Detailed phenotyping and next-generation sequencing for characterization of rare overgrowth syndromes

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.

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