Rubinstein-Taybi syndrome (RSTS) and Menke-Hennekam syndrome (MKHK) are two rare Mendelian disorders presented with variable degrees of intellectual disability and different facial dysmorphism. They are caused by loss-of-function (LOF) variants or missense/inframe deletion variants in the exon 30 and 31 of the CREBBP gene respectively. This study aimed to refine the phenotype and provide characterization of genome-wide DNA methylation (DNAm) in RSTS and MKHK. We integrated and analyzed clinical data of 151 patients with RSTS and 36 patients with MKHK from this study and literatures. Meanwhile, genome-wide DNAm analysis were carried out on 51 blood samples (RSTS n = 9, MKHK n = 8, control n = 33), and 21 human induced pluripotent cell (hiPSC) samples (RSTS n = 5, MKHK n = 4, control n = 12). Phenotype analysis showed that patients with RSTS variants downstream the last 50 nt of the penultimate exon had atypical facial malformation and severer medical problems compared to the classical RSTS caused by LOF CREBBP variants. Individuals with MKHK variants in intrinsically disordered region (IDR) showed resemblant features. Meanwhile, DNAm analysis identified two specific blood DNA methylation patterns (episignatures): RSTS and MKHK_IDR compared to matched normal controls. Samples with MKHK variants outside the IDR did not obey the MKHK_IDR episignature. By interrogating DNAm in hiPSCs of patients with RSTS and MKHK, we observed differentially methylated genes play a role in embryonic development and organogenesis. In conclusion, our results suggest that phenotypic features and DNA methylation episignatures may differ for each genomic region.

Rubinstein-Taybi syndrome (RSTS) is a rare autosomal dominant neurodevelopmental disorder linked to haploinsufficiency of CREBBP (RSTS1) and EP300 (RSTS2) genes. Characteristic features often include distinctive facial traits, broad thumbs and toes, short stature, and various degrees of intellectual disability. The clinical presentation of RSTS is notably variable, making it challenging to establish a clear genotype-phenotype correlation, except for specific variants which cause the allelic Menke-Hennekam syndrome. Trio exome analysis, data collection via networking and GeneMatcher platforms, transcript processing analysis, and DNA methylation profiling were performed. We identified two unrelated patients with de novo variants in EP300 (NM_001429.4: c.3671+5G>C; c.3671+5_3671+8delGTAA) predicted to cause in-frame exon 20 skipping, confirmed in one patient. In silico 3D protein modeling suggested that exon 20 deletion (comprising 27 amino acids) likely alters the structural conformation between the RING_CBP-p300 and HAT-KAT11 domains. Clinically, both patients displayed severe RSTS2-like clinical features, including autism spectrum disorder, speech delay, hearing loss, microcephaly, developmental delay, and intellectual disability, alongside ocular, respiratory, and cardiovascular abnormalities. Additionally, one patient developed early-onset colorectal cancer. DNA methylation profiling in Subject #1 confirmed RSTS but did not align with the specific episignatures for RSTS1 or RSTS2. We propose that skipping of exon 20 in EP300 is associated with a distinct form of Rubinstein-Taybi syndrome featuring clinical characteristics not fully aligning with RSTS1 or RSTS2. Our findings increase the understanding of RSTS genetic and molecular basis and stress the need for further research to establish definitive genotype-phenotype correlations.

Two closely related acetyltransferases, p300 and its paralog CBP, are important regulators of gene expression and protein modulators in higher eukaryotes, influencing a wide array of cellular processes, including cell division, growth, DNA replication and repair, and apoptosis. The broad cellular impact is underscored by p300/CBP's capacity to interact with hundreds of proteins through various domains and the capability to acetylate numerous substrates and ubiquitinate selected targets. This intricate network of interactions and modifications highlights the essential role of p300/CBP in orchestrating cellular responses to pathological and physiological stimuli, thereby necessitating precise regulatory mechanisms to maintain their activity and substrate specificity. The regulation of p300/CBP is primarily governed by protein interactions and posttranslational modifications, including acetylation and ubiquitination, with autoregulation serving as a vital component in sustaining their enzymatic functions. The significance of tightly controlled p300/CBP activity is further emphasized by its association with various diseases, including Rubinstein-Taybi syndrome, Menke-Hennekam syndrome, and numerous cancers. Furthermore, the potential of p300/CBP as a therapeutic target has sparked interest in developing specific inhibitors. This review aims to elucidate the complex regulatory mechanisms of p300/CBP, focusing on posttranslational modifications, intermolecular interactions, and their implications in disease. A comprehensive understanding of the molecular foundations of p300/CBP regulation is essential for unraveling their roles in cellular processes and advancing targeted therapeutic strategies.

Menke-Hennekam syndrome (MKHK) is a recently described rare autosomal dominant disorder caused by loss-of-function variants in exon 30 or 31 of CREBBP (CREB-binding protein) or EP300 genes. These genes encode transcriptional coactivators with a key role in chromatin remodeling and regulation of gene expression. Herein, we report the identification and characterization of a novel missense variant in CREBBP, NM_004380.3:c.5368T>C p.(Cys1790Arg), in a 4-year-old male. The clinical presentation of the patient included global developmental delay, intellectual disability, growth retardation, and distinct craniofacial dysmorphisms, resembling known MKHK subtypes, but also exhibiting less common or unique features such as excessive palmar skin and the absence of recurrent infections and autism spectrum behaviors. Genetic analysis via trio-based clinical exome sequencing confirmed the de novo origin of the CREBBP variant, which was classified as pathogenic based on ACGS guidelines 2020. Structural modeling predicted that the NP_004371.2:p.(Cys1790Arg) substitution may disrupt the tertiary structure of the CBP TAZ2 domain (amino acids 1772-1840) when interacting with STAT1 but not with adenovirus E1A, potentially affecting transcription factor binding and disease phenotype. The findings contribute to the evolving classification of MKHK subtypes and to deciphering the complexity of genotype-phenotype relationships in MKHK.

Pathogenic germline missense and in-frame indel variants in exons 30 or 31 of the EP300 gene are associated with Menke-Hennekam syndrome-2 (MKHK2). The phenotypic spectrum associated with MKHK2 is variable, including neurodevelopmental, respiratory, skeletal, and immunological impairments. Based on their genetic, clinical, and DNA methylation profiles, a recent study proposed three domain-specific subtypes of MKHK: MKHK-ZZ, MKHK-TAZ2, and MKHK-ID4. In somatic cells, EP300 variants have been reported in lymphoma, leukemia, and various solid tumors. We present an African American girl with global developmental delay, failure to thrive, microcephaly, seizure, osteopenia, and T-cell acute lymphoblastic leukemia (T-ALL). We performed karyotype, FISH, chromosomal microarray, and exome sequencing with probands bone marrow, blood, and buccal swab. Comprehensive genetic studies using multiple tissues detected somatic complex cytogenomic changes in blood cells and a de novo germline missense variant (NM_001429.4: c.5258G>A, p.Cys1753Tyr) in the TAZ2 domain of EP300 from her buccal swab, which is consistent with a diagnosis of MKHK2. While in our patient we observed phenotypic overlaps with affected individuals harboring variants in the TAZ2 domain, some phenotypes such as osteopenia and alopecia have not been reported previously. The hematolymphoid malignancy of our patient also raises the question of whether germline EP300 variants are associated with a genetic predisposition to cancer. Together, this case expands the growing body of knowledge regarding the clinical and genetic spectrum of MKHK2. This is the first MKHK individual reported in the literature in an underrepresented population of African American ancestry.