The Polycomb Repressive Complex 2 is an epigenetic reader/writer that methylates histone H3K27. Rare germline partial loss-of-function (pLoF) variants in core members of the complex (EZH2, EED, and SUZ12) cause overgrowth and intellectual disability syndromes, whereas somatic variants are implicated in cancer. However, up to 1% of the general population will have a rare variant in one of these genes, most of which would be classified as variants of uncertain significance (VUS). Towards screening these VUS for partial LoF alleles that may contribute to disease, here we report functional assays in Drosophila to interrogate Embryonic Ectoderm Development (EED) missense variants. We mimicked the amino acid change(s) of EED variants into its Drosophila ortholog, esc, and tested their function. Known likely benign variants functioned wildtype and known pathogenic variants were LoF. We further demonstrate the utility of this calibrated assay as a scalable approach to assist clinical interpretation of human EED VUS.

EED is a core component of polycomb repressive complex 2 (PRC2) with EZH2 and SUZ12. PRC2 has H3K27 methyltransferase activity (HMTase) that catalyzes the addition of up to three methyl groups on histone 3 at lysine residue 27 (H3K27). Germline heterozygous variants in EED, SUZ12, and EZH2 have been identified in patients with overgrowth and multiple dysmorphic features. The clinical manifestations of these syndromes significantly overlap: generalized overgrowth, intellectual disability, and scoliosis. To date, 11 unrelated patients have been published with missense variants in EED at highly conserved amino acids. We report three affected members in a family with a previously reported missense variant. All three affected members manifested very similarly, and this represents a homogenous clinical phenotype associated with EED related intellectual disability and overgrowth. This disorder is appropriately called Cohen-Gibson syndrome.

The SUZ12 gene encodes a subunit of polycomb repressive complex 2 (PRC2) that is essential for development by silencing the expression of multiple genes. Germline heterozygous variants in SUZ12 have been found in Imagawa-Matsumoto syndrome (IMMAS) characterized by overgrowth and multiple dysmorphic features. Similarly, both EZH2 and EED also encode a subunit of PRC2 each and their pathogenic variants cause Weaver syndrome and Cohen-Gibson syndrome, respectively. Clinical manifestations of these syndromes significantly overlap, although their different prevalence rates have recently been noted: generalized overgrowth, intellectual disability, scoliosis, and excessive loose skin appear to be less prevalent in IMMAS than in the other two syndromes. We could not determine any apparent genotype-phenotype correlation in IMMAS. The phenotype of neurofibromatosis type 1 arising from NF1 deletion was also shown to be modified by the deletion of SUZ12, 560 kb away. This review deepens our understanding of the clinical and genetic characteristics of IMMAS together with other overgrowth syndromes related to PRC2. We also report on a novel IMMAS patient carrying a splicing variant (c.1023+1G>C) in SUZ12. This patient had a milder phenotype than other previously reported IMMAS cases, with no macrocephaly or overgrowth phenotypes, highlighting the clinical variation in IMMAS.

Cohen-Gibson syndrome is a rare genetic disorder, characterized by fetal or early childhood overgrowth and mild to severe intellectual disability. It is caused by heterozygous aberrations in EED, which encodes an evolutionary conserved polycomb group (PcG) protein that forms the polycomb repressive complex-2 (PRC2) together with EZH2, SUZ12, and RBBP7/4. In total, 11 affected individuals with heterozygous pathogenic variants in EED were reported, so far. All variants affect a few key residues within the EED WD40 repeat domain. By trio exome sequencing, we identified the heterozygous missense variant c.581A > G, p.(Asn194Ser) in exon 6 of the EED-gene in an individual with moderate intellectual disability, overgrowth, and epilepsy. The same pathogenic variant was detected in 2 of the 11 previously reported cases. Epilepsy, however, was only diagnosed in one other individual with Cohen-Gibson syndrome before. Our findings further confirm that the WD40 repeat domain represents a mutational hotspot; they also expand the clinical spectrum of Cohen-Gibson syndrome and highlight the clinical variability even in individuals with the same pathogenic variant. Furthermore, they indicate a possible association between Cohen-Gibson syndrome and epilepsy.

The Polycomb repressive complex 2 is an epigenetic writer and recruiter with a role in transcriptional silencing. Constitutional pathogenic variants in its component proteins have been found to cause two established overgrowth syndromes: Weaver syndrome (EZH2-related overgrowth) and Cohen-Gibson syndrome (EED-related overgrowth). Imagawa et al. (2017) initially reported a singleton female with a Weaver-like phenotype with a rare coding SUZ12 variant-the same group subsequently reported two additional affected patients. Here we describe a further 10 patients (from nine families) with rare heterozygous SUZ12 variants who present with a Weaver-like phenotype. We report four frameshift, two missense, one nonsense, and two splice site variants. The affected patients demonstrate variable pre- and postnatal overgrowth, dysmorphic features, musculoskeletal abnormalities and developmental delay/intellectual disability. Some patients have genitourinary and structural brain abnormalities, and there may be an association with respiratory issues. The addition of these 10 patients makes a compelling argument that rare pathogenic SUZ12 variants frequently cause overgrowth, physical abnormalities, and abnormal neurodevelopmental outcomes in the heterozygous state. Pathogenic SUZ12 variants may be de novo or inherited, and are sometimes inherited from a mildly-affected parent. Larger samples sizes will be needed to elucidate whether one or more clinically-recognizable syndromes emerge from different variant subtypes.