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.
Abstract
Truncating variants in the human Additional sex combs (asx) ASXL genes are frequent in clonal hematopoiesis and severe dominant neurodevelopmental syndromes, yet are assumed to represent loss-of-function (LOF) alleles. However, numerous LOF alleles are documented in healthy individuals. Here we show ASXL3 patient truncations in neurodevelopmental condition Bainbridge-Ropers syndrome (BRS), by virtue of their distinct location in the gene body, instead 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. Patient-derived cell lines partnered with CRISPR knock-in of patient versus population truncations excluded simple haploinsufficiency, and instead support this two-hit GOF mechanism. Deletion mapping identified a broad C-terminal destabilizing region, explaining the 3-prime clustering of benign truncations. Haploinsufficiency was further excluded by forced expression of full length ASXL3 in patient lines, which failed to rescue the disease-associated differential gene expression signature. By contrast, antisense oligonucleotides targeting ASXL3 largely normalized this signature, providing a mechanistic rationale for knockdown therapies in ASXL associated disease.
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.
N. Mor, I. Shomer, S. Raviv et al.· medRxiv· 0 citations
Precise regulation of pre-mRNA splicing is essential for normal development, and its disruption represents an important but frequently underrecognized mechanism of human disease. The C-type natriuretic peptide (CNP) receptor NPR2 is a critical regulator of growth plate chondrocyte proliferation and differentiation, and loss-of-function variants in NPR2 cause acromesomelic dysplasia, Maroteaux type (AMDM). Here, we identify a homozygous synonymous NPR2 variant (NM_003995.4:c.2484C > T) in an individual with AMDM and demonstrate its pathogenic mechanism at the RNA level. Although predicted to be silent at the protein level, in silico analysis suggested splice donor gain. Functional analysis using patient-derived leukocyte RNA revealed aberrant splicing leading to partial exon truncation, frameshift, and premature termination of NPR2 which is predicted to trigger nonsense-mediated mRNA decay given its position upstream of multiple downstream exon-exon junctions. Heterozygous family members expressed both normal and aberrant transcripts, whereas the affected individual showed exclusive expression of the aberrant isoform, consistent with a dosage-dependent loss-of-function mechanism. These findings establish aberrant splicing induced by a synonymous variant as a disease-causing mechanism affecting a core developmental signaling pathway. Our study highlights the importance of transcript-level functional analysis in the interpretation of rare variants and underscores the central role of precise RNA processing in skeletal development and human disease.
N. B. Acikgoz, Hasan Basri Kılıç, Gizem Urel Demir et al.· Differentiation; research in...· 0 citations
Rare variants in SETD1A, encoding a histone H3K4 methyltransferase, are among the strongest genetic risk factors for schizophrenia. Exome sequencing (n=3,736) revealed a population-enriched SETD1A missense variant (P596L) in the Lancaster Old Order Amish founder population, presenting a unique opportunity to elucidate variant-specific, multi-scale mechanisms. Psychiatric and cognitive phenotyping revealed nearly two-fold increased risk for bipolar disorder, accompanied by allele dose-dependent cognitive deficits in adulthood. Induced pluripotent stem cells (iPSCs) from homozygous carriers exhibited signatures of SETD1A hypofunction, including reduced proliferation and heightened susceptibility to replication stress and DNA double-strand breaks. During forebrain-directed differentiation, homozygous mutant cells displayed premature activation of neurodevelopmental transcriptional programs but impaired neural rosette formation, reduced neurite complexity, and early progenitor senescence. Multi-omic profiling revealed dysregulation of gene modules converging on replication stress pathways and neuronal regulatory networks enriched for autism and psychiatric risk genes. Pharmacologic inhibition of the H3K4 demethylase KDM5 partially rescued replication stress and neurite deficits, supporting an epigenetic mechanism and suggesting therapeutic tractability. Together, these findings link a population-enriched missense variant to disrupted chromatin regulation, genome stability, and neurodevelopmental timing, bridging human genetic risk with cellular pathophysiology.
R. Lease, Rediet T. Oshone, Yumna Ahmed et al.· Research Square· 0 citations
Using patient-derived iPSCs and multi-omics profiling, it is demonstrated that early-truncating variants cause loss-of-function via nonsense-mediated decay (NMD), while late-truncating variants that escape NMD cause gain-of-function effects.
A. Nava, Y. Pérez-Rodríguez, T. Hsieh et al.· medRxiv· 0 citations
Alternative splicing coupled to nonsense-mediated mRNA decay (AS-NMD) evolved as a master regulator of gene expression. Dysregulated AS-NMD has been identified as the root of many human maladies, from developmental defects to deadly cancer. Poison exons (PEs) are highly conserved alternative exons that contain a premature termination codon and elicit AS-NMD when included in a transcript. Cancer cells often exploit the inclusion of PEs to downregulate tumor suppressors or the exclusion of PEs to upregulate oncoproteins. Therefore, PEs have drawn significant attention as a novel therapeutic avenue for cancer and other diseases. Here, we examine a therapeutic proof-of-concept for manipulating PE-mediated oncogenic AS-NMD using a CRISPR-based approach. Using paired guide RNA, we successfully deleted a PE of a tumor suppressor (EZH2) from the genome of SRSF2-mutated leukemia. This editing resulted in EZH2 mRNAs without a PE, escaped AS-NMD, and restored the protein expression. This subsequently reinstated H3K27 histone methylation and rescued defective chromatin regulation associated with impaired hematopoietic stem cell differentiation. Finally, we showed the preferential advantages of CRISPR over the antisense technology we recently developed targeting the PE of EZH2. Therefore, the CRISPR strategy shows compelling evidence as a therapeutic approach targeting PE in cancer and other human diseases.
Summary One of the seminal discoveries from genetic studies of autism spectrum disorder and related neurodevelopmental disorders (NDDs) has been that loss-of-function (LoF) mutations in genes that impact transcriptional regulation confer substantial liability to NDDs. Haploinsufficiency of the epigenetic regulator POGZ represents one of the strongest such associations; however, little is known about the mechanisms by which POGZ LoF alters early neuronal development. Here, we created an allelic series of CRISPR-engineered human induced pluripotent stem cell (hiPSC) clones harboring mono- and bi-allelic POGZ deletions. In hiPSC-derived neural stem cells (NSCs) and Neurogenin-2-induced neurons (iNs), POGZ LoF altered the expression of genes associated with synaptic and intracellular signaling and extracellular matrix organization. Our multiomics profiling also showed altered footprinting of critical transcription factors (e.g., activator protein 1 complexes) that were enriched at promoters of differentially expressed genes associated with synaptic function. To further interrogate the shared molecular changes associated with NDDs, we compared our results to deletions of the transcription factor MEF2C and the sodium channel gene SCN2A that we generated in these same isogenic iNs. These analyses revealed strong enrichment of extracellular matrix and intracellular signaling disruption associated with POGZ and MEF2C deletion, whereas POGZ and SCN2A haploinsufficiency exhibited shared transcriptional effects on gene modules enriched for NDD-associated genes with opposing regulatory effects. Notably, we also observed alterations to synaptic firing rate and neurite extension with bi-allelic deletions. These shared molecular consequences suggest key points of convergence that connect gene regulation to neuronal function in the etiology of neurodevelopmental pathologies.
M. Moyses-Oliveira, Yating Liu, Serkan Erdin et al.· HGG advances· 1 citation