Human molecular genetics最新文献

Antisense oligonucleotides (AONs) are small pieces of chemically modified DNA or RNA that bind to RNA in a sequence-specific manner based on Watson-Crick base-pairing. Splice-switching AONs are designed to modulate pre-mRNA splicing, thereby for instance restoring protein expression or modifying the eventual protein to restore its function or reduce its toxicity. Given the current lack of in silico methods that adequately predict off-target splicing events, assessment of off-target effects of AONs in human cells using RNAseq could be a promising approach. The identification and prioritization of potential off-target effects for validation and further investigation into the biological relevance would contribute to the development of safe and effective AONs. In this study, we used three different splice-switching AONs targeting three different human transcripts to study their transcriptome-wide, hybridization-dependent off-target effects with short read RNAseq. Using the computational tools rMATS and Whippet, we identified differential splicing events of which only a minority could be explained by hybridization, illustrating the difficulty of predicting off-target effects based on sequence homology. The main splicing events could all be validated with RT-PCR. Furthermore, from the three AONs studied, one AON induced considerably more changes in gene expression and splicing compared to the two other AONs assessed, which was confirmed in a validation experiment. Our study demonstrates that interpretation of short read RNAseq data to determine off-target effects is challenging. Nonetheless, valuable results can be obtained as it allows the comparison of toxicity between different AONs within an experiment and identification of AON-specific off-target profiles.

Congenital myasthenic syndromes (CMS) arise from mutations to proteins involved in neuromuscular junction (NMJ) development, maintenance, and neurotransmission. To date, mutations in more than 35 genes have been linked to CMS development. Glutamine fructose-6-phosphate transaminase 1 (GFPT1/Gfpt1) serves as the rate-limiting enzyme of the hexosamine biosynthetic pathway (HBP), producing the byproduct (UDP-GlcNAc) necessary for protein glycosylation. Gfpt1-deficient models have impaired protein glycosylation, impacting key proteins at the NMJ. The Leloir pathway is a galactose metabolizing pathway which produces UDP-GalNAc as its final product. The enzyme UDP-GalNAc Epimerase (GALE) can also convert excess UDP-GalNAc into UDP-GlcNAc, the byproduct of the HBP. We hypothesized that treatment with galactose both in vitro and in vivo in Gfpt1-deficient models would rescue impaired protein O-GlcNAcylation and reverse the glycosylation status of key NMJ-associated proteins. We show that galactose treatment in vitro activated the Leloir pathway and rescued protein O-GlcNAcylation in Gfpt1-deficient C2C12 myoblasts. In addition, we demonstrated that galactose therapy rescued neuromuscular deficits, improved muscle fatigue and restored NMJ morphology in a skeletal muscle-specific Gfpt1 knockout mouse model. Lastly, we showed that galactose treatment rescued protein O-GlcNAcylation in skeletal muscle, preserving the glycosylation status of the delta (δ) subunit of the acetylcholine receptor (AChRδ). Taken together, we suggest that galactose supplementation can be further explored as a therapy for GFPT1-CMS patients.

β-propeller protein-associated neurodegeneration (BPAN) is characterized by global developmental delay, intellectual disability, and epileptic encephalopathies in infancy or early childhood caused by WDR45/WIPI4 gene mutations. WDR45 depletion disrupted autophagy, leading to iron accumulation in the brain and contributing to neuronal apoptosis. The impact on neuron performance remains unknown. Our previous study established the iPSC cell line derived from a girl patient with a de novo variant c.344 + 5G > T in WDR45 (FDHPIi001). This study demonstrated that this intron 6 mutation impairs RNA splicing, resulting in a 28 bp insertion and nonsense-mediated mRNA decay (NMD) of truncated WDR45. Upon differentiating the iPSCs into dopaminergic neurons, we observed significantly shorter neuronal axons using high-intensity imaging analysis. Additionally, there was significant ferritin accumulation in the induced neurons but not in the iPSCs from the same patient. This research has elucidated the pathogenicity of a non-canonical splice site mutation in WDR45 and has provided deeper insights into the pathologies of neurodegenerative diseases caused by WDR45 defects.

Co-occurrence of double heterozygosity in TARDBP and C9ORF72 is exceedingly rare in amyotrophic lateral sclerosis. While TARDBP mutations and C9ORF72 hexanucleotide repeat expansions have each been independently implicated in disease pathogenesis, their combined effect on disease progression and neuropathology remains unclear. We present the first study documenting a patient harboring both a TARDBP mutation and a C9ORF72 expansion, with comprehensive postmortem data available, to elucidate any additive or synergistic effects on disease course and pathological burden. Detailed clinical assessments tracked the patient's progression, and neuropathological examination was performed postmortem. The presence and extent of TDP-43 pathology and other hallmark features were evaluated and compared to known patterns in carriers of isolated C9ORF72 mutations. The patient's clinical trajectory and pathological findings did not show evidence of a more aggressive disease course or heightened pathological burden attributable to the additional TARDBP mutation. Instead, the disease manifested in a manner consistent with other C9ORF72 carriers, suggesting that double heterozygosity do not necessarily exacerbate ALS pathology.

Leber's hereditary optic neuropathy (LHON) is characterized by painless and rapidly progressive central vision loss, caused by various mutations in mitochondrial DNA, leading to a high genetic and phenotypic heterogeneity. Currently, the only approved therapy is idebenone, a CoQ10 synthetic analogue, that improved visual acuity in some LHON patients; however, results are highly variable due its dependency on functional NAD(P)H oxidoreductase I (NQO1) protein levels, thus limiting broader applicability. Targeting the biochemical respiratory chain defect and mitigating reactive oxygen species emission using alternative treatments which act independent of NQO1 protein content, represent a promising therapeutic strategy for all LHON patients. Here, we first characterized mitochondrial biology of three distinct LHON mutations in patient-derived fibroblasts and evaluated the effects of a nutraceutical combination treatment in addressing these shared pathophysiological mechanisms. We identified a range of mitochondrial characteristics common among various LHON mutations, including higher ROS levels, altered autophagy programming, and reduced mitochondrial bioenergetics. Repeated antioxidant and creatine-based treatment (ACT) conferred a favorable stress-resistant phenotype in LHON cells, which was similar to, and in some cases superior to, the effects observed with idebenone treatment, irrespective of NQO1 protein expression. This phenotype was associated with enhanced mitochondrial biology, as evidenced by reduced reactive oxygen species levels, increased cellular respiration, and correction of autophagic flux. Overall, our findings reveal both common and divergent mitochondrial phenotypes among LHON-related mutations and highlight the potential of accessible multi-ingredient nutraceutical interventions that could benefit all LHON patients.

A de novo mutation in the transcription factor Nucleus accumbens associated protein 1 (NACC1) gene (c.892C > T p.R298W) causes a rare, severe neurodevelopmental disorder which manifests postnatally. Genome editing was used to generate human isogenic ESCs (control, mutant heterozygote and homozygote lines) which were differentiated to cortical neurons. Mutant neurons expressed higher levels of NACC1 protein by western blot. RNAseq, GO term and SynGO analysis revealed altered expression of transcripts involved with pre- and postsynaptic signaling, neurotransmission, extracellular matrix, and adhesion. Western blot revealed increased expression of the presynaptic proteins SNAP25 and VAMP2 and the postsynaptic protein SYNGAP1. A functional assay showed increased adhesion of neural stem cells to collagen 1 and 4. The mutation also changed levels of transcripts measured by qPCR involved with dorsal ventral patterning to favor a ventral signature. These results suggest that the NACC1 R298W mutation causes molecular changes in an embryonic cell model that may impact postnatal development of cortical neurons.

Ras-GTPase-activating protein-binding protein 1 (G3BP1) is a core component and key regulatory switch of stress granules. It influences cell proliferation, differentiation, apoptosis, and RNA metabolism and is implicated in the pathogenesis of various diseases, including neurodegenerative disorders and myocardial hypertrophy, as well as in the regulation of innate immune responses. G3BP1 also plays a crucial role in the proliferation, invasion, and metastasis of cancer cells. However, a comprehensive pan-cancer analysis investigating its roles in diagnosis, prognosis, and immunological prediction has not yet been conducted. In this study, we integrated multiple databases to explore the potential carcinogenic functions of G3BP1. We analysed its expression patterns, prognostic relevance, immune cell infiltration, gene mutations, DNA methylation levels across various tumours, and functional states at the single-cell level and performed functional enrichment analysis of G3BP1-associated genes. Additionally, we examined G3BP1's interactions with chemicals and other genes. Our findings showed that G3BP1 is highly expressed in most cancers and is associated with poor patient prognosis. Moreover, G3BP1 demonstrated early diagnostic value in 10 tumour types and showed positive and negative correlations with prognosis depending on the tumour. Regarding tumour immunity, high G3BP1 expression was linked to an immunosuppressive tumour microenvironment and indicators of immunotherapy response. G3BP1 expression was negatively correlated with the infiltration of most immune cells but showed significant positive correlations with T helper cells and central memory T cells. Furthermore, we validated the overexpression of G3BP1 in cancer cells through in vitro experiments. Our study suggests that G3BP1 may serve as a diagnostic, prognostic, and immune-related biomarker in various cancers and could represent a potential target for tumour immunotherapy.

Circular RNAs (circRNAs) are emerging as important regulatory molecules in both host and viral systems, acting as microRNA sponges, protein decoys or scaffolds, and templates for protein translation. Host-derived circRNAs are increasingly recognized for their roles in immune responses, while virus-encoded circRNAs, especially those from DNA viruses, have been shown to modulate host cellular machinery to favor viral replication and immune evasion. Recently, RNA virus-encoded circRNAs were also discovered, but evidence suggests that they might be generated using a different mechanism compared to the circRNAs produced from the host and DNA viruses. This review highlights recent advances in our understanding of both host and virus-derived circRNAs, with a focus on their biological roles and contributions to pathogenesis. Furthermore, we discuss the potential of circRNAs as biomarkers and their application as therapeutic targets or scaffolds for RNA-based therapies. Understanding the roles of circRNAs in host-virus interactions offers novel insights into RNA biology and opens new avenues for therapeutic strategies against viral diseases and associated cancers.

The stimulator of interferon gene (STING) is an important innate immune mediator of the cytoplasmic DNA sensing pathway. As a mediator known for its role in the immune response to infections, STING is also surprisingly at the center of a variety of non-infectious human diseases, including cancer, autoimmune diseases and neurodegenerative diseases. Recent studies have shown that STING has many signaling activities, including type I interferon (IFN-I) and other IFN-independent activities, many of which are poorly understood. STING also has the unique property of being continuous transported from the ER to the Golgi then to the lysosome. Mutations of STING or trafficking cofactors are associated with human diseases affecting multiple immune and non-immune organs. Here, we review recent advances in STING trafficking and signaling mechanisms based in part on studies of STING-associated monogenic inborn error diseases.

Neurodegenerative disorders (NDDs), characterized by a progressive loss of neurons and cognitive function, are a severe burden to human health and mental fitness worldwide. A hallmark of NDDs such as Alzheimer's disease, Huntington's disease, Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS) and prion diseases is disturbed cellular proteostasis, resulting in pathogenic deposition of aggregated protein species. Autophagy is a major cellular process maintaining proteostasis and integral to innate immune defenses that mediates lysosomal protein turnover. Defects in autophagy are thus frequently associated with NDDs. In this review, we discuss the interplay between NDDs associated proteins and autophagy and provide an overview over recent discoveries in inborn errors in canonical autophagy proteins that are associated with NDDs. While mutations in autophagy receptors seems to be associated mainly with the development of ALS, errors in mitophagy are mainly found to promote PD. Finally, we argue whether autophagy may impact progress and onset of the disease, as well as the potential of targeting autophagy as a therapeutic approach. Concludingly, understanding disorders due to inborn errors in autophagy-"autophagopathies"-will help to unravel underlying NDD pathomechanisms and provide unique insights into the neuroprotective role of autophagy, thus potentially paving the way for novel therapeutic interventions.