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Abstract Alternative splicing plays a crucial role in increasing the diversity of mRNAs expressed in the genome. Serine/arginine-rich splicing factor 3 (SRSF3) is responsible for regulating the alternative splicing of its own mRNA and ensuring that its expression is balanced to maintain homeostasis. Moreover, the exon skipping of SRSF3 leads to the production of a truncated protein instead of a frameshift mutation that generates a premature termination codon (PTC). However, the precise regulatory mechanism involved in the splicing of SRSF3 remains unclear. In this study, we first established a platform for coexpressing full-length SRSF3 (SRSF3-FL) and SRSF3-PTC and further identified a specific antibody against the SRSF3-FL and truncated SRSF3 (SRSF3-TR) proteins. Next, we found that exogenously overexpressing SRSF3-FL or SRSF3-PTC failed to reverse the effects of digoxin, caffeine, or both in combination on this molecule and its targets. Endoplasmic reticulum-related pathways, transcription factors, and chemicals such as palmitic acid and phosphate were found to be involved in the regulation of SRSF3 expression. The downregulation of SRSF3-FL by palmitic acid and phosphate was mediated via different regulatory mechanisms in HeLa cells. In summary, we provide new insights into the altered expression of the SRSF3-FL and SRSF3-TR proteins for the identification of the functions of SRSF3 in cells.

Simple Summary: PTEN is an essential tumor suppressor gene whose alterations are causative of cancer and neurodevelopmental disorders, grouped as PTEN hamartoma tumor syndrome (PHTS). Here, we present translational readthrough as a potential therapeutic approach to restore the function of PTEN isoforms in patients harboring nonsense mutations at PTEN that cause aberrant PTEN biosynthesis. We have found that aminoglycosides and protein synthesis inducers reconstitute PTEN isoform expression and the function of PTEN variants containing nonsense mutations. Our results provide insights with which to optimize readthrough-based therapies for patients with cancer and PHTS. The PTEN tumor suppressor is frequently targeted in tumors and patients with PTEN hamartoma tumor syndrome (PHTS) through nonsense mutations generating premature termination codons (PTC) that may cause the translation of truncated non-functional PTEN proteins. We have previously described a global analysis of the readthrough reconstitution of the protein translation and function of the human canonical PTEN isoform by aminoglycosides. Here, we report the efficient functional readthrough reconstitution of the PTEN translational isoform PTEN-L, which displays a minimal number of PTC in its specific N-terminal extension in association with disease. We illustrate the importance of the specific PTC and its nucleotide proximal sequence for optimal readthrough and show that the more frequent human PTEN PTC variants and their mouse PTEN PTC equivalents display similar patterns of readthrough efficiency. The heterogeneous readthrough response of the different PTEN PTC variants was independent of the length of the PTEN protein being reconstituted, and we found a correlation between the amount of PTEN protein being synthesized and the PTEN readthrough efficiency. Furthermore, combination of aminoglycosides and protein synthesis inducers increased the readthrough response of specific PTEN PTC. Our results provide insights with which to improve the functional reconstitution of human-disease-related PTC pathogenic variants from PTEN isoforms by increasing protein synthesis coupled to translational readthrough. [ABSTRACT FROM AUTHOR]

Background: BMPR1A‐mediated signaling transduction plays an essential role in intestinal growth. Variations of BMPR1A lead to a rare autosomal dominant inherited juvenile polyposis syndrome (JPS) with high probability of developing into colorectal cancer (CRC). Nonsense and frameshift variations, generating premature termination codons (PTCs), are the most pathogenic variants in the BMPR1A gene. Objective: This study aimed to investigate the molecular genetic etiology in a Chinese family with three generations of CRC. Methods: Pathogenic variants of 18 known CRC susceptibility genes were examined in a Chinese CRC family through multigene panel testing using the next‐generation sequencing platform. The candidate gene variant was validated in the family members by Sanger sequencing. Potential biological functions of the gene variant were further investigated in the RKO colon cancer cell line. Results: A novel nonsense variant (c.1114A > T, p.Lys372*) of BMPR1A was identified in the CRC family. This variant generated a PTC at the kinase domain and caused nonsense‐mediated mRNA decay. Read‐through inducing reagents G418 and PTC124 partially restored BMPR1A expression and its following signaling pathway. Conclusion: The identification of the novel BMPR1A variant enriched the genotype–phenotype spectrum of BMPR1A. Meanwhile, our finding also provided support for future PTC‐targeting therapy for BMPR1A‐mediated JPS and CRC. [ABSTRACT FROM AUTHOR]

Alternative splicing plays a crucial role in increasing the diversity of mRNAs expressed in the genome. Serine/arginine-rich splicing factor 3 (SRSF3) is responsible for regulating the alternative splicing of its own mRNA and ensuring that its expression is balanced to maintain homeostasis. Moreover, the exon skipping of SRSF3 leads to the production of a truncated protein instead of a frameshift mutation that generates a premature termination codon (PTC). However, the precise regulatory mechanism involved in the splicing of SRSF3 remains unclear. In this study, we first established a platform for coexpressing full-length SRSF3 (SRSF3-FL) and SRSF3-PTC and further identified a specific antibody against the SRSF3-FL and truncated SRSF3 (SRSF3-TR) proteins. Next, we found that exogenously overexpressing SRSF3-FL or SRSF3-PTC failed to reverse the effects of digoxin, caffeine, or both in combination on this molecule and its targets. Endoplasmic reticulum-related pathways, transcription factors, and chemicals such as palmitic acid and phosphate were found to be involved in the regulation of SRSF3 expression. The downregulation of SRSF3-FL by palmitic acid and phosphate was mediated via different regulatory mechanisms in HeLa cells. In summary, we provide new insights into the altered expression of the SRSF3-FL and SRSF3-TR proteins for the identification of the functions of SRSF3 in cells. [ABSTRACT FROM AUTHOR]

In the context of rare genetic diseases caused by nonsense mutations, the concept of induced stop codon readthrough (SCR) represents an attractive avenue in the ongoing search for improved treatment options. Epidermolysis bullosa (EB)—exemplary for this group of diseases—describes a diverse group of rare, blistering genodermatoses. Characterized by extreme skin fragility upon minor mechanical trauma, the most severe forms often result from nonsense mutations that lead to premature translation termination and loss of function of essential proteins at the dermo‐epidermal junction. Since no curative interventions are currently available, medical care is mainly limited to alleviating symptoms and preventing complications. Complementary to attempts of gene, cell and protein therapy in EB, SCR represents a promising medical alternative. While gentamicin has already been examined in several clinical trials involving EB, other potent SCR inducers, such as ataluren, may also show promise in treating the hitherto non‐curative disease. In addition to the extensively studied aminoglycosides and their derivatives, several other substance classes—non‐aminoglycoside antibiotics and non‐aminoglycoside compounds—are currently under investigation. The extensive data gathered in numerous in vitro experiments and the perspectives they reveal in the clinical setting will be discussed in this review. [ABSTRACT FROM AUTHOR]

Adeno-associated virus (AAV)-based gene therapy is a clinical stage therapeutic modality for neurological disorders. A common genetic defect in myriad monogenic neurological disorders is nonsense mutations that account for about 11% of all human pathogenic mutations. Stop codon readthrough by suppressor transfer RNA (sup-tRNA) has long been sought as a potential gene therapy approach to target nonsense mutations, but hindered by inefficient in vivo delivery. The rapid advances in AAV delivery technology have not only powered gene therapy development but also enabled in vivo preclinical assessment of a range of nucleic acid therapeutics, such as sup-tRNA. Compared with conventional AAV gene therapy that delivers a transgene to produce therapeutic proteins, AAV-delivered sup-tRNA has several advantages, such as small gene sizes and operating within the endogenous gene expression regulation, which are important considerations for treating some neurological disorders. This review will first examine sup-tRNA designs and delivery by AAV vectors. We will then analyze how AAV-delivered sup-tRNA can potentially address some neurological disorders that are challenging to conventional gene therapy, followed by discussing available mouse models of neurological diseases for in vivo preclinical testing. Potential challenges for AAV-delivered sup-tRNA to achieve therapeutic efficacy and safety will also be discussed.

BackgroundFrontotemporal lobar degeneration (FTLD) is a devastating and progressive disorder, and a common cause of early onset dementia. Progranulin (PGRN) haploinsufficiency due to autosomal dominant mutations in the progranulin gene (GRN) is an important cause of FTLD (FTLD-GRN), and nearly a quarter of these genetic cases are due to a nonsense mutation. Premature termination codons (PTC) can be therapeutically targeted by compounds allowing readthrough, and aminoglycoside antibiotics are known to be potent PTC readthrough drugs. Restoring endogenous PGRN through PTC readthrough has not previously been explored as a therapeutic intervention in FTLD.MethodsWe studied whether the aminoglycoside G418 could increase PGRN expression in HEK293 and human induced pluripotent stem cell (hiPSC)-derived neurons bearing the heterozygous S116X, R418X, and R493X pathogenic GRN nonsense mutations. We further tested a novel substituted phthalimide PTC readthrough enhancer in combination with G418 in our cellular models. We next generated a homozygous R493X knock-in hiPSC isogenic line (R493X-/- KI), assessing whether combination treatment in hiPSC-derived neurons and astrocytes could increase PGRN and ameliorate lysosomal dysfunction relevant to FTLD-GRN. To provide in vivo proof-of-concept of our approach, we measured brain PGRN after intracerebroventricular administration of G418 in mice expressing the V5-tagged GRN nonsense mutation R493X.ResultsThe R418X and R493X mutant GRN cell lines responded to PTC readthrough with G418, and treatments increased PGRN levels in R493X-/- KI hiPSC-derived neurons and astrocytes. Combining G418 with a PTC readthrough enhancer increased PGRN levels over G418 treatment alone in vitro. PGRN deficiency has been shown to impair lysosomal function, and the mature form of the lysosomal protease cathepsin D is overexpressed in R493X-/- KI neurons. Increasing PGRN through G418-mediated PTC readthrough normalized this abnormal lysosomal phenotype in R493X-/- KI neuronal cultures. A single intracerebroventricular injection of G418 induced GRN PTC readthrough in 6-week-old AAV-GRN-R493X-V5 mice.ConclusionsTaken together, our findings suggest that PTC readthrough may be a potential therapeutic strategy for FTLD caused by GRN nonsense mutations.

Long QT syndrome type 2 (LQT2) is a genetic disorder caused by mutations in the KCNH2 gene, also known as the human ether-a-go-go-related gene (HERG). More than 30% of HERG mutations result in a premature termination codon that triggers a process called nonsense-mediated messenger RNA (mRNA) decay (NMD), where the mRNA transcript is degraded. NMD is a quality control mechanism that removes faulty mRNA to prevent the translation of truncated proteins. Recent advances in antisense oligonucleotide (ASO) technology in the field of cystic fibrosis (CF) have yielded significant progress, including the ASO-mediated comprehensive characterization of key NMD factors and exon-skipping therapy. These advances have contributed to our understanding of the role of premature termination codon–containing mutations in disease phenotypes and have also led to the development of potentially useful therapeutic strategies. Historically, studies of CF have provided valuable insights for the research on LQT2, particularly concerning increasing the expression of HERG. In this article, we outline the current state of knowledge regarding ASO, NMD, and HERG and discuss the introduction of ASO technology in the CF to elucidate the pathogenic mechanisms through targeting NMD. We also discuss the potential clinical therapeutic benefits and limitations of ASO for the management of LQT2. By drawing on lessons learned from CF research, we explore the potential translational values of these advances into LQT2 studies. [ABSTRACT FROM AUTHOR]

Introduction: Asthenoteratozoospermia is one of the most common causes of male infertility. Several genes have been identified as genetic causative factors, but there is a considerable genetic heterogeneity underlying asthenoteratozoospermia. In this study, we performed a genetic analysis of two brothers from a consanguineous Uighur family in China to identify gene mutations causative for asthenoteratozoospermia-related male infertility. Methods: Two related patients with asthenoteratozoospermia from a large consanguineous family were sequenced by whole-exome sequencing and Sanger sequencing to identify disease-causing genes. Scanning and transmission electronmicroscopy analysis revealed ultrastructural abnormalities of spermatozoa. Quantitative real-time PCR (qRT-PCR) analysis and immunofluorescence (IF) analysis were used to assess the expression of the mutant messenger RNA (mRNA) and protein. Results: A novel homozygous frameshift mutation (c.2823dupT, p. Val942Cysfs*21) in DNAH6 was identified in both affected individuals and was predicted to be pathogenic. Papanicolaou staining and electron microscopy revealed multiple morphological and ultrastructural abnormalities of affected spermatozoa. qRTPCR and IF analysis showed abnormal expression of DNAH6 in affected sperm, probably due to premature termination code and decay of abnormal 3' untranslated region (UTR) region of mRNA. Furthermore, intracytoplasmic sperminjection could achieve successful fertilization in infertile men with DNAH6 mutations. Discussion: The novel frameshift mutation identified in DNAH6 may contribute to asthenoteratozoospermia. These findings expand the spectrum of genetic mutations and phenotypes associated with asthenoteratozoospermia and may be useful for genetic and reproductive counseling in male infertility. [ABSTRACT FROM AUTHOR]

Cellular protein–protein interactions are largely dependent on the activities of signaling proteins. Here, we present a technique to tune gene expression at translation level based on G418-inducible readthrough premature termination codon (PTC-on). To demonstrate how this PTC-on can control the expression level of a cellular signaling protein to regulate signal transduction, we settled a p53 PTC-on system in p53-null H1299 cells. After treating with G418, the cells expressed full-length p53 protein in a dose-dependent manner. We further demonstrated to use this PTC-on approach to dissect p53-dependent and p53-independent apoptosis in response to the DNA double strand breaks in H1299 cells. In principle, the PTC-on can be used as a general approach for exploring the functions of any other signaling proteins.

Nonsense mutations or premature termination codons (PTCs) comprise ∼11% of all genetic lesions, which result in over 7,000 distinct genetic diseases. Due to their outsized impact on human health, considerable effort has been made to find therapies for nonsense-associated diseases. Suppressor tRNAs have long been identified as a possible therapeutic for nonsense-associated diseases; however, their ability to inhibit nonsense-mediated mRNA decay (NMD) and support significant protein translation from endogenous transcripts has not been determined in mammalian cells. Here, we investigated the ability of anticodon edited (ACE)-tRNAs to suppress cystic fibrosis (CF) causing PTCs in the cystic fibrosis transmembrane regulator (CFTR) gene in gene-edited immortalized human bronchial epithelial (16HBEge) cells. Delivery of ACE-tRNAs to 16HBEge cells harboring three common CF mutations G542XUGA-, R1162XUGA-, and W1282XUGA-CFTR PTCs significantly inhibited NMD and rescued endogenous mRNA expression. Furthermore, delivery of our highly active leucine-encoding ACE-tRNA resulted in rescue of W1282X-CFTR channel function to levels that significantly exceed the necessary CFTR channel function for therapeutic relevance. This study establishes the ACE-tRNA approach as a potential standalone therapeutic for nonsense-associated diseases due to its ability to rescue both mRNA and full-length protein expression from PTC-containing endogenous genes.

Rind color is an economically important agronomic trait in eggplant that impacts consumer preferences. In this study, bulked segregant analysis and competitive allele-specific PCR were employed to identify the candidate gene for eggplant rind color through constructing a 2794 F2 population generated from a cross between "BL01" (green pericarp) and "B1" (white pericarp). Genetic analysis of rind color revealed that a single dominant gene controls green color of eggplant peel. Pigment content measurement and cytological observations demonstrated that chlorophyll content and chloroplast number in BL01 were higher than in B1. A candidate gene (EGP19168.1) was fine-mapped to a 20.36 Kb interval on chromosome 8, which was predicted to encode the two-component response regulator-like protein Arabidopsis pseudo-response regulator2 (APRR2). Subsequently, allelic sequence analysis revealed that a SNP deletion (ACT→AT) in white-skinned eggplant led to a premature termination codon. Genotypic validation of 113 breeding lines using the Indel marker closely linked to SmAPRR2 could predict the skin color (green/white) trait with an accuracy of 92.9%. This study will be valuable for molecular marker-assisted selection in eggplant breeding and provides theoretical foundation for analyzing the formation mechanism of eggplant peel color. [ABSTRACT FROM AUTHOR]

Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren's mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren's binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren's low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity. [ABSTRACT FROM AUTHOR]

[Display omitted] • The reactive oligonucleotide modifies the adenosine of the prematurue termination codon. • The adenosine of the natural termination codon is not modified. • The A-modified mRNA induced readthrough in a reconstituted E. coli translation system. • Readthrough is caused by inhibiting the RF2 function. Nonsense mutations in the coding region turn amino acid codons into termination codons, resulting in premature termination codons (PTCs). In the case of the in-frame PTC, if translation does not stop at the PTC but continues to the natural termination codon (NTC) with the insertion of an amino acid, known as readthrough, the full-length peptide is formed, albeit with a single amino acid mutation. We have previously developed the functionality-transfer oligonucleotide (FT-Probe), which forms a hybrid complex with RNA of a complementary sequence to transfer the functional group, resulting in modification of the 4-amino group of cytosine or the 6-amino group of adenine. In this study, the FT-Probe was used to chemically modify the adenosines of the PTC (UAA, UAG, and UGA) of mRNA, which were assayed for the readthrough in a reconstituted Escherichia coli translation system. The third adenosine-modified UAA produced three readthrough peptides incorporating tyrosine, glutamine and lysine at the UAA site. It should be noted that the additional modification with a cyclodextrin only induced glutamine incorporation. The adenosine modified UGA induced readthrough very efficiently with selective tryptophan incorporation. Readthrough of the modified UGA is caused by inhibition of the RF2 function. This study has demonstrated that the chemical modification of the adenosine 6-amino group of the PTC is a strategy for effective readthrough in a prokaryotic translation system. [ABSTRACT FROM AUTHOR]

Background: Currently, there is no cure for epidermolysis bullosa (EB) but few studies have explored the role of aminoglycosides in promoting collagen 7 expression in recessive dystrophic EB (RDEB). Materials and Methods: Consecutive patients aged >1 year with a confirmed diagnosis of dystrophic EB (DEB) were advised to apply 0.1% w/w gentamicin cream in a collagen base (Derbriment GTM) twice daily on a representative area on right lower limb (RLL) and paraffin gauze dressings on the corresponding opposite side on the left lower limb (LLL). Skin lesions were evaluated clinically during the 12-week treatment period at the end of which a repeat skin biopsy was sent for immunofluorescence antigen mapping (IFM). Results: Twelve patients with DEB were recruited but only eight completed the study and were analyzed. The mean fluorescence intensity (MFI) of the study cohort increased from 2765 ± 1732.07 (263–4845) at baseline to 5412.75 ± 3937.64 (2100–13536) at 12 weeks; a 95.75% (range 5.34%–775.14%) increase in the MFI of collagen 7 from baseline (P = 0.06). Among patients with a known termination codon mutation (n = 3), the percentage increase in MFI was greater among patients with known premature termination codon (PTC) mutations compared to those with unknown mutations. The clinical severity did not change significantly in terms of the mean number of blisters, erosions, and scarring during the study period. None of the parents reported any adverse effect. Conclusions: Topical gentamicin 0.1% w/w is a safe and effective way to promote the expression of COL7A1 in DEB patients, especially those carrying PTC mutations.

Premature termination codons (PTCs) account for 10 to 20% of genetic diseases in humans. The gene inactivation resulting from PTCs can be counteracted by the use of drugs stimulating PTC readthrough, thereby restoring production of the full-length protein. However, a greater chemical variety of readthrough inducers is required to broaden the medical applications of this therapeutic strategy. In this study, we developed a reporter cell line and performed high-throughput screening (HTS) to identify potential readthrough inducers. After three successive assays, we isolated 2-guanidinoquinazoline (TLN468). We assessed the clinical potential of this drug as a potent readthrough inducer on the 40 PTCs most frequently responsible for Duchenne muscular dystrophy (DMD). We found that TLN468 was more efficient than gentamicin, and acted on a broader range of sequences, without inducing the readthrough of normal stop codons (TC). [ABSTRACT FROM AUTHOR]

Background: Currently, there is no cure for epidermolysis bullosa (EB) but few studies have explored the role of aminoglycosides in promoting collagen 7 expression in recessive dystrophic EB (RDEB). Materials and Methods: Consecutive patients aged >1 year with a confirmed diagnosis of dystrophic EB (DEB) were advised to apply 0.1% w/w gentamicin cream in a collagen base (Derbriment GTM) twice daily on a representative area on right lower limb (RLL) and paraffin gauze dressings on the corresponding opposite side on the left lower limb (LLL). Skin lesions were evaluated clinically during the 12-week treatment period at the end of which a repeat skin biopsy was sent for immunofluorescence antigen mapping (IFM). Results: Twelve patients with DEB were recruited but only eight completed the study and were analyzed. The mean fluorescence intensity (MFI) of the study cohort increased from 2765 ± 1732.07 (263–4845) at baseline to 5412.75 ± 3937.64 (2100–13536) at 12 weeks; a 95.75% (range 5.34%–775.14%) increase in the MFI of collagen 7 from baseline (P = 0.06). Among patients with a known termination codon mutation (n = 3), the percentage increase in MFI was greater among patients with known premature termination codon (PTC) mutations compared to those with unknown mutations. The clinical severity did not change significantly in terms of the mean number of blisters, erosions, and scarring during the study period. None of the parents reported any adverse effect. Conclusions: Topical gentamicin 0.1% w/w is a safe and effective way to promote the expression of COL7A1 in DEB patients, especially those carrying PTC mutations. [ABSTRACT FROM AUTHOR]

Cystic fibrosis (CF) is a lethal hereditary disease caused by loss-of-function mutations of the chloride channel cystic fibrosis transmembrane conductance regulator (CFTR). With the development of small-molecule CFTR modulators, including correctors that facilitate protein folding and expression and potentiators that promote channel activity, about 90% of the CF patients are now receiving efficacious target therapies. G542X-CFTR, a premature termination codon (PTC) mutation, is the most common disease-associated mutation found in the remaining 10% of patients that await effective drugs to rectify the fundamental defects caused by PTC. In this study, we employed biophysical and biochemical techniques to characterize the pharmacological responses of the translational products of G542X-CFTR to a range of new CFTR modulators. Specifically, we identified two different proteins translated from the G542X-CFTR cDNA using western blotting: the C-terminus truncated protein that responds to the C1 corrector which binds to the N-terminal part of the protein and a full-length CFTR protein through the read-through process. Electrophysiological data suggest that the read-through protein, but not the C-terminus truncated one, is functional and responds well to CFTR potentiators despite a lower open probability compared to wild-type CFTR. As the expression of the read-through products can be increased synergistically with the read-through reagent G418 and C1 corrector, but not with combinations of different types of correctors, we concluded that an efficacious read-through reagent is a prerequisite for mitigating the deficits of G542X-CFTR. Moreover, the CFTR potentiators may help improve the effectiveness of future combinational therapy for patients carrying PTCs such as G542X.

Degradation of eukaryotic RNAs that contain premature termination codons (PTC) during nonsense‐mediated mRNA decay (NMD) is initiated by RNA decapping or endonucleolytic cleavage driven by conserved factors. Models for NMD mechanisms, including recognition of PTCs or the timing and role of protein phosphorylation for RNA degradation are challenged by new results. For example, the depletion of the SMG5/7 heterodimer, thought to activate RNA degradation by decapping, leads to a phenotype showing a defect of endonucleolytic activity of NMD complexes. This phenotype is not correlated to a decreased binding of the endonuclease SMG6 with the core NMD factor UPF1, suggesting that it is the result of an imbalance between active (e.g., in polysomes) and inactive (e.g., in RNA‐protein condensates) states of NMD complexes. Such imbalance between multiple complexes is not restricted to NMD and should be taken into account when establishing causal links between gene function perturbation and observed phenotypes. [ABSTRACT FROM AUTHOR]

• •This study provide evidence that c.683T > A and c.844C > T in SMN1 leading to SMA trigger NMD using a minigene system. • •The nonsense mutation c.43C > T in SMN1 minigene escapes NMD. • •This study highlights the importance of considering NMD when exploring the pathogenetic mechanisms for these mutations. Spinal muscular atrophy (SMA) is an autosomal recessive neurodegenerative disorder caused by SMN1 gene mutations. About 40% of SMN1 subtle mutations produced premature termination codons (PTC). This study aims to determine the capacity of these PTCs to trigger nonsense-mediated mRNA decay (NMD) pathway. Three nonsense mutations in SMN1 , including c.43C > T, c.683T > A and c.844C > T, were investigated by using a minigene system and in vivo splicing assays. Two strategies were supplied: administration of cycloheximide (NMD inhibitor) and knockdown of UPF1 (a key NMD factor) in the cells carrying different minigenes. The wild-type minigene exclusively produced correctly spliced transcripts (FL -SMN1). Both the 683T > A and 844C > T expressed remarkably lower FL -SMN1 than the wild-type cells. After cycloheximide treatment, the FL -SMN1 levels in both the 683T > A and 844C > T were increased significantly compared with that of untreated cells. UPF1 knockdown in both the mutant 683T > A and 844C > T caused a dramatically augmentation of FL -SMN1 as compared to that in the cells treated with non-specific control siRNAs. Our data provide evidence that c.683T > A and c.844C > T, but not c.43C > T, in SMN1 leading to SMA trigger NMD using a minigene system. Therefore, NMD should be taken into consideration when exploring the pathogenetic mechanisms for these mutations. [ABSTRACT FROM AUTHOR]

• Pharmacological repair of CFTR function beyond F508del/F508del-VX809/VX770 in intestinal organoids with homozygous PTC mutations. • The read-through agent ELXds-02, the NMD-inhibitor SMG1i and the CFTR modulators therapy VX-661/VX-445/VX-770 were required for maximal efficacy. • W1282X-CFTR function is partially rescued with the NMD-inhibitor SMG1i combined with the CFTR-modulators VX-661/VX-445/VX770. Background: Pharmacotherapies for people with cystic fibrosis (pwCF) who have premature termination codons (PTCs) in the cystic fibrosis transmembrane conductance regulator (CFTR) gene are under development. Thus far, clinical studies focused on compounds that induce translational readthrough (RT) at the mRNA PTC location. Recent studies using primary airway cells showed that PTC functional restoration can be achieved through combining compounds with multiple mode-of-actions. Here, we assessed induction of CFTR function in PTC-containing intestinal organoids using compounds targeting RT, nonsense mRNA mediated decay (NMD) and CFTR protein modulation. Methods: Rescue of PTC CFTR protein was assessed by forskolin-induced swelling of 12 intestinal organoid cultures carrying distinct PTC mutations. Effects of compounds on mRNA CFTR level was assessed by RT-qPCRs. Results: Whilst response varied between donors, significant rescue of CFTR function was achieved for most donors with the quintuple combination of a commercially available pharmacological equivalent of the RT compound (ELX-02-disulfate or ELX-02ds), NMD inhibitor SMG1i, correctors VX-445 and VX-661 and potentiator VX-770. The quintuple combination of pharmacotherapies reached swelling quantities higher than the mean swelling of three VX-809/VX-770-rescued F508del/F508del organoid cultures, indicating level of rescue is of clinical relevance as VX-770/VX-809-mediated F508del/F508del rescue in organoids correlate with substantial improvement of clinical outcome. Conclusions: Whilst variation in efficacy was observed between genotypes as well as within genotypes, the data suggests that strong pharmacological rescue of PTC requires a combination of drugs that target RT, NMD and protein function. [ABSTRACT FROM AUTHOR]

Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNAPyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNAPyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine. [ABSTRACT FROM AUTHOR]

Abstract Background Frontotemporal lobar degeneration (FTLD) is a devastating and progressive disorder, and a common cause of early onset dementia. Progranulin (PGRN) haploinsufficiency due to autosomal dominant mutations in the progranulin gene (GRN) is an important cause of FTLD (FTLD-GRN), and nearly a quarter of these genetic cases are due to a nonsense mutation. Premature termination codons (PTC) can be therapeutically targeted by compounds allowing readthrough, and aminoglycoside antibiotics are known to be potent PTC readthrough drugs. Restoring endogenous PGRN through PTC readthrough has not previously been explored as a therapeutic intervention in FTLD. Methods We studied whether the aminoglycoside G418 could increase PGRN expression in HEK293 and human induced pluripotent stem cell (hiPSC)-derived neurons bearing the heterozygous S116X, R418X, and R493X pathogenic GRN nonsense mutations. We further tested a novel substituted phthalimide PTC readthrough enhancer in combination with G418 in our cellular models. We next generated a homozygous R493X knock-in hiPSC isogenic line (R493X−/− KI), assessing whether combination treatment in hiPSC-derived neurons and astrocytes could increase PGRN and ameliorate lysosomal dysfunction relevant to FTLD-GRN. To provide in vivo proof-of-concept of our approach, we measured brain PGRN after intracerebroventricular administration of G418 in mice expressing the V5-tagged GRN nonsense mutation R493X. Results The R418X and R493X mutant GRN cell lines responded to PTC readthrough with G418, and treatments increased PGRN levels in R493X−/− KI hiPSC-derived neurons and astrocytes. Combining G418 with a PTC readthrough enhancer increased PGRN levels over G418 treatment alone in vitro. PGRN deficiency has been shown to impair lysosomal function, and the mature form of the lysosomal protease cathepsin D is overexpressed in R493X−/− KI neurons. Increasing PGRN through G418-mediated PTC readthrough normalized this abnormal lysosomal phenotype in R493X−/− KI neuronal cultures. A single intracerebroventricular injection of G418 induced GRN PTC readthrough in 6-week-old AAV-GRN-R493X-V5 mice. Conclusions Taken together, our findings suggest that PTC readthrough may be a potential therapeutic strategy for FTLD caused by GRN nonsense mutations.

Rind color is an economically important agronomic trait in eggplant that impacts consumer preferences. In this study, bulked segregant analysis and competitive allele-specific PCR were employed to identify the candidate gene for eggplant rind color through constructing a 2794 F2 population generated from a cross between “BL01” (green pericarp) and “B1” (white pericarp). Genetic analysis of rind color revealed that a single dominant gene controls green color of eggplant peel. Pigment content measurement and cytological observations demonstrated that chlorophyll content and chloroplast number in BL01 were higher than in B1. A candidate gene (EGP19168.1) was fine-mapped to a 20.36 Kb interval on chromosome 8, which was predicted to encode the two-component response regulator-like protein Arabidopsis pseudo-response regulator2 (APRR2). Subsequently, allelic sequence analysis revealed that a SNP deletion (ACT→AT) in white-skinned eggplant led to a premature termination codon. Genotypic validation of 113 breeding lines using the Indel marker closely linked to SmAPRR2 could predict the skin color (green/white) trait with an accuracy of 92.9%. This study will be valuable for molecular marker-assisted selection in eggplant breeding and provides theoretical foundation for analyzing the formation mechanism of eggplant peel color.

Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren’s mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren’s binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren’s low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity.

Nonsense mutations account for 12 % of cystic fibrosis (CF) cases. The presence of a premature termination codon (PTC) leads to gene inactivation, which can be countered by the use of drugs stimulating PTC readthrough, restoring production of the full-length protein. We recently identified a new readthrough inducer, TLN468, more efficient than gentamicin. We measured the readthrough induced by these two drugs with different cystic fibrosis transmembrane conductance regulator (CFTR) PTCs. We then determined the amino acids inserted at the S1196X, G542X, W846X and E1417X PTCs of CFTR during readthrough induced by gentamicin or TLN468. TLN468 significantly promoted the incorporation of one specific amino acid, whereas gentamicin did not greatly modify the proportions of the various amino acids incorporated relative to basal conditions. The function of the engineered missense CFTR channels corresponding to these four PTCs was assessed with and without potentiator. For the recoded CFTR, except for E1417Q and G542W, the PTC readthrough induced by TLN468 allowed the expression of CFTR variants that were correctly processed and had significant activity that was enhanced by CFTR modulators. These results suggest that it would be relevant to assess the therapeutic benefit of TLN468 PTC suppression in combination with CFTR modulators in preclinical assays., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Author(s). Published by Elsevier B.V. All rights reserved.)

Introduction: Epidermolysis bullosa (EB) describes a family of rare genetic blistering skin disorders. Various subtypes are clinically and genetically heterogeneous, and a lethal postpartum form of EB is the generalized severe junctional EB (gs-JEB). gs-JEB is mainly caused by premature termination codon (PTC) mutations in the skin anchor protein LAMB3 (laminin subunit beta-3) gene. The ribosome in majority of translational reads of LAMB3PTC mRNA aborts protein synthesis at the PTC signal, with production of a truncated, nonfunctional protein. This leaves an endogenous readthrough mechanism needed for production of functional full-length Lamb3 protein albeit at insufficient levels. Here, we report on the development of drugs targeting ribosomal protein L35 (rpL35), a ribosomal modifier for customized increase in production of full-length Lamb3 protein from a LAMB3PTC mRNA. Methods: Molecular docking studies were employed to identify small molecules binding to human rpL35. Molecular determinants of small molecule binding to rpL35 were further characterized by titration of the protein with these ligands as monitored by nuclear magnetic resonance (NMR) spectroscopy in solution. Changes in NMR chemical shifts were used to map the docking sites for small molecules onto the 3D structure of the rpL35. Results: Molecular docking studies identified 2 FDA-approved drugs, atazanavir and artesunate, as candidate small-molecule binders of rpL35. Molecular interaction studies predicted several binding clusters for both compounds scattered throughout the rpL35 structure. NMR titration studies identified the amino acids participating in the ligand interaction. Combining docking predictions for atazanavir and artesunate with rpL35 and NMR analysis of rpL35 ligand interaction, one binding cluster located near the N-terminus of rpL35 was identified. In this region, the nonidentical binding sites for atazanavir and artesunate overlap and are accessible when rpL35 is integrated in its natural ribosomal environment. Conclusion: Atazanavir and artesunate were identified as candidate compounds binding to ribosomal protein rpL35 and may now be tested for their potential to trigger a rpL35 ribosomal switch to increase production of full-length Lamb3 protein from a LAMB3PTC mRNA for targeted systemic therapy in treating gs-JEB. [ABSTRACT FROM AUTHOR]

Mutations in the MMADHC gene cause cobalamin D disorder (cblD), an autosomal recessive inborn disease with defects in intracellular cobalamin (cbl, vitamin B12) metabolism. CblD patients present methylmalonic aciduria (MMA), homocystinuria (HC), or combined MMA/HC, and usually suffer developmental delay and cognitive deficits. The most frequent MMADHC genetic alterations associated with disease generate MMADHC truncated proteins, in many cases due to mutations that create premature termination codons (PTC). In this study, we have performed a comprehensive and global characterization of MMADHC protein variants generated by all annotated MMADHC PTC mutations in cblD patients, and analyzed the potential of inducible translational PTC readthrough to reconstitute MMADHC biosynthesis. MMADHC protein truncation caused by disease-associated PTC differentially affected the alternative usage of translation initiation sites, protein abundance, and subcellular localization of MMADHC. Aminoglycoside compounds induced translational PTC readthrough of MMADHC truncated variants, allowing the biosynthesis of full-length MMADHC in a PTC-specific manner. Our results suggest that translational PTC readthrough-based interventions could complement current therapies for cblD patients carrying specific MMADHC PTC mutations.

The PTEN tumor suppressor gene is mutated with high incidence in tumors and in the germline of patients with cancer predisposition or with macrocephaly associated with autism. PTEN nonsense mutations generating premature termination codons (PTC) and producing nonfunctional truncated PTEN proteins are frequent in association with human disease. However, there are no studies addressing the restoration of full‐length PTEN proteins from the PTC‐mutated PTEN gene by translational readthrough. Here, we have performed a global translational and functional readthrough analysis of the complete collection of PTEN PTC somatic or hereditary mutations found in tumors or in the germline of patients (disease‐associated PTEN PTCome), and we set standards for the analysis of the potential of readthrough functional reconstitution in disease‐relevant genes. Our analysis indicates that prevalent pathogenic PTEN PTC mutations are susceptible to PTEN functional restoration in response to readthrough‐inducing compounds. Comprehensive readthrough analyses of disease‐associated PTComes will be valuable tools for the implementation of readthrough‐based precision interventions in specific groups of patients. [ABSTRACT FROM AUTHOR]

Despite many efforts and diverse approaches, developing an effective herpesvirus vaccine remains a great challenge. Traditional inactivated and live-attenuated vaccines always raise efficacy or safety concerns. This study used Pseudorabies virus (PRV), a swine herpes virus, as a model. We attempted to develop a live but replication-incompetent PRV by genetic code expansion (GCE) technology. Premature termination codon (PTC) harboring PRV was successfully rescued in the presence of orthogonal system MbpylRS/tRNAPyl pair and unnatural amino acids (UAA). However, UAA incorporating efficacy seemed extremely low in our engineered PRV PTC virus. Furthermore, we failed to establish a stable transgenic cell line containing orthogonal translation machinery for PTC virus replication, and we demonstrated that orthogonal tRNAPyl is a key limiting factor. This study is the first to demonstrate that orthogonal translation system-mediated amber codon suppression strategy could precisely control PRV-PTC engineered virus replication. To our knowledge, this is the first reported PTC herpesvirus generated by GCE technology. Our work provides a proof-of-concept for generating UAAs-controlled PRV-PTC virus, which can be used as a safe and effective vaccine.

Recognition of the stop codon by the translation machinery is essential to terminating translation at the right position and to synthesizing a protein of the correct size. Under certain conditions, the stop codon can be recognized as a coding codon promoting translation, which then terminates at a later stop codon. This event, called stop codon readthrough, occurs either by error, due to a dedicated regulatory environment leading to generation of different protein isoforms, or through the action of a readthrough compound. This review focuses on the mechanisms of stop codon readthrough, the nucleotide and protein environments that facilitate or inhibit it, and the therapeutic interest of stop codon readthrough in the treatment of genetic diseases caused by nonsense mutations. [ABSTRACT FROM AUTHOR]

McArdle disease is an autosomal recessive disorder caused by the absence of muscle glycogen phosphorylase, which leads to blocked muscle glycogen breakdown. We used three different cellular models to evaluate the efficiency of different read-through agents (including amlexanox, Ataluren, RTC13 and G418) in McArdle disease. The first model consisted of HeLa cells transfected with two different GFP-PYGM constructs presenting the Pygm p.R50X mutation (GFP-PYGM p.R50X and PYGM Ex1-GFP p.R50X). The second cellular model was based on the creation of HEK293T cell lines stably expressing the PYGM Ex1-GFP p.R50X construct. As these plasmids encode murine Pygm cDNA without any intron sequence, their transfection in cells would allow for analysis of the efficacy of read-through agents with no concomitant nonsense-mediated decay interference. The third model consisted of skeletal muscle cultures derived from the McArdle mouse model (knock-in for the p.R50X mutation in the Pygm gene). We found no evidence of read-through at detectable levels in any of the models evaluated. We performed a literature search and compared the premature termination codon context sequences with reported positive and negative read-through induction, identifying a potential role for nucleotide positions −9, −8, −3, −2, +13 and +14 (the first nucleotide of the stop codon is assigned as +1). The Pygm p.R50X mutation presents TGA as a stop codon, G nucleotides at positions −1 and −9, and a C nucleotide at −3, which potentially generate a good context for read-through induction, counteracted by the presence of C at −2 and its absence at +4.

Abstract Background The chloroplast of eukaryotic microalgae such as Chlamydomonas reinhardtii is a potential platform for metabolic engineering and the production of recombinant proteins. In industrial biotechnology, inducible expression is often used so that the translation or function of the heterologous protein does not interfere with biomass accumulation during the growth stage. However, the existing systems used in bacterial or fungal platforms do not transfer well to the microalgal chloroplast. We sought to develop a simple inducible expression system for the microalgal chloroplast, exploiting an unused stop codon (TGA) in the plastid genome. We have previously shown that this codon can be translated as tryptophan when we introduce into the chloroplast genome a trnW UCA gene encoding a plastidial transfer RNA with a modified anticodon sequence, UCA. Results A mutated version of our trnW UCA gene was developed that encodes a temperature-sensitive variant of the tRNA. This allows transgenes that have been modified to contain one or more internal TGA codons to be translated differentially according to the culture temperature, with a gradient of recombinant protein accumulation from 35 °C (low/off) to 15 °C (high). We have named this the CITRIC system, an acronym for cold-inducible translational readthrough in chloroplasts. The exact induction behaviour can be tailored by altering the number of TGA codons within the transgene. Conclusions CITRIC adds to the suite of genetic engineering tools available for the microalgal chloroplast, allowing a greater degree of control over the timing of heterologous protein expression. It could also be used as a heat-repressible system for studying the function of essential native genes in the chloroplast. The genetic components of CITRIC are entirely plastid-based, so no engineering of the nuclear genome is required.

Abstract Premature termination codons (PTCs) in the coding regions of mRNA lead to the incorrect termination of translation and generation of non-functional, truncated proteins. Translational readthrough of PTCs induced by pharmaceutical compounds is a promising way of restoring functional protein expression and reducing disease symptoms, without affecting the genome or transcriptome of the patient. While in some cases proven effective, the clinical use of readthrough-inducing compounds is still associated with many risks and difficulties. This review focuses on problems directly associated with compounds used to stimulate PTC readthrough, such as their interactions with the cell and organism, their toxicity and bioavailability (cell permeability; tissue deposition etc.). Various strategies designed to overcome these problems are presented.

Background: BReast CAncer gene 2 (BRCA2), a tumor suppressor gene located on chromosome 13q, encodes a 384-kDa protein which activates homologous recombination pathway to repair ssDNA damage. Aims and Objectives: Keeping in view the high prevalence of breast cancer in Pakistan and significant association of BRCA2 with breast cancer, this project was initiated to investigate the mutational status of BRCA2 in Pakistani breast cancer patients. Materials and Methods: For this purpose, blood samples of 45 individuals, including 24 female patients (infiltrating ductal carcinoma of breast), who visited Institute of Nuclear Medicine, Oncology and Radiotherapy Hospital and Ayub Medical Complex, Abbottabad, Khyber Pakhtunkhwa, and 21 normal female residents of the area were collected and processed to extract deoxyribonucleic acid (DNA). Different regions of BRCA2 exon 11 were amplified through polymerase chain reaction (PCR), and PCR-amplified products of one (NF45) normal sample and four cancerous (205BC, 215BC, 218BC, 222BC) samples were subjected to DNA sequence analysis. Results: Analysis of retrieved sequences revealed one novel nonsense mutation in sample 205BC. The observed mutation (delA21587) shifted the normal frame of amino acid (N905I, T906L, K907R, E908N, L909F, H910M, E911K, T912Q, and D913T) in encoded mutant protein and converted L914 into premature termination codon. In case of sample 222BC, another novel substitution mutation (A>G24962) was observed, which altered codon of I (isoleucine) into the codon for M (methionine) at position 2040 in resultant mutant protein. Conclusion: The results reflect the unique mutational profile of BRCA2 in Pakistani infiltrating ductal carcinoma patients and suggest an extension of study on a large scale. [ABSTRACT FROM AUTHOR]

Alternative splicing (AS) plays pivotal roles in regulating plant growth and development, flowering, biological rhythms, signal transduction, and stress responses. However, no studies on AS have been performed in Liriodendron chinense , a deciduous tree species that has high economic and ecological value. In this study, we used multiple tools and algorithms to analyze transcriptome data derived from seven tissues via hybrid sequencing. Although only 17.56% (8,503/48,408) of genes in L. chinense were alternatively spliced, these AS genes occurred in 37,844 AS events. Among these events, intron retention was the most frequent AS event, producing 1,656 PTC-containing and 3,310 non-PTC-containing transcripts. Moreover, 183 long noncoding RNAs (lncRNAs) also underwent AS events. Furthermore, weighted gene coexpression network analysis (WGCNA) revealed that there were great differences in the activities of transcription and post-transcriptional regulation between pistils and leaves, and AS had an impact on many physiological and biochemical processes in L. chinense , such as photosynthesis, sphingolipid metabolism, fatty acid biosynthesis and metabolism. Moreover, our analysis showed that the features of genes may affect AS, as AS genes and non-AS genes had differences in the exon/intron length, transcript length, and number of exons/introns. In addition, the structure of AS genes may impact the frequencies and types of AS because AS genes with more exons or introns tended to exhibit more AS events, and shorter introns tended to be retained, whereas shorter exons tended to be skipped. Furthermore, eight AS genes were verified, and the results were consistent with our analysis. Overall, this study reveals that AS and gene interaction are mutual—on one hand, AS can affect gene expression and translation, while on the other hand, the structural characteristics of the gene can also affect AS. This work is the first to comprehensively report on AS in L. chinense , and it can provide a reference for further research on AS in L. chinense. [ABSTRACT FROM AUTHOR]

The readthrough of premature termination codon (PTC) by ribosome sometimes produces full-length proteins. We previously reported a readthrough of PTC of glycosyltransferase gene B4GALNT1 with hereditary spastic paraplegia (HSP). Here we featured the readthrough of B4GALNT1 of two mutants, M4 and M2 with PTC by immunoblotting and flow cytometry after transfection of B4GALNT1 cDNAs into cells. Immunoblotting showed a faint band of full-length mutant protein of M4 but not M2 at a similar position with that of wild-type B4GALNT1. AGC sequences at immediately before and after the PTC in M4 were critical for the readthrough. Treatment of cells transfected with mutant M4 cDNA with aminoglycosides resulted in increased readthrough of PTC. Furthermore, treatment of transfectants of mutant M2 cDNA with G418 also resulted in the induction of readthrough of PTC. Both M4 and M2 cDNA transfectants showed increased/induced bands in immunoblotting and GM2 expression in a dose-dependent manner of aminoglycosides. Results of mass spectrometry supported this effect. Here, we showed for the first time the induction and/or enhancement of the readthrough of PTCs of B4GALNT1 by aminoglycoside treatment, suggesting that aminoglycosides are efficient for patients with HSP caused by PTC of B4GALNT1 , in which gradual neurological disorders emerged with aging. [ABSTRACT FROM AUTHOR]

• The compound heterozygous mutations c.3722_3749dup[p.V1252fs*23] and c.3355 + 5 G > A at CUL7 gene were detected. • The mutation c.3355 + 5 G > A was a novel mutation within intron 17 of the CUL7 gene. • The results showed that the mutation could result in the appearance of premature termination codon. • Our study enriched gene mutations of 3 M syndrome. A couple with five adverse pregnancy history required prenatal diagnosis. The fetus of this study was their fifth pregnancy. The fetus was found NT thickening at 12 weeks and 4 days gestation and the average long bone of limbs retardation 4SD at 27 weeks and 4 days gestation. Karyotype was normal. The next-generation sequencing (NGS) and Sanger sequencing were conducted of this fetus. The compound heterozygous mutations c.3722_3749dup[p.V1252fs*23] and c.3355 + 5 G > A at CUL7 gene were detected. The mutation c.3355 + 5 G > A was a novel mutation within intron 17 of the CUL7 gene. Minigene array was used to verify whether the novel mutation c.3355 + 5 G > A really affected the splicing of CUL7gene. The results showed that the mutation could result in the appearance of premature termination codon. The fetus could be diagnosed as 3 M syndrome. We suggested that close attention needed to be paid to fetuses with intrauterine growth restriction only by ultrasonic and avoid misdiagnosis and missed diagnosis of 3 M syndrome. In addition, our study enriched gene mutations of 3 M syndrome. [ABSTRACT FROM AUTHOR]

Hirschsprung disease (HSCR) is a genetic disorder characterized by the absence of ganglion cells in the gut. RET is considered to be the main susceptibility gene. In our previous screening of 83 HSCR patients, targeted exome sequencing identified nine rare variants of RET, most of which were new discoveries. Here, we performed in vitro arrays with functional studies to investigate their effects. Two variants (p.R77C and p.R67insL) were demonstrated to disrupt the glycosylation of RET and affect its subcellular localization. Three nonsense mutations (p.W85X, p.E252X, and p.Y263X) could not produce detectable RET full-length protein, and the other three mutations (p.R770X, p.Q860X, and p.V778Afs*1) were translated into truncated proteins of predicted sizes. One canonical splice acceptor site mutation (c.2802-2 A > G) was verified to affect gene regulation through aberrant splicing. In addition, we explored the effects of read-through reagents on RET nonsense mutations and showed that G418 significantly increased the full-length RET protein expression of p.Y263X in a dose-dependent manner, together with a mild recovery of p-ERK and p-STAT3. Our data provide a functional analysis of novel RET mutations and suggest that all of the rare variants detected from patients with clinically severe HSCR are indeed pathogenic. Thus, our findings have implications for proper genetic counseling.

Junctional epidermolysis bullosa (JEB) is a hereditary blistering disease caused by reduced dermal-epidermal adhesion due to deficiencies of one of the proteins, laminin-332, type XVII collagen, integrin α6β4 or integrin α3. Significant progress has been achieved in the development of therapies for EB, such as bone-marrow transplantation, local or systemic injections with fibroblasts or mesenchymal stromal cells, readthrough of premature termination codons, or exon skipping. These were tailored in particular for dystrophic EB, which is caused by type VII collagen deficiency and have not yet reached broad clinical practice. Recently, pioneering combined gene and stem cell therapy was successful in treating one boy with junctional EB. Beside these exclusive approaches, no specific therapy to amend the major clinical features, skin and mucosal blistering and non-healing wounds is available to date. Here we extend the mutational spectrum of junctional EB, provide a stratification of COL17A1 mutations and discuss potential molecular therapeutic approaches.

Nonsense mutations generating premature termination codons (PTCs) in various genes are frequently associated with somatic cancer and hereditary human diseases since PTCs commonly generate truncated proteins with defective or altered function. Induced translational readthrough during protein biosynthesis facilitates the incorporation of an amino acid at the position of a PTC, allowing the synthesis of a complete protein. This may evade the pathological effect of the PTC mutation and provide new therapeutic opportunities. Several protein tyrosine phosphatases (PTPs) genes are targeted by PTC in human disease, the tumor suppressor PTEN being the more prominent paradigm. Here, using PTEN and laforin as examples, two PTPs from the dual-specificity phosphatase subfamily, we describe methodologies to analyze in silico the distribution and frequency of pathogenic PTC in PTP genes. We also summarize laboratory protocols and technical notes to study the induced translational readthrough reconstitution of the synthesis of PTP targeted by PTC in association with disease in cellular models., (© 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Primary ciliary dyskinesia (PCD) is a rare disease with autosomal recessive inheritance, caused mostly by bi-allelic gene mutations that impair motile cilia structure and function. Currently, there are no causal treatments for PCD. In many disease models, translational readthrough of premature termination codons (PTC-readthrough) induced by aminoglycosides has been proposed as an effective way of restoring functional protein expression and reducing disease symptoms. However, variable outcomes of pre-clinical trials and toxicity associated with long-term use of aminoglycosides prompt the search for other compounds that might overcome these problems. Because a high proportion of PCD-causing variants are nonsense mutations, readthrough therapies are an attractive option. We tested a group of chemical compounds with known PTC-readthrough potential (ataluren, azithromycin, tylosin, amlexanox, and the experimental compound TC007), collectively referred to as non-aminoglycosides (NAGs). We investigated their PTC-readthrough efficiency in six PTC mutations found in Polish PCD patients, in the context of cell and cilia health, and in comparison to the previously tested aminoglycosides. The NAGs did not compromise the viability of the primary nasal respiratory epithelial cells, and the ciliary beat frequency was retained, similar to what was observed for gentamicin. In HEK293 cells transfected with six PTC-containing inserts, the tested compounds stimulated PTC-readthrough but with lower efficiency than aminoglycosides. The study allowed us to select compounds with minimal negative impact on cell viability and function but still the potential to induce PTC-readthrough.

Hirschsprung disease (HSCR) is a genetic disorder characterized by the absence of ganglion cells in the gut. RET is considered to be the main susceptibility gene. In our previous screening of 83 HSCR patients, targeted exome sequencing identified nine rare variants of RET , most of which were new discoveries. Here, we performed in vitro arrays with functional studies to investigate their effects. Two variants (p.R77C and p.R67insL) were demonstrated to disrupt the glycosylation of RET and affect its subcellular localization. Three nonsense mutations (p.W85X, p.E252X, and p.Y263X) could not produce detectable RET full-length protein, and the other three mutations (p.R770X, p.Q860X, and p.V778Afs*1) were translated into truncated proteins of predicted sizes. One canonical splice acceptor site mutation (c.2802-2 A > G) was verified to affect gene regulation through aberrant splicing. In addition, we explored the effects of read-through reagents on RET nonsense mutations and showed that G418 significantly increased the full-length RET protein expression of p.Y263X in a dose-dependent manner, together with a mild recovery of p-ERK and p-STAT3. Our data provide a functional analysis of novel RET mutations and suggest that all of the rare variants detected from patients with clinically severe HSCR are indeed pathogenic. Thus, our findings have implications for proper genetic counseling. [ABSTRACT FROM AUTHOR]

Assessment of approved drugs and developmental drug candidates for rare cystic fibrosis (CF)-causing variants of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) requires abundant material from relevant models. Isogenic cell lines harboring CFTR variants in the native genomic context were created through the development and utilization of a footprint-less, CRISPR/Cas9 gene editing pipeline in 16HBE14o- immortalized bronchial epithelial cells. Isogenic, homozygous cell lines for three CFTR variants (F508del and the two most common CF-causing nonsense variants, G542X and W1282X) were established and characterized. The F508del model recapitulates the known molecular pathology and pharmacology. The two models of nonsense variants (G542X and W1282X) are sensitive to Nonsense Mediated mRNA Decay (NMD) and responsive to reference compounds that inhibit NMD and promote ribosomal readthrough. We present a versatile, efficient gene editing pipeline that can be used to create CFTR variants in the native genomic context and the utilization of this pipeline to create homozygous cell models for the CF-causing variants F508del, G542X, and W1282X. The resulting cell lines provide a virtually unlimited source of material with specific pathogenic mutations that can be used in a variety of assays, including functional assays. • A gene editing pipeline was developed to model CFTR variants in the genomic context. • The model of F508del/M470 responds to known pharmacological agents. • The G542X and W1282X models recapitulate non-sense mediated mRNA decay. • The G542X and W1282X models are sensitive to small molecule induced read-through. [ABSTRACT FROM AUTHOR]