Overlapping complementary DNA clones were isolated from epithelial cell libraries with a genomic DNA segment containing a
portion of the putative cystic fibrosis (CF) locus, which is on chromosome 7. Transcripts, approximately 6500 nucleotides
in size, were detectable in the tissues affected in patients with CF. The predicted protein consists of two similar motifs,
each with (i) a domain having properties consistent with membrane association and (ii) a domain believed to be involved in
ATP (adenosine triphosphate) binding. A deletion of three base pairs that results in the omission of a phenylalanine residue
at the center of the first predicted nucleotide-binding domain was detected in CF patients.
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"Other symptoms include pancreatic insufficiency , elevated sweat electrolytes, gastrointestinal disorders , reproductive abnormalities, etc.[3,4]. CFTR is a cAMP/cGMP-regulated chloride /bicarbonate channel primarily expressed at the apical membrane of epithelial cells lining airway, gut, and exocrine glands, where it is responsible for transepithelial fluid secretion and homeostasis567. CFTR channel function is determined by the number of channels at the cell surface, the open probability of the channels, and the single channel conductance. Mutations in the CFTR gene alter one or more of these parameters, causing the impairment or loss of the channel activity. "
[Show abstract][Hide abstract] ABSTRACT: Background:
The aims of this study were to characterize clinical features of a pediatric African-American cystic fibrosis (CF) patient heterozygous for F508del and a novel c.3623G > A mutation, and to identify the molecular defect(s) associated with c.3623G > A mutation.
The medical record of this patient was analyzed retrospectively. Western blotting and iodide efflux assay were used to study mutant CFTR protein expression level, maturation status, channel function, and the effects of CFTR modulation on these characteristics.
The encoding protein of c.3623G > A mutation, G1208D-CFTR, has a moderate processing defect and exhibits impaired channel function, which were partially rescued by using VX-809 or exposed to low temperature (28 °C). The patient has mild CF disease manifestations.
Our biochemical findings correlate with the clinical phenotype and suggest that c.3623G > A is a CF-causing mutation. The study helps expand our knowledge of rare CFTR mutations in a minority population and may have important clinical implications for personalized therapeutic intervention.
Full-text · Article · Jan 2016 · Respiratory Research
"In humans, there are 48 known ABC transporters (Dean et al., 2001) that exhibit a wide range of substrate specificity, including nutrients, toxins, ions, and lipids. Some of them are known to play critical roles in cellular and biochemical processes and their abnormal function may lead to diseases such as cystic fibrosis (such as ABCC7/CFTR (Riordan et al., 1989)) or phenomena such as multidrug-resistant cancer (Szakacs et al., 2006). Since these transporters facilitate transport of their substrates against a concentration gradient, their expression is usually polarized. "
[Show abstract][Hide abstract] ABSTRACT: The ATP-binding cassette (ABC) transporter superfamily includes several membrane-bound proteins that are critical to drug pharmacokinetics and disposition. Pharmacological evaluation of these proteins in vitro still remains a challenge. In this study, human ABC transporters were expressed in polarized epithelial cell monolayers transduced using the recombinant BacMam baculovirus system. The purpose of the study was to evaluate the efficacy of BacMam vectors to transduce cells grown in monolayers. In LLC-PK1 cells, baculoviral transduction is only successful via the apical side of a polarized monolayer. We observed that recombinant ABC transporters were expressed on the cell surface with post-translational modification. Furthermore, sodium butyrate played a critical role in recombinant protein expression, and pre-incubation in the presence of tunicamycin or thapsigargin enhanced protein expression. Cells overexpressing human P-glycoprotein (P-gp) showed vectorial basolateral-to-apical transport of [(3)H]-paclitaxel, which could be reversed by the inhibitor tariquidar. Similarly, co-expression of human P-gp and ABCG2 in LLC-PK1 cells resulted in higher transport of mitoxantrone, which is a substrate for both transporters, than in either P-gp- or ABCG2-expressing cells alone. Taken together, our results indicate that a high level of expression of efflux transporters in a polarized cell monolayer is technically feasible with the BacMam baculovirus system.
No preview · Article · Nov 2015 · Drug metabolism and disposition: the biological fate of chemicals
"Cystic fibrosis (CF; MIM #219700) is a chronic, multisystem disease affecting epithelia of the respiratory tract, exocrine pancreas, intestine, hepatobiliary system, exocrine sweat glands, and male genital tract. CF results from mutations in the CF transmembrane conductance regulator gene, CFTR (MIM * 602421), which affects chloride transport and flow of water into and out of cells [Riordan et al., 1989; Rommens et al., 1989]. Thick, sticky mucus accumulates and clogs organs, leading to, among other chronic problems, persistent respiratory infections and pancreatic obstruction, hindering pancreatic enzymes from breaking down food and absorbing nutrients . "
[Show abstract][Hide abstract] ABSTRACT: Infants are screened for Cystic Fibrosis (CF) in New York State (NYS) using an IRT-DNA algorithm. The purpose of this study was to validate and assess clinical validity of the FDA-cleared Illumina MiSeqDx CF 139-Variant Assay (139-VA) in the diverse NYS CF population. The study included 439 infants with CF identified via newborn screening (NBS) from 2002-2012. All had been screened using the Abbott Molecular CF Genotyping Assay or the Hologic InPlex CF Molecular Test. All with CF and 0-1 mutations were tested using the 139-VA. DNA extracted from dried blood spots was reliably and accurately genotyped using the 139-VA. Sixty-three additional mutations were identified. Clinical sensitivity of three panels ranged from 76.2% (23 mutations recommended for screening by ACMG/ACOG), to 79.7% (current NYS 39-mutation InPlex panel), up to 86.0% for the 139-VA. For all, sensitivity was highest in Whites and lowest in the Black population. Although the sample size was small, there was a nearly 20% increase in sensitivity for the Black CF population using the 139-VA (68.2%) over the ACMG/ACOG and InPlex panels (both 50.0%). Overall, the 139-VA is more sensitive than other commercially-available panels, and could be considered for NBS, clinical or research laboratories conducting CF screening. This article is protected by copyright. All rights reserved.