Search Results (47)

Epidermolysis Bullosa (EB) comprises a group of inherited blistering disorders caused by pathogenic variants in genes essential for skin and mucosal integrity. Nonsense mutations, which generate premature termination codons (PTCs), result in reduced or absent protein expression and contribute to severe disease phenotypes in EB. Readthrough therapies, which may continue translation past PTCs to restore full-length functional proteins, have emerged as promising approaches. This review summarizes findings from preclinical studies investigating readthrough therapies in EB models, clinical studies demonstrating efficacy in EB patients, and emerging readthrough agents with potential application to EB. Preclinical and clinical studies with gentamicin have demonstrated restored type VII collagen and laminin-332 expression, leading to measurable clinical improvements. Parallel development of novel compounds—including aminoglycoside analogs (e.g., ELX-02), translation termination factor degraders (e.g., CC-90009, SRI-41315, SJ6986), tRNA post-transcriptional inhibitors (e.g., 2,6-diaminopurine, NV848), and nucleoside analogs (e.g., clitocine)—has expanded the therapeutic pipeline. Although challenges remain regarding toxicity, codon specificity, and variable protein restoration thresholds, continued advances in molecular targeting and combination therapies offer the potential to establish readthrough therapies as localized or systemic treatments addressing both cutaneous and extracutaneous disease manifestations in EB. Full article

Osteogenesis imperfecta type VI is a rare autosomal recessive disorder characterized by bone fragility and defective mineralization, caused by pathogenic variants in the SERPINF1 gene. This study aimed to expand the understanding of OI type VI by analyzing clinical, radiological, and molecular findings in four patients from three unrelated families. Genotyping revealed two novel SERPINF1 variants, c.185G>T (p.Gly62Val) and c.992_993insCA (p.Glu331Asnfs), in a compound heterozygous state in one patient, and a known pathogenic variant, c.261_265dup (p.Leu89Argfs26), in a homozygous form in three patients. Clinical manifestations included early-onset fractures, severe skeletal deformities, impaired mobility, and growth failure. Radiological assessments revealed multilevel and multiplanar bone deformities and metaphyseal widening. RNA analysis demonstrated that the c.992_993insCA variant results in a truncated PEDF protein without triggering nonsense-mediated decay. Population screening identified a carrier frequency of 0.0044 for the c.261_265dup variant, suggesting a founder effect in the Tuvinian population. These findings expand the mutational spectrum of the SERPINF1 gene and provide new insights into the phenotypic variability of OI type VI. Our results highlight the importance of genetic screening in isolated populations and emphasize the need for further research to develop more effective therapeutic approaches for patients with limited response to bisphosphonate therapy. Full article

Inherited epidermolysis bullosa (EB) comprises a group of genetic disorders characterized by fragile skin that blisters easily. Targeted therapies for EB necessitate personalized approaches, underscoring the importance of precise diagnostics through genetic analysis and skin biopsy using transmission electron microscopy and/or immunohistochemistry. This study highlights the application of whole-exome sequencing (WES) to identify key pathogenic variants associated with EB. Most identified variants were associated with the recessive form of dystrophic EB, including four novel COL7A1 mutations: p.Leu1488ArgfsTer222, c.7759-3C>G, p.Gln1886Ter, and c.6501+6T>C, as well as recurrent variants p.Lys142Arg and p.Gly2049Glu. Additionally, variants were detected in KRT5 (c.971T>C, p.Val324Ala), associated with EB simplex, and in LAMB3 (c.2500C>T, p.Gln834Ter) in the homozygous state, associated with junctional EB. In silico splice prediction tools suggested disrupted splicing in both cases. One patient received topical gentamicin therapy targeting the nonsense mutation p.Gln1886Ter. These findings underscore the utility of WES in EB diagnostics, broaden the mutation spectrum, and contribute to the understanding of genotype–phenotype correlations in adult patients with EB. Full article

Multiple myeloma (MM) first-line treatment algorithms include immuno-chemotherapy (ICT) induction, high-dose chemotherapy (HDCT) and autologous stem cell transplant (ASCT) consolidation, followed by lenalidomide maintenance. After these initial therapies, most patients suffer a disease relapse and require subsequent treatment lines including ICT, additional HDCT and ASCT, or novel immunotherapies. The presence of somatic mutations in peripheral blood cells has been associated with adverse outcomes in a variety of hematological malignancies. Nonsense and frameshift mutations in the PPM1D gene, a frequent driver alteration in clonal hematopoiesis (CH), lead to the gain-of-function of Wip1 phosphatase, which may impair the p53-dependent G1 checkpoint and promote cell proliferation. Here, we determined the presence of PPM1D gene mutations in peripheral blood cells of 75 subsequent myeloma patients in remission after first or second HDCT/ASCT. The prevalence of truncating PPM1D gene mutations emerged at 1.3% after first HDCT/ASCT, and 7.3% after second HDCT/ASCT, with variant allele frequencies (VAF) of 0.01 to 0.05. Clinical outcomes were inferior in the PPM1D-mutated (PPM1Dmut) subset with median progression-free survival (PFS) of 15 vs. 37 months (p = 0.0002) and median overall survival (OS) of 36 vs. 156 months (p = 0.001) for the PPM1Dmut and PPM1Dwt population, respectively. Our data suggest that the occurrence of PPM1D gene mutations in peripheral blood cells correlates with inferior outcomes after ASCT in patients with multiple myeloma. Full article

Nonsense-mediated mRNA decay (NMD) is a quality control mechanism that degrades mRNAs carrying a premature termination codon. Its inhibition, alone or in combination with other approaches, could be exploited to develop therapies for genetic diseases caused by a nonsense mutation. This, however, requires molecules capable of inhibiting NMD effectively without inducing toxicity. We have built a new screening system and used it to identify and validate two new molecules that can inhibit NMD at least as effectively as cycloheximide, a reference NMD inhibitor molecule. These new NMD inhibitors show no cellular toxicity at tested concentrations and have a working concentration between 6.2 and 12.5 µM. We have further validated this NMD-inhibiting property in a physiopathological model of lung cancer in which the TP53 gene carries a nonsense mutation. These new molecules may potentially be of interest in the development of therapies for genetic diseases caused by a nonsense mutation. Full article

Nonsense mutations cause several genetic diseases such as cystic fibrosis, Duchenne muscular dystrophy, β-thalassemia, and Shwachman–Diamond syndrome. These mutations induce the formation of a premature termination codon (PTC) inside the mRNA sequence, resulting in the synthesis of truncated polypeptides. Nonsense suppression therapy mediated by translational readthrough-inducing drugs (TRIDs) is a promising approach to correct these genetic defects. TRIDs generate a ribosome miscoding of the PTC named “translational readthrough” and restore the synthesis of full-length and potentially functional proteins. The new oxadiazole-core TRIDs NV848, NV914, and NV930 (NV) showed translational readthrough activity in nonsense-related in vitro systems. In this work, the possible off-target effect of NV molecules on natural termination codons (NTCs) was investigated. Two different in vitro approaches were used to assess if the NV molecule treatment induces NTC readthrough: (1) a study of the translational-induced p53 molecular weight and functionality; (2) the evaluation of two housekeeping proteins’ (Cys-C and β2M) molecular weights. Our results showed that the treatment with NV848, NV914, or NV930 did not induce any translation alterations in both experimental systems. The data suggested that NV molecules have a specific action for the PTCs and an undetectable effect on the NTCs. Full article

Nonsense mutations are involved in multiple peripheral neuropathies. These mutations induce the presence of a premature termination codon (PTC) at the mRNA level. As a result, a dysfunctional or truncated protein is synthesized, or even absent linked to nonsense-mediated mRNA degradation (NMD) system activation. Readthrough molecules or NMD inhibitors could be innovative therapies in these hereditary neuropathies, particularly molecules harboring the dual activity as amlexanox. Charcot–Marie–Tooth (CMT) is the most common inherited pathology of the peripheral nervous system, affecting 1 in 2500 people worldwide. Nonsense mutations in the GDAP1 gene have been associated with a severe form of CMT, prompting us to investigate the effect of readthrough and NMD inhibitor molecules. Although not clearly defined, GDAP1 could be involved in mitochondrial functions, such as mitophagy. We focused on the homozygous c.581C>G (p.Ser194*) mutation inducing CMT2H using patient human induced pluripotent stem cell (hiPSC)-derived neuronal cells. Treatment during 20 h with 100 µM of amlexanox on this cell model stabilized GDAP1 mRNAs carrying UGA-PTC and induced a restoration of the mitochondrial morphology. These results highlight the potential of readthrough molecules associated to NMD inhibitors for the treatment of genetic alterations in CMT, opening the way for future investigations and a potential therapy. Full article

Rett syndrome (RTT), a severe X-linked neurodevelopmental disorder, is primarily caused by mutations in the methyl CpG binding protein 2 gene (MECP2). Over 35% RTT patients carry nonsense mutation in MECP2, making it a suitable candidate disease for nonsense suppression therapy. In our previous study, gentamicin was found to induce readthrough of MECP2 nonsense mutations with modest efficiency. Given the recent discovery of readthrough enhancers, CDX compounds, we herein evaluated the potentiation effect of CDX5-1, CDX5-288, and CDX6-180 on gentamicin-mediated readthrough efficiency in transfected HeLa cell lines bearing the four most common MECP2 nonsense mutations. We showed that all three CDX compounds potentiated gentamicin-mediated readthrough and increased full-length MeCP2 protein levels in cells expressing the R168X, R255X, R270X, and R294X nonsense mutations. Among all three CDX compounds, CDX5-288 was the most potent enhancer and enabled the use of reduced doses of gentamicin, thus mitigating the toxicity. Furthermore, we successfully demonstrated the upregulation of full-length Mecp2 protein expression in fibroblasts derived from Mecp2^R255X/Y mice through combinatorial treatment. Taken together, findings demonstrate the feasibility of this combinatorial approach to nonsense suppression therapy for a subset of RTT patients. Full article

Infantile parkinsonism-dystonia due to dopamine transporter deficiency syndrome (DTDS) is an ultrarare childhood movement disorder caused by biallelic loss-of-function mutations in the SLC6A3 gene. Advances in genomic analysis have revealed an evolving spectrum of SLC6A3-related neurological and neuropsychiatric disorders. Since the initial clinical and genetic characterisation of DTDS in 2009, there have been thirty-one published cases with a variety of protein-truncating variants (nonsense variants, splice-site changes, and deletions) and missense changes. Amino acid substitutions result in mutant proteins with impaired dopamine transporter function due to reduced transporter activity, impaired dopamine binding, reduced cell-surface expression, and aberrant posttranslational protein modification with impaired glycosylation. In this review, we provide an overview of the expanding clinical phenotype of DTDS and the precision therapies in development, including pharmacochaperones and gene therapy. Full article

(1) Background: Focal and segmental glomerulosclerosis (FSGS) is a pattern of injury that results from podocyte loss in the setting of a wide variety of injurious mechanisms. These include both acquired and genetic as well as primary and secondary causes, or a combination thereof, without optimal therapy, and a high rate of patients develop end-stage renal disease (ESRD). Genetic studies have helped improve the global understanding of FSGS syndrome; thus, we hypothesize that patients with primary FSGS may have underlying alterations in adhesion molecules or extracellular matrix glycoproteins related to previously unreported mutations that may be studied through next-generation sequencing (NGS). (2) Methods: We developed an NGS panel with 29 genes related to adhesion and extracellular matrix glycoproteins. DNA was extracted from twenty-three FSGS patients diagnosed by renal biopsy; (3) Results: The average number of accumulated variants in FSGS patients was high. We describe the missense variant ITGB3c.1199G>A, which is considered pathogenic; in addition, we discovered the nonsense variant CDH1c.499G>T, which lacks a Reference SNP (rs) Report and is considered likely pathogenic. (4) Conclusions: To the best of our knowledge, this is the first account of a high rate of change in extracellular matrix glycoproteins and adhesion molecules in individuals with adult-onset FSGS. The combined effect of all these variations may result in a genotype that is vulnerable to the pathogenesis of glomerulopathy. Full article

Around 11% of all known gene lesions causing human genetic diseases are nonsense mutations that introduce a premature stop codon (PTC) into the protein-coding gene sequence. Drug-induced PTC readthrough is a promising therapeutic strategy for treating hereditary diseases caused by nonsense mutations. To date, it has been found that more than 50 small-molecular compounds can promote PTC readthrough, known as translational readthrough-inducing drugs (TRIDs), and can be divided into two major categories: aminoglycosides and non-aminoglycosides. This review summarizes the pharmacodynamics and clinical application potential of the main TRIDs discovered so far, especially some newly discovered TRIDs in the past decade. The discovery of these TRIDs brings hope for treating nonsense mutations in various genetic diseases. Further research is still needed to deeply understand the mechanism of eukaryotic cell termination and drug-induced PTC readthrough so that patients can achieve the greatest benefit from the various TRID treatments. Full article

Nonsense mutations trigger premature translation termination and often give rise to prevalent and rare genetic diseases. Consequently, the pharmacological suppression of an unscheduled stop codon represents an attractive treatment option and is of high clinical relevance. At the molecular level, the ability of the ribosome to continue translation past a stop codon is designated stop codon readthrough (SCR). SCR of disease-causing premature termination codons (PTCs) is minimal but small molecule interventions, such as treatment with aminoglycoside antibiotics, can enhance its frequency. In this review, we summarize the current understanding of translation termination (both at PTCs and at cognate stop codons) and highlight recently discovered pathways that influence its fidelity. We describe the mechanisms involved in the recognition and readthrough of PTCs and report on SCR-inducing compounds currently explored in preclinical research and clinical trials. We conclude by reviewing the ongoing attempts of personalized nonsense suppression therapy in different disease contexts, including the genetic skin condition epidermolysis bullosa. Full article

Mucopolysaccharidosis I-Hurler (MPS I-H) is caused by the loss of α-L-iduronidase, a lysosomal enzyme that degrades glycosaminoglycans. Current therapies cannot treat many MPS I-H manifestations. In this study, triamterene, an FDA-approved, antihypertensive diuretic, was found to suppress translation termination at a nonsense mutation associated with MPS I-H. Triamterene rescued enough α-L-iduronidase function to normalize glycosaminoglycan storage in cell and animal models. This new function of triamterene operates through premature termination codon (PTC) dependent mechanisms that are unaffected by epithelial sodium channel activity, the target of triamterene’s diuretic function. Triamterene represents a potential non-invasive treatment for MPS I-H patients carrying a PTC. Full article

Approximately 11% of genetic human diseases are caused by nonsense mutations that introduce a premature termination codon (PTC) into the coding sequence. The PTC results in the production of a potentially harmful shortened polypeptide and activation of a nonsense-mediated decay (NMD) pathway. The NMD pathway reduces the burden of unproductive protein synthesis by lowering the level of PTC mRNA. There is an endogenous rescue mechanism that produces a full-length protein from a PTC mRNA. Nonsense suppression therapies aim to increase readthrough, suppress NMD, or are a combination of both strategies. Therefore, treatment with translational readthrough-inducing drugs (TRIDs) and NMD inhibitors may increase the effectiveness of PTC suppression. Here we discuss the mechanism of PTC readthrough and the development of novel approaches to PTC suppression. We also discuss the toxicity and bioavailability of therapeutics used to stimulate PTC readthrough. Full article

The prognosis of pancreatic ductal adenocarcinoma (PDAC) is exceedingly poor. Although surgical resection is the only curative treatment option, multimodal treatment is of the utmost importance, as only about 20% of tumors are primarily resectable at the time of diagnosis. The choice of chemotherapeutic treatment regimens involving gemcitabine and FOLFIRINOX is currently solely based on the patient’s performance status, but, ideally, it should be based on the tumors’ individual biology. We established two novel patient-derived primary cell lines from surgical PDAC specimens. LuPanc-1 and LuPanc-2 were derived from a pT3, pN1, G2 and a pT3, pN2, G3 tumor, respectively, and the clinical follow-up was fully annotated. STR-genotyping revealed a unique profile for both cell lines. The population doubling time of LuPanc-2 was substantially longer than that of LuPanc-1 (84 vs. 44 h). Both cell lines exhibited a typical epithelial morphology and expressed moderate levels of CK7 and E-cadherin. LuPanc-1, but not LuPanc-2, co-expressed E-cadherin and vimentin at the single-cell level, suggesting a mixed epithelial-mesenchymal differentiation. LuPanc-1 had a missense mutation (p.R282W) and LuPanc-2 had a frameshift deletion (p.P89X) in TP53. BRCA2 was nonsense-mutated (p.Q780*) and CREBBP was missense-mutated (p.P279R) in LuPanc-1. CDKN2A was missense-mutated (p.H83Y) in LuPanc-2. Notably, only LuPanc-2 harbored a partial or complete deletion of DPC4. LuPanc-1 cells exhibited high basal and transforming growth factor (TGF)-β1-induced migratory activity in real-time cell migration assays, while LuPanc-2 was refractory. Both LuPanc-1 and LuPanc-2 cells responded to treatment with TGF-β1 with the activation of SMAD2; however, only LuPanc-1 cells were able to induce TGF-β1 target genes, which is consistent with the absence of DPC4 in LuPanc-2 cells. Both cell lines were able to form spheres in a semi-solid medium and in cell viability assays, LuPanc-1 cells were more sensitive than LuPanc-2 cells to treatment with gemcitabine and FOLFIRINOX. In summary, both patient-derived cell lines show distinct molecular phenotypes reflecting their individual tumor biology, with a unique clinical annotation of the respective patients. These preclinical ex vivo models can be further explored for potential new treatment strategies and might help in developing personalized (targeted) therapy regimens. Full article