Readthrough Activators and Nonsense-Mediated mRNA Decay Inhibitor Molecules: Real Potential in Many Genetic Diseases Harboring Premature Termination Codons.

Nonsense mutations that generate a premature termination codon (PTC) can induce both the accelerated degradation of mutated mRNA compared with the wild type version of the mRNA or the production of a truncated protein. One of the considered therapeutic strategies to bypass PTCs is their "readthrough" based on small-molecule drugs. These molecules promote the incorporation of a near-cognate tRNA at the PTC position through the native polypeptide chain. In this review, we detailed the various existing strategies organized according to pharmacological molecule types through their different mechanisms. The positive results that followed readthrough molecule testing in multiple neuromuscular disorder models indicate the potential of this approach in peripheral neuropathies.

Amlexanox: Readthrough Induction and Nonsense-Mediated mRNA Decay Inhibition in a Charcot-Marie-Tooth Model of hiPSCs-Derived Neuronal Cells Harboring a Nonsense Mutation in GDAP1 Gene.

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.

Pharmaceuticals Promoting Premature Termination Codon Readthrough: Progress in Development.

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.

Emerging Personalized Opportunities for Enhancing Translational Readthrough in Rare Genetic Diseases and Beyond.

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.

Nonsense codons suppression. An acute toxicity study of three optimized TRIDs in murine model, safety and tolerability evaluation.

Stop mutations cause 11% of the genetic diseases, due to the introduction of a premature termination codon (PTC) in the mRNA, followed by the production of a truncated protein. A promising therapeutic approach is the suppression therapy by Translational Readthrough Inducing Drugs (TRIDs), restoring the expression of the protein. Recently, three new TRIDs (NV848, NV914, NV930) have been proposed, and validated by several in vitro assays, for the rescue of the CFTR protein, involved in Cystic Fibrosis disease. In this work, an acute toxicological study for the three TRIDs was conducted in vivo on mice, according to the OECD No.420 guidelines. Animals were divided into groups and treated with a single dose of TRIDs molecules or Ataluren, an FDA-approved TRID molecule, as control. Mice were observed continuously for the first day post-drugs administration and the behavioral changes were recorded. On the 15th day, animals were sacrificed for histological examinations. The results showed that acute administration of 2000 mg/kg of NV914 and Ataluren and 300 mg/kg of NV848 or NV930, did not induce any mortality within 14 days. Moreover, histopathological analysis of treated mice showed no differences when compared to the experimental controls. In summary, our results suggest a good tolerability for the three molecules, and include NV848 and NV930 in a category 4 and NV914 in a category 5 of the Globally Harmonized System (GHS) of Classification and Labeling of Chemicals, classifying these compounds in a low-risk scale for health.

Case Report: Identification Pathogenic Abnormal Splicing of BBS1 Causing Bardet-Biedl Syndrome Type I (BBS1) due to Missense Mutation.

Conventionally, protein features affected by missense mutation was attributed to destroy an important domain with amino acid alternation, and it was difficult to clearly specify the pathogenicity of a novel missense mutation. Nevertheless, the associations between missense mutations and abnormal splicing are nowadays increasingly reported. Rarely, some missense mutations, locating at the non-canonical splicing sites, are observed to damage the splicing process. In this study, a couple has three adverse pregnancy history that the affected fetus presented typical polydactyly, renal abnormalities, and cerebral ventriculomegaly. To identify its genetic etiology, whole-exome sequencing (WES) was performed and a missense mutation c.1339G > A was identified, which was located at the non-canonical splicing sites of the BBS1 gene. Then, reverse transcription polymerase chain reaction was carried out and demonstrated extra 115bp originating from intron 13 cut into cDNA, which generated a predicted premature termination codon (PTC) in the BBS1 protein. Further expression analysis by using real-time reverse-transcribed PCR confirmed the occurrence of nonsense-mediated decay (NMD). Therefore, the pathogenicity of the missense mutation c.1339G > A was explicit and our study helped to extend the spectrum of pathogenic mutations in Bardet-Biedl syndrome type I.

Premature Termination Codon in 5' Region of Desmoplakin and Plakoglobin Genes May Escape Nonsense-Mediated Decay through the Reinitiation of Translation.

Arrhythmogenic cardiomyopathy is a heritable heart disease associated with desmosomal mutations, especially premature termination codon (PTC) variants. It is known that PTC triggers the nonsense-mediated decay (NMD) mechanism. It is also accepted that PTC in the last exon escapes NMD; however, the mechanisms involving NMD escaping in 5'-PTC, such as reinitiation of translation, are less known. The main objective of the present study is to evaluate the likelihood that desmosomal genes carrying 5'-PTC will trigger reinitiation. HL1 cell lines were edited by CRISPR/Cas9 to generate isogenic clones carrying 5'-PTC for each of the five desmosomal genes. The genomic context of the ATG in-frame in the 5' region of desmosomal genes was evaluated by in silico predictions. The expression levels of the edited genes were assessed by Western blot and real-time PCR. Our results indicate that the 5'-PTC in PKP2, DSG2 and DSC2 acts as a null allele with no expression, whereas in the DSP and JUP gene, N-truncated protein is expressed. In concordance with this, the genomic context of the 5'-region of DSP and JUP presents an ATG in-frame with an optimal context for the reinitiation of translation. Thus, 5'-PTC triggers NMD in the PKP2, DSG2* and DSC2 genes, whereas it may escape NMD through the reinitiation of the translation in DSP and JUP genes, with no major effects on ACM-related gene expression.

Exploring the Diverse Functional and Regulatory Consequences of Alternative Splicing in Development and Disease.

Alternative splicing is a fundamental mechanism of eukaryotic RNA regulation that increases the transcriptomic and proteomic complexity within an organism. Moreover, alternative splicing provides a framework for generating unique yet complex tissue- and cell type-specific gene expression profiles, despite using a limited number of genes. Recent efforts to understand the negative consequences of aberrant splicing have increased our understanding of developmental and neurodegenerative diseases such as spinal muscular atrophy, frontotemporal dementia and Parkinsonism linked to chromosome 17, myotonic dystrophy, and amyotrophic lateral sclerosis. Moreover, these studies have led to the development of innovative therapeutic treatments for diseases caused by aberrant splicing, also known as spliceopathies. Despite this, a paucity of information exists on the physiological roles and specific functions of distinct transcript spliceforms for a given gene. Here, we will highlight work that has specifically explored the distinct functions of protein-coding spliceforms during development. Moreover, we will discuss the use of alternative splicing of noncoding exons to regulate the stability and localization of RNA transcripts.

mRNA Analysis of Frameshift Mutations with Stop Codon in the Last Exon: The Case of Hemoglobins Campania [α1 cod95 (-C)] and Sciacca [α1 cod109 (-C)].

An insertion or deletion of a nucleotide (nt) in the penultimate or the last exon can result in a frameshift and premature termination codon (PTC), giving rise to an unstable protein variant, showing a dominant phenotype. We described two α-globin mutants created by the deletion of a nucleotide in the penultimate or the last exon of the α1-globin gene: the Hb Campania or α1 cod95 (-C), causing a frameshift resulting in a PTC at codon 102, and the Hb Sciacca or α1 cod109 (-C), causing a frameshift and formation of a PTC at codon 133. The carriers showed α-thalassemia alterations (mild microcytosis with normal Hb A2) and lacked hemoglobin variants. The 3D model indicated the α-chain variants' instability, due to the severe structural alterations with impairment of the chaperone alpha-hemoglobin stabilizing protein (AHSP) interaction. The qualitative and semiquantitative analyses of the α1mRNA from the reticulocytes of carriers highlighted a reduction in the variant cDNAs that constituted 34% (Hb Campania) and 15% (Hb Sciacca) of the total α1-globin cDNA, respectively. We developed a workflow for the in silico analysis of mechanisms triggering no-go decay, and its results suggested that the reduction in the variant mRNA was likely due to no-go decay caused by the presence of a rare triplet, and, in the case of Hb Sciacca, also by the mRNA's secondary structure variation. It would be interesting to correlate the phenotype with the quantity of other frameshift mRNA variants, but very few data concerning α- and ß-globin variants are available.

Amber codon is genetically unstable in generation of premature termination codon (PTC)-harbouring Foot-and-mouth disease virus (FMDV) via genetic code expansion.

The foot-and-mouth disease virus (FMDV) is the causative agent of FMD, a highly infectious and devastating viral disease of domestic and wild cloven-hoofed animals. FMD affects livestock and animal products' national and international trade, causing severe economic losses and social consequences. Currently, inactivated vaccines play a vital role in FMD control, but they have several limitations. The genetic code expansion technology provides powerful strategies for generating premature termination codon (PTC)-harbouring virus as a live but replication-incompetent viral vaccine. However, this technology has not been explored for the design and development of new FMD vaccines. In this study, we first expanded the genetic code of the FMDV genome via a transgenic cell line containing an orthogonal translation machinery. We demonstrated that the transgenic cells stably integrated the orthogonal pyltRNA/pylRS pair into the genome and enabled efficient, homogeneous incorporation of unnatural amino acids into target proteins in mammalian cells. Next, we constructed 129 single-PTC FMDV mutants and four dual-PTC FMDV mutants after considering the tolerance, location, and potential functions of those mutated sites. Amber stop codons individually substituted the selected amino acid codons in four viral proteins (3D, L, VP1, and VP4) of FMDV. We successfully rescued PTC-FMDV mutants, but the amber codon unexpectedly showed a highly degree of mutation rate during PTC-FMDV packaging and replication. Our findings highlight that the genetic code expansion technology for the generation of PTC-FMD vaccines needs to be further improved and that the genetic stability of amber codons during the packaging and replication of FMDV is a concern.

Nonsense suppression therapies in human genetic diseases.

About 11% of all human disease-associated gene lesions are nonsense mutations, resulting in the introduction of an in-frame premature translation-termination codon (PTC) into the protein-coding gene sequence. When translated, PTC-containing mRNAs originate truncated and often dysfunctional proteins that might be non-functional or have gain-of-function or dominant-negative effects. Therapeutic strategies aimed at suppressing PTCs to restore deficient protein function-the so-called nonsense suppression (or PTC readthrough) therapies-have the potential to provide a therapeutic benefit for many patients and in a broad range of genetic disorders, including cancer. These therapeutic approaches comprise the use of translational readthrough-inducing compounds that make the translational machinery recode an in-frame PTC into a sense codon. However, most of the mRNAs carrying a PTC can be rapidly degraded by the surveillance mechanism of nonsense-mediated decay (NMD), thus decreasing the levels of PTC-containing mRNAs in the cell and their availability for PTC readthrough. Accordingly, the use of NMD inhibitors, or readthrough-compound potentiators, may enhance the efficiency of PTC suppression. Here, we review the mechanisms of PTC readthrough and their regulation, as well as the recent advances in the development of novel approaches for PTC suppression, and their role in personalized medicine.

Premature Termination Codon-Bearing mRNA Mediates Genetic Compensation Response.

The genetic compensation response (GCR), triggered by deleterious mutations but not by gene knockdown, has been proposed to explain many phenotypic discrepancies between gene-knockout and gene-knockdown models. GCRs have been observed in many model organisms from mice to Arabidopsis. Although the GCR is beneficial for organism survival, it impedes the exploration of gene function as many knockout mutants do not display discernible phenotypes due to the GCR. Uncovering how the mechanism of GCR operates is not only a fundamental goal in biology but also may provide a key solution in the unmasking of phenotypes in mutants displaying GCRs. Using zebrafish as the model, two recent studies have provided a molecular basis to explain this genetic paradox by demonstrating that the nonsense-mediated mRNA decay pathway is essential for nonsense mRNA to upregulate the expression of its homologous genes through an enhancement of histone H3 Lys4 trimethylation (H3K4me3) at the transcription start site regions of the compensatory genes. Here, we summarize the progress on the molecular mechanism of the GCR and make suggestions on how to overcome GCRs in the generation of genetic mutants.

The Effect of Nonsense Mediated Decay on Transcriptional Activity Within the Novel ß-Thalassemia Mutation HBB: c.129delT.

Premature termination codons (PTCs) are caused by mutations in the coding sequences of functional genes resulting in an incorrect assignment of a stop codon. Abnormal and truncated proteins are prevented from being translated due to the rapid degradation of mRNA carrying these mutations by an RNA surveillance mechanism referred to as nonsense mediated decay (NMD). Recently, a novel mutation in a patient from Thailand with the clinical diagnosis of Hb E (HBB: c.79G > A)/ß(0)-thalassemia (Hb E/ß(0)-thal) and whose molecular analysis demonstrated a novel mutation in the ß-globin gene, HBB: c.129delT, was reported. The result of this deletion is a frameshift (FSC) resulting in a PTC at codon 60. We have analyzed the impact of this mutation on transcription and translation of the affected ß-globin gene using an in vitro model. The quantitative real-time polymerase chain reaction (qReTi-PCR) analysis revealed that this nucleotide mutation resulted in marked mRNA degradation, which we attributed to the NMD mechanism and as such, the expected deleterious truncated HBB was not generated. This result highlights a valuable application of our in vitro gene expression model that can be used to predict possible molecular pathology for any given nucleotide mutations.

Molecular characterization of Hb Hamilton Hill (HBA2: c.388delC), a novel HBA2 variant generating a premature termination codon and truncated HBA2 chain.

In recent years, the identification of α-thalassemias caused by nondeletional mutations has increased significantly due to the advancement of sensitive molecular genetics tools. We report clinical and experimental data for a novel frameshift mutation caused by a single base deletion at position 388 in exon 3 of the α2-globin gene (HBA2: c.388delC; Hb Hamilton Hill), resulting in the phenotype of α-thalassemia (α-thal). Hb Hamilton Hill was identified in an adult female of unknown ethnicity investigated for unexplained microcytosis. Direct DNA sequencing of the HBA2 gene revealed a heterozygous mutation, HBA2: c.388delC, and the molecular effect of this mutation was assessed experimentally using our previously described in vitro model. The experimental analysis involved transfection of a human bladder carcinoma (5637) cell line with expression vectors carrying either HBA2-wild type (HBA2-WT) or HBA2: c.388delC followed by total RNA purification and cDNA synthesis. Both wild type and mutant gene expression was studied and compared at the transcriptional and translational levels using quantitative real time polymerase chain reaction (qReTi-PCR) and immunofluorochemistry (IFC), respectively. Our experimental data showed a significant reduction by 25.0% (p = 0.04) in the transcriptional activity generated from HBA2: c.388delC compared to HBA2-WT. As a result of this base deletion, a frameshift in the open reading frame generates a premature termination codon (PTC) at codon 132 of exon 3 resulting in the formation of a truncated α-globin chain. The truncated α-globin chain, observed by the IFC technique, is most likely unstable and undergoes a rapid turnover resulting in the thalassemic phenotype.

pDsRed-EGFPmtag-, an effective dual fluorescent reporter system for cell-based screens of premature termination codon.

A large number of inherited diseases are caused by premature termination codon (PTC) mutations that lead to the degradation of mRNA template. In this report, we developed a dual fluorescent reporter that relied the feature of fluorescent protein coding region to express a fusion protein from pDsRed-EGFPmtag-. Expression of the fusion protein from a single reporter provides a sensitive approach for high-throughput screening of cell-specific PTC events in mixed cell cultures. Results from the read-through analysis of COS7 cells carrying the nonsense mutation pDsRed-EGFPmtag-Y445X treated by PTC 124 showed EGFP transcript level was increased in the COS7 cells treated by PTC124 in a dose-dependent manner. This novel reporter system was applicable to the majority of different PTC patterns and could be used to quantify efficiency of read-through within a single cell or select cells carrying PTC.

Identification of a PTC-containing DlRan transcript and its differential expression during somatic embryogenesis in Dimocarpus longan.

RAN (Ras-related nuclear protein) plays crucial roles in multiple cellular processes in yeast, animals and plants. Here we present a DlRan gene and its alternative splicing transcripts containing premature terminator codons (PTCs), identified from embryogenic cultures in longan. Multiple alignment and splicing pattern analyses indicated that DlRan-1 transcript harboring PTC was the consequence of alternative splicing. The accumulation of DlRan PTC-containing transcripts increased significantly when the embryogenic calli were treated with the translation inhibitor, cycloheximide, indicating that DlRan-1 may be targeted by NMD. The analysis of expression profiles of DlRan transcripts revealed that differential expression levels of the alternative spliced DlRan transcripts occurred during the development of embryogenic callus, globular-shaped embryos, and cotyledon-shaped embryos, respectively, in the longan somatic embryogenesis, and were in consistent with the embryo development in corresponding wild-type transcripts. The present work offers evidence to speculate that the alternatively spliced PTC-containing transcripts can be functional and may shed light on expression regulation of DlRan during development of the longan somatic embryos.