scholarly journals A Comprehensive Analysis and Splicing Characterization of Naturally Occurring Synonymous Variants in the ATP7B Gene

2021 ◽  
Vol 11 ◽  
Author(s):  
Xiaoying Zhou ◽  
Wei Zhou ◽  
Chunli Wang ◽  
Lan Wang ◽  
Yu Jin ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Genetic Diseases ◽  
Genetic Diagnosis ◽  
Exon Skipping ◽  
Computational Prediction ◽  
Regulatory Elements ◽  
Naturally Occurring ◽  
Splicing Regulatory Elements ◽  
The Impact

Next-generation sequencing is effective for the molecular diagnosis of genetic diseases. However, the identification of the clinical significance of synonymous variants remains a challenge. Our previous study showed that some synonymous variants in ATP7B gene produced splicing disruptions, leading to Wilson disease (WD). To test the hypothesis that synonymous variants of ATP7B cause abnormal splicing by disrupting authentic splice sites or splicing regulatory elements, we used computational tools and minigene assays to characterize 253 naturally occurring ATP7B gene synonymous variants in this study. Human Splicing Finder (HSF) and ESE Finder 3.0 were used to predict the impact of these rare synonymous variants on pre-mRNA splicing. Then, we cloned 14 different wild-type Minigene_ATP7B_ex constructs for in vitro minigene assay, including 16 exons of ATP7B gene. After computational prediction, 85 candidate variants were selected to be introduced into the corresponding Minigene_ATP7B_ex constructs for splicing assays. Using this two-step procedure, we demonstrated that 11 synonymous variants in ExAc database (c.1620C>T, c.3888C>T, c.1554C>T, c.1677C>T, c.1830G>A, c.1875T>A, c.2826C>A, c.4098G>A, c.2994C>T, c.3243G>A, and c.3747G>A) disrupted RNA splicing in vitro, and two (c.1620C>T and c.3243G>A) of these caused a complete exon skipping. The results not only provided a reliable experimental basis for the genetic diagnosis of WD patients but also offered some new insights into the pathogenicity of synonymous variants in genetic diseases.

scholarly journals Identification of eight exonic variants in the SLC4A1, ATP6V1B1 and ATP6V0A4 gene that alter RNA splicing by minigene assay

2021 ◽  
Author(s):  
Ruixiao Zhang ◽  
Zeqing Chen ◽  
Qijing Song ◽  
Sai Wang ◽  
Zhiying Liu ◽  
...  
Keyword(s):  
Rna Splicing ◽  
Mrna Level ◽  
Exon Skipping ◽  
Regulatory Elements ◽  
Mrna Splicing ◽  
Splicing Regulatory Elements ◽  
Renal Tubular ◽  
Splicing Site ◽  
Minigene Assay

Primary distal renal tubular acidosis (dRTA) is a rare tubular disease associated with variants in SLC4A1, ATP6V0A4, ATP6V1B1, FOXⅠ1 or WDR72 genes. Currently, there is growing evidence that all types of exonic variants can alter splicing regulatory elements, affecting the pre-mRNA splicing process. This study was to determine the consequences of variants associated with dRTA on pre-mRNA splicing combined with predictive bioinformatics tools and minigene assay. As a result, among the 15 candidate variants, 8 variants distributed in SLC4A1 (c.1765C>T, p.Arg589Cys), ATP6V1B1( c.368G>T, p.Gly123Val; c.370C>T, p.Arg124Trp; c.484G>T, p.Glu162* and c.1102G>A, p.Glu368Lys) and ATP6V0A4 genes (c.322C>T, p.Gln108*; c.1571C>T, p.Pro524Leu and c.1572G>A, p.Pro524Pro) were identified to result in whole or part of exon skipping by either disruption of ESEs and generation of ESSs, or interference with the recognition of the classic splicing site, or both. To our knowledge, this is the first study on pre-mRNA splicing of exonic variants in the dRTA-related genes. These results highlight the importance of assessing the effects of exonic variants at the mRNA level and suggest that minigene analysis is an effective tool for evaluating the effects of splicing on variants in vitro

scholarly journals Minigene splicing assays reveal new insights into exonic variants of the SLC12A3 gene in Gitelman Syndrome.

2021 ◽  
Author(s):  
Xiaomeng Shi ◽  
Qihua Liu ◽  
Ruixiao Zhang ◽  
Zhiying Liu ◽  
Wencong Guo ◽  
...  
Keyword(s):  
Mrna Level ◽  
Exon Skipping ◽  
Regulatory Elements ◽  
Mrna Splicing ◽  
Gitelman Syndrome ◽  
Splice Sites ◽  
Functional Assays ◽  
Slc12a3 Gene ◽  
Splicing Regulatory Elements

Gitelman syndrome (GS) is a kind of salt-losing tubular disease, most of which is caused by SLC12A3 gene variants, and missense variants account for the majority. Recently, the phenomenon of exon skipping, in which exonic variants disrupt normal pre-mRNA splicing, has been related to a variety of diseases. The purpose of this study was to identify the effect of previously presumed missense SLC12A3 variants on pre-mRNA splicing using bioinformatics tools and minigenes. The results revealed that, among ten candidate variants, six variants (c.602G>A, c.602G>T, c.1667C>T, c.1925G>A, c.2548G>C and c.2549G>C) led to complete or incomplete exon skipping by affecting exonic splicing regulatory elements and/or disturbing canonical splice sites. It is worth mentioning that this is the largest study on pre-mRNA splicing of SLC12A3 exonic variants. In addition, our study emphasizes the importance of detecting splicing function at the mRNA level in GS and indicates that minigene analysis is a valuable tool for splicing functional assays of variants in vitro.

Legal restrictions on pre-implantation genetic diagnostics in the framework of in vitro fertilization procedure: foreign experience and Russian perspective

2019 ◽  
Author(s):  
N.A. Altinnik , S.S. Zenin , V.V. Komarova et all
Keyword(s):  
In Vitro Fertilization ◽  
Assisted Reproductive Technologies ◽  
Genetic Diseases ◽  
Genetic Diagnosis ◽  
Reproductive Technologies ◽  
Legal Regulation ◽  
Genetic Diagnostics ◽  
Vitro Fertilization ◽  
Medical Indications

The article discusses the factors that determine the content of the legal limitations of pre-implantation genetic diagnosis in the framework of the in vitro fertilization procedure, taking into account international experience and modern domestic regulatory legal regulation of the field of assisted reproductive technologies. The authors substantiates the conclusion that it is necessary to legislate a list of medical indications for preimplantation genetic diagnosis, as well as the categories of hereditary or other genetic diseases diagnosed in the framework of this procedure.

scholarly journals Adenovirus-mediated delivery into myocytes of muscle glycogen phosphorylase, the enzyme deficient in patients with glycogen-storage disease type V

1994 ◽  
Vol 304 (3) ◽  
pp. 1009-1014 ◽  
Author(s):  
S Baqué ◽  
C B Newgard ◽  
R D Gerard ◽  
J J Guinovart ◽  
A M Gómez-Foix
Keyword(s):  
Muscle Glycogen ◽  
Glycogen Phosphorylase ◽  
Recombinant Adenovirus ◽  
Genetic Diseases ◽  
C2c12 Myoblast ◽  
Mrna Levels ◽  
Phosphorylase Activity ◽  
Muscle Phosphorylase ◽  
The Impact

The feasibility of using adenovirus as a vector for the introduction of glycogen phosphorylase activity into myocytes has been examined. We used the C2C12 myoblast cell line to assay the impact of phosphorylase gene transfer on myocyte glycogen metabolism and to reproduce in vitro the two strategies proposed for the treatment of muscle genetic diseases, myoblast transplantation and direct DNA delivery. In this study, a recombinant adenovirus containing the muscle glycogen phosphorylase cDNA transcribed from the cytomegalovirus promoter (AdCMV-MGP) was used to transduce both differentiating myoblasts and nondividing mature myotube cells. Muscle glycogen phosphorylase mRNA levels and total phosphorylase activity were increased in both cell types after viral treatment although more efficiently in the differentiated myotubes. The increase in phosphorylase activity was transient (15 days) in myoblasts whereas in myotubes higher levels of phosphorylase gene expression and activity were reached, which remained above control levels for the duration of the study (20 days). The introduction of muscle phosphorylase into myotubes enhanced their glycogenolytic capacity. AdCMV MGP-transduced myotubes had lower glycogen levels under basal conditions. In addition, these engineered cells showed more extensive glycogenolysis in response to both adrenaline, which stimulates glycogen phosphorylase phosphorylation, and carbonyl cyanide m-chlorophenylhydrazone, a metabolic uncoupler. In conclusion, transfer of the muscle glycogen phosphorylase cDNA into myotubes confers an enhanced and regulatable glycogenolytic capacity. Thus this system might be useful for delivery of muscle glycogen phosphorylase and restoration of glycogenolysis in muscle cells from patients with muscle phosphorylase deficiency (McArdle's disease).

scholarly journals Evolutionary Emergence of a Novel Splice Variant with an Opposite Effect on the Cell Cycle

2015 ◽  
Vol 35 (12) ◽  
pp. 2203-2214 ◽  
Author(s):  
Muhammad Sohail ◽  
Jiuyong Xie
Keyword(s):  
Cell Cycle ◽  
Splice Variant ◽  
Splice Variants ◽  
Exon Skipping ◽  
Regulatory Elements ◽  
Cellular Processes ◽  
Human Genes ◽  
Splicing Regulatory Elements ◽  
Evolutionary Emergence ◽  
Nuclear Ribonucleoprotein

Alternative splicing contributes greatly to the diversification of mammalian proteomes, but the molecular basis for the evolutionary emergence of splice variants remains poorly understood. We have recently found a novel class of splicing regulatory elements between the polypyrimidine tract (Py) and 3′ AG (REPA) at intron ends in many human genes, including the multifunctionalPRMT5(for protein arginine methyltransferase 5) gene. The PRMT5 element is comprised of two G tracts that arise in most mammals and accompany significant exon skipping in human transcripts. The G tracts inhibit splicing by recruiting heterogeneous nuclear ribonucleoprotein (hnRNP) H and F (H/F) to reduce U2AF65 binding to the Py, causing exon skipping. The resulting novel shorter variant PRMT5S exhibits a histone H4R3 methylation effect similar to that seen with the original longer PRMT5L isoform but exhibits a distinct localization and preferential control of critical genes for cell cycle arrest at interphase in comparison to PRMT5L. This report thus provides a molecular mechanism for the evolutionary emergence of a novel splice variant with an opposite function in a fundamental cell process. The presence of REPA elements in a large group of genes implies their wider impact on different cellular processes for increased protein diversity in humans.

scholarly journals In vivo recognition of a vertebrate mini-exon as an exon-intron-exon unit.

1993 ◽  
Vol 13 (5) ◽  
pp. 2677-2687 ◽  
Author(s):  
D A Sterner ◽  
S M Berget
Keyword(s):  
Splice Site ◽  
Rna Splicing ◽  
Troponin I ◽  
Exon Skipping ◽  
Splice Sites ◽  
Small Vertebrate ◽  
Upstream Exon ◽  
I Gene

Very small vertebrate exons are problematic for RNA splicing because of the proximity of their 3' and 5' splice sites. In this study, we investigated the recognition of a constitutive 7-nucleotide mini-exon from the troponin I gene that resides quite close to the adjacent upstream exon. The mini-exon failed to be included in spliced RNA when placed in a heterologous gene unless accompanied by the upstream exon. The requirement for the upstream exon disappeared when the mini-exon was internally expanded, suggesting that the splice sites bordering the mini-exon are compatible with those of other constitutive vertebrate exons and that the small size of the exon impaired inclusion. Mutation of the 5' splice site of the natural upstream exon did not result in either exon skipping or activation of a cryptic 5' splice site, the normal vertebrate phenotypes for such mutants. Instead, a spliced RNA accumulated that still contained the upstream intron. In vitro, the mini-exon failed to assemble into spliceosome complexes unless either internally expanded or accompanied by the upstream exon. Thus, impaired usage of the mini-exon in vivo was accompanied by impaired recognition in vitro, and recognition of the mini-exon was facilitated by the presence of the upstream exon in vivo and in vitro. Cumulatively, the atypical in vivo and in vitro properties of the troponin exons suggest a mechanism for the recognition of this mini-exon in which initial recognition of an exon-intron-exon unit is followed by subsequent recognition of the intron.

scholarly journals Potential usefulness of preimplantation genetic diagnosis in the control and prevention of genetic diseases

1999 ◽  
Vol 5 (6) ◽  
pp. 1134-1139
Author(s):  
M. A. El Hazmi
Keyword(s):  
Prenatal Diagnosis ◽  
Preimplantation Genetic Diagnosis ◽  
Genetic Disorder ◽  
Genetic Diseases ◽  
Genetic Diagnosis ◽  
Potential Usefulness ◽  
Vitro Fertilization ◽  
Polar Bodies ◽  
Control And Prevention

Prenatal diagnosis of molecular mutations can be of immense value, since diagnosis followed by genetic counselling provides the most appropriate approach to genetic diseases control and prevention. However, ethical, psychosocial and religious considerations hamper adoption of prenatal diagnosis in communities where termination of a pregnancy may not be acceptable. Recently, preimplantation genetic diagnosis has attracted considerable interest. This involves in vitro fertilization, followed by genetic disorder diagnosis using polar bodies or cells extracted from a blastomere stage. The normal blastomere is implanted in the womb and pregnancy proceeds naturally. If an abnormality is diagnosed, the blastomere is not implanted, thus preventing pregnancy with the affected fetus. This paper outlines the potential usefulness of preimplantation genetic diagnosis in the control and prevention of genetic disease in our part of the world

scholarly journals Exploring thermosonication as non-chemical disinfection technology for strawberries

2021 ◽  
Author(s):  
Iolanda Nicolau-Lapeña ◽  
Ingrid Aguiló-Aguayo ◽  
Marina Anguera ◽  
Inmaculada Viñas ◽  
Maribel Abadias
Keyword(s):  
Fruit Quality ◽  
Listeria Innocua ◽  
Naturally Occurring ◽  
Effects Of Temperature ◽  
Yeasts And Molds ◽  
The Impact ◽  
Detrimental Impact ◽  
Chemical Disinfection ◽  
Main Factor

AbstractThe scope of this work was to study the efficacy of the combination of sonication at 35 or 130 kHz with three temperature treatments: 20, 50 and 55 ºC, on the population of artificially inoculated Listeria innocua in strawberries, and on their overall quality. Prior in vitro results showed that temperature was the main factor in decreasing L. innocua population: a maximum of 3.8 log reductions was obtained with sonication at 130 kHz and 55 ºC for 15 min Treatments combining—or not—sonication at 130 kHz with mild temperatures (50 and 55 ºC) for 5 or 10 min were able to decrease about 3 log units of artificially inoculated L. innocua in strawberries and about 2 log units of total aerobic mesophilic and yeasts and molds populations naturally occurring in strawberries. Thermosonication treatments did not exert a detrimental impact on fruit quality, except for those at the higher temperatures and times, which caused a change in color to more purplish and a little softening of the strawberries, which were proposed to be assessed for further processing other than fresh commercialization. Overall, the impact of sonication in fresh strawberries needs to be further investigated to find the adequate conditions to enhance the effects of temperature itself.

scholarly journals Chromatin accessibility dynamics of Chlamydia-infected epithelial cells

2019 ◽  
Author(s):  
Regan J. Hayward ◽  
James W. Marsh ◽  
Michael S. Humphrys ◽  
Wilhelmina M. Huston ◽  
Garry S.A. Myers
Keyword(s):  
Epithelial Cells ◽  
Host Cell ◽  
Intracellular Signaling ◽  
Chlamydial Infection ◽  
Regulatory Elements ◽  
Chromatin Accessibility ◽  
Developmental Cycle ◽  
Transmitted Infection ◽  
The Impact

AbstractChlamydia are Gram-negative, obligate intracellular bacterial pathogens responsible for a broad spectrum of human and animal diseases. In humans, Chlamydia trachomatis is the most prevalent bacterial sexually transmitted infection worldwide and is the causative agent of trachoma (infectious blindness) in disadvantaged populations. Over the course of its developmental cycle, Chlamydia extensively remodels its intracellular niche and parasitises the host cell for nutrients, with substantial resulting changes to the host cell transcriptome and proteome. However, little information is available on the impact of chlamydial infection on the host cell epigenome and global gene regulation. Regions of open eukaryotic chromatin correspond to nucleosome-depleted regions, which in turn are associated with regulatory functions and transcription factor binding. We applied Formaldehyde-Assisted Isolation of Regulatory Elements enrichment followed by sequencing (FAIRE-Seq) to generate temporal chromatin maps of C. trachomatis-infected human epithelial cells in vitro over the chlamydial developmental cycle. We detected both conserved and distinct temporal changes to genome-wide chromatin accessibility associated with C. trachomatis infection. The observed differentially accessible chromatin regions, including several Clusters of Open Regulatory Elements (COREs) and temporally-enriched sets of transcription factors, may help shape the host cell response to infection. These regions and motifs were linked to genomic features and genes associated with immune responses, re-direction of host cell nutrients, intracellular signaling, cell-cell adhesion, extracellular matrix, metabolism and apoptosis. This work provides another perspective to the complex response to chlamydial infection, and will inform further studies of transcriptional regulation and the epigenome in Chlamydia-infected human cells and tissues

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