scholarly journals The novel mutation c.1210-3C>G in cis with a poly-T tract of 5T affects CFTR mRNA splicing in a Chinese patient with cystic fibrosis

2020 ◽  
Author(s):  
Xinyue Zhao ◽  
Keqiang Liu ◽  
Wenshuai Xu ◽  
Meng Xiao ◽  
Qianli Zhang ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Sanger Sequencing ◽  
Novel Mutation ◽  
Chinese Patient ◽  
Wild Type ◽  
Mutant Type ◽  
Pathogenic Mutations ◽  
In Cis ◽  
Exon 10

Abstract Purpose To identify potential pathogenic mutations in a Chinese patient with cystic fibrosis (CF) and subsequently study its splicing effect on cystic fibrosis transmembrane conductance regulator (CFTR) mRNA in vitro. Methods Genomic DNA was extracted from peripheral blood leukocytes of the patient and his parents. To detect the possible pathogenic mutations in this patient, Sanger sequencing was conducted on all 27 coding exons of CFTR and their flanking intronic regions. Minigene constructs of the wild type and the identified mutant type were produced and transfected into HEK293T cells. Total RNA was extracted and reverse-transcribed into cDNA, with which as the template polymerase chain reaction (PCR) was performed to amplify the corresponding region. Original TA cloning and Sanger sequencing of the resultant PCR products were performed to analyze their splicing patterns. Results The patient is a compound heterozygote of c.2909G>A, p.Gly970Asp in exon 18 and c.1210-3C>G in cis with a poly-T of 5T (T5) sequence, 3 bp upstream in intron 9. As reported, c.2909G>A, p.Gly970Asp is considered to be the most frequent CFTR mutation among Chinese CF patients. c.1210-3C>G, a variant adjacent to the 3’ splice site, may affect splicing and reduce the levels of normal mRNA. We validated this hypothesis by a minigene assay in vitro, which showed that the wild-type plasmid containing c.1210-3C together with the T7 sequence produced a normal transcript as well as a partial exon 10-skipping transcript, whereas the mutant plasmid containing c.1210-3G in cis with the T5 sequence caused almost all mRNA to skip exon 10. Conclusion c.1210-3C>G, newly identified in our patient, in combination with the T5 sequence in cis affects CFTR gene splicing and produces nearly no normal transcripts in vitro, which makes it a pathogenic mutation in this patient. Moreover, this patient carries a p.Gly970Asp mutation, which reinforces the high frequency of this mutation in Chinese CF patients.

Bioelectric characteristics of exon 10 insertional cystic fibrosis mouse: comparison with humans

1995 ◽  
Vol 268 (2) ◽  
pp. C297-C307 ◽  
Author(s):  
S. N. Smith ◽  
D. M. Steel ◽  
P. G. Middleton ◽  
F. M. Munkonge ◽  
D. M. Geddes ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Intestinal Tract ◽  
Chloride Secretion ◽  
Sodium Absorption ◽  
Wild Type ◽  
Nasal Potential Difference ◽  
New Treatments ◽  
Exon 10

Two important issues that can be addressed by animal models are disease pathogenesis and the testing of new treatments, including gene therapy. How closely these models mimic the relevant disorder in humans will determine their usefulness. This study examines how closely the characteristic bioelectric features of cystic fibrosis (CF) are reproduced in the airways and intestinal tract of the exon 10 insertional mutant mouse (cf/cf). In agreement with CF subjects these cf/cf mutant mice demonstrate the following: 1) reduced adenosine 3',5'-cyclic monophosphate-related chloride secretion throughout the respiratory and intestinal tracts both in vivo and in vitro, 2) calcium-related chloride secretion that is preserved in the airways but reduced in the intestine, and 3) a more negative nasal potential difference and increased amiloride response compared with wild-type animals, likely to relate to increased sodium absorption. In contrast to humans, sodium absorption is not increased in the small intestine and is reduced in the trachea of the cf/cf mice. We conclude that the majority of the salient electrophysiological features of CF required for studies of pathogenesis or testing of new treatments are present in these cf/cf mice.

scholarly journals A Missense Mutation rs781536408 (c.2395G>A) of TYK2 Affects Splicing and Causes Skipping of Exon18 in vivo

2021 ◽  
Vol 12 ◽  
Author(s):  
Suqing Chen ◽  
Peilin Wu ◽  
Bin Wu ◽  
Chenye Lin ◽  
Junhong Chen ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Disease Onset ◽  
Hek293t Cell ◽  
Pcr Amplification ◽  
Wild Type ◽  
Mutant Type ◽  
Whole Exome ◽  
Construction Strategies

TYK2 variants can impact disease onset or progression. In our previous study, we identified abnormal splicing that happened near rs781536408 in the TYK2 gene. The purpose of this research was to examine the effect of the mutation on alternative splicing in vivo and in vitro. Whole exome sequencing was performed to identify the mutations followed by bidirectional Sanger sequencing. Then the minigene analysis was carried out based on HeLa and HEK293T cell lines. The results showed that rs781536408 (c.2395G>A, p.G799R) was homozygous in the patient, but heterozygous in parents. PCR amplification confirmed the abnormal splicing in the somatic cells of the patients, but not in the parents. Sanger sequencing results showed that there was a skipping of exon18 near the mutation. For minigene analysis, there was no difference between the wild-type and the mutant type in the two minigene construction strategies, indicating that mutation c.2395G>A had no effect on splicing in vitro. Combining the results of in vivo, we speculated that the effect of the mutation on splicing was not absolute, but rather in degree.

Abstract 34: Acetylation of GATA 4 Enhanced Direct Cardiac Reprogramming of Induced Cardiomyocytes

2017 ◽  
Vol 121 (suppl_1) ◽  
Author(s):  
Vivek P Singh ◽  
Megumi Mathison ◽  
Jaya P Pinnamaneni ◽  
Deepthi Sanagasetti ◽  
Narasimhaswamy S Belaguli ◽  
...  
Keyword(s):  
Ventricular Function ◽  
Troponin T ◽  
Novel Mutation ◽  
Cardiac Regeneration ◽  
Negative Control ◽  
Direct Reprogramming ◽  
Wild Type ◽  
Reprogramming Efficiency

Objective: Direct reprogramming of fibroblasts into induced cardiomyocytes (iCMs) by forced expression of cardiomyogenic factors, GMT (GATA4, Mef2c and Tbx5), has recently been demonstrated, suggesting a promising statregy for cardiac regeneration. However, the efficiency of direct reprogramming is usually relatively low and requires extensive epigenetic redesigning, although the underlying mechanism are largely unknown. Methods: In a recent study, we created a novel mutation in rat GATA 4 by replacing lysine residue with glutamine at position 299 i.e. (K299Q), to mimic constitutive acetylation and examined whether constitutive acetylation of GATA4, when compared with wild type GATA4, further enhance GMT-mediated direct reprogramming efficiency of induced cardiomyocytes in vitro and accordingly ventricular function after myocardial infarction in rat, in vivo . Results: We found that acetylated GATA 4 (K299Q), in the presence of Mef2c and Tbx5 upregulated cardiac-specific markers, suppressed fibroblast genes, in rat cardiac fibroblasts (RCFs) more efficiently when compared with Mef2c, Tbx5 plus wild type GATA4. FACS analyses revealed that G(K299Q) MT induced significantly more cardiomyocyte marker cardiac troponin T (cTnT) expression compared with GMT alone. Mechanistic studies demonstrated that the K299Q substitution, resulting in enriched p300 occupancy at the GATA 4 promoter, induced acetylation of Histine 3, decreased HDAC expression. In addition, substitution augmented the increase in an acetylated form of GATA-4 and its DNA binding and transcriptional activity, compared with wildtype GATA 4. In agreement with upregulated cTNT gene expression in vitro , echocardiographic analysis demonstrate that the acetylated G(K299Q) MT vectors have improved effect in enhancing ventricular function than GMT vectors from postinfarct baselines as compared to negative control [G(K299Q) MT, 15.6% ± 2.7%; G(WT)MT, 12.8% ± 1.7%; GFP, -2.3% ± 1.1%]. Conclusions: Collectivily, these data indicate that acetylated GATA4 (K299Q) significantly increases reprogramming efficiency of induced cardiomyocytes (iCMs), in vitro and in vivo, and provide new insight into the molecular mechanism underlying cardiac regeneration.

Autoinflammatory disease with corneal and mucosal dyskeratosis caused by a novel NLRP1 variant

Rheumatology ◽  
2019 ◽  
Vol 59 (9) ◽  
pp. 2334-2339 ◽  
Author(s):  
Troels Herlin ◽  
Sofie E Jørgensen ◽  
Christian Høst ◽  
Patrick S Mitchell ◽  
Maria Hønholt Christensen ◽  
...  
Keyword(s):  
Sanger Sequencing ◽  
Mononuclear Cells ◽  
Novel Mutation ◽  
Mucosal Inflammation ◽  
Inflammasome Activation ◽  
Incomplete Penetrance ◽  
Peripheral Blood Mononuclear ◽  
Pyrin Domain ◽  
Patient Pbmcs

Abstract Objectives Here we investigated a patient with inflammatory corneal intraepithelial dyskeratosis, mucosal inflammation, tooth abnormalities and, eczema to uncover the genetic and immunological basis of the disease. Methods On suspicion of an autoinflammatory condition, Sanger sequencing of nucleotide-binding oligomerization domain-like, leucine-rich repeat pyrin domain containing 1 (NLRP1) was performed and combined with an in vitro inflammasome reconstitution assay to measure caspase-1-mediated IL-1β cleavage, stimulation of patient peripheral blood mononuclear cells (PBMCs) and whole blood to measure IL-1β, IL-18 production and quantification of apoptosis-associated speck-like protein containing CARD (ASC) speck formation as a measure of inflammasome activation by flow cytometry. Results Sanger sequencing revealed a novel mutation (c.175G>C, p.A59P; NM_33004.4) in the inflammasome molecule NLRP1 segregating with disease, although with incomplete penetrance, in three generations. We found that patient PBMCs produced increased IL-1β in response to inflammatory stimuli, as well as increased constitutive levels of IL-18. Moreover, we demonstrate that expression of the identified NLRP1 A59P variant caused spontaneous IL-1β cleavage to mature IL-1β. In addition, patient PBMCs responded to NLRP1 stimulation with increased ASC speck formation as a reflection of elevated inflammasome activity. Conclusion We demonstrate that this novel NLRP1 A59P variant caused increased activation of the NLRP1 inflammasome, resulting in constitutively and inducibly elevated IL-1β and IL-18 synthesis. We suggest the NLRP1 mutation underlies the pathogenesis of this rare autoinflammatory dyskeratotic disease inherited in an autosomal dominant manner with incomplete penetrance in the patient and within the family for several generations.

A New BCR-ABL1 Mutation (L248R) Is Highly Resistant to Imatinib, Bosutinib, Nilotinib and Dasatinib, but Can Be Inhibited by AP-24534 and DCC-2036.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 3398-3398
Author(s):  
Sara Redaelli ◽  
Luca Mologni ◽  
Roberta Rostagno ◽  
Rocco Piazza ◽  
Michela Viltadi ◽  
...  
Keyword(s):  
Kinase Inhibitors ◽  
Novel Mutation ◽  
Clinical Sample ◽  
Tritiated Thymidine ◽  
Blast Crisis ◽  
Point Mutations ◽  
Molecular Response ◽  
Wild Type ◽  
Activity Profile

Abstract Abstract 3398 Chronic Myeloid Leukemia (CML) treatment was radically modified by the discovery of imatinib (IM), a selective inhibitor of the fusion kinase Bcr-Abl. Second generation ATP-competitive tyrosine kinase inhibitors (TKIs) bosutinib (BOS), nilotinib (NIL) and dasatinib (DAS) further improved CML therapy. However, resistance to TKIs occurs in a variable proportion of patients; it can arise from different mechanisms but in the majority of cases it is due to Bcr-Abl point mutations that alter directly or indirectly the drug-protein binding. Over 70 mutations have been described in patients, with T315I showing resistance to IM, NIL, BOS and DAS. Here we report the discovery of a new P-loop mutant (L248R) that is highly resistant to all currently available inhibitors. L248R was identified in a lymphoid Blast Crisis CML patient. The patient initially presented with an IM-resistant F359I mutation. A cytogenetic and molecular response was obtained with BOS, but after 1 year haematological relapse developed; at this point a F359I/L248R clone was identified. L248R was previously reported only in an in vitro during a mutagenesis screen involving IM (Bradeen et al. 2006) and in a study with the T315I inhibitor SGX393 (O'Hare et al. 2008). L248R was never isolated from a clinical sample. Activity profile of BOS, IM, DAS and NIL against L248R, F359I and F359I/L248R was performed (Table). Stable transfectant Ba/F3 cells were generated and the TKIs anti-proliferative activity was determined. The relative IC50 increase over wild type Bcr-Abl (Relative Resistance, RR) was calculated. We classified RR values in four categories: sensitive (RR≤ 2), moderately resistant (RR between 2.1 and 4), resistant (RR between 4.1 and 10) or highly resistant (RR>10). In all cases a RR >10 was obtained, for L248R. Recently new compounds were developed to overcome resistance generated by the T315I mutant. Among them AP-24534 (AP), a panBcr-Abl inhibitor (O'Hare et al. 2009) and the switch pocket inhibitor DCC-2036 (DCC) were reported as potently active against the T315I mutation. Both compounds showed activity against L248R (RR 6.2 and 0.4), although the activity against the double mutant F359I/L248R was reduced especially for AP (RR 17.7 and 1.0). The activity profile of AP and DCC against L248R and a panel of 26 mutated forms of Bcr/Abl covering the most common mutations is also presented (Table). Activity of BOS, IM, DAS and NIL is also reported for comparison. According to our data, only mutation E255V is classified as highly resistant to AP, in addition to F359I/L248R: 6/26 mutations are considered “resistant” to AP (L248R, G250E, G252H, E255K, F359V, H396R) and 3/26 to DCC (D276G, E279K, F317V). The huge difference in the IC50 values for AP between Bcr/Abl wild type (2.1 nM) and parental Ba/F3 cells (>1000 nM) could render some of the mutants with high RR values to AP still sensitive to this inhibitor. It is also important to note that, according to our data, every mutation analysed shows sensitivity to at least one of the tested TKIs. In conclusion we describe a novel mutation that is highly resistant to the commonly used Bcr-Abl TKIs but which is inhibited by AP and DCC. Modelling data on the L248R mutant in complex with different TKIs will also be presented IC 50 values are based on tritiated thymidine incorporation assay. Results are an average of at least 3 independent experiments Disclosures: Wise: Deciphera Pharmaceuticals: Employment. Flynn:Deciphera Pharmaceuticals: Employment. Gambacorti-Passerini:Pfizer pharmaceuticals: Research Funding.

scholarly journals Edition of Prostaglandin E2 Receptors EP2 and EP4 by CRISPR/Cas9 Technology in Equine Adipose Mesenchymal Stem Cells

Animals ◽  
2020 ◽  
Vol 10 (6) ◽  
pp. 1078
Author(s):  
Ana Carolina Furlanetto Mançanares ◽  
Joel Cabezas ◽  
José Manríquez ◽  
Vanessa Cristina de Oliveira ◽  
Yat Sen Wong Alvaro ◽  
...  
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Prostaglandin E2 ◽  
Sanger Sequencing ◽  
Surface Markers ◽  
Wild Type ◽  
Migration Ability ◽  
Knock Out ◽  
Directed Mutation

In mesenchymal stem cells (MSCs), it has been reported that prostaglandin E2 (PGE2) stimulation of EP2 and EP4 receptors triggers processes such as migration, self-renewal, survival, and proliferation, and their activation is involved in homing. The aim of this work was to establish a genetically modified adipose (aMSC) model in which receptor genes EP2 and EP4 were edited separately using the CRISPR/Cas9 system. After edition, the genes were evaluated as to if the expression of MSC surface markers was affected, as well as the migration capacity in vitro of the generated cells. Adipose MSCs were obtained from Chilean breed horses and cultured in DMEM High Glucose with 10% fetal bovine serum (FBS). sgRNA were cloned into a linearized LentiCRISPRv2GFP vector and transfected into HEK293FT cells for producing viral particles that were used to transduce aMSCs. GFP-expressing cells were separated by sorting to obtain individual clones. Genomic DNA was amplified, and the site-directed mutation frequency was assessed by T7E1, followed by Sanger sequencing. We selected 11 clones of EP2 and 10 clones of EP4, and by Sanger sequencing we confirmed 1 clone knock-out to aMSC/EP2 and one heterozygous mutant clone of aMSC/EP4. Both edited cells had decreased expression of EP2 and EP4 receptors when compared to the wild type, and the edition of EP2 and EP4 did not affect the expression of MSC surface markers, showing the same pattern in filling the scratch. We can conclude that the edition of these receptors in aMSCs does not affect their surface marker phenotype and migration ability when compared to wild-type cells.

Long-term survival of the exon 10 insertional cystic fibrosis mutant mouse is a consequence of low level residual wild-type Cftr gene expression

1994 ◽  
Vol 5 (8) ◽  
pp. 465-472 ◽  
Author(s):  
J. R. Dorin ◽  
B. J. Stevenson ◽  
S. Fleming ◽  
E. W. F. W. Alton ◽  
P. Dickinson ◽  
...  
Keyword(s):  
Gene Expression ◽  
Cystic Fibrosis ◽  
Mutant Mouse ◽  
Cftr Gene ◽  
Wild Type ◽  
Term Survival ◽  
Long Term Survival ◽  
Low Level ◽  
Exon 10

scholarly journals Expression Analysis of a Highly Adherent and Cytotoxic Small Colony Variant of Pseudomonas aeruginosa Isolated from a Lung of a Patient with Cystic Fibrosis

2004 ◽  
Vol 186 (12) ◽  
pp. 3837-3847 ◽  
Author(s):  
Franz von Götz ◽  
Susanne Häussler ◽  
Doris Jordan ◽  
Senthil Selvan Saravanamuthu ◽  
Dirk Wehmhöner ◽  
...  
Keyword(s):  
Cystic Fibrosis ◽  
Pseudomonas Aeruginosa ◽  
Dna Microarrays ◽  
Effector Proteins ◽  
Wild Type ◽  
Small Colony Variant ◽  
A Genome ◽  
Small Colony ◽  
Type Iii Protein Secretion

ABSTRACT The heterogeneous environment of the lung of the cystic fibrosis (CF) patient gives rise to Pseudomonas aeruginosa small colony variants (SCVs) with increased antibiotic resistance, autoaggregative growth behavior, and an enhanced ability to form biofilms. In this study, oligonucleotide DNA microarrays were used to perform a genome-wide expression study of autoaggregative and highly adherent P. aeruginosa SCV 20265 isolated from a CF patient's lung in comparison with its clonal wild type and a revertant generated in vitro from the SCV population. Most strikingly, SCV 20265 showed a pronounced upregulation of the type III protein secretion system (TTSS) and the respective effector proteins. This differential expression was shown to be biologically meaningful, as SCV 20265 and other hyperpiliated and autoaggregative SCVs with increased TTSS expression were significantly more cytotoxic for macrophages in vitro and were more virulent in a mouse model of respiratory tract infection than the wild type. The observed cytotoxicity and virulence of SCV 20265 required exsA, an important transcriptional activator of the TTSS. Thus, the prevailing assumption that P. aeruginosa is subject to selection towards reduced cytotoxicity and attenuated virulence during chronic CF lung infection might not apply to all clonal variants.

scholarly journals A novel mutation of SATB2 inhibits odontogenesis of human dental pulp stem cells through Wnt/β-catenin signaling pathway

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Tianyi Xin ◽  
Qian Li ◽  
Rushui Bai ◽  
Ting Zhang ◽  
Yanheng Zhou ◽  
...  
Keyword(s):  
Stem Cells ◽  
Signaling Pathway ◽  
Dental Pulp ◽  
Frameshift Mutation ◽  
Novel Mutation ◽  
Chinese Patient ◽  
Histone Demethylase ◽  
Tooth Agenesis ◽  
Wild Type ◽  
Human Dental Pulp

Abstract Background SATB2-associated syndrome (SAS) is a multisystem disorder caused by mutation of human SATB2 gene. Tooth agenesis is one of the most common phenotypes observed in SAS. Our study aimed at identifying novel variant of SATB2 in a patient with SAS, and to investigate the cellular and molecular mechanism of tooth agenesis caused by SATB2 mutation. Methods We applied whole exome sequencing (WES) to identify the novel mutation of SATB2 in a Chinese patient with SAS. Construction and overexpression of wild-type and the mutant vector was performed, followed by functional analysis including flow cytometry assay, fluorescent immunocytochemistry, western blot, quantitative real-time PCR and Alizarin Red S staining to investigate its impact on hDPSCs and the underlying mechanisms. Results As a result, we identified a novel frameshift mutation of SATB2 (c. 376_378delinsTT) in a patient with SAS exhibiting tooth agenesis. Human DPSCs transfected with mutant SATB2 showed decreased cell proliferation and odontogenic differentiation capacity compared with hDPSCs transfected with wild-type SATB2 plasmid. Mechanistically, mutant SATB2 failed to translocate into nucleus and distributed in the cytoplasm, failing to activate Wnt/β-catenin signaling pathway, whereas the wild-type SATB2 translocated into the nucleus and upregulated the expression of active β-catenin. When we used Wnt inhibitor XAV939 to treat hDPSCs transfected with wild-type SATB2 plasmid, the increased odontogenic differentiation capacity was attenuated. Furthermore, we found that SATB2 mutation resulted in the upregulation of DKK1 and histone demethylase JHDM1D to inhibit Wnt/β-catenin signaling pathway. Conclusion We identified a novel frameshift mutation of SATB2 (c.376_378delinsTT, p.Leu126SerfsX6) in a Chinese patient with SATB2-associated syndrome (SAS) exhibiting tooth agenesis. Mechanistically, SATB2 regulated osteo/odontogenesis of human dental pulp stem cells through Wnt/β-catenin signaling pathway by regulating DKK1 and histone demethylase JHDM1D.

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