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.
Abstract
Chromatin remodelers are increasingly recognized as key contributors to neurodevelopmental disorders, usually through de novo dominant variants. Mortality Factor 4 Like 1 (MORF4L1) is recognized for its role in chromatin organization and transcriptional regulation. We identified a consanguineous Middle Eastern family in which a neurodevelopmental disorder with a distinctive dysmorphic phenotype segregated with a homozygous variant in MORF4L1. The autosomal recessive inheritance sets it apart from most other chromatin remodeling disorders. Consistent with its epigenetic role, we also observed a disruption in DNA methylation patterns in patient blood compared to controls. We modelled both the patient variant and a predicted loss of function variant in zebrafish and found that biallelic loss of function fish exhibited growth restriction and craniofacial/skeletal defects consistent with the patients' phenotypes. Surprisingly, larvae with knock in of the mutant morf4l1KI/KI appeared phenotypically normal with respect to jaw development suggesting that the impact of the variant is less severe in zebrafish. 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. The tolerance of zebrafish to the human variant provides a cautionary tale about overreliance on animal models as a screening tool for variant classification.
Overall, EIF1AX is a novel gene for which loss-of-function variants appear to produce syndromic neurodevelopmental disorders in males, and its pathogenicity was evaluated using a molecular dynamic simulation and transgenic Drosophila models.
Kazuyuki Komatsu, Atsushi Sugie, Yohei Nitta et al.· European Journal of Human Ge...· 1 citation
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
Disruption of chromatin organization is a common pathogenic mechanism in neurodevelopmental disorders, yet how changes in 3D genome architecture relate to transcriptional dysfunction in the developing brain remains unclear. POGZ, a transposase-derived chromatin regulator mutated in White–Sutton syndrome and autism spectrum disorder (ASD) has been linked to both heterochromatin and accessible regulatory DNA, but its in vivo function in the brain is unresolved. Using immunoprecipitation–mass spectrometry in embryonic day 13.5 (E13.5) mouse cortex, we identify the H3K9 methyltransferases G9a/GLP as principal POGZ interactors, placing POGZ within the core H3K9 methylation machinery in vivo. We find that POGZ loss in embryonic mouse cortex drives bidirectional, megabase-scale redistribution of H3K9me3, with ectopic losses and gains over discrete neuronal gene loci. In developing Pogz-/-cortex, regions with H3K9me3 gains are repositioned to the nuclear lamina and exhibit strengthened B-compartment scores by Micro-C, locus-restricted erosion of TAD architecture, weakened boundary insulation, and reduced CTCF occupancy. Within these domains, nascent RNA synthesis at neuronal genes is markedly diminished. 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. Major Points POGZ interacts with the G9a/GLP H3K9 methyltransferase complex in the developing mouse cortex, identified by IP–mass spectrometry. Loss of POGZ drives megabase-scale redistribution of H3K9me3 and increases compartmentalization and nuclear-lamina association of neurodevelopmental gene loci, shown by ChIP-seq, Micro-C, and DNA FISH. Micro-C reveals disrupted TAD architecture at specific neuronal gene loci in Pogz⁻/⁻.PRO-seq shows POGZ is required to maintain nascent RNA synthesis of neurodevelopmental genes within disrupted chromatin domains.
N. Mariano, Katerina J. Williams, Katie Munechika et al.· bioRxiv· 0 citations
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
The findings implicate DCLK1 in a previously unrecognized progressive neurodevelopmental disorder and demonstrate the power of integrative cross-species functional genomics in resolving ultra-rare disease variants.
Stephen C. Pak, David Butler, Wei-Xi Yuan et al.· Research Square· 0 citations
Pathogenic rewiring of the three-dimensional (3D) genome architecture is increasingly being identified as the cause of genetic diseases, but recognizing the cis-regulatory effects of structural variation remains a challenge. The Xq27.1 region contains a quasi-palindrome identified as a pleiotropic hotspot for disease-causing interchromosomal insertions. In a large Danish family affected by X-linked recessive complex spastic paraplegia, we identified the segregation of a 149-kb interchromosomal insertion at Xq27.1 originating from 4q24. To understand the disease mechanism, we generated induced pluripotent stem cells (iPSCs) from affected individuals. Using CRISPR perturbation and neural differentiation experiments combined with high-throughput chromatin conformation capture (Hi-C) and transcriptomic analyses, we identify a 3D regulatory rewiring of SOX3 and transcriptional dysregulation of SOX3 targets in iPSC-derived neurons. Consistent with regulatory partitioning of the SOX3 topologically associating domain (TAD) in affected individuals, our experiments show that upstream cis-regulatory elements have a reduced ability to activate SOX3 expression and that the observed dysregulation depends on CTCF-binding sites within the insertion. This work provides mechanistic evidence that a position effect at the SOX3 locus can cause hereditary spastic paraplegia.
T. Terkelsen, V. Yumiceba, Joshua Kim et al.· American Journal of Human Ge...· 0 citations