Weaver syndrome is a rare neurodevelopmental disorder that encompasses macrocephaly, tall stature, obesity, brain anomalies, intellectual disability, and increased susceptibility to cancer. This dominant monogenic disorder is caused by germline variants in enhancer of zeste 2 polycomb repressive complex 2 subunit (EZH2), a key epigenetic writer. Unfortunately, there are no effective treatments for Weaver syndrome. However, preclinical results support the potential for therapeutic gains, despite the prenatal onset. Thus, for the first time, we tested whether CRISPR/Cas9 gene-editing strategies may be able to "correct" a Weaver syndrome variant at the DNA level. We initiated these preclinical studies by humanizing the region surrounding the most-common recurring patient-variant location in mouse embryonic stem cells (ESCs). Humanization ensures that DNA-binding strategies will be directly translatable to human cells and patients. We then introduced into ESCs the humanized region, but now carrying the Weaver syndrome EZH2 variant c.2035C>T p.Arg684Cys, and characterized the enzymatic properties of this missense variant. Our data showed a significant and dramatic reduction in EZH2-enzymatic activity, supporting previous cell-free studies of this variant as well as in vitro and in vivo mouse work by other teams. Intriguingly, this most-common variant does not create a complete loss-of-function, but rather is a hypomorphic allele. Together with prior reports describing hypomorphic effects of missense EZH2 variants, these results demonstrate that the etiology of Weaver syndrome does not require complete loss of EZH2 enzymatic activity. Toward therapy, we tested four CRISPR gene-editing strategies. We demonstrated that Streptococcus pyogenes Cas9 (SpCas9) showed the highest variant correction (70.5%), but unfortunately also the highest alteration of the nonvariant allele (21.1-26.2%), an important consideration for gene-editing treatment of a dominant syndrome. However, Staphylococcus aureus Cas9 (SaCas9) gave a variant correction (52.5%) that was not significantly different than SpCas9, and encouragingly the lowest alteration of the nonvariant allele (2.0%). Thus, the therapeutic strategy using the small SaCas9 enzyme, a size that allows flexibility in therapeutic delivery, was the most optimal for targeting the Weaver syndrome EZH2 variant c.2035C>T p.Arg684Cys.

Weaver syndrome is a rare congenital overgrowth disorder caused by pathogenic EZH2 variants. This study reports a novel EZH2 variant associated with atypical manifestations, including severe bilateral camptodactyly and complex brain malformations. A 4-year-old Taiwanese female exhibited classical Weaver syndrome features including macrosomia, macrocephaly, hypertelorism, and developmental delay, plus atypical findings of severe bilateral camptodactyly and complex brain malformations. Neuroimaging revealed corpus callosum dysgenesis with rostral agenesis and genu hypoplasia, bilateral frontal lobe hypoplasia, and an arachnoid cyst. The patient demonstrated global developmental delay with marked motor impairment but less severely affected speech and cognition, consistent with mild intellectual disability. Whole-exome sequencing identified a novel de novo pathogenic variant in EZH2: c.449T>C (p.Ile150Thr), affecting a highly conserved amino acid within the SANT domain. This case broadens the clinical spectrum of Weaver syndrome by highlighting severe camptodactyly and complex brain malformations as possible EZH2-related manifestations. The corpus callosum dysgenesis suggests a wider role of EZH2 in neurodevelopment than previously recognized. The novel SANT domain variant may explain the severe phenotypic presentation. The novel EZH2 variant c.449T>C (p.Ile150Thr) expands the molecular and phenotypic spectrum of Weaver syndrome. These findings underscore the importance of comprehensive neuroimaging and molecular genetic testing in suspected cases, particularly atypical presentations.

Weaver syndrome is a rare congenital overgrowth disorder characterized by a wide spectrum of clinical manifestations that often overlap with other overgrowth syndromes. It is primarily caused by pathogenic variants in the Enhancer of Zeste Homolog 2 (EZH2) gene on chromosome 7q36.1. Globally, fewer than 70 cases have been reported, with only a few documented in the Chinese population. We report a 13-day-old Chinese male infant, born with macrosomia (birth weight: 5.04 kg), who was admitted for persistent neonatal jaundice. Physical examination and subsequent follow-up revealed accelerated postnatal growth and characteristic craniofacial features, including a broad forehead, hypertelorism, epicanthal folds, a flat nasal bridge, and low-set ears. His length, weight, and head circumference consistently plotted above the 97th percentile for his age. Additional findings included large hands and feet. The child was ultimately diagnosed with "Weaver syndrome." DNA nanoballs were prepared with a universal sequencing reaction kit and subjected to paired-end sequencing on the MGISEQ-2000 platform. The resulting reads were aligned to the human reference genome hg19 (GRCh37). After removing PCR duplicates, single nucleotide polymorphisms and insertions/deletions were identified and annotated against established variant databases. The potential pathogenicity of the identified variants and their structural impact on the protein were evaluated using computational prediction tools. This analysis revealed a missense variant in the EZH2 gene (NM_004456.4:c.2050C>T) in the proband, resulting in an arginine-to-cysteine substitution at codon 684 (p.Arg684Cys). In accordance with American College of Medical Genetics and Genomics guidelines, this variant was classified as pathogenic. Subsequent Sanger sequencing confirmed it as a de novo mutation. The patient received multidisciplinary guidance for neurodevelopmental, speech, and behavioral therapy. He remains under regular follow-up to monitor his growth and development. This report documents a new case of Weaver syndrome in China harboring a de novo EZH2 mutation, expanding the genotypic and phenotypic spectrum of this disorder in the Chinese population. Our findings underscore the critical role of genetic testing in achieving a definitive diagnosis for rare overgrowth syndromes, facilitating early intervention and appropriate management.

Heterozygous missense mutations in EZH2 cause Weaver syndrome (WS), a developmental disorder characterized by intellectual disability and overgrowth. EZH2 encodes the enzymatic subunit of Polycomb repressive complex 2 (PRC2), which mediates monomethylation, dimethylation, and trimethylation of histone H3 lysine 27 (H3K27me1/2/3). Most WS-associated EZH2 variants lack functional characterization but are presumed loss-of-function. However, the lack of early truncating mutations in EZH2 led us to hypothesize a dominant-negative mechanism for WS, which was supported by our structural analysis of all known WS-associated EZH2 variants. We isogenically modeled 10 representative variants in embryonic stem cells and showed that they reduce global H3K27me2/3 with concomitant increases in H3K27ac and chromatin decompaction. Notably, the pattern of H3K27me2/3 reductions indicated dominant-negative interference on PRC2 activity even when WS variants were expressed at low levels. RNA-seq identified weakly Polycomb-bound genes that lose canonical PRC1 (cPRC1) occupancy and become derepressed, including several phenotypically relevant growth control genes. Comparative analysis of a gain-of-function EZH2 variant causing growth restriction revealed reciprocal chromatin and transcriptional changes compared with WS-associated variants. Taken together, our findings support a model in which EZH2 variants associated with opposing developmental growth syndromes affect not only H3K27me3 but also intergenic H3K27me2, chromatin architecture, and cPRC1 recruitment.

Pathogenic mutations in DNMT3A cause Tatton-Brown-Rahman Syndrome (TBRS), a disorder characterized by somatic overgrowth of multiple tissues including the brain and intellectual disability (OGID). Here, we investigated TBRS etiology using new human pluripotent stem cell models, modeling varying levels of TBRS-associated loss of DNMT3A function. We identified lineage-specific overgrowth in TBRS ventral forebrain medial ganglionic eminence (MGE)-like progenitors, due in part to increased signaling through the PIK3/AKT/mTOR pathway that could be modulated to ameliorate this phenotype. By contrast, reduced DNA methylation during MGE-like progenitor differentiation into GABAergic interneurons caused premature expression of neuronal and synaptic genes, triggering precocious neuronal maturation. As a result, TBRS GABAergic neurons exhibited hyperactivity sufficient to alter the development and structure of neuronal networks, likely contributing to the intellectual disability and autism spectrum disorder common to TBRS patients. Together, this work elucidates new roles for DNMT3A-mediated gene repression in human cortical development, identifying critical requirements for regulating GABAergic neuron production and neuronal network function. These findings also provide evidence for interrelated pathogenic mechanisms underlying TBRS and other OGIDs, including PIK3CA-related overgrowth syndrome and Weaver Syndrome, providing a foundation and rationale for future studies to identify common paradigms to treat these related disorders. EZH2-related overgrowth is a variable overgrowth syndrome characterized by tall stature, macrocephaly, variable intellect (ranging from normal intellect to severe intellectual disability), characteristic facial appearance, and a range of associated clinical features including advanced bone age, poor coordination, soft, doughy skin, camptodactyly of the fingers and/or toes, umbilical hernia, abnormal tone, and hoarse, low cry in infancy. Brain MRI has identified abnormalities in a few individuals with EZH2-related overgrowth. Neuroblastoma occurs at an increased frequency in individuals with a heterozygous EZH2 pathogenic variant. There is currently no evidence that additional malignancies (including hematologic malignancies) occur with increased frequency, though a few have been reported. The diagnosis of EZH2-related overgrowth is based on detection of a heterozygous germline EZH2 pathogenic variant on molecular genetic testing. Treatment of manifestations: For individuals with developmental delay and/or learning disability, referral for learning/behavior/speech assessment and support may be indicated. Occasionally, toe camptodactyly may require surgical release. Physiotherapy may be of benefit to those experiencing joint pain secondary to ligamentous laxity or joint contractures. Standard treatment with appropriate specialist referral(s) is indicated for epilepsy, scoliosis, and other clinical issues. Surveillance: Regular medical follow up of young children with EZH2-related overgrowth to monitor developmental progress, camptodactyly (for resolution/improvement), and/or hypotonia; medical follow up of older children/teenagers who do not have medical complications may be less frequent. If scoliosis is present, monitoring per the recommendations of an orthopedist. Clinicians should have a low threshold for investigating any possible tumor-related symptoms. There are no internationally ratified surveillance guidelines for screening of neuroblastoma in individuals carrying inactivating EZH2 pathogenic variants, but US guidelines recommend imaging and urine biochemistry surveillance until age 10 years. Pregnancy management: Families and their health care providers should be made aware that an affected baby may be large so that appropriate delivery plans can be made. EZH2-related overgrowth is inherited in an autosomal dominant manner. Some individuals diagnosed with EZH2-related overgrowth have an affected parent; some individuals diagnosed with EZH2-related overgrowth have the disorder as the result of a de novo EZH2 pathogenic variant. The proportion of individuals with EZH2-related overgrowth caused by a de novo pathogenic variant is unknown. Each child of an individual with EZH2-related overgrowth has a 50% chance of inheriting the pathogenic variant; the phenotype in individuals who inherit a familial EZH2 pathogenic variant cannot be predicted. Once the pathogenic variant has been identified in an affected family member, prenatal and preimplantation genetic testing are possible.