Hyperammonemia (HA) is a life-threatening metabolic emergency with diverse genetic etiologies. While classical causes include the urea-cycle and organic acid disorders, the complete mutational spectrum remains incompletely defined. This study aimed to identify the underlying molecular diagnoses in pediatric patients presenting with hyperammonemia as a prominent metabolic manifestation, using whole-exome sequencing (WES). Five pediatric patients with hyperammonemia were retrospectively analyzed using WES followed by Sanger sequencing. Variants were classified according to the ACMG guidelines, and clinical data were reviewed to establish genotype–phenotype correlations. WES identified variants in five genes: FBXL4, GLDC, FUCA1, BSND, and COL6A2. Three novel variants, FBXL4 c.1702G>A; p. (Gly568Arg), BSND c.85G>T; p. (Asp29Tyr), and COL6A2 c.1999C>T; p. (His667Tyr), were identified. The affected genes were related to mitochondrial function, glycine cleavage, lysosomal degradation, renal tubular transport, and extracellular matrix organization. All variants were biallelic or homozygous and were confirmed by segregation analysis. This study highlights the diagnostic power of WES in identifying unexpected molecular causes of pediatric hyperammonemia and expanding the spectrum of implicated genes. The identification of these three novel variants emphasizes the genetic heterogeneity of HA and supports early genomic testing in patients with unexplained or atypical biochemical profiles.
Off-target activity remains a significant challenge in the clinical development of CRISPR-based therapies. As programmable nucleases advance from experimental tools toward approved medicines, the ability to predict, detect, and mitigate unintended genomic editing events has acquired direct translational importance. This review examines the molecular basis of off-target cleavage across three nuclease platforms—zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and CRISPR-Cas systems—with emphasis on mechanistic and structural factors that govern mismatch tolerance, including PAM sampling, seed region thermodynamics, and chromatin accessibility. In silico prediction methods are surveyed from early alignment-based approaches through feature-engineered machine learning models to more recent deep learning architectures, with critical attention to training data limitations, cross-platform generalisability, and the distinction between exhaustive genomic search tools and predictive scoring models. The experimental detection landscape is reviewed with expanded coverage of established and newer unbiased genome-wide assays, including their biological principles, sensitivity characteristics, and suitability for structural variant detection. Particular attention is given to next-generation editing modalities—base editors, prime editors, and Cas12/Cas13 systems—and to epigenome editing approaches using non-cleaving CRISPR platforms, which avoid double-strand breaks but retain specificity concerns. The review also addresses delivery-related off-target risks, including tissue-level biodistribution and germline exposure, alongside mitigation strategies. It closes by outlining directions for improving standardisation, expanding structural variation surveillance, and extending safety characterisation to the full diversity of editing platforms now approaching clinical use.
Spermidine synthase (SPDS) is a key enzyme in polyamine biosynthesis, a metabolic pathway that plays a critical regulatory role in pathogen–host interactions. In this study, we characterized spermidine synthase in Verticillium dahliae (VdSPDS) by generating gene knockout and complementation mutants to elucidate its role in stress responses and pathogenicity. The spermidine synthase deletion mutant (ΔVdSPDS) completely lost the ability to undergo vegetative growth and conidiation. These defects were restored by exogenous spermidine (Spd) supplementation. Deletion of VdSPDS also resulted in sparse mycelial growth and increased sensitivity to osmotic, membrane, cell wall, oxidative, and nitrosative stresses. Moreover, the ΔVdSPDS mutant exhibited reduced cellophane penetration and impaired production of cell wall–degrading enzymes. Loss of VdSPDS significantly decreased pathogenicity toward cotton. Collectively, these results demonstrate that VdSPDS is essential for fungal growth, development, stress tolerance, and virulence in Verticillium dahliae.
4-methylcyclohexane methanol (MCHM), an alicyclic alcohol, is a synthetic hydrotrope that induces a starvation response by upregulation of biosynthetic pathways despite the availability of nutrients. To investigate how Saccharomyces cerevisiae adapts its metabolism to tolerate MCHM, we evolved MCHM-resistant strains in In-Lab Evolutions (ILEs). We sequenced the parental (YJM789) with long read sequencing and the evolved strain using short sequencing, increasing the quality of the YJM789 genome. We identified thousands of SNPs and indel variants per ILE strain, which was a consistent number between strains that evolved resistance and control strains that remained sensitive. However, one gene, PDR3, was consistently mutated in all resistant strains. Because it controls the pleiotropic drug response, recurrent mutations in PDR3 across resistant strains indicate that it is a key driver of adaptive resistance to MCHM in yeast. While many of the evolved alleles of PDR3 would likely produce functional proteins, a PDR3 knockout in the parent YJM789 strain was sufficient to reproduce resistance to MCHM. We found that the pdr3 resistance is mediated through Med15, a component of the Mediator complex which regulates activation by mediating interactions between transcription factors of RNA Pol II. Pdr3 can homodimerize or dimerize with Pdr1, another transcription factor paralog and loss of Pdr1 also confers MCHM resistance. The pleiotropic drug response (PDR) pathway facilitates the export of amino acid catabolites, and deletion of PDR3 disrupted glutathione metabolism and its intermediates. Thus, mutations in PDR3 represent the first identified mechanism conferring resistance to this novel hydrotropic chemical.
Trichoderma virens, a plant- beneficial fungus, has the ability to produce volatile and non-volatile secondary metabolites that possess antimicrobial properties and contribute to promoting plant growth and development. Gliovirin, viridin, viridiol, and heptelidic acid are among its major non-volatile metabolites. In our previous study, we observed that the deletion of heptelidic acid synthase (has1), a terpene cyclase, abolished the biosynthesis of major non-volatile metabolites, in addition to heptelidic acid. Conversely, deletion of the Tex7 gene, a non-ribosomal peptide synthetase, led to an increase in heptelidic acid production, along with other major secondary metabolites. Additionally, disruption of a GAPDH gene located within a secondary metabolite gene cluster abolished heptelidic acid biosynthesis and, consequently, all major secondary metabolites. These findings suggest that heptelidic acid might act as a microbial hormone that regulates secondary metabolism. To investigate this further, we conducted transcriptomic and metabolomic comparisons between the wild-type strain and a Δhas1 mutant lacking heptelidic acid biosynthesis. Transcriptome analysis revealed a major proportion of transcripts were down regulated. Of them, nine transcripts belonged to gliovirin biosynthesis cluster, 14 for viridin cluster and five for volatile sesquiterpene and heptelidic acid biosynthesis cluster; metabolite analysis couldn't detect any heptelidic acid along with viridin, viridiol and gliovirin in the Δhas1 mutant. Additionally, several other secondary metabolites were significantly down regulated. Our results are indicative of a possibility that heptelidic acid might function as a microbial hormone regulating secondary metabolism in this biotechnologically important fungus.
