Functional analysis of the p.[Arg74Trp;Val201Met;Asp1270Asn]/p.Phe508del <i>CFTR</i> mutation genotype in human native colon

Schucht, Sylvia; Minso, Rebecca; Lex, Christiane; Reiss, Jochen; Stanke, Frauke; Tamm, Stephanie; Barneveld, Andrea van; Tümmler, Burkhard

doi:10.1002/mgg3.526

Cited by 5 publications

(3 citation statements)

References 22 publications

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“…The two index cases were affected by mild CF. 19 Discordant CFTR biomarker phenotypes were noted for carriers of the p.Arg117His mutant. The three p.Phe508del /p.Arg117His compound heterozygotes in our cohort showed CFTR dysfunction in the CF range, whereas the p.Arg117His-7T homozygote exhibited normal CFTR activity consistent with a previous report of normal clinical features and normal CFTR activity in two homozygous p.Arg117His-7T adults.…”

Section: Cftr Activity Of Individuals With Cftr Genotypes Of Unknown mentioning

confidence: 99%

Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusiveCFTRgenetics and sweat test

Minso

Schulz

Dopfer³

et al. 2020

BMJ Open Resp Res

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BackgroundNasal potential difference (NPD) and intestinal current measurements (ICM) are cystic fibrosis transmembrane conductance regulator (CFTR) biomarkers recommended to make a diagnosis in individuals with inconclusive sweat test and CFTR genetics and a clinical suspicion for cystic fibrosis (CF) or CFTR-related disorder (CFTR-RD).MethodsNPD and ICM were measured according to standard operating procedures of the European Cystic Fibrosis Society Diagnostic Network Working Group.ResultsWe assessed 219 individuals by NPD or ICM who had been referred to our laboratory due to clinical symptoms suggestive of CF, but inconclusive sweat test and CFTR genetics (median age: 16.3 years, range 0.4 to 76 years). CF or CFTR-related disorder was diagnosed in 22 of 29 patients (76%) with a CFTR genotype of unknown or variable clinical significance and in 51 of 190 carriers (27%) of one (35/42) or no (16/148) identified CFTR mutation. If two CFTR sequence variants had been identified, the outcome of NPD and ICM was consistent with the classification of the CFTR2 database. Moreover, a suspected false-positive diagnosis of CF was confirmed in seven and withdrawn in eight patients. Of 26 individuals assessed by both NPD and ICM, eleven individuals exhibited discordant tracings of ICM and NPD, with one measurement being in the CF range and the other in the normal range.ConclusionThe majority of patients whom we diagnosed with CF or CFTR-RD by extended electrophysiology are carriers of the wild-type CFTR coding sequence on at least one of their CF alleles. The disease-causing genetic lesions should reside in the non-coding region of CFTR or elsewhere in the genome, affecting the regulation of CFTR expression in a tissue-depending fashion which may explain the large within-group variability of CFTR activity in the respiratory and intestinal epithelium seen in this group.

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Section: Cftr Activity Of Individuals With Cftr Genotypes Of Unknown mentioning

confidence: 99%

Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusiveCFTRgenetics and sweat test

Minso

Schulz

Dopfer³

et al. 2020

BMJ Open Resp Res

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“…In our hands, such signals were regularly unmasked by PNgase digestion or peptide competition as false-positive immunoreactive signals not corresponding to the CFTR protein. Apart from the monoclonal CFTR antibodies provided by Tim Jensen, Chapel Hill via the antibody distribution program for this work [ 34 ], we also tested a collection of 10 customized rabbit polyclonal antibodies (Eurogentec) generated to detect CFTR [ 35 , 36 , 37 ] via immunoblotting. With three of these customized antibodies, we detected a lege artis CFTR signal from 16HBE14o- positive control lysates.…”

Section: Discussionmentioning

confidence: 99%

Analysis of CFTR mRNA and Protein in Peripheral Blood Mononuclear Cells via Quantitative Real-Time PCR and Western Blot

Schnell,

Tamm,

Hedtfeld

et al. 2024

IJMS

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The Cystic Fibrosis Conductance Transmembrane Regulator gene encodes for the CFTR ion channel, which is responsible for the transport of chloride and bicarbonate across the plasma membrane. Mutations in the gene result in impaired ion transport, subsequently leading to perturbed secretion in all exocrine glands and, therefore, the multi-organ disease cystic fibrosis (CF). In recent years, several studies have reported on CFTR expression in immune cells as demonstrated by immunofluorescence, flow cytometry, and immunoblotting. However, these data are mainly restricted to single-cell populations and show significant variation depending on the methodology used. Here, we investigated CFTR transcription and protein expression using standardized protocols in a comprehensive panel of immune cells. Methods: We applied a high-resolution Western blot protocol using a combination of highly specific monoclonal CFTR antibodies that have been optimized for the detection of CFTR in epithelial cells and healthy primary immune cell subpopulations sorted by flow cytometry and used immortalized cell lines as controls. The specificity of CFTR protein detection was controlled by peptide competition and enzymatic Peptide-N-Glycosidase-F (PNGase) digest. CFTR transcripts were analyzed using quantitative real-time PCR and normalized to the level of epithelial T84 cells as a reference. Results: CFTR mRNA expression could be shown for primary CD4+ T cells, NK cells, as well as differentiated THP-1 and Jurkat T cells. In contrast, we failed to detect CFTR transcripts for CD14+ monocytes and undifferentiated THP-1 cells, as well as for B cells and CD8+ T cells. Prominent immunoreactive bands were detectable by immunoblotting with the combination of four CFTR antibodies targeting different epitopes of the CFTR protein. However, in biosamples of non-epithelial origin, these CFTR-like protein bands could be unmasked as false positives through peptide competition or PNGase digest, meaning that the observed mRNA transcripts were not necessarily translated into CFTR proteins, which could be detected via immunoblotting. Our results confirm that mRNA expression in immune cells is many times lower than in that cells of epithelial origin. The immunoreactive signals in immune cells turned out to be false positives, and may be provoked by the presence of a high-affinity protein with a similar epitope. Non-specific binding (e.g., Fab-interaction with glycosyl branches) might also contribute to false positive signals. Our findings highlight the necessity of accurate controls, such as CFTR-negative cells, as well as peptide competition and glycolytic digest in order to identify genuine CFTR protein by immunoblotting. Our data suggest, furthermore, that CFTR protein expression data from techniques such as histology, for which the absence of a molecular weight or other independent control prevents the unmasking of false positive immunoreactive signals, must be interpreted carefully as well.

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“…Normal (younger brother) and subnormal (older brother) chloride secretion measured by ICM were associated with normal CFTR protein formation and a fourfold decrease in its amount, respectively. Thus, the authors found differences in the same family in phenotypic manifestations with the same genotypes [ 37 ].…”

Section: The Influence Of Genetic Variants In the Composition Of A Co...mentioning

confidence: 99%

The Effect of Complex Alleles of the CFTR Gene on the Clinical Manifestations of Cystic Fibrosis and the Effectiveness of Targeted Therapy

Krasnova,

Efremova,

Bukhonin

et al. 2023

IJMS

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The authors of this article analyzed the available literature with the results of studying the prevalence of complex alleles of the CFTR gene among patients with cystic fibrosis, and their pathogenicity and influence on targeted therapy with CFTR modulators. Cystic fibrosis (CF) is a multisystemic autosomal recessive disease caused by a defect in the expression of the CFTR protein, and more than 2000 genetic variants are known. Clinically significant variants are divided into seven classes. Information about the frequency of complex alleles appears in a number of registers, along with the traditional presentation of data on genetic variants. Complex alleles (those with the presence of more than two nucleotide variants on one allele) can complicate the diagnosis of the disease, and change the clinical manifestations of cystic fibrosis and the response to treatment, since each variant in the complex allele can contribute to the functional activity of the CFTR protein, changing it both in terms of increasing and decreasing function. The role of complex alleles is often underestimated, and their frequency has not been studied. At the moment, characteristic frequently encountered complex alleles have been found for several populations of patients with cystic fibrosis, but the prevalence and pathogenicity of newly detected complex alleles require additional research. In this review, more than 35 complex alleles of the CFTR gene from existing research studies were analyzed, and an analysis of their influence on the manifestations of the disease and the effectiveness of CFTR modulators was also described.

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Functional analysis of the p.[Arg74Trp;Val201Met;Asp1270Asn]/p.Phe508del CFTR mutation genotype in human native colon

Cited by 5 publications

References 22 publications

Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusiveCFTRgenetics and sweat test

Intestinal current measurement and nasal potential difference to make a diagnosis of cases with inconclusiveCFTRgenetics and sweat test

Analysis of CFTR mRNA and Protein in Peripheral Blood Mononuclear Cells via Quantitative Real-Time PCR and Western Blot

The Effect of Complex Alleles of the CFTR Gene on the Clinical Manifestations of Cystic Fibrosis and the Effectiveness of Targeted Therapy

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