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1.
J Cyst Fibros ; 20(3): 464-472, 2021 05.
Article in English | MEDLINE | ID: mdl-33341408

ABSTRACT

BACKGROUND: Minigenes and in silico prediction tools are commonly used to assess the impact on splicing of CFTR variants. Exon skipping is often neglected though it could impact the efficacy of targeted therapies. The aim of the study was to identify exon skipping associated with CFTR variants and to evaluate in silico predictions of seven freely available software. METHODS: CFTR basal exon skipping was evaluated on endogenous mRNA extracted from non-CF nasal cells and on two CFTR minigene banks. In silico tools and minigene systems were used to evaluate the impact of CFTR exonic variants on exon skipping. RESULTS: Data showed that out of 65 CFTR variants tested, 26 enhanced exon skipping and that in silico prediction efficacy was of 50%-66%. Some in silico tools presented predictions with a bias towards the occurrence of splicing events while others presented a bias towards the absence of splicing events (non-detection including true negatives and false negatives). Classification of exons depending on their basal exon skipping level increased prediction rates up to 80%. CONCLUSION: This study indicates that taking basal exon skipping into account could orientate the choice of the in silico tools to improve prediction rates. It also highlights the need to validate effects using in vitro assays or mRNA studies in patients. Eventually, it shows that variant-guided therapy should also target exon skipping associated with variants.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/genetics , Exons , Alternative Splicing , Cells, Cultured , Humans , Nasal Mucosa/cytology , RNA Splice Sites , Sequence Deletion
2.
Chemosphere ; 264(Pt 2): 128573, 2021 Feb.
Article in English | MEDLINE | ID: mdl-33059281

ABSTRACT

In this study, an electro-replacement/precipitation/deposition/direct reduction (ERPDD) process with scrap iron packed in a Ti mesh cage as a sacrificial anode was investigated for the treatment of wastewater containing CuEDTA complexes. The ERPDD mechanisms were responsible for the removal of Cu from CuEDTA complexes and were verified by a series of experiments using either iron or carbon plates as anodes for the Cu-containing solutions with and without EDTA. A complete Cu removal was achieved with electrical current density applied (1.18-2.36 mA/cm2), whereas only 60% of the Cu was removed without electricity. Dissolved oxygen (DO) was found to have a significant impact on Cu removal. Aeration reduced Cu removal (i.e., only 60% of the Cu was removed), whereas complete Cu removal was achieved with negligible DO concentration under mechanical mixing and N2 purging conditions. Compared to chemical replacement/precipitation (CRP) process, the ERPDD was able to save approximately 60-75% of the total operational costs during the treatment of CuEDTA-containing wastewater, due to the electrochemically controlled dosing of inexpensive sacrificial scrap iron and additional removal mechanisms not found in the CRP process.


Subject(s)
Wastewater , Water Pollutants, Chemical , Chemical Precipitation , Copper , Electrodes , Iron , Wastewater/analysis , Water Pollutants, Chemical/analysis
3.
ERJ Open Res ; 4(1)2018 Jan.
Article in English | MEDLINE | ID: mdl-29497617

ABSTRACT

Premature termination codons (PTCs) are generally associated with severe forms of genetic diseases. Readthrough of in-frame PTCs using small molecules is a promising therapeutic approach. Nonetheless, the outcome of preclinical studies has been low and variable. Treatment efficacy depends on: 1) the level of drug-induced readthrough, 2) the amount of target transcripts, and 3) the activity of the recoded protein. The aim of the present study was to identify, in the cystic fibrosis transmembrane conductance regulator (CFTR) model, recoded channels from readthrough therapy that may be enhanced using CFTR modulators. First, drug-induced readthrough of 15 PTCs was measured using a dual reporter system under basal conditions and in response to gentamicin and negamycin. Secondly, exon skipping associated with these PTCs was evaluated with a minigene system. Finally, incorporated amino acids were identified by mass spectrometry and the function of the predicted recoded CFTR channels corresponding to these 15 PTCs was measured. Nonfunctional channels were subjected to CFTR-directed ivacaftor-lumacaftor treatments. The results demonstrated that CFTR modulators increased activity of recoded channels, which could also be confirmed in cells derived from a patient. In conclusion, this work will provide a framework to adapt treatments to the patient's genotype by identifying the most efficient molecule for each PTC and the recoded channels needing co-therapies to rescue channel function.

4.
Hum Mutat ; 39(4): 506-514, 2018 04.
Article in English | MEDLINE | ID: mdl-29271547

ABSTRACT

Molecules correcting the trafficking (correctors) and gating defects (potentiators) of the cystic fibrosis causing mutation c.1521_1523delCTT (p.Phe508del) begin to be a useful treatment for CF patients bearing p.Phe508del. This mutation has been identified in different genetic contexts, alone or in combination with variants in cis. Until now, 21 exonic variants in cis of p.Phe508del have been identified, albeit at a low frequency. The aim of this study was to evaluate their impact on the efficacy of CFTR-directed corrector/potentiator therapy (Orkambi). The analysis by minigene showed that two out of 15 cis variants tested increased exon skipping (c.609C > T and c.2770G > A). Four cis variants were studied functionally in the absence of p.Phe508del, one of which was found to be deleterious for protein maturation c.1399C > T (p.Leu467Phe). In the presence of p.Phe508del, this variant was the only to prevent the response to Orkambi treatment. This study showed that some patients carrying p.Phe508del complex alleles are predicted to poorly respond to corrector/potentiator treatments. Our results underline the importance to validate treatment efficacy in the context of complex alleles.


Subject(s)
Aminophenols/therapeutic use , Aminopyridines/therapeutic use , Benzodioxoles/therapeutic use , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Cystic Fibrosis/drug therapy , Cystic Fibrosis/genetics , Quinolones/therapeutic use , Alleles , Drug Combinations , Humans , Mutation , Phenylalanine/genetics
5.
Clin Case Rep ; 5(5): 658-663, 2017 05.
Article in English | MEDLINE | ID: mdl-28469871

ABSTRACT

In vitro functional tests aimed to investigate CFTR dysfunction appear critical to help elucidate the functional impact of new variants of uncertain clinical significance and solve inconclusive cases, especially in early deceased newborns.

6.
Hum Mutat ; 35(7): 805-8, 2014 Jul.
Article in English | MEDLINE | ID: mdl-24633926

ABSTRACT

Cystic fibrosis may be revealed by nasal polyposis (NP) starting early in life. We performed cystic fibrosis transmembrane conductance regulator (CFTR) DNA and mRNA analyses in the family of a 12-year-old boy presenting with NP and a normal sweat test. Routine DNA analysis only showed the heterozygous c.2551C>T (p.Arg851*) mutation in the child and the father. mRNA analysis showed partial exon skipping due to c.2551C>T and a significant increase in total CFTR mRNA in the patient and the mother, which was attributable to the heterozygous c. -2954G>A variant in the distant promoter region, as demonstrated by in vitro luciferase assays. The 5' rapid amplification of cDNA ends analysis showed the presence of a novel transcript, where the canonical exon 1 was replaced by an alternative exon called 1a-Long. This case report could represent the first description of a CFTR-related disorder associated with the presence of a 5' alternative, probably nonfunctional transcript, similar to those of fetal origin.


Subject(s)
Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Mutation , Nasal Polyps/genetics , RNA Isoforms , RNA, Messenger/genetics , 5' Untranslated Regions , Alternative Splicing , Child , DNA Mutational Analysis , Gene Order , Humans , Male , Nasal Polyps/diagnosis , Pedigree
7.
Infect Genet Evol ; 20: 16-25, 2013 Dec.
Article in English | MEDLINE | ID: mdl-23932959

ABSTRACT

INTRODUCTION: Infants born to mothers with placental malaria at delivery develop Plasmodium falciparum parasitemia earlier than those born to mothers without placental infection. This phenomenon may be explained by the development of immune tolerance due to exposure to P. falciparum antigens in utero. The hypothesis of this study is that this increased susceptibility might be related to infections by parasites expressing the same blood stage allele's antigens as those to which the infants were exposed in utero. METHODS: The comparison of P.falciparum msp2 (3D7 and FC27) and glurp gene polymorphisms of infected mothers at delivery to those of their offspring's infections during infancy was realized and the possible associations of the different polymorphisms with clinical outcomes were assessed. A second approach consisted in the use of a Geographic Information System to determine whether the antigen alleles were homogeneously distributed in the area of study. This was necessary to analyze whether the biological observations were due to high exposure to a particular antigen allelic form in the environment or to high infant permissiveness to the same allelic antigen polymorphism as the placental one. RESULTS: Infants born to mothers with placental malaria at delivery were more susceptible to infections by parasites carrying the same glurp allele as encountered in utero compared to distinct alleles, independently of their geographic distribution. CONCLUSION: The increased permissiveness of infants to plasmodial infections with shared placental-infant glurp alleles sheds light on the role that P. falciparum blood stage antigen polymorphisms may play in the first plasmodial infections in infancy.


Subject(s)
Antigens, Protozoan/genetics , Infectious Disease Transmission, Vertical , Malaria, Falciparum/transmission , Placenta/parasitology , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , Alleles , DNA, Protozoan/genetics , Female , Genetic Variation , Humans , Infant , Infant, Newborn , Malaria, Falciparum/parasitology , Male , Placenta Diseases/parasitology , Plasmodium falciparum/immunology , Polymorphism, Single Nucleotide , Pregnancy , Pregnancy Complications, Infectious/parasitology , Sequence Analysis, DNA
8.
Hum Mutat ; 34(6): 873-81, 2013 Jun.
Article in English | MEDLINE | ID: mdl-23420618

ABSTRACT

With the increased number of identified nucleotide sequence variations in genes, the current challenge is to classify them as disease causing or neutral. These variants of unknown clinical significance can alter multiple processes, from gene transcription to RNA splicing or protein function. Using an approach combining several in silico tools, we identified some exons presenting weaker splicing motifs than other exons in the Cystic Fibrosis Transmembrane conductance Regulator (CFTR) gene. These exons exhibit higher rates of basal skipping than exons harboring no identifiable weak splicing signals using minigene assays. We then screened 19 described mutations in three different exons, and identified exon-skipping substitutions. These substitutions induced higher skipping levels in exons having one or more weak splicing motifs. Indeed, this level remained under 2% for exons with strong splicing motifs and could reach 40% for exons having at least one weak motif. Further analysis revealed a functional exon splicing enhancer within exon 3 that was associated with the SR protein SF2/ASF and whose disruption induced exon skipping. Exon skipping was confirmed in vivo in two nasal epithelial cell brushing samples. Our approach, which point out exons with some splicing signals weaknesses, will help spot splicing mutations of clinical relevance.


Subject(s)
Alternative Splicing , Computational Biology , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Exons , Computational Biology/methods , Computer Simulation , Humans , Models, Biological , Mutation, Missense , Nuclear Proteins/metabolism , RNA-Binding Proteins/metabolism , Regulatory Sequences, Nucleic Acid , Serine-Arginine Splicing Factors , Transcription, Genetic
9.
Hum Mutat ; 34(2): 287-91, 2013 Feb.
Article in English | MEDLINE | ID: mdl-23065710

ABSTRACT

The correction of premature termination codons (PTCs) by agents that promote readthrough represents a promising emerging tool for the treatment of many genetic diseases. The efficiency of the treatment, however, varies depending on the stop codon itself and the amount of correctible transcripts related to the efficiency of nonsense-mediated decay. In the current study, a screen by in vitro minigene assay of all six PTCs described in exon 15 of the CFTR gene demonstrated alternative splicing to differing degrees for five of them. Of the five, PTC mutations c.2537G>A (p.Trp846*(UAG) ) and c.2551C>T (p.Arg851*) cause the greatest proportion of transcripts lacking exon 15; both mutations altering exonic splicing regulatory elements. In order to increase the amount of full-length transcripts, different pharmacological treatments were performed showing both negative and positive effects on exon inclusion for the same mutation. Therefore, the total amount of transcripts together with the splicing profile should be assessed to anticipate and improve efficacy of readthrough therapy.


Subject(s)
Alternative Splicing/genetics , Codon, Nonsense/genetics , Exons , Reading Frames/genetics , Bronchi/cytology , Bronchi/metabolism , Cell Line , Cystic Fibrosis/genetics , Cystic Fibrosis/therapy , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Epithelial Cells , Humans , RNA, Messenger/genetics , RNA, Messenger/metabolism
10.
PLoS Genet ; 6(10)2010 Oct 07.
Article in English | MEDLINE | ID: mdl-20949073

ABSTRACT

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies.


Subject(s)
Alternative Splicing , Cystic Fibrosis Transmembrane Conductance Regulator/genetics , Mutation, Missense , RNA Splice Sites/genetics , Base Sequence , Cystic Fibrosis/genetics , Cystic Fibrosis/pathology , DNA Mutational Analysis , Female , Genotype , HEK293 Cells , HeLa Cells , Humans , INDEL Mutation , Luminescent Proteins/genetics , Luminescent Proteins/metabolism , Male , Pedigree , Phenotype , Transfection
12.
Blood Cells Mol Dis ; 43(3): 226-9, 2009.
Article in English | MEDLINE | ID: mdl-19632868

ABSTRACT

We report the second documented observation of a rare class-III variant, we named G6PD Pyrgos, [c.383 T>G, p.128Leu>Arg] found in a Greek family. A 3-dimensional structure model for the enzyme shows that the region modified by the substitution is identical to that modified in G6PD A(-) (68Val>Met, 126Asn>Asp), suggesting a common underlying pathophysiological mechanism. Observation of this mutation in different Mediterranean regions suggests that it might be more widespread that initially supposed and, in the absence of molecular characterization, could be confused with other frequent variants.


Subject(s)
Glucosephosphate Dehydrogenase/genetics , Mutation , Amino Acid Substitution/genetics , Exons/genetics , Female , Genetic Variation/genetics , Glucosephosphate Dehydrogenase/chemistry , Greece , Humans , Male , Protein Conformation
13.
Hum Mutat ; 24(3): 215-24, 2004 Sep.
Article in English | MEDLINE | ID: mdl-15300849

ABSTRACT

von Hippel-Lindau (VHL) disease arises from mutations in the VHL gene and predisposes patients to develop a variety of tumors in different organs. In the kidney, single or multiple cysts and renal cell carcinomas (RCC) may occur. Both inter- and intrafamilial heterogeneity in clinical expression are well recognized. To identify VHL-dependent genetic factors, we investigated the renal phenotype in 274 individuals from 126 unrelated VHL families in whom 92 different VHL mutations were characterized. The incidence of renal involvement was increased in families with mutations leading to truncated protein (MLTP) or large rearrangement, as compared to families with missense changes (81 vs. 63%, respectively; P=0.03). In the latter group, we identified two mutation cluster regions (MCRs) associated with a high risk of harboring renal lesions: MCR-1 (codons 74-90) and MCR-2 (codons 130-136). In addition, the incidence of RCC was higher in families with MLTP than in families with missense changes (75 vs. 57%; P=0.04). Furthermore, mutations within MCR-1 but not MCR-2 conferred genetic susceptibility to develop RCC. Overall, our data argued for a substantial contribution of the genetic change in the VHL gene to susceptibility to renal phenotype in VHL patients.


Subject(s)
Carcinoma, Renal Cell/genetics , Kidney Diseases, Cystic/genetics , Kidney Neoplasms/genetics , Tumor Suppressor Proteins/genetics , Ubiquitin-Protein Ligases/genetics , von Hippel-Lindau Disease/genetics , Adult , Amino Acid Substitution , Carcinoma, Renal Cell/epidemiology , Carcinoma, Renal Cell/pathology , Codon , Codon, Nonsense , Codon, Terminator , Exons/genetics , Female , France/epidemiology , Genotype , Humans , Incidence , Kidney Diseases, Cystic/epidemiology , Kidney Diseases, Cystic/pathology , Kidney Neoplasms/epidemiology , Kidney Neoplasms/pathology , Male , Middle Aged , Mutation, Missense , Phenotype , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Protein Structure, Tertiary , Risk , Sequence Deletion , Tumor Suppressor Proteins/chemistry , Ubiquitin-Protein Ligases/chemistry , Von Hippel-Lindau Tumor Suppressor Protein , von Hippel-Lindau Disease/pathology
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