drosophila melanogaster to develop inexpensive and robust assays with the capacity to screen hundreds of variants. We use a combination of strategies including CRISPR/cas9 editing, ΦC31-mediated Recombinase-mediated cassette exchange and ΦC31-transgenesis to express human and orthologous Drosophila transgenes to interrogate human PRC2 variants. Despite the conservation of PRC2 between humans and Drosophila, we show here that expression of EZH2 or EED variant human proteins within Drosophila is not very effective in quickly or accurately interrogating variant function. However, creating EED or EZH2 mimetic or analogous human mutations in the fly’s orthologous gene or cDNA is a successful strategy that can be used to investigate loss-of-function (LoF) effects of variants. Moreover, we present EED assays that can accurately discern between LoF EED variants and benign variants.

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