Spectrum of Mutations in the <i>CFTR</i> Gene in Cystic Fibrosis Patients of Spanish Ancestry

Alonso, María J.; Heine-Suñer, Damián; Calvo, M.; Rosell, Jordi; Giménez, Joaquím; Ramos, María Dolores Burguete; Orriols, Juan José Tellería; Palacio, Ana; Estivill, Xavier; Casals, Teresa

doi:10.1111/j.1469-1809.2006.00310.x

Cited by 52 publications

(52 citation statements)

References 33 publications

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“…In addition, Corr-4a was able to enhance the accumulation of CFTR 837X V232D and to increase levels of the mature, highly glycosylated form of CFTR 837-1480 with CFTR 837X V232D. The V232D mutant presents a mild form of CF (Alonso et al, 2007); however, the molecular basis for CF associated with this mutation has not been welldefined. The V232D mutation is proposed to form an aberrant hydrogen bond in MSD1 (Therien et al, 2001;Choi et al, 2004) that may hinder formation of interdomain contacts between MSD1 and MSD2 that are proposed to occur in the folded CFTR channel (Dawson and Locher, 2006;.…”

Section: Discussionmentioning

confidence: 99%

Mechanisms for Rescue of Correctable Folding Defects in CFTRΔF508

Grove

Rosser

Ren

et al. 2009

MBoC

108

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Premature degradation of CFTR⌬F508 causes cystic fibrosis (CF). CFTR⌬F508 folding defects are conditional and folding correctors are being developed as CF therapeutics. How the cellular environment impacts CFTR⌬F508 folding efficiency and the identity of CFTR⌬F508's correctable folding defects is unclear. We report that inactivation of the RMA1 or CHIP ubiquitin ligase permits a pool of CFTR⌬F508 to escape the endoplasmic reticulum. Combined RMA1 or CHIP inactivation and Corr-4a treatment enhanced CFTR⌬F508 folding to 3-7-fold greater levels than those elicited by Corr-4a. Some, but not all, folding defects in CFTR⌬F508 are correctable. CHIP and RMA1 recognize different regions of CFTR and a large pool of nascent CFTR⌬F508 is ubiquitinated by RMA1 before Corr-4a action. RMA1 recognizes defects in CFTR⌬F508 related to misassembly of a complex that contains MSD1, NBD1, and the R-domain. Corr-4a acts on CFTR⌬F508 after MSD2 synthesis and was ineffective at rescue of ⌬F508 dependent folding defects in amino-terminal regions. In contrast, misfolding caused by the rare CF-causing mutation V232D in MSD1 was highly correctable by Corr-4a. Overall, correction of folding defects recognized by RMA1 and/or global modulation of ER quality control has the potential to increase CFTR⌬F508 folding and provide a therapeutic approach for CF. INTRODUCTIONCystic fibrosis (CF) is a lethal inherited disorder that is caused by mutations in the gene coding for the CF transmembrane conductance regulator (CFTR; Riordan et al., 1989). The CFTR protein is a cAMP-regulated chloride channel that is localized to the apical surface of epithelial cells (Li et al., 1988;Anderson et al., 1991) where it functions to regulate water and salt homeostasis. Inheritance of the CFTR⌬F508 gene, in which phenylalanine 508 is deleted from the nucleotide binding domain 1 (NBD1), is the major cause of CF. This prevalent mutation is found in ϳ90% of patients afflicted with CF (Bobadilla et al., 2002). Loss of functional CFTR alters the hydration of airway epithelial and gives rise to microbial infections, which can ultimately lead to lung failure and death (Rowe et al., 2005).CFTR, an ATP-binding cassette (ABC) transporter superfamily member (Hyde et al., 1990), is a 1480-residue polytopic membrane glycoprotein that is predicted to contain two membrane-spanning domains (MSD), two nucleotide-binding domains (NBD), and a regulatory domain (R; Riordan et al., 1989). The folding of CFTR into a functional chloride channel is complex because it requires the coordinated folding and assembly of its membrane and cytoplasmic domains Cui et al., 2007). Consequently, CFTR folding is a relatively inefficient process with ϳ70% of the wild type and 99% of the ⌬F508 mutant being partitioned from a folding to a degradation pathway (Ward and Kopito, 1994). However, although early stage biogenic intermediates of CFTR and CFTR⌬F508 appear to have similar conformations (Zhang et al., 1998), the folding of CFTR⌬F508 becomes arrested at a poorly defined step. Interestingly, deletion ...

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Section: Discussionmentioning

confidence: 99%

Mechanisms for Rescue of Correctable Folding Defects in CFTRΔF508

Grove

Rosser

Ren

et al. 2009

MBoC

108

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“…Fifty-three individuals previously characterized 10,11 were enrolled in this study. Samples were grouped considering four genotypes: (a) CF patients homozygous for p.Phe508del (CF-F508del), n ¼ 7; (b) compound heterozygous CF patients carrying one of the two different splicing mutations: the c.580-1G4T, n ¼ 4 [p.Phe508del (2), p.Gly542* (1), p.Ala399Asp (1) Table S1).…”

Section: Materials and Methods Individualsmentioning

confidence: 99%

Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis

et al. 2013

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The major purpose of the present study was to quantify correctly spliced CFTR transcripts in human nasal epithelial cells from cystic fibrosis (CF) patients carrying the splicing mutations c.580-1G4T (712-1G4T) and c.2657 þ 5G4A (2789 þ 5G4A) and to assess the applicability of this model in CFTR therapeutic approaches. We performed the relative quantification of CFTR mRNA by reverse transcription quantitative PCR (RT-qPCR) of these splicing mutations in four groups (wild type, CF-F508del controls, CF patients and CF carriers) of individuals. In addition, in vitro assays using minigene constructs were performed to evaluate the effect of a new CF complex allele c.[2657 þ 5G4A; 2562T4G]. Ex vivo qPCR data show that the primary consequence of both mutations at the RNA level is the skipping of their neighboring exon (6 and 16, respectively). The CFTR minigenes results mimicked the ex vivo data, as exon 16 skipping is the main aberrant transcript, and the correctly spliced transcript level was observed in a similar proportion when the c.2657 þ 5G4A mutation is present. In summary, we provide evidence that ex vivo quantitative transcripts analysis using RT-qPCR is a robust technology that could be useful for measuring the efficacy of therapeutic approaches that attempt to achieve an increase in CFTR gene expression.

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“…Corr-4a had modest effects on misfolding of most mutants tested but could correct misfolding of CFTRV232D to wild-type levels in human embryonic kidney (HEK-293) cells. CFTRV232D is a rare CF-causing mutation located in the fourth transmembrane (TM)-spanning domain of the channel protein and is prevalent in CF patients of Spanish origin (2). The folding defect caused by the V232D mutation appears to be due to the introduction of a charged residue into a region of CFTR that is embedded in the lipid bilayer of the endoplasmic reticulum (ER) membrane (17,24).…”

mentioning

confidence: 99%

Increased folding and channel activity of a rare cystic fibrosis mutant with CFTR modulators

Caldwell

Grove

Houck

et al. 2011

American Journal of Physiology-Lung Cellular and Molecular Phys

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Cystic fibrosis (CF) is a lethal recessive genetic disease caused by mutations in the CFTR gene. The gene product is a PKA-regulated anion channel that is important for fluid and electrolyte transport in the epithelia of lung, gut, and ducts of the pancreas and sweat glands. The most common CFTR mutation, ΔF508, causes a severe, but correctable, folding defect and gating abnormality, resulting in negligible CFTR function and disease. There are also a large number of rare CF-related mutations where disease is caused by CFTR misfolding. Yet the extent to which defective biogenesis of these CFTR mutants can be corrected is not clear. CFTRV232D is one such mutant that exhibits defective folding and trafficking. CFTRΔF508 misfolding is difficult to correct, but defective biogenesis of CFTRV232D is corrected to near wild-type levels by small-molecule folding correctors in development as CF therapeutics. To determine if CFTRV232D protein is competent as a Cl− channel, we utilized single-channel recordings from transfected human embryonic kidney (HEK-293) cells. After PKA stimulation, CFTRV232D channels were detected in patches with a unitary Cl− conductance indistinguishable from that of CFTR. Yet the frequency of detecting CFTRV232D channels was reduced to ∼20% of patches compared with 60% for CFTR. The folding corrector Corr-4a increased the CFTRV232D channel detection rate and activity to levels similar to CFTR. CFTRV232D-corrected channels were inhibited with CFTRinh-172 and stimulated fourfold by the CFTR channel potentiator VRT-532. These data suggest that CF patients with rare mutations that cause CFTR misfolding, such as CFTRV232D, may benefit from treatment with folding correctors and channel potentiators in development to restore CFTRΔF508 function.

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Spectrum of Mutations in the CFTR Gene in Cystic Fibrosis Patients of Spanish Ancestry

Cited by 52 publications

References 33 publications

Mechanisms for Rescue of Correctable Folding Defects in CFTRΔF508

Mechanisms for Rescue of Correctable Folding Defects in CFTRΔF508

Assessing the residual CFTR gene expression in human nasal epithelium cells bearing CFTR splicing mutations causing cystic fibrosis

Increased folding and channel activity of a rare cystic fibrosis mutant with CFTR modulators

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