ABCMdb

ABCC7 p.Asn287Tyr

ClinVar:	c.861C>G , p.Asn287Lys ? , not provided c.859A>T , p.Asn287Tyr ? , not provided
CF databases:	c.859A>T , p.Asn287Tyr (CFTR1) ? , This individual was a compound heterozygote for [delta]F508. He was diagnosed at 3 1/2 years of age when referred because of rectal prolapse; his sweat Cl was 75 and 81 mEq/L. He had been totally healthy since birth with normal growth and no significant gastrointestinal complaints. He had 4-5 upper respiratory infections per year. Formal pancreatic function testing was within normal limits. A vas deferens was identified on the side of the herniorraphy. He has grown along the fiftieth percentile for weight and the ninetieth for height without the use of pancreatic enzyme supplements. N287Y was originally identified by SSCA and HA and was subsequently detected by artificial Dra I site generating PCR amplification. c.861C>G , p.Asn287Lys (CFTR1) ? ,
Predicted by SNAP2:	A: D (63%), C: D (75%), D: D (66%), E: D (75%), F: D (80%), G: D (63%), H: D (71%), I: D (80%), K: N (57%), L: D (80%), M: D (85%), P: D (80%), Q: D (66%), R: D (80%), S: D (53%), T: D (53%), V: D (75%), W: D (91%), Y: N (53%),
Predicted by PROVEAN:	A: N, C: D, D: N, E: N, F: D, G: N, H: N, I: D, K: N, L: D, M: N, P: D, Q: N, R: N, S: N, T: N, V: N, W: D, Y: N,

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Publications
[hide] A mutation in the cystic fibrosis transmembrane co... J Biol Chem. 2003 Mar 28;278(13):11554-60. Epub 2003 Jan 15. Silvis MR, Picciano JA, Bertrand C, Weixel K, Bridges RJ, Bradbury NA
A mutation in the cystic fibrosis transmembrane conductance regulator generates a novel internalization sequence and enhances endocytic rates.
J Biol Chem. 2003 Mar 28;278(13):11554-60. Epub 2003 Jan 15., 2003-03-28 [PMID:12529365]

Abstract [show]
Cystic fibrosis is a common lethal genetic disease among Caucasians. The cystic fibrosis gene encodes a cyclic adenosine monophosphate-activated chloride channel (cystic fibrosis transmembrane conductance regulator (CFTR)) that mediates electrolyte transport across the luminal surfaces of a variety of epithelial cells. Mutations in CFTR fall into two broad categories; those that affect protein biosynthesis/stability and traffic to the cell surface and those that cause altered channel kinetics in proteins that reach the cell surface. Here we report a novel mechanism by which mutations in CFTR give rise to disease. N287Y, a mutation within an intracellular loop of CFTR, increases channel endocytosis from the cell surface without affecting either biosynthesis or channel gating. The sole consequence of this novel mutation is to generate a novel tyrosine-based endocytic sequence within an intracellular loop in CFTR leading to increased removal from the cell surface and a reduction in the steady-state level of CFTR at the cell surface.

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Sentences [show]
No. Sentence Comment
4 N287Y, a mutation within an intracellular loop of CFTR, increases channel endocytosis from the cell surface without affecting either biosynthesis or channel gating. Login to comment
21 Recently an individual with mutation N287Y (991A3T) was identified based on a diagnosis of elevated sweat electrolytes (18). Login to comment
22 Since tyrosine-based signals are important in endocytic targeting, we hypothesized that the N287Y mutation generated a novel additional internalization signal in CFTR (Fig. 1a), leading to reduced cell surface expression of CFTR as a result of increased endocytic activity. Login to comment
23 Using site-directed mutagenesis in conjunction with morphological, biochemical, and functional assays, we demonstrate that N287Y CFTR generates a novel endocytic sequence enhancing the endocytic rate of CFTR compared with wild type. Login to comment
40 The N287Y mutation was introduced into the pFRT CFTR wild-type vector using the QuikChange Site-Directed mutagenesis kit (Stratagene). Login to comment
41 The sequences of the pcDNA5/FRT-CFTR wild type and N287Y were verified prior to use for expression. Login to comment
42 Cell Lines and Transfections-293 and CHO Flp-InTM (Invitrogen) isogenic cell lines expressing wild-type, ⌬F508, and N287Y CFTR from the same genomic locus were generated according to the manufacturer`s instructions. Login to comment
45 Confluent monolayers of Madin-Darby canine kidney cells (type II) grown on permeable filter supports were transiently transfected with either wild-type or N287Y CFTR using calcium phosphate. Login to comment
58 Patch Clamp Analysis-Whole cell and cell-attached patch clamp studies were performed on CHO Flp-In cells expressing wt CFTR or N287Y CFTR. Login to comment
70 RESULTS ER Export and Polarity of CFTR Distribution Is Preserved in N287Y CFTR-The cellular distribution of N287Y CFTR was initially examined since intracellular retention of mutant CFTRs by the ER quality control and subsequent failure to mature to a complex-glycosylated form is the most prevalent form of CF. Login to comment
72 Schematic representation of CFTR and immunofluorescence localization of ⌬F508, wild-type, and N287Y CFTR. Login to comment
75 The shaded circle shows the N287Y mutated residue within the second intracellular loop (CL-2). Login to comment
78 b-d, Flp 293 cells were stably transfected with wild-type (b), ⌬F508 (c), or N287Y (d) CFTR or mutant CFTR. Login to comment
80 type and N287Y CFTR were seen at the cell surface (Fig. 1, b and d). Login to comment
81 Similar results were obtained for N287Y CFTR stably expressed in CHO cells (data not shown). Login to comment
82 These results suggest that intracellular retention is unlikely to account for the clinical phenotype associated with N287Y CFTR. Login to comment
83 To determine whether the N287Y mutation altered the polarization of CFTR in epithelial cells, we transiently expressed wild-type and N287Y CFTR in Madin-Darby canine kidney cells. Confocal immunofluorescence microscopy demonstrated that wild-type CFTR was polarized to the apical plasma membrane (Fig. 2a) with little or no staining of the basal membrane (Fig. 2e). Login to comment
85 Similarly N287Y CFTR was also polarized to the apical plasma membrane (Fig. 2b). Login to comment
86 The ratio of N287Y CFTR in the apical membrane relative to the basolateral membrane was 8.1 Ϯ 3.1 as determined by domain-selective cell surface biotinylation. Login to comment
88 Steady-state levels and cell surface expression of wild-type and N287Y CFTR. Login to comment
90 Equal amounts of metabolically labeled 293 cells expressing wild-type or N287Y CFTR were subject to immunoprecipitation using the M3A7 anti-CFTR antibody followed by phosphorimage analysis (top). Login to comment
94 b, the expression level of wt and N287Y CFTR at the cell surface (biotinylated) and post-ER compartments (complex-glycosylated). Login to comment
96 c, glycosidase sensitivity of wild-type and N287Y CFTR. Login to comment
101 Polarized distribution of wild-type and N287Y CFTR in Madin-Darby canine kidney cells. Confocal micrograph of Madin-Darby canine kidney II cells transiently expressing wild-type and N287Y CFTR. Login to comment
103 Images show confocal sections taken at the plane of the apical membrane (a and b), plane of the tight junction (c and d), and plane of the basal membrane (e and f) for wild-type (a, c, and e) and N287Y (b, d, and f) CFTR. Login to comment
104 g, immunoblot of cells expressing wild-type and N287Y CFTR following domain-selective cell surface biotinylation. Login to comment
107 Thus, abnormalities in the polarized distribution of N287Y CFTR compared with wild-type CFTR cannot account for the disease phenotype associated with this mutation. Login to comment
108 N287Y CFTR Shows Altered Cellular Distribution-Quantitative immunoblot analysis of whole cell lysates from isogenic wild-type and N287Y CFTR-expressing cells revealed that steady-state levels of protein expression were identical in each cell line (Fig. 3, a and b). Login to comment
109 Moreover the ratio of fully glycosylated mature band C CFTR to immature core-glycosylated band B CFTR was not altered by the N287Y mutation compared with wild-type CFTR (7.2 Ϯ 0.5 and 6.9 Ϯ 0.5, mean Ϯ S.E., n ϭ 4 for wild-type and N287Y CFTR, respectively). Login to comment
110 In addition, the complex- and core-glycosylated forms of wild-type and N287Y CFTR could be distinguished by their sensitivity to endoglycosidases, which was not different between the two cell lines (Fig. 3c). Login to comment
112 In both wild-type and N287Y CFTR-expressing cell lines, biotinylated CFTR was detected as a single band of ϳ170 kDa, consistent with the presence of mature fully glycosylated CFTR at the cell surface. Login to comment
113 However, densitometric analysis revealed that the level of biotinylated N287Y CFTR was only ϳ50% of that for wild-type CFTR (Fig. 3, a and b). Login to comment
114 The detection of the mature complex-glycosylated form of wild-type and N287Y CFTR in immunoblots and at the cell surface demonstrates that biosynthesis and intracellular transport occurred. Login to comment
115 Biosynthesis of N287Y CFTR Is Not Impaired-To better evaluate the efficiency of N287Y CFTR biosynthesis, pulse-chase experiments were performed (Fig. 4, a and b). Login to comment
121 Biosynthetic maturation, cell surface targeting, and stability of wild-type and N287Y CFTR. Login to comment
128 Following pulse labeling of wt or N287Y CFTR for 25 min, plasma membrane insertion of the ion channels was determined by biotinylation during a 1-h chase at 37 °C using freshly dissolved sulfo-NHS-SS-biotin (1 mg/ml) every 15 min. Login to comment
131 d, integrated densities of wt and N287Y CFTR were obtained by phosphorimage analysis and represent mean Ϯ S.E. (n ϭ 3). Login to comment
139 The apparently normal biosynthesis of mature fully glycosylated N287Y CFTR suggests that targeting to the plasma membrane is largely intact. Login to comment
140 Wild-type and N287Y CFTR were pulse-labeled with [35 S]methionine, and those molecules that arrived at the cell surface were biotinylated throughout the subsequent chase. Login to comment
142 The cell surface targeting efficiency of N287Y CFTR was 91 Ϯ 4% (mean Ϯ S.E., n ϭ 3) of wild-type CFTR, suggesting that biosynthesis and plasma membrane delivery of N287Y CFTR is largely uncompromised. Login to comment
143 The turnover of complex-glycosylated wild-type and N287Y CFTR was assessed by pulse-chase labeling. Login to comment
145 The stability of complex-glycosylated N287Y CFTR stably expressed in 293 cells was not significantly different from that observed for wild-type CFTR. Login to comment
146 N287Y CFTR Displays Abnormal Endocytic Trafficking- Since biosynthesis and membrane insertion of N287Y CFTR was uncompromised, we hypothesized that the reduction in cell surface N287Y CFTR compared with the wild type was likely due to an increase in endocytic retrieval from the plasma membrane. Login to comment
148 Direct evidence for enhanced endocytosis of N287Y CFTR was obtained by determining the amount of thiol-resistant biotinylated CFTR with respect to time. Login to comment
149 The rapid increase in thiol-resistant biotinylated wild-type and N287Y CFTR indicates that both constructs are internalized with high efficiency (Fig. 5). Login to comment
151 In contrast, the internalization rate of N287Y CFTR was nearly 2-fold faster (12.4 Ϯ 0.4%/min, n ϭ 4), implying that the tyrosine substitution enhanced the endocytic activity of CFTR. Login to comment
152 Channel Density, but Not Single Channel Properties, Is Altered in N287Y CFTR-expressing Cells-We have documented abnormalities in the endocytic, but not biosynthetic, traffic of N287Y CFTR that result in a decrease in biotinylatable CFTR at the cell surface. Login to comment
154 However, it is formally possible that there are also alterations in the biophysical fingerprint of N287Y CFTR compared with wild type. Login to comment
155 To confirm that N287Y CFTR is functional at the plasma membrane, electrophysiological recordings were performed in the whole cell and cell-attached patch configurations on CHO cells stably expressing either wild-type or N287Y CFTR. Login to comment
157 Expression of both wild-type and N287Y CFTR conferred cAMP-stimulated whole cell currents in CHO cells (Fig. 6) with no change in base-line current in the absence of cAMP. Login to comment
158 The cAMP-stimulated whole cell conductance was 2.6-fold higher in wt CFTR-expressing cells compared with N287Y CFTR-expressing cells. Login to comment
159 Cell-attached patch studies revealed the single channel properties of the N287Y CFTR were nearly identical to that of wt CFTR. Login to comment
160 The single channel conductance`s were 9.7 Ϯ 0.35 versus 9.8 Ϯ 0.39 picosiemens and open probabilities were 0.48 Ϯ 0.04 versus 0.46 Ϯ 0.05 for N287Y CFTR and wt CFTR, respectively. Login to comment
161 These results strongly support the conclusion that the lower whole cell conductance of the N287Y CFTR-expressing cells is the result of a lower density of functional channels in the plasma membrane. Login to comment
162 DISCUSSION We have demonstrated that the N287Y mutation in CFTR causes clinical disease by dramatically increasing the rate at which CFTR is sequestered from the plasma membrane without altering its channel properties. Login to comment
173 Internalization efficiency of CFTR is increased by the N287Y mutation. Login to comment
174 The rate of removal of CFTR from the cell surface was monitored as an increase in biotinylated CFTR resistant to thiol cleavage of the biotin moiety as described under ''Experimental Procedures.`` Cells stably expressing wild-type (filled circle) or N287Y (open circle) CFTR were subjected to cell surface biotinylation at 4 °C, washed, and left to internalize CFTR at 37 °C. Login to comment
179 The N287Y mutation, a mutation in the second intracellular loop, results in a channel that is biosynthetically and biophysically normal but has greater endocytosis kinetics compared with wild-type CFTR. Login to comment
181 Similarly targeting of CFTR to the apical plasma membrane domain of polarized epithelial cells is unaffected by the N287Y mutation. Login to comment
182 In contrast to other mutations in the second intracellular loop, the N287Y mutation has no functional consequence on the gating kinetics of CFTR. Login to comment
184 The only physiological consequence of the N287Y mutation is therefore to produce a more rapidly endocytosed protein, reducing steady-state levels in the plasma membrane. Login to comment
185 The genotype of the initial patient reported to have the N287Y mutation was ⌬F508/N287Y (18). Login to comment
186 Since little or no cell surface CFTR is produced by the ⌬F508 allele, the only cell surface CFTR protein is produced by the N287Y allele. Login to comment
188 The most parsimonious interpretation of our data is that the N287Y mutation generates a novel tyrosine-based endocytic motif. Login to comment
191 Thus it is possible that the N287Y mutation results in the generation of a sequence that displays modest affinity for the AP-2 clathrin adaptor complex. Login to comment
192 Although we have focused upon the role of the N287Y mutation in mediating enhanced endocytosis of CFTR, it is still a formal possibility that the N287Y mutation could also reduce the endocytic recycling rate. Login to comment
194 It is of interest to note that the N287Y mutation is a gain of function mutation and appears to generate an endocytic signal that is present within the body of the protein rather than at the termini of the protein, a localization not previously identified in polytopic membrane proteins. Login to comment

[hide] The role of regulated CFTR trafficking in epitheli... Am J Physiol Cell Physiol. 2003 Jul;285(1):C1-18. Bertrand CA, Frizzell RA
The role of regulated CFTR trafficking in epithelial secretion.
Am J Physiol Cell Physiol. 2003 Jul;285(1):C1-18., [PMID:12777252]

Abstract [show]
The focus of this review is the regulated trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) in distal compartments of the protein secretory pathway and the question of how changes in CFTR cellular distribution may impact on the functions of polarized epithelial cells. We summarize data concerning the cellular localization and activity of CFTR and attempt to synthesize often conflicting results from functional studies of regulated endocytosis and exocytosis in CFTR-expressing cells. In some instances, findings that are inconsistent with regulated CFTR trafficking may result from the use of overexpression systems or nonphysiological experimental conditions. Nevertheless, judging from data on other transporters, an appropriate cellular context is necessary to support regulated CFTR trafficking, even in epithelial cells. The discovery that disease mutations can influence CFTR trafficking in distal secretory and recycling compartments provides support for the concept that regulated CFTR recycling contributes to normal epithelial function, including the control of apical CFTR channel density and epithelial protein secretion. Finally, we propose molecular mechanisms for regulated CFTR endocytosis and exocytosis that are based on CFTR interactions with other proteins, particularly those whose primary function is membrane trafficking. These models provide testable hypotheses that may lead to elucidation of CFTR trafficking mechanisms and permit their experimental manipulation in polarized epithelial cells.

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Sentences [show]
No. Sentence Comment
404 The N287Y mutant resides in the second intracellular loop of CFTR and results in mild disease when expressed in combination with ⌬F508 CFTR. Login to comment
405 This mutant did not exhibit a folding defect, because the N287Y and wt CFTRs showed similar maturation kinetics. Login to comment
408 Cl transport was reduced in proportion to altered cell surface CFTR, but the single-channel properties of N287Y were similar to those of wt CFTR. Login to comment
409 Biotinylation experiments showed that N287Y CFTR was internalized approximately twice as fast as wt CFTR, which is expected to alter its distribution between plasma membrane and intracellular compartments. Login to comment

[hide] Mutations in the amino terminus of the cystic fibr... J Biol Chem. 2006 Feb 10;281(6):3329-34. Epub 2005 Dec 8. Jurkuvenaite A, Varga K, Nowotarski K, Kirk KL, Sorscher EJ, Li Y, Clancy JP, Bebok Z, Collawn JF
Mutations in the amino terminus of the cystic fibrosis transmembrane conductance regulator enhance endocytosis.
J Biol Chem. 2006 Feb 10;281(6):3329-34. Epub 2005 Dec 8., 2006-02-10 [PMID:16339147]

Abstract [show]
Efficient endocytosis of the cystic fibrosis transmembrane conductance regulator (CFTR) is mediated by a tyrosine-based internalization signal in the CFTR carboxyl-terminal tail 1424YDSI1427. In the present studies, two naturally occurring cystic fibrosis mutations in the amino terminus of CFTR, R31C, and R31L were examined. To determine the defect that these mutations cause, the Arg-31 mutants were expressed in COS-7 cells and their biogenesis and trafficking to the cell surface tested in metabolic pulse-chase and surface biotinylation assays, respectively. The results indicated that both Arg-31 mutants were processed to band C at approximately 50% the efficiency of the wild-type protein. However, once processed and delivered to the cell surface, their half-lives were the same as wild-type protein. Interestingly, indirect immunofluorescence and cell surface biotinylation indicated that the surface pool was much smaller than could be accounted for based on the biogenesis defect alone. Therefore, the Arg-31 mutants were tested in internalization assays and found to be internalized at 2x the rate of the wild-type protein. Patch clamp and 6-methoxy-N-(3-sulfopropyl)quinolinium analysis confirmed reduced amounts of functional Arg-31 channels at the cell surface. Together, the results suggest that both R31C and R31L mutations compromise biogenesis and enhance internalization of CFTR. These two additive effects contribute to the loss of surface expression and the associated defect in chloride conductance that is consistent with a disease phenotype.

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Sentences [show]
No. Sentence Comment
222 Another CFTR mutation was recently identified in the second intracellular loop, N287Y, which affects endocytosis over wild-type levels (34). Login to comment
223 Interestingly, the N287Y mutation does not introduce a consensus tyrosine-based signal and did not affect biogenesis of CFTR. Login to comment
232 Analysis of the ⌬F508, N287Y, and R31L and R31C indicate that alterations in the transport of CFTR at the cell surface, whether it is enhanced internalization or compromised recycling, can result in a disease phenotype. Login to comment

[hide] Direct interaction with filamins modulates the sta... J Clin Invest. 2007 Feb;117(2):364-74. Epub 2007 Jan 18. Thelin WR, Chen Y, Gentzsch M, Kreda SM, Sallee JL, Scarlett CO, Borchers CH, Jacobson K, Stutts MJ, Milgram SL
Direct interaction with filamins modulates the stability and plasma membrane expression of CFTR.
J Clin Invest. 2007 Feb;117(2):364-74. Epub 2007 Jan 18., [PMID:17235394]

Abstract [show]
The role of the cystic fibrosis transmembrane conductance regulator (CFTR) as a cAMP-dependent chloride channel on the apical membrane of epithelia is well established. However, the processes by which CFTR is regulated on the cell surface are not clear. Here we report the identification of a protein-protein interaction between CFTR and the cytoskeletal filamin proteins. Using proteomic approaches, we identified filamins as proteins that associate with the extreme CFTR N terminus. Furthermore, we identified a disease-causing missense mutation in CFTR, serine 13 to phenylalanine (S13F), which disrupted this interaction. In cells, filamins tethered plasma membrane CFTR to the underlying actin network. This interaction stabilized CFTR at the cell surface and regulated the plasma membrane dynamics and confinement of the channel. In the absence of filamin binding, CFTR was internalized from the cell surface, where it prematurely accumulated in lysosomes and was ultimately degraded. Our data demonstrate what we believe to be a previously unrecognized role for the CFTR N terminus in the regulation of the plasma membrane stability and metabolic stability of CFTR. In addition, we elucidate the molecular defect associated with the S13F mutation.

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Sentences [show]
No. Sentence Comment
258 For example, the R31L or N287Y mutations may introduce a nonnative internalization motif in CFTR and result in increased plasma membrane internalization (8, 9). Login to comment

[hide] Spectrum of mutations and variants/haplotypes of C... Clin Genet. 2007 Jun;71(6):530-9. Chang MC, Chang YT, Wei SC, Tien YW, Liang PC, Jan IS, Su YN, Wong JM
Spectrum of mutations and variants/haplotypes of CFTR and genotype-phenotype correlation in idiopathic chronic pancreatitis and controls in Chinese by complete analysis.
Clin Genet. 2007 Jun;71(6):530-9., [PMID:17539902]

Abstract [show]
Mutations in cystic fibrosis transmembrane conductance regulator (CFTR) gene have been reported in patients with chronic pancreatitis. The authors examine whether the mutations and haplotypes of CFTR will increase the risk of developing idiopathic chronic pancreatitis (ICP) in Chinese and their genotype and phenotype correlations. Seventy-eight patients with ICP and 200 geographically and ethnically matched controls in Taiwan were analyzed. The entire 27 coding and intronic regions of the CFTR gene were identified using heteroduplex analytical techniques and confirmed by sequencing analysis. The presence of 125G/C, 1001+10C>T, IVSTn(TG)m, 1540A>G, c2694T>G, and c4521G>A were determined by directing sequencing. Abnormal CFTR allele was found to be thrice as frequent in ICP patients as in controls (22/156 vs 19/400, p < 0.0001). T5 allele was associated with early onset of ICP. In six-loci haplotype analysis, 13 common haplotypes were assembled in the 278 individuals tested. The 125G/1001+11C/TG12/470M/2694T/4521G haplotype was associated with risk of ICP (odds ratio 11.3; 95% confidence interval 2.3-54.6, p = 0.008) in Chinese. The mutation spectrum is different from other ethnic groups. A population-specific panel of CFTR changes should be recommended for targeted populations including ICP in Chinese. It is important to design suitable screening programs for different populations.

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Sentences [show]
No. Sentence Comment
97 These mutations include I556V, G to A 3849145, N287Y, I125T, E217G, S895N, G1O69R, and Q1352H that have been found in patients with CP or CBAVD (http://www.genet. Login to comment
109 (%) in ICP Controls (%) I556V Exon 11 A to G 1798 Amino acid substitution 7 (8.9) 2 (1) IVS8-5T Intron 8 deletaion of 2T between 1342-12 and 1342-6 Aberrant splicing 6 (7.7) 14 (7) G to A 3849145 Intron 19 G to A at 3849145 mRNA splicing defect 3 (3.8) 2 (1) N287Y Exon 6b A to T 991 Amino acid substitution 2 (2.6) 00 (0) I125T Exon 4 T to C 506 Amino acid substitution 1 (1.3) 00 (0) E217G Exon 6a A to G 782 Amino acid substitution 1 (1.3) 00 (0) S895N Exon 15 G to A 2816 Missense mutation 1 (1.3) 00 (0) G1O69R Exon 17b G to A 3337 Amino acid substitution 1 (1.3) 00 (0) Q1352H Exon 22 G to C at 4188 Amino acid substitution 0 (0.0) 1 (0.5) ICP, idiopathic chronic pancreatitis. Login to comment
157 All our mutations are belonging to Ômild` mutations compatible with previous studies (6), including I556V, G to A 3849145, I125T, E217G, N287Y, S895N, G1O69R, and Q1352H. Login to comment

[hide] Localization studies of rare missense mutations in... Hum Mutat. 2008 Nov;29(11):1364-72. Krasnov KV, Tzetis M, Cheng J, Guggino WB, Cutting GR
Localization studies of rare missense mutations in cystic fibrosis transmembrane conductance regulator (CFTR) facilitate interpretation of genotype-phenotype relationships.
Hum Mutat. 2008 Nov;29(11):1364-72., [PMID:18951463]

Abstract [show]
We have been investigating the functional consequences of rare disease-associated amino acid substitutions in the cystic fibrosis transmembrane conductance regulator (CFTR). Mutations of the arginine residue at codon 1070 have been associated with different disease consequences; R1070P and R1070Q with "severe" pancreatic insufficient cystic fibrosis (CF) and R1070W with "mild" pancreatic sufficient CF or congenital bilateral absence of the vas deferens. Intriguingly, CFTR bearing each of these mutations is functional when expressed in nonpolarized cells. To determine whether R1070 mutations cause disease by affecting CFTR localization, we created polarized Madin Darby canine kidney (MDCK) cell lines that express either wild-type or mutant CFTR from the same genomic integration site. Confocal microscopy and biotinylation studies revealed that R1070P was not inserted into the apical membrane, R1070W was inserted at levels reduced from wild-type while R1070Q was present in the apical membrane at levels comparable to wild-type. The abnormal localization of CFTR bearing R1070P and R1070W was consistent with deleterious consequences in patients; however, the profile of CFTR R1070Q was inconsistent with a "severe" phenotype. Reanalysis of 16 patients with the R1070Q mutation revealed that 11 carried an in cis nonsense mutation, S466X. All 11 patients carrying the complex allele R1070Q-S466X had severe disease, while 4 out of 5 patients with R1070Q had "mild" disease, thereby reconciling the apparent discrepancy between the localization studies of R1070Q and the phenotype of patients bearing this mutation. Our results emphasize that localization studies in relevant model systems can greatly assist the interpretation of the disease-causing potential of rare missense mutations.

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Sentences [show]
No. Sentence Comment
197 Partial mislocalization of CFTR R1070W may be due to alteration in the rate of membrane recycling, as has previously been observed with N287Y, a CFTR cytoplasmic loop 2 mutant [Silvis et al., 2003]. Login to comment

[hide] CFTR: folding, misfolding and correcting the Delta... Trends Mol Med. 2012 Feb;18(2):81-91. Epub 2011 Dec 3. Lukacs GL, Verkman AS
CFTR: folding, misfolding and correcting the DeltaF508 conformational defect.
Trends Mol Med. 2012 Feb;18(2):81-91. Epub 2011 Dec 3., [PMID:22138491]

Abstract [show]
Cystic fibrosis (CF), the most common lethal genetic disease in the Caucasian population, is caused by loss-of-function mutations of the CF transmembrane conductance regulator (CFTR), a cyclic AMP-regulated plasma membrane chloride channel. The most common mutation, deletion of phenylalanine 508 (DeltaF508), impairs CFTR folding and, consequently, its biosynthetic and endocytic processing as well as chloride channel function. Pharmacological treatments may target the DeltaF508 CFTR structural defect directly by binding to the mutant protein and/or indirectly by altering cellular protein homeostasis (proteostasis) to promote DeltaF508 CFTR plasma membrane targeting and stability. This review discusses recent basic research aimed at elucidating the structural and trafficking defects of DeltaF508 CFTR, a prerequisite for the rational design of CF therapy to correct the loss-of-function phenotype.

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Sentences [show]
No. Sentence Comment
112 Conversely, trafficking mutants may have no defect in gating function, as illustrated by the N287Y mutation within the second intracellular loop of CFTR in which channel endocytosis from the PM is increased without impairment in channel gating [72]. Login to comment

[hide] Endocytic trafficking of CFTR in health and diseas... J Cyst Fibros. 2007 Jan;6(1):1-14. Epub 2006 Nov 13. Ameen N, Silvis M, Bradbury NA
Endocytic trafficking of CFTR in health and disease.
J Cyst Fibros. 2007 Jan;6(1):1-14. Epub 2006 Nov 13., [PMID:17098482]

Abstract [show]
The cystic fibrosis transmembrane conductance regulator (CFTR) is a Cl-selective anion channel expressed in epithelial tissues. Mutations in CFTR lead to the genetic disease cystic fibrosis (CF). Within each epithelial cell, CFTR interacts with a large number of transient macromolecular complexes, many of which are involved in the trafficking and targeting of CFTR. Understanding how these complexes regulate the trafficking and fate of CFTR, provides a singular insight not only into the patho-physiology of cystic fibrosis, but also provides potential drug targets to help cure this debilitating disease.

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Sentences [show]
No. Sentence Comment
671 A patient with a mutation N287Y (991A→T) was identified based on a diagnosis of elevated sweat electrolytes [70]. Login to comment
674 Biosynthesis and delivery of N287Y CFTR to the cell surface is unaffected, but endocytosis of N287Y CFTR from the cell surface is markedly enhanced (~twice the rate of wild-type CFTR), resulting in a 50% reduction in steady-state levels of CFTR at the cell surface. Login to comment
675 In contrast to many mutations in CFTR, the single channel properties of N287Y CFTR and wild-type CFTR are indistinguishable arguing that disease cannot be attributed to conductance or gating defects, but rather to enhanced CFTR endocytosis. Login to comment
676 The genotype of the identified patient was ΔF508/N287Y [70]. Login to comment
678 Such data would argue that at least 25% of wild-type CFTR activity is required to ameliorate the disease symptoms in CF patients. It is of interest to note that the N287Y mutation is a gain of function mutation and appears to generate an endocytic signal that is present within the body of the protein rather than at the termini of the protein, a location not previously identified in polytopic membrane proteins. Login to comment
679 In the broader context of molecular mechanisms underlying the pathology of human genetic diseases, the significance of the observations on N287Y CFTR lies in the recognition that mutations can reduce the expression level of a membrane protein, not only by impairing its biosynthesis or stability (or in the case of ion channels their biophysical fingerprint), but also by accelerating endocytic retrieval from the plasma membrane. Login to comment
680 R31C and R31L are CFTR mutations that also give rise to a mild clinical phenotype [71]. Login to comment
672 A patient with a mutation N287Y (991A࢐T) was identified based on a diagnosis of elevated sweat electrolytes [70]. Login to comment
677 The genotype of the identified patient was ƊF508/N287Y [70]. Login to comment

[hide] Genotyping microarray for the detection of more th... J Mol Diagn. 2005 Aug;7(3):375-87. Schrijver I, Oitmaa E, Metspalu A, Gardner P
Genotyping microarray for the detection of more than 200 CFTR mutations in ethnically diverse populations.
J Mol Diagn. 2005 Aug;7(3):375-87., [PMID:16049310]

Abstract [show]
Cystic fibrosis (CF), which is due to mutations in the cystic fibrosis transmembrane conductance regulator gene, is a common life-shortening disease. Although CF occurs with the highest incidence in Caucasians, it also occurs in other ethnicities with variable frequency. Recent national guidelines suggest that all couples contemplating pregnancy should be informed of molecular screening for CF carrier status for purposes of genetic counseling. Commercially available CF carrier screening panels offer a limited panel of mutations, however, making them insufficiently sensitive for certain groups within an ethnically diverse population. This discrepancy is even more pronounced when such carrier screening panels are used for diagnostic purposes. By means of arrayed primer extension technology, we have designed a genotyping microarray with 204 probe sites for CF transmembrane conductance regulator gene mutation detection. The arrayed primer extension array, based on a platform technology for disease detection with multiple applications, is a robust, cost-effective, and easily modifiable assay suitable for CF carrier screening and disease detection.

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51 Complete List of Mutations Detectable with the CF APEX Assay CFTR location Amino acid change Nucleotide change 1 E 1 Frameshift 175delC 2 E 2,3 Frameshift del E2, E3 3 E 2 W19C 189 GϾT 4 E 2 Q39X 247 CϾT 5 IVS 2 Possible splicing defect 296 ϩ 12 TϾC 6 E 3 Frameshift 359insT 7 E 3 Frameshift 394delTT 8 E 3 W57X (TAG) 302GϾA 9 E 3 W57X (TGA) 303GϾA 10 E 3 E60X 310GϾT 11 E 3 P67L 332CϾT 12 E 3 R74Q 353GϾA 13 E 3 R75X 355CϾT 14 E 3 G85E 386GϾA 15 E 3 G91R 403GϾA 16 IVS 3 Splicing defect 405 ϩ 1GϾA 17 IVS 3 Possible splicing defect 405 ϩ 3AϾC 18 IVS 3 Splicing defect 406 - 1GϾA 19 E 4 E92X 406GϾT 20 E 4 E92K 406GϾA 21 E 4 Q98R 425AϾG 22 E 4 Q98P 425AϾC 23 E 4 Frameshift 444delA 24 E 4 Frameshift 457TATϾG 25 E 4 R117C 481CϾT 26 E 4 R117H 482GϾA 27 E 4 R117P 482GϾC 28 E 4 R117L 482GϾT 29 E 4 Y122X 498TϾA 30 E 4 Frameshift 574delA 31 E 4 I148T 575TϾC 32 E 4 Splicing defect 621GϾA 33 IVS 4 Splicing defect 621 ϩ 1GϾT 34 IVS 4 Splicing defect 621 ϩ 3AϾG 35 E 5 Frameshift 624delT 36 E 5 Frameshift 663delT 37 E 5 G178R 664GϾA 38 E 5 Q179K 667CϾA 39 IVS 5 Splicing defect 711 ϩ 1GϾT 40 IVS 5 Splicing defect 711 ϩ 1GϾA 41 IVS 5 Splicing defect 712 - 1GϾT 42 E 6a H199Y 727CϾT 43 E 6a P205S 745CϾT 44 E 6a L206W 749TϾG 45 E 6a Q220X 790CϾT 46 E 6b Frameshift 935delA 47 E 6b Frameshift 936delTA 48 E 6b N287Y 991AϾT 49 IVS 6b Splicing defect 1002 - 3TϾG 50 E 7 ⌬F311 3-bp del between nucleotides 1059 and 1069 51 E 7 Frameshift 1078delT 52 E 7 Frameshift 1119delA 53 E 7 G330X 1120GϾT 54 E 7 R334W 1132CϾT 55 E 7 I336K 1139TϾA 56 E 7 T338I 1145CϾT 57 E 7 Frameshift 1154insTC 58 E 7 Frameshift 1161delC 59 E 7 L346P 1169TϾC 60 E 7 R347H 1172GϾA 61 E 7 R347P 1172GϾC 62 E 7 R347L 1172GϾT 63 E 7 R352Q 1187GϾA 64 E 7 Q359K/T360K 1207CϾA and 1211CϾA 65 E 7 S364P 1222TϾC 66 E 8 Frameshift 1259insA 67 E 8 W401X (TAG) 1334GϾA 68 E 8 W401X (TGA) 1335GϾA 69 IVS 8 Splicing changes 1342 - 6 poly(T) variants 5T/7T/9T 70 IVS 8 Splicing defect 1342 - 2AϾC Table 1. Continued CFTR location Amino acid change Nucleotide change 71 E 9 A455E 1496CϾA 72 E 9 Frameshift 1504delG 73 E 10 G480C 1570GϾT 74 E 10 Q493X 1609CϾT 75 E 10 Frameshift 1609delCA 76 E 10 ⌬I507 3-bp del between nucleotides 1648 and 1653 77 E 10 ⌬F508 3-bp del between nucleotides 1652 and 1655 78 E 10 Frameshift 1677delTA 79 E 10 V520F 1690GϾT 80 E 10 C524X 1704CϾA 81 IVS 10 Possible splicing defect 1717 - 8GϾA 82 IVS 10 Splicing defect 1717 - 1GϾA 83 E 11 G542X 1756GϾT 84 E 11 G551D 1784GϾA 85 E 11 Frameshift 1784delG 86 E 11 S549R (AϾC) 1777AϾC 87 E 11 S549I 1778GϾT 88 E 11 S549N 1778GϾA 89 E 11 S549R (TϾG) 1779TϾG 90 E 11 Q552X 1786CϾT 91 E 11 R553X 1789CϾT 92 E 11 R553G 1789CϾG 93 E 11 R553Q 1790GϾA 94 E 11 L558S 1805TϾC 95 E 11 A559T 1807GϾA 96 E 11 R560T 1811GϾC 97 E 11 R560K 1811GϾA 98 IVS 11 Splicing defect 1811 ϩ 1.6 kb AϾG 99 IVS 11 Splicing defect 1812 - 1GϾA 100 E 12 Y563D 1819TϾG 101 E 12 Y563N 1819TϾA 102 E 12 Frameshift 1833delT 103 E 12 D572N 1846GϾA 104 E 12 P574H 1853CϾA 105 E 12 T582R 1877CϾG 106 E 12 E585X 1885GϾT 107 IVS 12 Splicing defect 1898 ϩ 5GϾT 108 IVS 12 Splicing defect 1898 ϩ 1GϾA 109 IVS 12 Splicing defect 1898 ϩ 1GϾC 110 IVS 12 Splicing defect 1898 ϩ 1GϾT 111 E 13 Frameshift 1924del7 112 E 13 del of 28 amino acids 1949del84 113 E 13 I618T 1985TϾC 114 E 13 Frameshift 2183AAϾG 115 E 13 Frameshift 2043delG 116 E 13 Frameshift 2055del9ϾA 117 E 13 D648V 2075TϾA 118 E 13 Frameshift 2105-2117 del13insAGAA 119 E 13 Frameshift 2108delA 120 E 13 R668C 2134CϾT 121 E 13 Frameshift 2143delT 122 E 13 Frameshift 2176insC 123 E 13 Frameshift 2184delA 124 E 13 Frameshift 2184insA 125 E 13 Q685X 2185CϾT 126 E 13 R709X 2257CϾT 127 E 13 K710X 2260AϾT 128 E 13 Frameshift 2307insA 129 E 13 V754M 2392GϾA 130 E 13 R764X 2422CϾT 131 E 14a W846X 2670GϾA 132 E 14a Frameshift 2734delGinsAT 133 E 14b Frameshift 2766del8 134 IVS 14b Splicing defect 2789 ϩ 5GϾA 135 IVS 14b Splicing defect 2790 - 2AϾG 136 E 15 Q890X 2800CϾT 137 E 15 Frameshift 2869insG 138 E 15 S945L 2966CϾT 139 E 15 Frameshift 2991del32 140 E 16 Splicing defect 3120GϾA interrogation: ACCAACATGTTTTCTTTGATCTTAC 3121-2A3G,T S; 5Ј-ACCAACATGTTTTCTTTGATCTTAC A GTTGTTATTAATTGTGATTGGAGCTATAG-3Ј; CAACAA- TAATTAACACTAACCTCGA 3121-2A3G,T AS. Login to comment
150 Primers Generated to Create Synthetic Templates That Serve As Positive Mutation Controls Primer name Sense strand 5Ј 3 3Ј Name Antisense strand 5Ј 3 3Ј 175delC synt F T(15)ATTTTTTTCAGGTGAGAAGGTGGCCA 175delC synt R T(15)ATTTGGAGACAACGCTGGCCTTTTCC W19C synt F T(15)TACCAGACCAATTTTGAGGAAAGGAT W19C synt R T(15)ACAGCTAAAATAAAGAGAGGAGGAAC Q39X synt F T(15)TAAATCCCTTCTGTTGATTCTGCTGA Q39X synt R T(15)AGTATATGTCTGACAATTCCAGGCGC 296 ϩ 12TϾC synt F T(15)CACATTGTTTAGTTGAAGAGAGAAAT 296 ϩ 12TϾC synt R T(15)GCATGAACATACCTTTCCAATTTTTC 359insT synt F T(15)TTTTTTTCTGGAGATTTATGTTCTAT 359insT synt R T(15)AAAAAAACATCGCCGAAGGGCATTAA E60X synt F T(15)TAGCTGGCTTCAAAGAAAAATCCTAA E60X synt R T(15)ATCTATCCCATTCTCTGCAAAAGAAT P67L synt F T(15)TTAAACTCATTAATGCCCTTCGGCGA P67L synt R T(15)AGATTTTTCTTTGAAGCCAGCTCTCT R74Q synt F T(15)AGCGATGTTTTTTCTGGAGATTTATG R74Q synt R T(15)TGAAGGGCATTAATGAGTTTAGGATT R75X synt F T(15)TGATGTTTTTTCTGGAGATTTATGTT R75X synt R T(15)ACCGAAGGGCATTAATGAGTTTAGGA W57X(TAG) synt F T(15)AGGATAGAGAGCTGGCTTCAAAGAAA W57X(TAG) synt R T(15)TATTCTCTGCAAAAGAATAAAAAGTG W57X(TGA) synt F T(15)AGATAGAGAGCTGGCTTCAAAGAAAA W57X(TGA) synt R T(15)TCATTCTCTGCAAAAGAATAAAAAGT G91R synt F T(15)AGGGTAAGGATCTCATTTGTACATTC G91R synt R T(15)TTAAATATAAAAAGATTCCATAGAAC 405 ϩ 1GϾA synt F T(15)ATAAGGATCTCATTTGTACATTCATT 405 ϩ 1GϾA synt R T(15)TCCCTAAATATAAAAAGATTCCATAG 405 ϩ 3AϾC synt F T(15)CAGGATCTCATTTGTACATTCATTAT 405 ϩ 3AϾC synt R T(15)GACCCCTAAATATAAAAAGATTCCAT 406 - 1GϾA synt F T(15)AGAAGTCACCAAAGCAGTACAGCCTC 406 - 1GϾA synt R T(15)TTACAAAAGGGGAAAAACAGAGAAAT E92X synt F T(15)TAAGTCACCAAAGCAGTACAGCCTCT E92X synt R T(15)ACTACAAAAGGGGAAAAACAGAGAAA E92K synt F T(15)AAAGTCACCAAAGCAGTACAGCCTCT E92K synt R T(15)TCTACAAAAGGGGAAAAACAGAGAAA 444delA synt F T(15)GATCATAGCTTCCTATGACCCGGATA 444delA synt R T(15)ATCTTCCCAGTAAGAGAGGCTGTACT 574delA synt F T(15)CTTGGAATGCAGATGAGAATAGCTAT 574delA synt R T(15)AGTGATGAAGGCCAAAAATGGCTGGG 621GϾA synt F T(15)AGTAATACTTCCTTGCACAGGCCCCA 621GϾA synt R T(15)TTTCTTATAAATCAAACTAAACATAG Q98P synt F T(15)CGCCTCTCTTACTGGGAAGAATCATA Q98P synt R T(15)GGTACTGCTTTGGTGACTTCCTACAA 457TATϾG synt F T(15)GGACCCGGATAACAAGGAGGAACGCT 457TATϾG synt R T(15)CGGAAGCTATGATTCTTCCCAGTAAG I148T synt F T(15)CTGGAATGCAGATGAGAATAGCTATG I148T synt R T(15)GTGTGATGAAGGCCAAAAATGGCTGG 624delT synt F T(15)CTTAAAGCTGTCAAGCCGTGTTCTAG 624delT synt R T(15)TAAGTCTAAAAGAAAAATGGAAAGTT 663delT synt F T(15)ATGGACAACTTGTTAGTCTCCTTTCC 663delT synt R T(15)CATACTTATTTTATCTAGAACACGGC G178R synt F T(15)AGACAACTTGTTAGTCTCCTTTCCAA G178R synt R T(15)TAATACTTATTTTATCTAGAACACGG Q179K synt F T(15)AAACTTGTTAGTCTCCTTTCCAACAA Q179K synt R T(15)TTCCAATACTTATTTTATCTAGAACA 711 ϩ 5GϾA synt F T(15)ATACCTATTGATTTAATCTTTTAGGC 711 ϩ 5GϾA synt R T(15)TTATACTTCATCAAATTTGTTCAGGT 712 - 1GϾT synt F T(15)TGGACTTGCATTGGCACATTTCGTGT 712 - 1GϾT synt R T(15)TATGGAAAATAAAAGCACAGCAAAAAC H199Y synt F T(15)TATTTCGTGTGGATCGCTCCTTTGCA H199Y synt R T(15)TATGCCAATGCTAGTCCCTGGAAAATA P205S synt F T(15)TCTTTGCAAGTGGCACTCCTCATGGG P205S synt R T(15)TAAGCGATCCACACGAAATGTGCCAAT L206W synt F T(15)GGCAAGTGGCACTCCTCATGGGGCTA L206W synt R T(15)TCAAGGAGCGATCCACACGAAATGTGC Q220X synt F T(15)TAGGCGTCTGCTTTCTGTGGACTTGG Q220X synt R T(15)TATAACAACTCCCAGATTAGCCCCATG 936delTA synt F T(15)AATCCAATCTGTTAAGGCATACTGCT 936delTA synt R T(15)TGATTTTCAATCATTTCTGAGGTAATC 935delA synt F T(15)GAAATATCCAATCTGTTAAGGCATAC 935delA synt R T(15)TATTTCAATCATTTCTGAGGTAATCAC N287Y synt F T(15)TACTTAAGACAGTAAGTTGTTCCAAT N287Y synt R T(15)TATTCAATCATTTTTTCCATTGCTTCT 1002 - 3TϾG synt F T(15)GAGAACAGAACTGAAACTGACTCGGA 1002 - 3TϾG synt R T(15)TCTAAAAAACAATAACAATAAAATTCA 1154insTC syntwt F T(15)ATCTCATTCTGCATTGTTCTGCGCAT 1154insTC syntwt R T(15)TTGAGATGGTGGTGAATATTTTCCGGA 1154insTC syntmt F T(15)TCTCTCATTCTGCATTGTTCTGCGCAT 1154insTC syntmt R T(15)TAGAGATGGTGGTGAATATTTTCCGGA DF311 mt syntV1 F T(15)CCTTCTTCTCAGGGTTCTTTGTGGTG dF311 mt syntV1 R T(15)GAGAAGAAGGCTGAGCTATTGAAGTATC G330X synt F T(15)TGAATCATCCTCCGGAAAATATTCAC G330X synt R T(15)ATTTGATTAGTGCATAGGGAAGCACA S364P synt F T(15)CCTCTTGGAGCAATAAACAAAATACA S364P synt R T(15)GGTCATACCATGTTTGTACAGCCCAG Q359K/T360K mt synt F T(15)AAAAAATGGTATGACTCTCTTGGAGC Q359K/T360K mt synt R T(15)TTTTTTACAGCCCAGGGAAATTGCCG 1078delT synt F T(15)CTTGTGGTGTTTTTATCTGTGCTTCC 1078delT synt R T(15)CAAGAACCCTGAGAAGAAGAAGGCTG 1119delA synt F T(15)CAAGGAATCATCCTCCGGAAAATATT 1119delA synt R T(15)CTTGATTAGTGCATAGGGAAGCACAG 1161delC synt F T(15)GATTGTTCTGCGCATGGCGGTCACTC 1161delC synt R T(15)TCAGAATGAGATGGTGGTGAATATTT T338I synt F T(15)TCACCATCTCATTCTGCATTGTTCTG T338I synt R T(15)ATGAATATTTTCCGGAGGATGATTCC R352Q synt F T(15)AGCAATTTCCCTGGGCTGTACAAACA R352Q synt R T(15)TGAGTGACCGCCATGCGCAGAACAAT L346P synt F T(15)CGCGCATGGCGGTCACTCGGCAATTT L346P synt R T(15)GGAACAATGCAGAATGAGATGGTGGT 1259insA synt F T(15)AAAAAGCAAGAATATAAGACATTGGA 1259insA synt R T(15)TTTTTGTAAGAAATCCTATTTATAAA W401X(TAG)mtsynt F T(15)AGGAGGAGGTCAGAATTTTTAAAAAA W401X(TAG)mtsynt R T(15)TAGAAGGCTGTTACATTCTCCATCAC W401X(TGA) synt F T(15)AGAGGAGGTCAGAATTTTTAAAAAAT W401X(TGA) synt R T(15)TCAGAAGGCTGTTACATTCTCCATCA 1342 - 2AϾC synt F T(15)CGGGATTTGGGGAATTATTTGAGAAA 1342 - 2AϾC synt R T(15)GGTTAAAAAAACACACACACACACAC 1504delG synt F T(15)TGATCCACTGTAGCAGGCAAGGTAGT 1504delG synt R T(15)TCAGCAACCGCCAACAACTGTCCTCT G480C synt F T(15)TGTAAAATTAAGCACAGTGGAAGAAT G480C synt R T(15)ACTCTGAAGGCTCCAGTTCTCCCATA C524X synt F T(15)ACAACTAGAAGAGGTAAGAAACTATG C524X synt R T(15)TCATGCTTTGATGACGCTTCTGTATC V520F synt F T(15)TTCATCAAAGCAAGCCAACTAGAAGA V520F synt R T(15)AGCTTCTGTATCTATATTCATCATAG 1609delCA synt F T(15)TGTTTTCCTGGATTATGCCTGGCACC 1609delCA synt R T(15)CAGAACAGAATGAAATTCTTCCACTG 1717 - 8GϾA synt F T(15)AGTAATAGGACATCTCCAAGTTTGCA 1717 - 8GϾA synt R T(15)TAAAAATAGAAAATTAGAGAGTCACT 1784delG synt F T(15)AGTCAACGAGCAAGAATTTCTTTAGC 1784delG synt R T(15)ACTCCACTCAGTGTGATTCCACCTTC A559T synt F T(15)ACAAGGTGAATAACTAATTATTGGTC A559T synt R T(15)TTAAAGAAATTCTTGCTCGTTGACCT Q552X synt F T(15)TAACGAGCAAGAATTTCTTTAGCAAG Q552X synt R T(15)AACCTCCACTCAGTGTGATTCCACCT S549R(AϾC) synt F T(15)CGTGGAGGTCAACGAGCAAGAATTTC S549R(AϾC) synt R T(15)GCAGTGTGATTCTACCTTCTCCAAGA S549R(TϾG) synt F T(15)GGGAGGTCAACGAGCAAGTATTTC S549R(TϾG) synt R T(15)CCTCAGTGTGATTCCACCTTCTCCAA L558S synt F T(15)CAGCAAGGTGAATAACTAATTATTGG L558S synt R T(15)GAAGAAATTCTCGCTCGTTGACCTCC 1811 ϩ 1.6 kb AϾG synt F T(15)GTAAGTAAGGTTACTATCAATCACAC 1811 ϩ 1.6 kb AϾG synt R T(15)CATCTCAAGTACATAGGATTCTCTGT 1812 - 1GϾA synt F T(15)AAGCAGTATACAAAGATGCTGATTTG 1812 - 1GϾA synt R T(15)TTAAAAAGAAAATGGAAATTAAATTA D572N synt F T(15)AACTCTCCTTTTGGATACCTAGATGT D572N synt R T(15)TTAATAAATACAAATCAGCATCTTTG P574H synt F T(15)ATTTTGGATACCTAGATGTTTTAACA P574H synt R T(15)TGAGAGTCTAATAAATACAAATCAGC 1833delT synt F T(15)ATTGTATTTATTAGACTCTCCTTTTG 1833delT synt R T(15)CAATCAGCATCTTTGTATACTGCTCT Table 4. Continued Primer name Sense strand 5Ј 3 3Ј Name Antisense strand 5Ј 3 3Ј Y563D synt F T(15)GACAAAGATGCTGATTTGTATTTATT Y563D synt R T(15)CTACTGCTCTAAAAAGAAAATGGAAA T582R synt F T(15)GAGAAAAAGAAATATTTGAAAGGTAT T582R synt R T(15)CTTAAAACATCTAGGTATCCAAAAGG E585X synt F T(15)TAAATATTTGAAAGGTATGTTCTTTG E585X synt R T(15)ATTTTTCTGTTAAAACATCTAGGTAT 1898 ϩ 5GϾT synt F T(15)TTTCTTTGAATACCTTACTTATATTG 1898 ϩ 5GϾT synt R T(15)AATACCTTTCAAATATTTCTTTTTCT 1924del7 synt F T(15)CAGGATTTTGGTCACTTCTAAAATGG 1924del7 synt R T(15)CTGTTAGCCATCAGTTTACAGACACA 2055del9ϾA synt F T(15)ACATGGGATGTGATTCTTTCGACCAA 2055del9ϾA synt R T(15)TCTAAAGTCTGGCTGTAGATTTTGGA D648V synt F T(15)TTTCTTTCGACCAATTTAGTGCAGAA D648V synt R T(15)ACACATCCCATGAGTTTTGAGCTAAA K710X synt F T(15)TAATTTTCCATTGTGCAAAAGACTCC K710X synt R T(15)ATCGTATAGAGTTGATTGGATTGAGA I618T synt F T(15)CTTTGCATGAAGGTAGCAGCTATTTT I618T synt R T(15)GTTAATATTTTGTCAGCTTTCTTTAA R764X synt F T(15)TGAAGGAGGCAGTCTGTCCTGAACCT R764X synt R T(15)ATGCCTGAAGCGTGGGGCCAGTGCTG Q685X synt F T(15)TAATCTTTTAAACAGACTGGAGAGTT Q685X synt R T(15)ATTTTTTTGTTTCTGTCCAGGAGACA R709X synt F T(15)TGAAAATTTTCCATTGTGCAAAAGAC R709X synt R T(15)ATATAGAGTTGATTGGATTGAGAATA V754M synt F T(15)ATGATCAGCACTGGCCCCACGCTTCA V754M synt R T(15)TGCTGATGCGAGGCAGTATCGCCTCT 1949del84 synt F T(15)AAAAATCTACAGCCAGACTTTATCTC 1949del84 synt R T(15)TTTTTAGAAGTGACCAAAATCCTAGT 2108delA synt F T(15)GAATTCAATCCTAACTGAGACCTTAC 2108delA synt R T(15)ATTCTTCTTTCTGCACTAAATTGGTC 2176insC synt F T(15)CCAAAAAAACAATCTTTTAAACAGACTGGAGAG 2176insC synt R T(15)GGTTTCTGTCCAGGAGACAGGAGCAT 2184delA synt F T(15)CAAAAAACAATCTTTTAAACAGACTGG 2184delA synt R T(15)GTTTTTTGTTTCTGTCCAGGAGACAG 2105-2117 del13 synt F T(15)AAACTGAGACCTTACACCGTTTCTCA 2105-2117 del13 synt R T(15)TTTCTTTCTGCACTAAATTGGTCGAA 2307insA synt F T(15)AAAGAGGATTCTGATGAGCCTTTAGA 2307insA synt R T(15)TTTCGATGCCATTCATTTGTAAGGGA W846X synt F T(15)AAACACATACCTTCGATATATTACTGTCCAC W846X synt R T(15)TCATGTAGTCACTGCTGGTATGCTCT 2734G/AT synt F T(15)TTAATTTTTCTGGCAGAGGTAAGAAT 2734G/AT synt R T(15)TTAAGCACCAAATTAGCACAAAAATT 2766del8 synt F T(15)GGTGGCTCCTTGGAAAGTGAGTATTC 2766del8 synt R T(15)CACCAAAGAAGCAGCCACCTGGAATGG 2790 - 2AϾG synt F T(15)GGCACTCCTCTTCAAGACAAAGGGAA 2790 - 2AϾG synt R T(15)CGTAAAGCAAATAGGAAATCGTTAAT 2991del32 synt F T(15)TTCAACACGTCGAAAGCAGGTACTTT 2991del32 synt R T(15)AAACATTTTGTGGTGTAAAATTTTCG Q890X synt F T(15)TAAGACAAAGGGAATAGTACTCATAG Q890X synt R T(15)AAAGAGGAGTGCTGTAAAGCAAATAG 2869insG synt F T(15)GATTATGTGTTTTACATTTACGTGGG 2869insG synt R T(15)CACGAACTGGTGCTGGTGATAATCAC 3120GϾA synt F T(15)AGTATGTAAAAATAAGTACCGTTAAG 3120GϾA synt R T(15)TTGGATGAAGTCAAATATGGTAAGAG 3121 - 2AϾT synt F T(15)TGTTGTTATTAATTGTGATTGGAGCT 3121 - 2AϾT synt R T(15)AGTAAGATCAAAGAAAACATGTTGGT 3132delTG synt F T(15)TTGATTGGAGCCATAGCAGTTGTCGC 3132delTG synt R T(15)AATTAATAACAACTGTAAGATCAAAG 3271delGG synt F T(15)ATATGACAGTGAATGTGCGATACTCA 3271delGG synt R T(15)ATTCAGATTCCAGTTGTTTGAGTTGC 3171delC synt F T(15)ACCTACATCTTTGTTGCAACAGTGCC 3171delC synt R T(15)AGGTTGTAAAACTGCGACAACTGCTA 3171insC synt F T(15)CCCCTACATCTTTGTTGCTACAGTGC 3171insC synt R T(15)GGGGTTGTAAAACTGCGACAACTGCT 3199del6 synt F T(15)GAGTGGCTTTTATTATGTTGAGAGCATAT 3199del6 synt R T(15)CCACTGGCACTGTTGCAACAAAGATG M1101K synt F T(15)AGAGAATAGAAATGATTTTTGTCATC M1101K synt R T(15)TTTTGGAACCAGCGCAGTGTTGACAG G1061R synt F T(15)CGACTATGGACACTTCGTGCCTTCGG G1061R synt R T(15)GTTTTAAGCTTGTAACAAGATGAGTG R1066L synt F T(15)TTGCCTTCGGACGGCAGCCTTACTTT R1066L synt R T(15)AGAAGTGTCCATAGTCCTTTTAAGCT R1070P synt F T(15)CGCAGCCTTACTTTGAAACTCTGTTC R1070P synt R T(15)GGTCCGAAGGCACGAAGTGTCCATAG L1077P synt F T(15)CGTTCCACAAAGCTCTGAATTTACAT L1077P synt R T(15)GGAGTTTCAAAGTAAGGCTGCCGTCC W1089X synt F T(15)AGTTCTTGTACCTGTCAACACTGCGC W1089X synt R T(15)TAGTTGGCAGTATGTAAATTCAGAGC L1093P synt F T(15)CGTCAACACTGCGCTGGTTCCAAATG L1093P synt R T(15)GGGTACAAGAACCAGTTGGCAGTATG W1098R synt F T(15)CGGTTCCAAATGAGAATAGAAATGAT W1098R synt R T(15)GGCGCAGTGTTGACAGGTACAAGAAC Q1100P synt F T(15)CAATGAGAATAGAAATGATTTTTGTC Q1100P synt R T(15)GGGAACCAGCGCAGTGTTGACAGGTA D1152H synt F T(15)CATGTGGATAGCTTGGTAAGTCTTAT D1152H synt R T(15)GTATGCTGGAGTTTACAGCCCACTGC R1158X synt F T(15)TGATCTGTGAGCCGAGTCTTTAAGTT R1158X synt R T(15)ACATCTGAAATAAAAATAACAACATT S1196X synt F T(15)GACACGTGAAGAAAGATGACATCTGG S1196X synt R T(15)CAATTCTCAATAATCATAACTTTCGA 3732delA synt F T(15)GGAGATGACATCTGGCCCTCAGGGGG 3732delA synt R T(15)CTCCTTCACGTGTGAATTCTCAATAA 3791delC synt F T(15)AAGAAGGTGGAAATGCCATATTAGAG 3791delC synt R T(15)TTGTATTTTGCTGTGAGATCTTTGAC 3821delT synt F T(15)ATTCCTTCTCAATAAGTCCTGGCCAG 3821delT synt R T(15)GAATGTTCTCTAATATGGCATTTCCA Q1238X synt F T(15)TAGAGGGTGAGATTTGAACACTGCTT Q1238X synt R T(15)AGCCAGGACTTATTGAGAAGGAAATG S1255X (ex19)synt F T(15)GTCTGGCCCTCAGGGGGCCAAATGAC S1255X (ex19) synt R T(15)CGTCATCTTTCTTCACGTGTGAATTC S1255X;L synt F T(15)AAGCTTTTTTGAGACTACTGAACACT S1255X;L synt R T(15)TATAACAAAGTAATCTTCCCTGATCC 3849 ϩ 4AϾG synt F T(15)GGATTTGAACACTGCTTGCTTTGTTA 3849 ϩ 4AϾG synt R T(15)CCACCCTCTGGCCAGGACTTATTGAG 3850 - 1GϾA synt F T(15)AGTGGGCCTCTTGGGAAGAACTGGAT 3850 - 1GϾA synt R T(15)TTATAAGGTAAAAGTGATGGGATCAC 3905insT synt F T(15)TTTTTTTGAGACTACTGAACACTGAA 3905insT synt R T(15)AAAAAAAGCTGATAACAAAGTACTCT 3876delA synt F T(15)CGGGAAGAGTACTTTGTTATCAGCTT 3876delA synt R T(15)CGATCCAGTTCTTCCCAAGAGGCCCA G1244V synt F T(15)TAAGAACTGGATCAGGGAAGAGTACT G1244V synt R T(15)ACCAAGAGGCCCACCTATAAGGTAAA G1249E synt F T(15)AGAAGAGTACTTTGTTATCAGCTTTT G1249E synt R T(15)TCTGATCCAGTTCTTCCCAAGAGGCC S1251N synt F T(15)ATACTTTGTTATCAGCTTTTTTGAGACTACTG S1251N synt R T(15)TTCTTCCCTGATCCAGTTCTTCCCAA S1252P synt F T(15)CCTTTGTTATCAGCTTTTTTGAGACT S1252P synt R T(15)GACTCTTCCCTGATCCAGTTCTTCCC D1270N synt F T(15)AATGGTGTGTCTTGGGATTCAATAAC D1270N synt R T(15)TGATCTGGATTTCTCCTTCAGTGTTC W1282R synt F T(15)CGGAGGAAAGCCTTTGGAGTGATACC W1282R synt R T(15)GCTGTTGCAAAGTTATTGAATCCCAA R1283K synt F T(15)AGAAAGCCTTTGGAGTGATACCACAG R1283K synt R T(15)TTCCACTGTTGCAAAGTTATTGAATC 4005 ϩ 1GϾA synt F T(15)ATGAGCAAAAGGACTTAGCCAGAAAA 4005 ϩ 1GϾA synt R T(15)TCTGTGGTATCACTCCAAAGGCTTTC 4010del4 synt F T(15)GTATTTTTTCTGGAACATTTAGAAAAAACTTGG 4010del4 synt R T(15)AAAATACTTTCTATAGCAAAAAAGAAAAGAAGAA 4016insT synt F T(15)TTTTTTTCTGGAACATTTAGAAAAAACTTGG 4016insT synt R T(15)AAAAAAATAAATACTTTCTATAGCAAAAAAGAAAAGAAGA CFTRdele21 synt F T(15)TAGGTAAGGCTGCTAACTGAAATGAT CFTRdele21 synt R T(15)CCTATAGCAAAAAAGAAAAGAAGAAGAAAGTATG 4382delA synt F T(15)GAGAGAACAAAGTGCGGCAGTACGAT 4382delA synt R T(15)CTCTATGACCTATGGAAATGGCTGTT Bold, mutation allele of interest; bold and italicized, modified nucleotide. 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[hide] Cystic fibrosis mutation frequencies in upstate Ne... Hum Mutat. 1997;10(6):436-42. Shrimpton AE, Borowitz D, Swender P
Cystic fibrosis mutation frequencies in upstate New York.
Hum Mutat. 1997;10(6):436-42., [PMID:9401006]

Abstract [show]
Upstate New York patients (100) with cystic fibrosis (i.e., 200 CF chromosomes), 72 from the CF center in Syracuse and 28 from a Buffalo CF center, were analyzed for their CF-causing mutations using restriction enzyme digest, single-strand conformation analysis (SSCA), and Heteroduplex (HA) analysis. Polymerase chain reaction (PCR) amplified products from all 27 CFTR exons using primers that included flanking intron junction sequence were investigated. More than 120 known cystic fibrosis transmembrane conductance regulator (CFTR) disease-causing mutations were screened. Four novel CFTR disease-causing mutations were identified (N287Y in exon 6b, 1259insA in exon 8, R1070P in exon 17b, and CF?20kbdel14b-18). A detection rate of 96% of the combined Syracuse and Buffalo population CF chromosomes was obtained.

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4 Four novel CFTR disease-causing mutations were identified (N287Y in exon 6b, 1259insA in exon 8, R1070P in exon 17b, and CF?20kbdel14b-18). Login to comment
82 Novel Disease-Causing Mutations N287Y (991A>T) This individual, who is of Italian and German/Irish extraction, was diagnosed at 3 1/2 years of age when referred because of rectal prolapse; his sweat Cl was TABLE 2. Login to comment
84 % Comment 3 G85E 1 1 0.5 4 R117H 1 1 0.5 i4 621 + 1,G>T 1 2 3 1.5 5 711 + 1,G>T 1 1 0.5 6b N287Y 1 1 0.5 Novel 7 1154insTC 2 2 1.0 8 1259insA 1 1 0.5 Novel 9 A455E 1 1 0.5 10 Delta F508 109 39 148 74.0 10 1609delCA 1 1 0.5 Spanish i10 1717-1,G>A 3 3 1.5 11 G542X 2 1 3 1.5 11 G551D 3 3 1.5 11 R553X 4 4 2.0 i12 1898+1,G>A 2 2 1.0 13 2143delT 1 1 0.5 13 2184delA+G>A 1 1 0.5 i14 2789+5,G>A 2 2 1.0 17b R1070P 1 1 0.5 Novel 17b Y1092X(C>A) 2 2 1.0 French Canadian (Rozen et al., 1992) 17b CF?20kbdel 14b-18 1 1 0.5 Novel (Shrimpton and Borowitz, 1997) i19 3849+10kb,C>T 1 1 0.5 20 W1282X 2 2 0.5 Ashkenazi 21 N1303K 3 3 6 3.0 Unknown 4/144 4/56 8/200 4.0 AL. 75 and 81 mMol/L. Login to comment
85 This patient was a compound heterozygote for the Delta F508 (Kerem et al., 1989) in exon 10 and a novel CF mutation N287Y (Asn to Tyr) in exon 6b, which could be detected by the use of a restriction site-generating primer. Login to comment
86 N287Y can be detected by artificial Dra I site-generating PCR amplification using the N287Ymis and 6B-i3 primers (Table 1b). Login to comment

[hide] Cystic fibrosis: toward personalized therapies. Int J Biochem Cell Biol. 2014 Jul;52:192-200. doi: 10.1016/j.biocel.2014.02.008. Epub 2014 Feb 20. Ikpa PT, Bijvelds MJ, de Jonge HR
Cystic fibrosis: toward personalized therapies.
Int J Biochem Cell Biol. 2014 Jul;52:192-200. doi: 10.1016/j.biocel.2014.02.008. Epub 2014 Feb 20., [PMID:24561283]

Abstract [show]
Cystic fibrosis (CF), the most common, life-threatening monogenetic disease in Caucasians, is caused by mutations in the CFTR gene, encoding a cAMP- and cGMP-regulated epithelial chloride channel. Symptomatic therapies treating end-organ manifestations have increased the life expectancy of CF patients toward a mean of 40 years. The recent development of CFTR-targeted drugs that emerged from high-throughput screening and are capable of correcting the basic defect promises to transform the therapeutic landscape from a trial-and-error prescription to personalized medicine. This stratified approach is tailored to a specific functional class of mutations in CFTR, but can be refined further to an individual level by exploiting recent advances in ex vivo drug testing methods. These tests range from CFTR functional measurements in rectal biopsies donated by a CF patient to the use of patient-derived intestinal or pulmonary organoids. Such organoids may serve as an inexhaustible source of epithelial cells that can be stored in biobanks and allow medium- to high-throughput screening of CFTR activators, correctors and potentiators on the basis of a simple microscopic assay monitoring organoid swelling. Thus the recent breakthrough in stem cell biology allowing the culturing of mini-organs from individual patients is not only relevant for future stem cell therapy, but may also allow the preclinical testing of new drugs or combinations that are optimally suited for an individual patient.

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1620 Most CFTR trafficking mutants, including F508del, with a few exceptions (e.g. G480C; N287Y), show additional defects in channel gating function, and most PCs and PRs identified thus far only partially normalize the channel conformational defect. Login to comment

[hide] Cystic fibrosis genetics: from molecular understan... Nat Rev Genet. 2015 Jan;16(1):45-56. doi: 10.1038/nrg3849. Epub 2014 Nov 18. Cutting GR
Cystic fibrosis genetics: from molecular understanding to clinical application.
Nat Rev Genet. 2015 Jan;16(1):45-56. doi: 10.1038/nrg3849. Epub 2014 Nov 18., [PMID:25404111]

Abstract [show]
The availability of the human genome sequence and tools for interrogating individual genomes provide an unprecedented opportunity to apply genetics to medicine. Mendelian conditions, which are caused by dysfunction of a single gene, offer powerful examples that illustrate how genetics can provide insights into disease. Cystic fibrosis, one of the more common lethal autosomal recessive Mendelian disorders, is presented here as an example. Recent progress in elucidating disease mechanism and causes of phenotypic variation, as well as in the development of treatments, demonstrates that genetics continues to play an important part in cystic fibrosis research 25 years after the discovery of the disease-causing gene.

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74 The processing of CFTR can be altered by variants that cause aberrant folding of the protein, leading to degradation (for example, p.Phe508del (legacy F508del))18 , or by variants that cause reduced membrane stability as a result of increased rates of endocytosis (for example, p.Asn287Tyr (legacy N287Y))143 . Login to comment