Biology subjects

Yoon, W. H.

Publications and source records attributed to Yoon, W. H..

5 recordsLinked to original sources

bioRxiv2026

Pathogenic mutations in ATAD3A cause dysregulation of RagC/D-TFEB axis and disrupt lysosomal homeostasis

We previously discovered that a de novo variant p.R528W in ATAD3A, encoding a mitochondrial membrane-anchored protein, causes a human neurological syndrome. While ATAD3A mutations induce aberrant lysosomal expansion accompanied by undigested material in the lysosomes, how mutant ATAD3A disrupts lysosomal homeostasis and whether this contributes to neurodevelopmental defects remain unknown. Here we show that pathogenic ATAD3A p.R528W expression disrupts the mTORC1-TFEB axis as revealed by dysregulation of mTORC1 substrate phosphorylation, TFEB nuclear localization, and CLEAR gene activation associated with lysosomal biogenesis. ATAD3A binds to lysosome-localized Rag C/D GTPases, which constitute a platform for TFEB recruitment, with pathogenic variants increasing this association and thereby decreasing lysosomal localization of Rag GTPases. Importantly, overexpression of RagC or RagD restores TFEB phosphorylation in human cells expressing p.R528W, and RagC-D overexpression or TFEB/Mitf knockdown rescues lysosomal expansion and neurodevelopmental defects in Drosophila. These data indicate that disrupted Rag GTPase recruitment to lysosomes and subsequent aberrant TFEB/Mitf activation contribute to neurodevelopmental and lysosomal phenotypes caused by pathogenic mutations in ATAD3A. Our work reveals a novel role for the mitochondrial resident protein ATAD3A in modulating lysosomal homeostasis through regulation of the mTORC1-TFEB axis, providing a mechanistic link between impaired mitochondrial and lysosomal homeostasis in a neurodevelopmental disorder.

genetics↗

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↗

bioRxiv2022

Clonally selected lines after CRISPR/Cas editing are not isogenic

The CRISPR-Cas9 system has enabled researchers to precisely modify/edit the sequence of a genome. A typical editing experiment consists of two steps: (i) editing cultured cells; (ii) cell cloning and selection of clones with and without intended edit, presumed to be isogenic. The application of CRISPR-Cas9 system may result in off-target edits, while cloning will reveal culture-acquired mutations. We analyzed the extent of the former and the latter by whole genome sequencing in three experiments involving separate genomic loci and conducted by three independent laboratories. In all experiments we hardly found any off-target edits, while detecting hundreds to thousands of single nucleotide mutations unique to each clone after relatively short culture of 10-20 passages. Notably, clones also differed in copy number alterations that were several kb to several mb in size and represented the largest source of genomic divergence among clones. We suggest that screening of clones for mutations and copy number alterations acquired in culture is a necessary step to allow correct interpretation of DNA editing experiments. Furthermore, since culture associated mutations are inevitable, we propose that experiments involving derivation of clonal lines should compare a mix of multiple unedited lines and a mix of multiple edited lines.

genomics↗

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↗