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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.

Studies on genetic forms of diabetes have been pivotal in understanding how genetic mutations impair pancreatic β cell function. We identified a patient who presented with early-onset diabetes similar to known monogenic forms of diabetes. The patient carries a microdeletion that removes a copy of EP300, a gene that encodes the transcriptional coactivator p300. EP300 mutations cause Rubinstein-Taybi syndrome, a rare genetic condition that has been associated with early-onset glucose dysregulation. Whether and how p300 loss may affect β cell function in vivo was not clear. Here, we show that expression of p300 regulates β cell development and function in vivo. By deleting p300 at different developmental stages in mouse β cells, we demonstrate that p300 is required for the proliferation and proper maturation of developing β cells. β cell development requires p300 to acetylate histone H3K27 across the genome and to coactivate transcription. In mature β cells, p300 maintains insulin granule biosynthesis and secretion. To regulate these processes transcriptionally, p300 and NeuroD1/Nkx6.1/Pdx1 co-occupy loci that are critical for β cell function, including insulin. In addition to the mouse studies, we have identified three additional probands who developed hyperinsulinism associated with their rare, potentially gain-of-function EP300 variants. Our data demonstrate a critical role of p300 as a transcriptional coactivator and a histone acetyltransferase in β cells. Taken together, our human data highlight the relevance of p300 to the pathogenesis of genetic forms of diabetes, and our mouse data provide mechanistic insights on how p300 deficiency may lead to glucose dysregulation.

Weaver syndrome (WS) is a rare overgrowth disorder characterized by tall stature, macrocephaly, advanced bone age, facial dysmorphism, intellectual disability and cancer susceptibility, and it is caused by constitutional mutations in the enhancer of zeste homolog 2 gene (EZH2). To expand our understanding of WS pathogenesis, we assembled a cohort of 66 individuals with Weaver-like features, and collected DNA together with detailed clinical information. Sanger sequencing identified eleven individuals with pathogenic mutations in EZH2 (equivalent to a 17% diagnostic rate). A further seven individuals carried mutations in the nuclear receptor-binding SET domain-containing protein 1 gene (NSD1), which cause a similar overgrowth disorder called Sotos syndrome (11% diagnostic rate). Furthermore, we expanded the phenotypic spectrum of WS to include neuronal migration disorders. EZH2 is a histone methyltransferase that acts as the catalytic agent of the Polycomb Repressive Complex 2 (PRC2) to maintain gene repression via methylation of lysine 27 on histone H3 (H3K27). Functional studies investigating the activity of mutant EZH2 from various cancers showed that both gain- and loss-of-function mechanisms exist, thus it was important to determine which mechanism is causing WS. Using a standard histone methyltransferase assay, we observed that WS-associated EZH2 mutations impair PRC2’s histone methyltransferase activity in vitro, suggesting a loss-of-function mechanism of disease. In addition, no correlation between degree of functional impairment and phenotypic severity was noted. Recognizing a clear role for chromatin modifications in the molecular pathophysiology of overgrowth syndromes, we hypothesized that mutations in other chromatin regulators might explain the phenotype observed in the remaining undiagnosed individuals. Using next-generation sequencing in combination with detailed phenotyping, we identified EED as a novel overgrowth gene. EED happens to be the main partner of EZH2 within PRC2, and is necessary for proper H3K27 methylation to occur. Altogether, we have expanded the phenotypic and mutational spectrums of WS, and begun to uncover the underlying mechanism of disease. We also discovered a novel overgrowth gene, EED, reinforcing a role for PRC2 in the regulation of human growth and development.