ARTICLE | doi:10.20944/preprints202103.0676.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Neural network; MET; exon skipping
Online: 26 March 2021 (16:29:10 CET)
Background: Disruption of alternative splicing (AS) is frequently observed in cancer and it might represent an important signature for tumor progression and therapy. Exon skipping (ES) represents one of the most frequent AS events and in non-small cell lung cancer (NSCLC) MET exon 14 skipping was shown to be targetable. Methods: We constructed a neural network (NN) specifically designed to detect MET exon 14 skipping events using RNAseq data. Furthermore, for discovery purpose we also developed a sparsely connected autoencoder to identify uncharacterized MET isoforms. Results: The NN had 100% Met exon 14 skipping detection rate, when tested on a manually curated set of 690 TCGA bronchus and lung samples. When globally applied to 2605 TCGA samples, we observed that the majority of false positives was characterized by a blurry coverage of exon 14, but interesting they share a common coverage peak in the second intron and we speculate that this event could be the transcription signature of a LINE1-MET fusion. Conclusions: Taken together our results indicate that neural networks can be an effective tool to provide a quick classification of pathological transcription events and sparsely connected autoencoders could represent the basis for the development of an effective discovery tool.
ARTICLE | doi:10.20944/preprints202101.0023.v1
Subject: Medicine And Pharmacology, Immunology And Allergy Keywords: Familial Adenomatous Polyposis; APC; Splicing; Exon Skipping; FAP Pathogenesis
Online: 4 January 2021 (11:59:26 CET)
Familial adenomatous polyposis (FAP) is caused by germline mutations in the tumor suppressor gene APC. To date, nearly 2000 APC mutations have been described in FAP, most of which are predicted to result in truncated protein products. Mutations leading to aberrant APC splicing have rarely been reported. Here, we characterized a novel germline heterozygous splice donor site mutation in APC exon 12 (NM_000038.5: c.1621_1626+7del) leading to exon 12 skipping in an Italian family with the attenuated FAP (AFAP) phenotype. Moreover, we performed a literature me-ta-analysis of APC splicing mutations. We found that 123 unique APC splice site mutations, in-cluding the one described here, have been reported in FAP patients, 69 of which have been char-acterized at the mRNA level. Among these, only a small proportion (9/69) results in an in-frame protein, with 4 mutations causing skipping of exon 12 and/or 13 with loss of armadillo repeat 2 (ARM2) and 3 (ARM3), and 5 mutations leading to skipping of exon 5, 7, 8, and (partially) 9 with loss of regions not encompassing known functional domains. The APC splicing mutations considered in this study cluster with the AFAP phenotype and delineate a novel molecular mechanism of pathogenesis in FAP disease.
ARTICLE | doi:10.20944/preprints202303.0389.v1
Subject: Computer Science And Mathematics, Artificial Intelligence And Machine Learning Keywords: antisense oligonucleotides; exon skipping; machine learning; ensemble learning; personalized medicine; n-of-1 therapy, splice switching; genetic disease; splicing; RNA
Online: 22 March 2023 (03:24:48 CET)
Antisense oligonucleotide (ASO)-mediated exon skipping has become a valuable tool for investigating gene function and developing gene therapy. Machine learning-based computational methods such as eSkip-Finder have been developed to predict the efficacy of ASOs via exon skipping. However, these methods are computationally demanding, and the accuracy of predictions remains suboptimal. In this study, we propose a new approach to reduce computational burden and improve prediction performance by using feature selection within machine learning algorithms and ensemble learning techniques. We evaluated our approach using a dataset of experimentally validated exon skipping events, dividing it into training and testing sets. Our results demonstrate that using a 3-way voting approach with random forest, gradient boosting, and XGBoost can significantly reduce computation time to under ten seconds while improving prediction performance, as measured by R2 for both 2’-O-methyl nucleotides (2OMe) and phosphorodiamidate morpholino oligomers (PMOs). Additionally, the feature importance ranking derived from our approach is in good agreement with previously published results. Our findings suggest that our approach has the potential to enhance the accuracy and efficiency of predicting ASO efficacy via exon skipping. It could also facilitate the development of novel therapeutic strategies. This study could contribute to the ongoing efforts to improve ASO design and optimize gene therapy approaches.
REVIEW | doi:10.20944/preprints201811.0018.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Duchenne muscular dystrophy (DMD); CRISPR/Cas9; exon skipping therapy; gene editing; human induced pluripotent stem cells (hiPSCs); immortalized patient muscle cells; mdx mice; humanized dystrophic mouse models; deltaE50-MD dog model
Online: 2 November 2018 (05:14:23 CET)
Duchenne muscular dystrophy (DMD) is a fatal X-linked recessive neuromuscular disease prevalent in 1 in 3500 to 5000 males worldwide. As a result of mutations that interrupt the reading frame of the dystrophin gene (DMD), DMD is characterized by a loss of dystrophin protein which leads to decreased muscle membrane integrity, which increases susceptibility to degeneration. CRISPR/Cas9 technology has garnered interest as an avenue for DMD therapy due to its potential for permanent exon skipping, which can restore the disrupted DMD reading frame in DMD and lead to dystrophin restoration. An RNA-guided DNA endonuclease system, CRISPR/Cas9 allows for the targeted editing of specific sequences in the genome. The efficacy and safety of CRISPR/Cas9 as a therapy for DMD has been evaluated by numerous studies in vitro and in vivo, with varying rates of success. Despite the potential of CRISPR/Cas9-mediated gene editing for the long-term treatment of DMD, its translation into the clinic is currently challenged by issues such as off-targeting, immune response activation, and sub-optimal in vivo delivery. Its nature as being mostly a personalized form of therapy also limits applicability to DMD patients, who exhibit a wide spectrum of mutations. This review summarizes the various CRISPR/Cas9 strategies that have been tested in vitro and in vivo for the treatment of DMD. Perspectives on the approach will be provided, and the challenges faced by CRISPR/Cas9 in its road to the clinic will be briefly discussed.
ARTICLE | doi:10.20944/preprints202303.0167.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: antisense oligonucleotides; exon skipping; machine learning; ensemble learning; personalized medicine; n-of-1 therapy; splice switching; genetic disease; splicing; RNA
Online: 9 March 2023 (04:43:55 CET)
Antisense oligonucleotide (ASO)-mediated exon skipping has emerged as a powerful tool for examining the function of genes and exons in basic research, as well as gene therapy. Computational methods, such as eSkip-Finder, have been developed to predict the efficacy of ASOs via exon skipping using machine learning. However, these methods can be computationally demanding and the prediction accuracy of the tool is not yet optimal. In this study, we propose an approach to reduce computational burden and improve prediction performance by utilizing feature selection within machine learning algorithms and employing ensemble learning techniques. The method was evaluated using a dataset of genes with experimentally validated exon skipping events. The dataset was divided into training and testing sets to assess the accuracy of the algorithm. Our results demonstrate that using a 3-way voting approach with random forest, gradient boosting, and XGBoost can significantly reduce computation time to under ten seconds while improving prediction performance, as measured by R2 for both 2’-O-methyl nucleotides (2OMe) and phosphorodiamidate morpholino oligomers (PMOs). Additionally, the feature importance ranking derived from our approach is in good agreement with previously published results. These findings suggest that this approach has the potential to enhance the efficiency and accuracy of predicting ASO efficacy via exon skipping, facilitating the development of novel therapeutic strategies.
ARTICLE | doi:10.20944/preprints202201.0390.v1
Subject: Medicine And Pharmacology, Oncology And Oncogenics Keywords: non-small cell lung cancer (NSCLC); liquid biopsy; cell-free nucleic acid (cfNA); next generation sequencing (NGS); CNA (copy number amplifications); fusions, exon-skipping.
Online: 26 January 2022 (03:21:12 CET)
Liquid biopsies are an integral part of the diagnosis of cancer. Here, we have extended previous validation studies of a new targeted NGS panel to include the detection of copy number amplifications (CNAs), fusions, and exon skipping variants. Detection of these gene classes included specimens from clinical and healthy donors and cell lines (fusions: ROS1, EML4-ALK, NTRK1), (exon skipping: MET exon 14), and (CNAs: HER2, CDK6, EGFR, MYC and MET). The limit of detection (LOD) for fusion/skipping was 42 copies (QC threshold = 3 copies) and was verified using 3 additional fusion/skipping variants. LOD for CNAs was 1.40-fold-change (QC threshold = 1.15-fold change) and was verified with 3 additional CNAs. In precision studies, all fusion/skipping variants were detected in all runs and all days of testing (n=18/18; 100%); average CV for repeatability was 20.5% (range 8.7% - 34.8%), and for reproducibility was 20.8% (range 15.7% - 30.5%). For CNAs, 28/29 (96.6%) copy gains were detected. For CNAs, average CV of 1.85% (range 0% to 5.49%) for repeatability and 6.59% (range 1.65% to 9.22%) for reproducibility. The test panel meets the criteria for being highly sensitive and specific and extends its utility for the detection of clinically relevant variants in cancer.
ARTICLE | doi:10.20944/preprints202106.0610.v1
Subject: Biology And Life Sciences, Anatomy And Physiology Keywords: phylogenomic analyses; exon sequence; Gadiformes; Lota lota
Online: 25 June 2021 (09:13:35 CEST)
Our understanding of phylogenetic relationships among Gadiformes fish is obtained through the analysis of a small number of genes, but uncertainty remains around critical nodes. A series of phylogenetic controversial exists at the suborder, family, subfamily, and species levels. A total of 1105 orthologous exon sequences and translated amino acid sequences from 36 genomes and 12 transcriptomes covering 33 species were applied to investigate the phylogenetic relationships within Gadiformes and address these problems. Phylogenetic trees reconstructed with the amino acid data set using different tree-building methods (RAxML and MrBayes) showed consistent topology. The monophyly of Gadifromes was confirmed in our study. However, the three suborders Muraenolepidoidei, Macrouroidei, and Gadoidei were not well recovered by our phy-logenomic study, rejecting the validity of suborder Muraenolepidoidei. Four major lineages were revealed in this study. The family Bregmacerotidae forming clade I was the basal lineage of Gadiformes. The family Merluciidae formed clade II. Clade III contained families Melanonidae, Muraenolepididae, Macrouridae (with subfamilies Trachyrincinae, Macrourinae, and Bathygadinae), and Moridae. Clade IV contained at least three families of suborder Gadoidei, i.e., Gadidae, Phycidae, and Ranicipitidae. The subspecies of Lota lota from Amur River were confirmed, indicating that exon markers were a valid high-resolution method for delimiting subspecies or distinct lineages within species level. The PSMC analysis of different populations of L. lota suggests a continuous decline since 2 Myr.
ARTICLE | doi:10.20944/preprints202008.0562.v1
Subject: Medicine And Pharmacology, Oncology And Oncogenics Keywords: mutations; exon; p53; regulation; DNA; tumour suppressor
Online: 26 August 2020 (09:10:14 CEST)
Introduction: The tumour suppressor protein p53 commonly referred to as guardian of the genome plays important role in preserving the genome through the regulation of programmed cell death, DNA repair, energy metabolism, cell cycle entry or exit and senescence. Mutations in p53 can either result to a loss of tumour suppressor function or gain of oncogenic properties. Hence, mutations in p53 are the most frequent genetic mutational alteration in human cancers, associated with worse prognosis and more aggressive disease outcome. Methods: To assess the mutational hotspots and conserved regions of p53, I analyzed 76 complete p53 protein sequences covering whole exons from the NCBI GenBank database. Multiple sequence alignment (MSA) was done using ClustalX version 2.1. Results: Thirty-five (19) mutations were identified with more frequent mutations in amino acid (aa) position 72 and 79 (Exon 4), amino acid deletion in codon 112-122 (Exon 4), codon 213 (Exon 6), codon 248 (Exon 7), codon 273 (Exon 8) and codon 278 (Exon 8). Mutations at amino acid position 79, 248, 278 located in the DNA-binding domain exhibited more than one alteration in same position. Conclusions: Findings from this study revealed the prevalence of mutations in the DNA binding domain of p53 and the structure-function effect of the mutations. Assessing the pattern and frequency of p53 alterations, and analyzing it thoroughly for each carrier, could help in identifying correlations between p53 status and outcome and possible candidate for gene therapy.
ARTICLE | doi:10.20944/preprints202007.0739.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Lung-molGPA; exon 19 deletion; Leu858Arg; rare mutation; prognostic implication
Online: 31 July 2020 (09:30:53 CEST)
EGFR mutations are heterogenous but all carry the same weighting in the Lung-molGPA. The aim of this study was to elucidate the different prognostic implications of molecular subtypes and frontline TKIs in EGFR-mutated lung adenocarcinoma with synchronous brain metastases (BM) using the Lung-molGPA. Medical records were searched in hospital databases from 2011 to 2015. Patients with EGFR-mutated adenocarcinoma and brain metastases who received TKIs were included. The Kaplan-Meier method was used to estimate survival, and multivariate Cox proportional hazard models were used to estimate adjusted hazard ratios (HRs) and 95% confidence intervals (CIs). A total of 256 patients were included with a median overall survival (OS) of 17.2 months. In multivariate analysis of OS, only age (70 versus <70 years, HR:1.71, 95% CI:1.25-2.35, p<0.001), KPS (<70 versus 70, HR:1.71, 95% CI:1.26-2.31, p<0.001), and rare mutations (other versus exon 19 deletions, HR:1.78, 95% CI:1.04-3.05, p=0.037) remained statistically significant. In patients with a Lung-molGPA score 2.5, EGFR molecular subtypes had different median OS (exon 19 deletions versus Leu858Arg versus other, 18.8 vs 12.4 vs 12.1 months, p=0.021). In conclusion, different molecular subtypes treated with frontline TKIs have different prognostic implications in the Lung-molGPA. Further prospective studies are warranted to validate these findings.
REVIEW | doi:10.20944/preprints202201.0265.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: CRISPR; gene editing; Duchenne muscular dystrophy (DMD); exon skipping; NHEJ; dystrophin
Online: 18 January 2022 (17:30:37 CET)
Duchenne muscular dystrophy (DMD) is an X-linked recessive neuromuscular disorder with a prevalence of approximately 1 in 3,500-5,000 males. DMD manifests as childhood-onset muscle degeneration, followed by loss of ambulation, cardiomyopathy, and death in early adulthood due to a lack of functional dystrophin protein. Out-of-frame mutations in the dystrophin gene are the most common underlying cause of DMD. Gene editing via the clustered regularly interspaced short palindromic repeats (CRISPR) system is a promising therapeutic for DMD, as it can permanently correct DMD mutations and thus restore the reading frame, allowing for the production of functional dystrophin. The specific mechanism of gene editing can vary based on a variety of factors such as the number of cuts generated by CRISPR, the presence of an exogenous DNA template, or the current cell cycle stage. CRISPR-mediated gene editing for DMD has been tested both in vitro and in vivo, with many of these studies discussed herein. Additionally, novel modifications to the CRISPR system such as base or prime editors allow for more precise gene editing. Despite recent advances, limitations remain including delivery efficiency, off-target mutagenesis, and long-term maintenance of dystrophin. Further studies focusing on safety and accuracy of the CRISPR system are necessary prior to clinical translation.
ARTICLE | doi:10.20944/preprints202205.0292.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: Meier-Gorlin syndrome; Jeune syndrome; ORC6; exon skipping variant; prenatal genetic testing
Online: 23 May 2022 (10:18:24 CEST)
Meier–Gorlin syndrome (MGS) is a rare genetic developmental disorder that causes primordial proportional dwarfism, microtia, absent or hypoplastic patellae and other skeletal anomalies. Overlapping skeletal symptoms make MGS difficult to diagnose clinically. We describe a 3-year-old boy with short stature, recurrent respiratory infections, short-rib dysplasia, tower head and facial dysmorphisms who was admitted to the Tomsk Genetic Clinic to verify a clinical diagnosis of Jeune syndrome. Clinical Exome sequencing revealed two variants (compound heterozygosity) in the ORC6 gene: c.2T>C(p.Met1Thr) and c.449+5G>A. In silico analysis showed the pathogenicity of these two mutations and predicted a decrease in donor splicing site strength for c.449+5G>A. An in vitro minigene assay demonstrated that variant c.449+5G>A causes a complete skipping of exon 4 in ORC6 gene. The parents asked for urgent prenatal testing for MGS for the next pregnancy, but it ended in a miscarriage. Thus, this case report may help to prevent MGS misdiagnosis in the future. We also performed in silico and functional analyses of ORC6 mutations and developed a restriction fragment length polymorphism and haplotype-based short-tandem-repeat assay for prenatal genetic testing for MGS. These findings should elucidate MGS aetiology and improve the quality of genetic counselling for affected families.
REVIEW | doi:10.20944/preprints202107.0202.v1
Subject: Biology And Life Sciences, Biochemistry And Molecular Biology Keywords: colorblindness; color vision; myopia; cone photopigment; exon skipping; X-linked cone dysfunction
Online: 8 July 2021 (13:27:17 CEST)
The first step in seeing is light absorption by photopigment molecules expressed in the photore-ceptors of the retina. There are two types of photoreceptors in the human retina that are respon-sible for image formation, rods and cones. Except at very low light levels when rods are active, all vision is based on cones. Cones mediate high acuity vision and color vision. Furthermore, they are critically important in the visual feedback mechanism that regulates refractive development of the eye during childhood. The human retina contains a mosaic of three cone types, short-wavelength (S), long-wavelength (L) and middle-wavelength (M); however, the vast major-ity (~94%) are L and M cones. The OPN1LW and OPN1MW genes, located on the X-chromosome at Xq28, encode the protein component of the light-sensitive photopigments. Here we review mechanism by which splicing defects in these genes cause vision disorders.
REVIEW | doi:10.20944/preprints202308.1470.v1
Subject: Biology And Life Sciences, Endocrinology And Metabolism Keywords: Growth hormone receptor (GHR); Human growth hormone; Deletion of exon 3 (d3GHR); Polymorphism; Growth and development; Hormone deficiency
Online: 21 August 2023 (11:59:26 CEST)
Growth hormone (GH) is a peptide hormone that plays a crucial role in controlling growth, development, and lifespan. Molecular regulation of GH is accomplished via the GH receptor (GHR) gene, which is the main factor influencing human development and is essential to optimal functioning of the GH/IGF-I axis. Two GHR isoforms have been studied, according to the presence (flGHR) or absence (d3GHR) of exon 3. The d3GHR variant, which has recently been related to longevity, is associated with enhanced signal transduction and higher receptor function. Many of these studies indicated that the growth response to GH treatment may be affected. Individuals carrying the d3GHR isoform have higher receptor activity, improved signal transduction, and alterations in the treatment response and efficacy compared with those carrying the WT isoform. Further, studies performed in patients with acromegaly, Prader-Willi syndrome, Turner syndrome, small for gestational age (SGA), and growth hormone deficiency (GHD) suggested that the d3GHR variant may have an impact on the relationship between GH and IGF-I levels, height, weight, BMI, and other variables. Other research, however, revealed inconsistent results, which might have been caused by confounding factors, including limited sample sizes and different experimental methods. In this review, we lay out the complexity of the GHR isoforms and provide an overview of the major pharmacogenetic research conducted on this ongoing and unresolved subject.
ARTICLE | doi:10.20944/preprints202306.1818.v1
Subject: Biology And Life Sciences, Biology And Biotechnology Keywords: Alternative Splicing; two-colour (GFP/RFP) Fluorescent Reporter; MAPT; Exon-Skipping; FTDP-17; High Content Screening; siRNA; Nucleic Acids Therapeutics; Drug discovery
Online: 26 June 2023 (13:55:26 CEST)
Nucleic acid therapeutics are witnessing an impressive acceleration in recent years. They work through multiple mechanisms of action, including downregulation of gene expression and modulation of RNA splicing. While several drugs based on the former mechanism have been approved, few target the latter, despite the promise of RNA splicing modulation. To improve our ability to discover novel RNA splicing-modulating therapies, we developed HCS-Splice, a robust cell-based High-Content Screening (HCS) assay. By implementing the use of a two-colour (GFP/RFP) fluorescent splicing reporter plasmid, we developed a versatile, effective, rapid, and robust high-throughput strategy for the identification of potent splicing-modulating molecules. The HCS-Splice strategy can also be used to functionally confirm splicing mutations in human genetic disorders or to screen drug candidates. As a proof-of-concept, we introduced a dementia-related splice-switching mutation in Microtubule-Associated Protein Tau (MAPT) exon 10 splicing reporter. We applied HCS-Splice to the wild-type and mutant reporters and measured the functional change in exon 10 inclusion. To demonstrate the applicability of the method to cell-based drug discovery, HCS-Splice was used to evaluate the efficacy of an exon 10-targeting siRNA, which was able to restore the correct alternative splicing balance.
REVIEW | doi:10.20944/preprints202311.0801.v1
Subject: Medicine And Pharmacology, Neuroscience And Neurology Keywords: Dysferlinopathy; limb-girdle muscular dystrophy recessive type 2 (LGMDR2); Miyoshi myopathy; distal myopathy with anterior tibial onset (DMAT); dysferlin; membrane resealing; genetic therapy; mini-dysferlin; exon skipping
Online: 13 November 2023 (10:41:28 CET)
Dysferlinopathies comprise a spectrum of muscular dystrophies characterized by progressive muscle weakness and degeneration due to mutations in the DYSF gene, which encodes the dysferlin protein critical for muscle membrane repair. This review delves into the clinical spectra of dysferlinopathies, their molecular mechanisms, and the spectrum of emerging therapeutic strategies. We explore the phenotypic heterogeneity of dysferlinopathies, highlight the incomplete understanding of genotype-phenotype correlations, and discuss the implications of various DYSF mutations. Furthermore, we examine the utility of animal models in elucidating disease mechanisms and the potential of symptomatic, pharmacological, molecular, and genetic therapies in mitigating the disease's progression. We also consider the roles of diet and metabolism in managing dysferlinopathies, as well as the impact of clinical trials on treatment paradigms. By culminating the complexities inherent in dysferlinopathies, this article emphasizes the need for multidisciplinary approaches, precision medicine, and extensive collaboration in research and clinical trial design to advance our understanding and treatment of these challenging disorders.