Biology subjects

Harel, T.

Publications and source records attributed to Harel, T..

8 recordsLinked to original sources

bioRxiv2026

Allele-specific antisense oligonucleotide treatment rescues atad3-associated phenotype in zebrafish

Pathogenic variants in ATAD3A cause a spectrum of multisystem disorders, with a recurrent dominant-negative variant (c.1582C>T; p.Arg528Trp) associated with neurodevelopmental disease. Given the tolerance of ATAD3A to heterozygous loss of function variants, allele-specific transcript reduction represents a promising therapeutic strategy. We designed and optimized allele-specific antisense oligonucleotides (ASOs) targeting the c.1582C>T transcript and evaluated their efficacy and specificity in affected fibroblasts using allele-specific primers and amplicon-based next generation sequencing. Therapeutic potential was further assessed in vivo in zebrafish embryos expressing human wild-type or mutant ATAD3A transcripts. An optimized gapmer ASO selectively reduced mutant ATAD3A transcripts while relatively sparing the wild-type allele. In addition to RNase H-mediated degradation, the ASO induced exon skipping, leading to degradation of the aberrant transcript without production of a truncated protein. In zebrafish, expression of mutant human ATAD3A in embryos caused developmental abnormalities including reduced eye size, which were robustly rescued by co-injection of the optimized ASO. Our findings provide proof of concept for allele-targeted ASO therapy for dominant-negative ATAD3A variants. This work highlights the therapeutic potential of ASOs for rare dominant disorders involving genes tolerant to heterozygous loss-of-function, and establishes zebrafish as a versatile platform for in vivo ASO optimization.

genetics↗

bioRxiv2025

Allele-specific correction of ATAD3A pathogenic variants via template-free CRISPR-Cas9 editing and gene conversion

Gene conversion is a specific form of homologous recombination (HR), involving the unidirectional transfer of genetic information from one genomic locus to another. CRISPR-Cas9-directed double strand breaks (DSBs) induce both interallelic and interlocus gene conversion in early human embryos and somatic cells, suggesting its potential for correcting pathogenic mutations. However, the key features in mitotic gene conversion, including its efficiency, the length of conversion track, and its dependency on specific recombination proteins, remain largely undefined. Here, we show that allele-specific CRISPR-Cas9-induced DSBs, without exogenous donor templates, can efficiently correct a heterozygous pathogenic variant (c.1582C>T; p.Arg528Trp) in the ATAD3A gene of patient-derived induced pluripotent stem cells (iPSCs). Amplicon-based next-generation sequencing (NGS) revealed that approximately 38%[~]53% of edited iPSCs carried two wild-type ATAD3A alleles. Notably, over 99% of the corrected alleles derived from the homologous chromosome, indicating that the repair occurred mainly via interallelic gene conversion. Long-range amplicon nanopore sequencing coupled with haplotype analysis showed that the majority of gene conversion tracts was less than 2 kilobases in length. Whole-genome sequencing of three corrected iPSC clones showed the absence of large deletions or structural rearrangements at the ATAD3A target site. However, one clone carried a heterozygous deletion in ATAD3B locus, suggesting that CRISPR-Cas9 can introduce off-target genomic alterations. Knockdown of key HR proteins, including RAD51, CtIP, and BRCA1/2, significantly reduced the correction efficiency, indicating that the gene conversion relies on a RAD51-dependent HR pathway. Together, our findings provide compelling evidence that template-free CRISPR-Cas9-mediated interallelic gene conversion can be harnessed to correct disease causing variants in human iPSCs.

genomics↗

bioRxiv2025

The Quest for a Universal Parkinson's Transcriptomics Signature is Derailed by Inherent Variability between Patients

ObjectiveTo systematically evaluate the reproducibility and clinical utility of published blood mRNA-based gene signatures for classifying PD from healthy controls, and to uncover the pitfalls that limit their performances. MethodsWe validated the classification performance of 13 gene signatures unique for PD (published 2015-2025) using the Parkinsons Progression Markers Initiative (PPMI) database. We further validated classification performance on data collected in a prospective clinical trial where demographic and clinical parameters were minimized and environmental confounders were strictly controlled. Sources of gene expression variability were studied by mean pairwise distance analysis. ResultsGene overlap between signatures was low (11/411, 2.7%) but statistically significant (P<0.001) and enriched with genes involved in lipid metabolism. The majority (10/13) of these signatures retained statistical significance when tested on the PPMI dataset, but their classification performance was modest (median AUC 59.7%), substantially lower than originally reported. Classification performance improved when comparing GBA1-associated PD to controls (median AUC 65.4%, P=0.006). A prospective trial (16 PD patients, 14 controls) with rigorous environmental standardization did not improve classification accuracy. Inter-individual expression variability is the dominant limiting factor in classifier performance. Variability was not impacted by long-term levodopa therapy. InterpretationWhile blood transcriptomic signatures unique to PD are reproducible and may elucidate PD pathophysiology, their broad clinical utility remains severely limited by inherent inter-individual variability. Future research should prioritize biomarkers of divergence rather than the pursuit of a universal PD signature.

bioinformatics↗

bioRxiv2024

Transcriptome analysis of atad3-null zebrafish embryos elucidates possible disease mechanisms

ATAD3A, a nuclear gene encoding the ATAD3A protein, has diverse roles in mitochondrial processes, encompassing mitochondrial dynamics, mitochondrial DNA maintenance, metabolic pathways and inter-organellar interactions. Pathogenic variants in this gene cause neurological diseases in humans with recognizable genotype-phenotype correlations. To further investigate the gene function and its implication in health and disease, we utilized CRISPR/Cas9 genome editing to generate a knockout (KO) model of the zebrafish ortholog gene, atad3. Analysis of atad3-null zebrafish embryos revealed microcephaly, small eyes, pericardial edema and musculature thinning, closely mirroring with the human rare disease phenotype. Larvae exhibited delayed hatching and embryonic lethality by 13 days post-fertilization (dpf). Locomotor activity, ATP content, mitochondrial content, and mitochondrial activity were all reduced in the mutant embryos. Transcriptome analysis at 3 dpf via RNA-sequencing indicated decline in most mitochondrial pathways, accompanied by a global upregulation of cytosolic tRNA synthetases, presumably secondary to mitochondrial stress and possibly endoplasmic reticulum (ER)-stress. Differential expression of select genes was corroborated in fibroblasts from an affected individual. The atad3-null zebrafish model emerges as a reliable representation of human ATAD3A-associated disorders, with similarities in differentially expressed pathways and processes. Furthermore, our study underscores mitochondrial dysfunction as the primary underlying pathogenic mechanism in ATAD3A-associated disorders and identifies potential readouts for therapeutic studies.

genetics↗

bioRxiv2023

Point mutations in IMPDH2 which cause early-onset neurodevelopmental disorders disrupt enzyme regulation and filament structure

Inosine 5 monophosphate dehydrogenase (IMPDH) is a critical regulatory enzyme in purine nucleotide biosynthesis that is inhibited by the downstream product GTP. Multiple point mutations in the human isoform IMPDH2 have recently been associated with dystonia and other neurodevelopmental disorders, but the effect of the mutations on enzyme function has not been described. Here, we report identification of two additional affected individuals with missense variants in IMPDH2 and show that all of the disease-associated mutations disrupt GTP regulation. Cryo-EM structures of one IMPDH2 mutant suggest this regulatory defect arises from a shift in the conformational equilibrium toward a more active state. This structural and functional analysis provides insight into IMPDH2-associated disease mechanisms that point to potential therapeutic approaches and raises new questions about fundamental aspects of IMPDH regulation. Significance StatementPoint mutations in the human enzyme IMPDH2, a critical regulator of nucleotide biosynthesis, are linked to neurodevelopmental disorders, such as dystonia. Here, we report two additional IMPDH2 point mutants associated with similar disorders. We investigate the effects of each mutation on IMPDH2 structure and function in vitro and find that all mutations are gain of function, preventing allosteric regulation of IMPDH2 activity. We report high resolution structures of one variant and present a structure-based hypothesis for its dysregulation. This work provides a biochemical basis for understanding diseases caused by IMPDH2 mutation and lays a foundation for future therapeutic development.

biochemistry↗

bioRxiv2020

Functional interpretation of ATAD3A variants in neuro-mitochondrial phenotypes

BackgroundThe ATPase family AAA-domain containing protein 3A (ATAD3A) is a nuclear-encoded mitochondrial membrane anchored protein involved in diverse processes including mitochondrial dynamics, mitochondrial DNA organization, and cholesterol metabolism. Biallelic deletions (null), recessive missense variants (hypomorph), and heterozygous missense variants or duplications (antimorph) in ATAD3A lead to neurological syndromes in humans. ObjectiveTo expand the mutational spectrum of ATAD3A variants and to provide functional interpretation of missense alleles in trans to deletion alleles. MethodsExome sequencing was used to identify single nucleotide variants (SNVs) and copy number variants (CNVs) in ATAD3A in individuals with neurological and mitochondrial phenotypes. A Drosophila Atad3A Gal4 trap null allele was generated using CRISPR-Cas9 genome editing technology to aid interpretation of variants. ResultsWe report 13 individuals from 8 unrelated families with biallelic ATAD3A variants. Four of the identified missense variants, p.(Leu77Val), p.(Phe50Leu), p.(Arg170Trp), p.(Gly236Val), were inherited in trans to loss-of-function alleles. A fifth missense variant, p.(Arg327Pro), was homozygous. Affected individuals exhibited findings previously associated with ATAD3A pathogenic variation, including developmental delay, hypotonia, congenital cataracts, hypertrophic cardiomyopathy, and cerebellar atrophy. Drosophila studies indicated that Phe50Leu, Gly236Val, and Arg327Pro are severe loss-of-function alleles leading to early developmental lethality and neurogenesis defects, whereas Leu77Val and Arg170Trp are partial loss of function alleles that cause progressive locomotion defects. Moreover, Leu77Val and Arg170Trp expression leads to an increase in autophagy and mitophagy in adult muscles. ConclusionOur findings expand the allelic spectrum of ATAD3A variants, and exemplify the use of a functional assay in Drosophila to aid variant interpretation.

genetics↗

bioRxiv2020

Mutations in MYLPF cause a novel segmental amyoplasia that manifests as distal arthrogryposis

We identified ten persons in six consanguineous families with Distal Arthrogryposis (DA) who had congenital contractures, scoliosis, and short stature. Exome sequencing revealed that each affected person was homozygous for one of two different rare variants (c.470G>T, p.(Cys157Phe) or c.469T>C, p.(Cys157Arg)) affecting the same residue of myosin light chain, phosphorylatable, fast skeletal muscle (MYLPF). In a seventh family, a c.487G>A, p.(Gly163Ser) variant in MYLPF arose de novo in a father, who transmitted it to his son. In an eighth family comprised of seven individuals with dominantly-inherited DA, a c.98C>T, p.(Ala33Val) variant segregated in all four persons tested. Variants in MYLPF underlie both dominant and recessively inherited DA. Mylpf protein models suggest that the residues associated with dominant DA interact with myosin whereas the residues altered in families with recessive DA only indirectly impair this interaction. Pathological and histological exam of a foot amputated from an affected child revealed complete absence of skeletal muscle (i.e., segmental amyoplasia). To investigate the mechanism for this finding, we generated an animal model for partial MYLPF impairment by knocking out zebrafish mylpfa. The mylpfa mutant had reduced trunk contractile force and complete pectoral fin paralysis, demonstrating that mylpf impairment most severely affects limb movement. mylpfa mutant muscle weakness was most pronounced in an appendicular muscle and was explained by reduced myosin activity and fiber degeneration. Collectively, our findings demonstrate that partial loss of MYLPF function can lead to congenital contractures, likely as a result of degeneration of skeletal muscle in the distal limb.

genetics↗

bioRxiv2018

Phenotypic expansion in DDX3X -- a common cause of intellectual disability in females

De novo variants in DDX3X account for 1-3% of unexplained intellectual disability (ID), one of the most common causes of ID, in females. Forty-seven patients (44 females, 3 males) have been described. We identified 29 additional individuals carrying 27 unique DDX3X variants in the setting of complex clinical presentations including developmental delay or ID. In addition to previously reported manifestations, rare or novel phenotypes were identified including respiratory problems, congenital heart disease, skeletal muscle mitochondrial DNA depletion, and late-onset neurologic decline. Our findings expand the spectrum of DNA variants and phenotypes associated with DDX3X disorders.

genetics↗