The results support a dominant-negative mechanism for BRS causing truncating mutations, offering a compelling rationale for allele-specific ASO therapeutic strategy and new venues for treatment.
Abstract
Bainbridge-Ropers syndrome (BRS) is a rare neurodevelopmental disorder caused by truncating mutations in the epigenetic regulator ASXL3. While traditionally considered a haploinsufficiency disorder, the precise molecular mechanisms driving BRS remain poorly understood. Here, we combine patient-derived cellular lines and novel mouse models to elucidate the molecular function of disease-associated ASXL3 variants. We show that several pathogenic ASXL3 variants escape nonsense-mediated decay (NMD), possibly leading to the accumulation of truncated protein, and widespread epigenetic changes, resulting in distinct transcriptomic and proteomic profiles. These changes include increased chromatin accessibility and global DNA hypomethylation, particularly at promoters and imprinted loci. A knock-in Asxl3 mouse model harboring a mutation corresponding to one diagnosed in BRS-patient recapitulated the molecular features BRS-patient derived cellular model, including the escape from NMD and Polycomb Repressive Complex 2 (PRC2)-related transcriptomic dysregulation. In contrast, heterozygous Asxl3 knockout mice and transient knockdown models showed no phenotype, indicating that truncated ASXL3 that may exert dominant-negative effects rather than simple loss of function. This molecular dissection offers new venues for treatment, including allele-specific Antisense Oligonucleotides (ASO), which were used by us in patient-derived cells to downregulate the expression of the mutated allele, and were able to induce partial recovery of the proteomic profile. Taken together, our results support a dominant-negative mechanism for BRS causing truncating mutations, offering a compelling rationale for allele-specific ASO therapeutic strategy.
ASXL3 patient truncations in neurodevelopmental condition Bainbridge-Ropers syndrome are shown to mediates gain-of-function (GOF) by escaping nonsense-mediated decay and Cullin 4-dependent degradation, resulting in aberrant protein accumulation, widespread transcriptional dysregulation, and altered chromatin accessibility.
Y. Nakamura, T. Nguyen, N. Mor et al.· medRxiv· 0 citations
Functional modelling in zebrafish confirms a loss‑of-function mechanism and highlights species‑dependent differences specifically in the impact of the missense variant on protein function, and provides a cautionary tale about overreliance on animal models as a screening tool for variant classification.
H. Shamseldin, Dana Marafi, Mohammed A Al-Muhaizea et al.· Scientific Reports· 0 citations
Bohring-Opitz syndrome (BOS, OMIM#605309) is a rare neurodevelopmental disorder caused by heterozygous and truncating variants in ASXL1 (Additional Sex Combs Like 1), a chromatin-associated epigenetic regulator that forms the catalytic PR-DUB complex with BAP1. Truncating ASXL1 variants are also recurrent somatic drivers in myeloid leukemia, yet the metabolic consequences of these mutations remain undefined. Using patient derived dermal fibroblasts, we show that truncating ASXL1 variants drive a Warburg-like metabolic state characterized by increased glycolytic flux, and accumulation of pyruvate and lactate. Truncated ASXL1 and BAP1 show aberrant co-occupancy at an H3K4me3-marked intronic regulatory element within MPC2 intron 1, with broadened ASXL1 occupancy extending beyond BRD4-defined regulatory boundaries while BRD4 positioning remains unchanged, consistent with aberrant PR-DUB complex spreading beyond its normally constrained chromatin territory. This altered occupancy is accompanied by modest but significant reduction in MPC2 transcript abundance and a disproportionately larger reduction in MPC1 and MPC2 protein levels, indicating that transcriptional dysregulation at this intronic element is amplified at the protein level through post-transcriptional mechanisms including impaired MPC1/MPC2 heterodimer stability. Pharmacologic MPC inhibition recapitulates both the metabolic and Wnt signaling phenotypes of BOS cells, while canonical Wnt activation increases glycolytic flux without reducing MPC abundance, establishing mitochondrial pyruvate restriction as causally upstream of signaling dysregulation. These findings define a previously unrecognized chromatin-to-metabolism axis connecting gain-of-function ASXL1 truncation to mitochondrial pyruvate transport, identifying MPC as a central mediator of epigenetic-metabolic crosstalk in both a rare developmental syndrome and ASXL1-mutant myeloid malignancy. Graphical Abstract Truncating and heterozygous ASXL1 variants cause a neurodevelopmental syndrome called Bohring-Opitz syndrome. (1) At an epigenetic level, we have shown that Truncating ASXL1 variants drive more open chromatin and aberrant activation of key developmental pathways. (2) Truncating ASXL1 mutations are sufficient to drive Decreased MPC1 and MPC2 protein levels. (3) Decreased MPC1 or MPC2 level or function are sufficient to drive increased glycolysis which is observed in BOS cells. (4) Truncating ASXL1 mutations drive Increased Wnt signaling via MPC depletion. * Increased Wnt signaling (4) is also sufficient to drive increased glycolysis (3), however Increased Wnt signaling does not drive Decreased MPC1 and MPC2 levels (2).
Isabella Lin, Michael Sigfrid S. Reyes, A. Krall et al.· bioRxiv· 0 citations
These results identify POGZ as a G9a/GLP-associated chromatin regulator that protects neurodevelopmental gene domains from heterochromatinization and perinuclear sequestering, preserving 3D architecture and transcription during cortical development.
N. Mariano, Katerina J. Williams, Katie Munechika et al.· bioRxiv· 0 citations
The PR-DUB complex is responsible for erasing the repressive histone modification, H2AK119ub1. ASXL1-3 proteins are mutually exclusive catalytic partners of BAP1 in the PR-DUB complex. Somatic heterozygous ASXL1-3 variants are associated with cancer, including myeloid malignancies, while de novo germline variants cause neurodevelopmental disorders such as Bohring-Opitz syndrome. These pathogenic variants are almost exclusively nonsense and frameshift and have been proposed to act as gain-of-function. However, the precise catalytic impact and mechanism of variant ASXL1-3 remains elusive. Using an isogenic embryonic stem cell model we have discovered that ASXL1 BOS variants drive reductions – but not global ablations – in H2AK119ub1, consistent with gain-of-function. This catalytic change occurs through the production of a truncated ASXL1 protein with enhanced stability. Hyper-stabilised ASXL1 drives a stoichiometric shift in PR-DUB assembly away from ASXL2 complexes. The drop in H2AK119ub1 levels ultimately reduces PRC2 binding and H3K27me3 deposition. Surprisingly, this phenotype is shared across PR-DUB loss-of-function models and indeed is emerging as a common phenotype across genetically and mechanistically distinct Polycomb-related chromatinopathies.
E. Doyle, Maeve Boyce, Sarah Buggle et al.· bioRxiv· 0 citations