Search

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. [ABSTRACT FROM AUTHOR]

This editorial discusses a study on the potential use of a drug called SZC for the treatment of crush injuries. The study found that SZC improved survival rates in rats with crush injuries, but did not have a significant impact on renal function. The authors suggest that SZC may be a promising treatment option for controlling early hyperkalemia in crush injuries. The study highlights the need for further research to explore the application of SZC in crush injury scenarios and its impact on overall rescue strategies. [Extracted from the 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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.

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.

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.

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.

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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.

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. [ABSTRACT FROM AUTHOR]

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.

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 β-globin variants are available.

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. [Display omitted] • Nonsense mutations represent the cause of different mendelian genetic diseases. • The suppression therapy is a promising approach to fight nonsense mutations. • Low toxicity is a pre-requisite for the development of nonsense specific therapy. • NV molecules result good candidates for the nonsense mutations suppression therapy. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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)/β0-thalassemia (Hb E/β0-thal) and whose molecular analysis demonstrated a novel mutation in the β-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 β-globin gene using anin vitromodel. 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 truncatedHBBwas not generated. This result highlights a valuable application of ourin vitrogene expression model that can be used to predict possible molecular pathology for any given nucleotide mutations. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: The pathological role of mutations that affect not conserved splicing regulatory sequences can be difficult to determine. In a patient with Fanconi anemia, we identified two unpredictable splicing mutations that act on either sides of FANCA exon 8. In patients-derived cells and in minigene splicing assay, we showed that both an apparently benign intronic c.710-5T>C transition and the nonsense c.790C>T substitution induce almost complete exon 8 skipping. Site-directed mutagenesis experiments indicated that the c.710-5T>C transition affects a polypyrimidine tract where most of the thymidines cannot be compensated by cytidines. The c.790C>T mutation located in position −3 relative to the donor site induce exon 8 skipping in an NMD-independent manner and complementation experiments with modified U1 snRNAs showed that U1 snRNP is only partially involved in the splicing defect. Our results highlight the importance of performing splicing functional assay for correct identification of disease-causing mechanism of genomic variants and provide mechanistic insights on how these two FANCA mutations affect exon 8 definition. [Copyright &y& Elsevier]

Abstract: More than 1900 different mutations in the CFTR gene have been reported. These are grouped into classes according to their effect on the synthesis and/or function of the CFTR protein. CFTR repair therapies that are mutation or mutation class specific are under development. To progress efficiently in the clinical phase of drug development, knowledge of the relative frequency of CFTR mutation classes in different populations is useful. Therefore, we describe the mutation class spectrum in 25,394 subjects with CF from 23 European countries. In 18/23 countries, 80% or more of the patients had at least one class II mutation, explained by F508del being by far the most frequent mutation. Overall 16.4% of European patients had at least one class I mutation but this varied from 3 countries with more than 30% to 4 countries with less than 10% of subjects. Overall only respectively 3.9, 3.3 and 3.0% of European subjects had at least one mutation of classes III, IV and V with again great variability: 14% of Irish patients had at least one class III mutation, 7% of Portuguese patients had at least one class IV mutation, and in 6 countries more than 5% of patients had at least one class V mutation. [Copyright &y& Elsevier]

Abstract: Cystic fibrosis (CF) is an autosomal recessive disease caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene, which encodes an epithelial anion channel. Since the identification of the disease in 1938 and up until 2012, CF patients have been treated exclusively with medications aimed at bettering their respiratory, digestive, inflammatory and infectious symptoms. The identification of the CFTR gene in 1989 gave hopes of rapidly finding a cure for the disease, for which over 1950 mutations have been identified. Since 2012, recent approaches have enabled the identification of small molecules targeting either the CFTR protein directly or its key processing steps, giving rise to novel promising therapeutic tools. This review presents the current CFTR mutation classifications according to their clinical consequences and to their effect on the structure and function of the CFTR channel. How these classifications are essential in the establishment of mutation-targeted therapeutic strategies is then discussed. The future of CFTR-targeted treatment lies in combinatory therapies that will enable CF patients to receive a customized treatment. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances. [Copyright &y& Elsevier]

Abstract: Defective epithelial ion transport is the hallmark of the life-limiting genetic disease cystic fibrosis (CF). This abnormality is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR), the ATP-binding cassette transporter that functions as a ligand-gated anion channel. Since the identification of the CFTR gene, almost 2000 disease-causing mutations associated with a spectrum of clinical phenotypes have been reported, but the majority remain poorly characterised. Studies of a small number of mutations including the most common, F508del-CFTR, have identified six general mechanisms of CFTR dysfunction. Here, we review selectively progress to understand how CF mutations disrupt CFTR processing, stability and function. We explore CFTR structure and function to explain the molecular mechanisms of CFTR dysfunction and highlight new knowledge of disease pathophysiology emerging from large animal models of CF. Understanding CFTR dysfunction is crucial to the development of transformational therapies for CF patients. This article is part of a Directed Issue entitled: Cystic Fibrosis: From o-mics to cell biology, physiology, and therapeutic advances. [Copyright &y& Elsevier]

Abstract: The degradation of human ether-a-go-go-related gene (hERG, KCNH2) transcripts containing premature termination codon (PTC) mutations by nonsense-mediated mRNA decay (NMD) is an important mechanism of long QT syndrome type 2 (LQT2). The mechanisms governing the recognition of PTC-containing hERG transcripts as NMD substrates have not been established. We used a minigene system to study two frameshift mutations, R1032Gfs*25 and D1037Rfs*82. R1032Gfs*25 introduces a PTC in exon 14, whereas D1037Rfs*82 causes a PTC in the last exon (exon 15). We showed that R1032Gfs*25, but not D1037Rfs*82, reduced the level of mutant mRNA compared to the wild-type minigene in an NMD-dependent manner. The deletion of intron 14 prevented degradation of R1032Gfs*25 mRNA indicating that a downstream intron is required for NMD. The recognition and elimination of PTC-containing transcripts by NMD required that the mutation be positioned >54–60nt upstream of the 3′-most exon–exon junction. Finally, we used a full-length hERG splicing-competent construct to show that inhibition of downstream intron splicing by antisense morpholino oligonucleotides inhibited NMD and rescued the functional expression of a third LQT2 mutation, Y1078*. The present study defines the positional requirements for the susceptibility of LQT2 mutations to NMD and posits that the majority of reported LQT2 nonsense and frameshift mutations are potential targets of NMD. [Copyright &y& Elsevier]

Abstract: Huntington's disease (HD) is an inherited progressive neurodegenerative disorder caused by a pathological CAG trinucleotide repeat expansion in the large multi-exon gene, huntingtin (HTT). Although multiple pathogenic mechanisms have been proposed for HD, there is increasing interest in the RNA processing of the HTT gene. In mammals, most multi-exon genes are alternatively spliced; however, few alternative transcripts have been described for HTT. Given the numerous protein bands detected in mouse and human brain tissue by Western blotting using anti-huntingtin antibodies, we examined whether alternative splicing of HTT may account for some of these fragments. Using RT-PCR in mouse brain, we detected two novel splice variants of Htt that lacked the 111-bp exon 29 (Htt∆ex29) or retained a 57-bp portion of intron 28 (Htt+57in28) via use of a cryptic splice site. The alternative transcripts were present in wild-type and homozygous Hdh(Q150/Q150) mouse brain at all ages and in all brain regions and peripheral tissues studied. Differential splicing of Htt∆ex29 was found in the cerebellum of Hdh(Q150/Q150) mice with a significant reduction in transcript levels in mutant animals. In human brain, we detected similar splice variants lacking exons 28 and 29. The ability of alternatively spliced transcripts to encode different protein isoforms with individual functions in the cell, combined with the known role of splicing in disease, renders these novel transcripts of interest in the context of HD pathogenesis. [Copyright &y& Elsevier]

Background: Brugada syndrome (BrS) is an inherited cardiac arrhythmia associated with sudden death due to ventricular fibrillation. Mutations in genes related to the cardiac L-type calcium channel have been reported to be causative of BrS. Generally, the messenger RNA (mRNA) that contains a nonsense mutation is rapidly degraded via its decay pathway, which is known as nonsense-mediated mRNA decay (NMD). Previously, we reported a male patient with BrS who carried c.1896G>A (the first nucleotide of CACNA1C exon 14), which caused a synonymous mutation, p.R632R. Objective: To examine how the synonymous CACNA1C mutation p.R632R produces the phenotype of BrS, with a special emphasis on the splicing error and NMD processes. Methods: We extracted mRNA from leukocytes of the proband and his 2 children and performed reverse transcription polymerase chain reaction. Complementary DNAs were checked by using direct sequencing and quantitative analysis. Results: The subsequent sequence electropherogram of the complementary DNAs did not show the substitution of the nucleotide identified in the genomic DNA of the proband. In the mRNA quantification analysis, we confirmed that reduction in the CACNA1C expression level was suspected to be caused by NMD. Conclusions: Mutant mRNA with a c.1896G>A substitution may be diminished by NMD, and the resultant decrease in CACNA1C message leads to a novel mechanism for inducing BrS that is distinct from that reported previously. [Copyright &y& Elsevier]

Abstract: 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. [Copyright &y& Elsevier]

Abstract: Marfan syndrome is an autosomal dominant disorder of connective tissue caused by mutations in the gene encoding fibrillin-1 (FBN1), a matrix component of microfibrils. Dural ectasia, i.e. enlargement of the neural canal mainly located in the lower lumbar and sacral region, frequently occurs in Marfan patients. The aim of our study was to investigate the role of dural ectasia in raising the diagnosis of Marfan syndrome and its association with FBN1 mutations. We studied 40 unrelated patients suspected for MFS, who underwent magnetic resonance imaging searching for dural ectasia. In all of them FBN1 gene analysis was also performed. Thirty-seven patients resulted affected by Marfan syndrome according to the '96 Ghent criteria; in 30 of them the diagnosis was confirmed when revaluated by the recently revised criteria (2010). Thirty-six patients resulted positive for dural ectasia. The degree of dural ectasia was grade 1 in 19 patients, grade 2 in 11 patients, and grade 3 in 6 patients. In 7 (24%) patients, the presence of dural ectasia allowed to reach a positive score for systemic feature criterion. Twenty-four patients carried an FBN1 mutation, that were represented by 13 missense (54%), and 11 (46%) mutations generating a premature termination codon (PTC, frameshifts and stop codons). No mutation was detected in the remaining 16 (6 patients with MFS and 10 with related disorders according to revised Ghent criteria). The prevalence of severe (grade 2 and grade 3) involvement of dura mater was higher in patients harbouring premature termination codon (PTC) mutations than those carrying missense-mutations (8/11 vs 2/13, P = 0.0111). Our data emphasizes the importance of dural ectasia screening to reach the diagnosis of Marfan syndrome especially when it is uncertain and indicates an association between PTC mutations and severe dural ectasia in Marfan patients. [Copyright &y& Elsevier]

Several hereditary cancer syndromes are associated with nonsense mutations that create premature termination codons (PTC). Therapeutic strategies involving readthrough induction partially restore expression of proteins with normal function from nonsense-mutated genes, and small molecules such as aminoglycosides and PTC124 have exhibited promising results for treating patients with cystic fibrosis and Duchenne muscular dystrophy. Transgenic expression of suppressor-tRNAs and depleting translation termination factors are, among others, potential strategies for treating PTC-associated diseases. In this review, the potential of using readthrough strategies as a therapy for cancer syndromes is discussed, and we consider the effect of nonsense-mediated decay and other factors on readthrough efficiency. [Copyright &y& Elsevier]

Abstract: Cyclin T1 (CCNT1), a gene containing nine exons, forms the positive transcription elongation factor b (P-TEFb) complex and regulates a wide variety of biological processes including transcription. We discovered a novel splice variant of CCNT1 that lacks exon 7 (dE7). RT-PCR analysis revealed that the dE7 transcript was detected in almost all tissues examined. The dE7/FL transcript ratio was high in quiescent peripheral blood mononuclear cells (PBMC) and in tissues poor in cell division; however, it was low in activated PBMC and in tissues with high cell proliferative potential. These results suggest that exon 7 skipping is linked to cell cycle progression. Increasing the dE7/FL transcript ratio resulted in the reduction of CCNT1 protein levels, indicating that the expression of CCNT1 protein is controlled by exon skipping. Exon 7 skipping yields a +1 frameshift at exon 8, which generates a premature termination codon (PTC). The dE7 transcript levels increased when cells were treated with the protein synthesis inhibitor cycloheximide (CHX) or a kinase inhibitor wortmannin (WORT), whilst the FL transcript levels were unchanged, suggesting that the dE7 transcript is a target of nonsense-mediated decay (NMD). Importantly, reduction of dE7 transcript by WORT correlated well with the decrement of CCNT1 protein expression. The dE7 transcript would produce an approximately 23kDa protein that covers approximately 70% of the cyclin box. The ectopically expressed dE7 protein physically interacted with CDK9 and competed with FL CCNT1 for CDK9, thus should act dominant-negatively on FL CCNT1. The replication of human immunodeficiency virus type 1 (HIV-1), heavily dependent on the CCNT1 function, was inhibited by dE7 protein through the attenuation of Tat/long terminal repeat (LTR)-driven transcription. Taken together, these results suggest that dE7 is a novel splice variant that regulates the expression and function of CCNT1. [Copyright &y& Elsevier]

The identification of α-thalassemia (α-thal) due to point mutations has been increasing significantly with the advancement of molecular diagnostic tools. We describe here the molecular and cellular characteristics of the thalassemia mutation HBA2:c.94A>C, a novel point mutation affecting the α2-globin gene, causing a mild α-thal phenotype in a male patient of undisclosed ethnicity, investigated for unexplained microcytosis. The detected mutation is located at the penultimate nucleotide (nt) of the first exon which we postulated might affect pre mRNA splicing. While an in silico analysis did not predict any aberrant splice variants, experimental analysis using our in vitro model for gene expression studies showed utilization of a cryptic splice site at codon 15 that resulted in an aberrant splice variant. As a result, a frameshift in the reading frame of the mature mRNA was produced, leading to the formation of a premature termination codon (PTC) between codons 48 and 49 in exon 2. This in turn leads to nonsense mediated mRNA decay (NMD) and the phenotype of α-thal. [ABSTRACT FROM AUTHOR]

In Arabidopsis, the NMD-defective mutants upf1-5 and upf3-1 are characterized by dwarfism, curly leaves and late flowering. These phenotypes are similar to those of mutants showing constitutive pathogenesis-related (PR) gene expression, salicylic acid (SA) accumulation and, subsequently, resistance to pathogens. The disease symptoms of upf1-5 and upf3-1 mutants were observed following infection with the virulent pathogen Pst DC3000 with the aim of determining whether the loss of nonsense-mediated mRNA decay (NMD) is involved in disease resistance. These mutant plants showed not only enhanced resistance to Pst DC3000, but also elevated levels of endogenous SA, PR gene transcripts and WRKY transcripts. UPF1 and UPF3 expression was down-regulated in Pst DC3000-infected Arabidopsis plants, but the expression of various NMD target genes was up-regulated. The expression of 10 defense-related genes was elevated in cycloheximide (CHX)-treated plants. The transcriptional ratios of eight of these 10 defense-related genes in CHX-treated to non-treated plants were lower in NMD-defective mutants than in the wild-type plants. These eight defense-related genes are possibly regulated by the NMD mechanism, and it is clear that an alternatively spliced transcript of WRKY62, which contains a premature termination codon, was regulated by this mechanism. Taken together, our results suggest that UPF1 and UPF3, which are key NMD factors, may act as defense-related regulators associated with plant immunity. [ABSTRACT FROM PUBLISHER]

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. [ABSTRACT FROM AUTHOR]

Abstract: Functional polymorphisms that alter gene expression and mRNA processing appear to play a critical role in shaping human phenotypic variability. Intensive studies on polymorphisms affecting drug response have revealed multiple modes of altered gene function, frequently involving cis-acting regulatory sequence variants. Experimental and in silico methods have advanced the search for such polymorphisms, but considerable challenges remain. Here, a survey of polymorphisms in drug-related genes indicates that: (a) a substantial proportion of genetic variability still remains unaccounted for; (b) a majority of these genes harbors known regulatory polymorphisms; and (c) a portion of polymorphisms affect splicing and mRNA turnover. Pharmacogenetic optimiziation of individual drug therapy may require a complete understanding of all functional sequence variants in key genes. This review surveys known noncoding polymorphisms in genes encoding cytochrome P450s and other drug-metabolizing enzymes, drug transporters, and drug targets and receptors. Current methods and challenges associated with the identification and characterization of functional polymorphisms are also discussed. [Copyright &y& Elsevier]

The ABC transporter gene family has evolved by a gene “birth-and-death” process; however, the number of ABC pseudogenes in the human genome is surprisingly small. On chromosome 21q11.2, spanning 90 kb, is an ABC gene-like sequence (recently annotated as ABCC13) with the highest similarity to ABCC2. Here we show that while comparative analysis and in silico prediction methods indicate the presence of at least 28 exons, the major ABCC13 transcript in humans consists of only 6 exons with a total length of 1.1 kb. The open reading frame of this transcript is capable of encoding a polypeptide of only 274 amino acids, compared to the more than 1500 amino acids of related ABC transporters. The truncated ABCC13 transcript shows tissue-specific expression, highest in fetal liver, bone marrow, and colon. Since the last exon of the ABCC13 transcript contains an apparent frameshift, we sequenced the respective region from several primates and found that the frameshift is due to an 11-bp deletion that is shared between human, chimpanzee, and gorilla, but is not found in monkeys. In addition, the human ABCC13 gene contains two other frameshift indels in the exons that encode the second nucleotide-binding domain, indicating that ABCC13 is not capable of encoding a functional ABC protein. In an attempt to identify an intact ABCC13 ortholog, we have sequenced the full-length cDNA from rhesus macaque, which contains an open reading frame of 1296 amino acids, producing an apparently functional ABC transporter. Although the mouse and rat genomes contain long-range similarity in the locus where Abcc13 is expected to reside, most of the Abcc13 exons in rodents are degraded below the threshold of sequence homology searches or have been deleted completely. [Copyright &y& Elsevier]

Alternative splicing is now commonly thought to affect more than half of all human genes. Recent studies have investigated not only the scope but also the biological impact of alternative splicing on a large scale, revealing that its role in generating proteome diversity may be augmented by a role in regulation. For instance, protein function can be regulated by the removal of interaction or localization domains by alternative splicing. Alternative splicing can also regulate gene expression by splicing transcripts into unproductive mRNAs targeted for degradation. To fully understand the scope of alternative splicing, we must also determine how many of the predicted splice variants represent functional forms. Comparisons of alternative splicing between human and mouse genes show that predominant splice variants are usually conserved, but rare variants are less commonly shared. Evolutionary conservation of splicing patterns suggests functional importance and provides insight into the evolutionary history of alternative splicing. [Copyright &y& Elsevier]

Although it is universally accepted that protein synthesis occurs in the cytoplasm, the possibility that translation can also take place in the nucleus has been hotly debated. Reports have been published claiming to demonstrate nuclear translation, but alternative explanations for these results have not been excluded, and other experiments argue against it. Much of the appeal of nuclear translation is that functional proofreading of newly made mRNAs in the nucleus would provide an efficient way to monitor mRNAs for the presence of premature termination codons, thereby avoiding the synthesis of deleterious proteins. mRNAs that are still in the nucleus-associated fraction of cells are subject to translational proofreading resulting in nonsense-mediated mRNA decay and perhaps nonsense-associated alternate splicing. However, these mRNAs are likely to be in the perinuclear cytoplasm rather than within the nucleus. Therefore, in the absence of additional evidence, we conclude that nuclear translation is unlikely to occur. [Copyright &y& Elsevier]

Over the past decade many studies have revealed a complex web of interconnections between the numerous steps required for eukaryotic gene expression. One set of interconnections link nuclear pre-mRNA splicing and the subsequent metabolism of the spliced mRNAs. It is now apparent that the means of connection is a set of proteins, collectively called the exon junction complex, which are deposited as a consequence of splicing upstream of mRNA exon–exon junctions. [Copyright &y& Elsevier]

A new HLA-B null allele has been identified within the B*51 group by combined serological and molecular typing of an Italian Caucasoid family. Serological data indicated that the proband typed homozygous for A2 and B60. Confirmatory typing using sequence specific oligonucleotide hybridization (SSPOH) detected a second B allele within the B*51 group. Allele specific typing (SSP) for B*51 subtypes, including the known B*5111N allele, was performed, and typing results were consistent with B*5101, suggesting the presence of a new null variant. Cloning and sequencing of this allele identified a B*5101 variant with a nonsense mutation in exon 3. This new null allele has been designated B*5127N. The combined use of serologic and DNA-based typing methods facilitates the identification of null and low-expression alleles. An overview of null alleles of class I HLA is presented. [ABSTRACT FROM AUTHOR]

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 β-globin variants are available. [ABSTRACT FROM AUTHOR]

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 β-globin variants are available.

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.