ABCC7 p.Pro67Ser
| ClinVar: |
c.200C>T
,
p.Pro67Leu
D
, Pathogenic
|
| CF databases: |
c.200C>T
,
p.Pro67Leu
D
, CF-causing ; CFTR1: P67L mutation was seen on one Caucasian CF chromosome of 48 screened. It was not detected on any of 181 non-CF Caucasian chromosomes by ASO analysis. The patient, a 15 year old female, carries [delta]F508 on the other allele. She was diagnosed at the age of 12. She is pancreatic sufficient with sweat chloride concentrations of 54 and 67 meq/L.
|
| Predicted by SNAP2: | A: D (85%), C: D (85%), D: D (95%), E: D (95%), F: D (91%), G: D (91%), H: D (91%), I: D (91%), K: D (95%), L: N (57%), M: D (91%), N: D (95%), Q: D (91%), R: D (95%), S: D (91%), T: D (91%), V: D (85%), W: D (91%), Y: D (91%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
[switch to compact view]
Comments [show]
None has been submitted yet.
[hide] Functional characterization of a novel CFTR mutati... Cell Physiol Biochem. 2007;19(5-6):239-48. Kraus C, Reis A, Naehrlich L, Dotsch J, Korbmacher C, Rauh R
Functional characterization of a novel CFTR mutation P67S identified in a patient with atypical cystic fibrosis.
Cell Physiol Biochem. 2007;19(5-6):239-48., [PMID:17495464]
Abstract [show]
Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene. CFTR serves as a cAMP-stimulated chloride channel in a wide range of epithelial tissues and its dysfunction is a hallmark of CF. Over 1400 mutations in the CFTR gene are known, but functional data exist only for a minority of the mutant channels. The aim of the present study was to functionally characterize a novel CFTR mutation identified in a patient with atypical CF. Full length sequencing of the patient's CFTR gene revealed a homozygous C to T transition at nucleotide position 331 (CCT>TCT), which results in a P67S amino acid substitution. Mutant and wild-type CFTR were heterologously expressed in Xenopus laevis oocytes. CFTR whole-cell currents were studied using the two-electrode voltage-clamp technique. Channel surface expression was assessed by a chemiluminescence assay. Expression of P67S-CFTR resulted in functional CFTR chloride channels. However, the CFTR chloride conductance observed in oocytes expressing the mutant channel averaged only 24% of that in oocytes expressing wild-type CFTR. Similarly, surface expression of the mutant channel was reduced. In contrast, the mutation did not alter the anion selectivity of the channel, and Western blot analysis indicated a similar protein expression level of mutant and wild-type CFTR. Our findings indicate that the P67S mutation reduces CFTR chloride channel function by reducing channel surface expression. The mild disease phenotype of the patient indicates that the residual function of the mutant channel is sufficient to prevent the development of severe CF symptoms.
Comments [show]
None has been submitted yet.
No. Sentence Comment
0 Original Paper Cell Physiol Biochem 2007;19:239-248 Accepted: December 04, 2006Cellular PhysiologyCellular PhysiologyCellular PhysiologyCellular PhysiologyCellular Physiology and Biochemistrand Biochemistrand Biochemistrand Biochemistrand Biochemistryyyyy Copyright (c) 2007 S. Karger AG, Basel Fax +41 61 306 12 34 E-Mail karger@karger.ch www.karger.com (c) 2007 S. Karger AG, Basel 1015-8987/07/0196-0239$23.50/0 Accessible online at: www.karger.com/cbp Functional Characterization of a Novel CFTR Mutation P67S Identified in a Patient with Atypical Cystic Fibrosis Cornelia Kraus1 , André Reis1 , Lutz Naehrlich2 , Jörg Dötsch2 , Christoph Korbmacher3 and Robert Rauh3 1 Institute of Human Genetics, University of Erlangen-Nuremberg, 2 University Hospital for Children and Adolescents, University of Erlangen-Nuremberg, 3 Institute of Cellular and Molecular Physiology, University of Erlangen-Nuremberg Prof.
X
ABCC7 p.Pro67Ser 17495464:0:509
status: NEW2 6, D-91054 Erlangen (Germany) Tel. +49 9131 85 22301, Fax +49 9131 85 22770 E-Mail christoph.korbmacher@physiologie2.med.uni-erlangen.de Key Words CFTR • P67S mutation • Cystic fibrosis • Pancreatic insufficiency • Xenopus laevis oocytes • Chloride channel • Electrophysiology • Surface expression Abstract Cystic fibrosis (CF) is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene.
X
ABCC7 p.Pro67Ser 17495464:2:161
status: NEW6 Full length sequencing of the patient`s CFTR gene revealed a homozygous C to T transition at nucleotide position 331 (CCT>TCT), which results in a P67S amino acid substitution.
X
ABCC7 p.Pro67Ser 17495464:6:147
status: NEW10 Expression of P67S-CFTR resulted in functional CFTR chloride channels.
X
ABCC7 p.Pro67Ser 17495464:10:14
status: NEW14 Our findings indicate that the P67S mutation reduces CFTR chloride channel function by reducing channel surface expression.
X
ABCC7 p.Pro67Ser 17495464:14:31
status: NEW36 However, full sequencing of the CFTR gene revealed a homozygous mutation, that leads to a substitution of a serine for a proline at position 67 (P67S) in the amino acid chain of the CFTR protein.
X
ABCC7 p.Pro67Ser 17495464:36:108
status: NEWX
ABCC7 p.Pro67Ser 17495464:36:145
status: NEW37 The aim of this study was to investigate the functional effect of P67S in the Xenopus laevis oocyte expression system using whole-cell current measurements and a chemiluminescence assay to assess channel surface expression.
X
ABCC7 p.Pro67Ser 17495464:37:66
status: NEW38 We found that P67S largely reduces cAMP stimulated CFTR chloride currents and channel surface expression without altering the ion selectivity of the channel.
X
ABCC7 p.Pro67Ser 17495464:38:14
status: NEW39 Our findings indicate that P67S represents a class II mutation of CFTR associated with abnormal or reduced channel trafficking.
X
ABCC7 p.Pro67Ser 17495464:39:27
status: NEW85 In a second step a P67S mutation was inserted into the wild-type CFTR clone (forward: AGC TGG CTT CAA AGA AAA ATT CTA AAC TCA TTA ATG CCC TTC G; reverse: CGA AGG GCA TTA ATG AGT TTA GAA TTT TTC TTT GAA GCC AGC T).
X
ABCC7 p.Pro67Ser 17495464:85:19
status: NEW101 Defolliculated stage V-VI oocytes were injected (Nanoject automatic injector, Drummond, Broomall, PA) with 0.25 ng cRNA of wild-type CFTR or P67S mutant or P67L mutant.
X
ABCC7 p.Pro67Ser 17495464:101:141
status: NEW123 Sequencing of exon 3 revealed a homozygous C to T transition at nucleotide position 331 (CCT>TCT), which results in a substitution of a serine for a proline at position 67 (P67S).
X
ABCC7 p.Pro67Ser 17495464:123:136
status: NEWX
ABCC7 p.Pro67Ser 17495464:123:173
status: NEW127 Therefore, we conclude that P67S is a novel CFTR mutation.
X
ABCC7 p.Pro67Ser 17495464:127:28
status: NEW129 To test the functional relevance of the mutation, we heterologously expressed wild-type and P67S mutant CFTR in Xenopus laevis oocytes.
X
ABCC7 p.Pro67Ser 17495464:129:92
status: NEW132 Sequencing analysis of CFTR exon 3 at the level of genomic DNA revealed a homozygous C>T transition at nucleotide position c.331 in the patient. This transition resulted in the substitution of a serine for a proline at position 67 (p.P67S).
X
ABCC7 p.Pro67Ser 17495464:132:195
status: NEWX
ABCC7 p.Pro67Ser 17495464:132:234
status: NEW134 Substitution of serine for proline at position 67 reduces CFTR whole-cell currents As previously described [21] we used a combination of 1mM IBMX and 1µM forskolin (IBMX/Fsk) to elevate intracellular cAMP and activate whole-cell CFTR Cl- currents (ΔIIBMX/Fsk ) in the oocyte expression system.
X
ABCC7 p.Pro67Ser 17495464:134:16
status: NEW135 Figure 2A shows typical whole-cell current recordings obtained from an oocyte expressing wild-type CFTR (upper trace) and from an oocyte expressing the P67S mutant CFTR (lower trace) at a holding potential of -60 mV.
X
ABCC7 p.Pro67Ser 17495464:135:152
status: NEW138 These findings indicate that expression of P67S mutant CFTR results in functional CFTR chloride channels.
X
ABCC7 p.Pro67Ser 17495464:138:43
status: NEW139 However, the CFTR whole-cell currents activated by IBMX/Fsk were much smaller in oocytes expressing the P67S mutant CFTR than in matched oocytes expressing wild-type CFTR.
X
ABCC7 p.Pro67Ser 17495464:139:104
status: NEW141 The P67S mutant CFTR channel is functional but at a largely reduced level.
X
ABCC7 p.Pro67Ser 17495464:141:4
status: NEW142 A: Representative whole-cell current traces recorded at a holding potential of -60 mV from an oocyte expressing wild-type CFTR (upper trace) or the P67S mutant CFTR (bottom trace).
X
ABCC7 p.Pro67Ser 17495464:142:148
status: NEW145 Voltage step protocols for current-voltage (I-V) plots were performed at times indicated by asterisks, but the complete current responses were omitted from the continuous current trace for clarity B: Average I-V plots of IBMX/Fsk induced CFTR whole-cell currents (ΔIIBMX/Fsk ) were obtained from experiments as shown in A using matched oocytes expressing wild-type CFTR (circles) or P67S mutant CFTR (diamonds) (N=7, n=52 per group).
X
ABCC7 p.Pro67Ser 17495464:145:389
status: NEW147 The steeper slope of the wild-type CFTR I-V curve compared to the slope of the P67S mutant CFTR I-V curve indicates a reduced CFTR whole-cell conductance in oocytes expressing the mutant channel.
X
ABCC7 p.Pro67Ser 17495464:147:79
status: NEW148 C: Representative Western blot analysis to detect CFTR protein expression in oocytes injected with 2ng of cRNA coding for wild-type (wt) or P67S mutant CFTR or in non-injected control oocytes (n.
X
ABCC7 p.Pro67Ser 17495464:148:140
status: NEW153 ΔGIBMX/ Fsk averaged 31.8 ± 4.3 µS (n = 52) in P67S mutant expressing oocytes and was significantly lower than ΔGIBMX/ Fsk in wild-type CFTR expressing oocytes (131.9 ± 12.5 µS, n = 52, p<0.0001).
X
ABCC7 p.Pro67Ser 17495464:153:63
status: NEW154 These results demonstrate that the P67S mutation does not abolish but largely reduces CFTR chloride channel function in the oocyte system.
X
ABCC7 p.Pro67Ser 17495464:154:35
status: NEW156 Thus, the reduced channel function of the P67S mutant compared to wild-type CFTR is unlikely to be caused by reduced protein synthesis of the mutant channel.
X
ABCC7 p.Pro67Ser 17495464:156:42
status: NEW159 Since the P67S mutation is not localized in the vicinity of channel regions thought to contribute to the pore of the CFTR channel or its gating mechanism [3], we hypothesized that the mutation was likely to affect channel surface expression.
X
ABCC7 p.Pro67Ser 17495464:159:10
status: NEW160 To investigate a possible effect of the P67S mutation on channel surface expression, we used HA-tagged CFTR constructs and a chemiluminescence assay as described previously to measure CFTR surface expression and whole-cell currents in parallel [21].
X
ABCC7 p.Pro67Ser 17495464:160:40
status: NEW161 In three independent experiments using different batches of oocytes we found a large reduction of the surface expression of the P67S mutant channel compared to wild-type CFTR.
X
ABCC7 p.Pro67Ser 17495464:161:128
status: NEW162 Our results indicate that a reduced surface expression of the P67S mutant CFTR can fully explain the reduction of ΔGIBMX/Fsk observed in oocytes expressing the mutant channel (figure 3).
X
ABCC7 p.Pro67Ser 17495464:162:62
status: NEW163 The P67S mutation does not alter the anion selectivity of CFTR It is well known that mutations in CFTR can influence the anion selectivity of the channel [26].
X
ABCC7 p.Pro67Ser 17495464:163:4
status: NEW164 Therefore, we wanted to test whether the P67S mutation affects the anion selectivity of the IBMX/Fsk induced whole-cell conductance.
X
ABCC7 p.Pro67Ser 17495464:164:41
status: NEW170 Surface expression of the P67S mutant CFTR channel is reduced.
X
ABCC7 p.Pro67Ser 17495464:170:26
status: NEW171 Surface expression (open columns) and IBMX/Fsk stimulated whole-cell conductance (closed columns) of HA-tagged wild-type and P67S mutant CFTR channels were assessed in matched groups of oocytes.
X
ABCC7 p.Pro67Ser 17495464:171:125
status: NEW181 These results indicate that the P67S mutation does not alter the anion selectivity of CFTR.
X
ABCC7 p.Pro67Ser 17495464:181:32
status: NEW182 The previously reported P67L mutation has a similar effect on CFTR function as the P67S mutation As shown above the P67S mutation reduces ΔGIBMX/ Fsk by decreasing the surface expression of the mutated CFTR channels.
X
ABCC7 p.Pro67Ser 17495464:182:83
status: NEWX
ABCC7 p.Pro67Ser 17495464:182:116
status: NEW185 A: Typical whole-cell recordings of two-electrode voltage-clamp measurements of a wild-type CFTR (top) and a P67S mutant (bottom) expressing oocyte after activation with IBMX/Fsk.
X
ABCC7 p.Pro67Ser 17495464:185:109
status: NEW189 Please note the different current scales for wild-type CFTR and the P67S mutant.
X
ABCC7 p.Pro67Ser 17495464:189:68
status: NEW193 There was no difference in the whole-cell conductance sequence of wild-type CFTR (open bars, n=3) and P67S mutant CFTR (grey bars, n=3).
X
ABCC7 p.Pro67Ser 17495464:193:102
status: NEW196 Therefore, we decided to investigate the P67L mutation to compare its effect with the effect of the P67S mutation.
X
ABCC7 p.Pro67Ser 17495464:196:100
status: NEW197 In two different batches of oocytes we found that ΔGIBMX/Fsk was similarly decreased in P67S CFTR (30.2 ± 2.8 µS, n=9) and P67L CFTR (14.3 ± 4.1 µS, n=9) expressing oocytes compared to ΔGIBMX/Fsk of wild-type CFTR (157.0 ± 24.4 µS, n=9, see figure 5) expressing oocytes.
X
ABCC7 p.Pro67Ser 17495464:197:94
status: NEW200 IBMX/forskolin activated whole-cell conductance (ΔGIBMX/Fsk ) of wild-type, P67S, and P67L CFTR.
X
ABCC7 p.Pro67Ser 17495464:200:82
status: NEW201 Experiments were essentially performed as described in figure 2 to determine ΔGIBMX/Fsk in matched oocytes from two different batches expressing either wild-type CFTR, P67S or P67L CFTR (N=2, n=9,** p<0.01, *** p<0.001).
X
ABCC7 p.Pro67Ser 17495464:201:174
status: NEW203 The following findings of this study support the hypothesis that this mutation is indeed pathophysiologically relevant: 1) Expression of the P67S mutant CFTR in Xenopus laevis oocytes resulted in a CFTR whole-cell conductance that averaged only 24 % of that of wild-type CFTR.
X
ABCC7 p.Pro67Ser 17495464:203:141
status: NEW204 2) The surface expression of the P67S mutant was also largely reduced compared to that of wild-type CFTR.
X
ABCC7 p.Pro67Ser 17495464:204:33
status: NEW205 3) The mutation P67L, a previously described dominant mild form of CFTR [29], reduced the CFTR whole-cell conductance to about the same level as the P67S mutation.
X
ABCC7 p.Pro67Ser 17495464:205:149
status: NEW206 Therefore we believe that P67S is a novel CFTR mutation which in homozygous patients can cause a mild phenotype of CF characterized by abdominal pain and pancreatic insufficiency in our patient.
X
ABCC7 p.Pro67Ser 17495464:206:26
status: NEW207 The combination of a reduced CFTR whole-cell conductance, decreased channel surface expression, and normal protein expression level indicates that the P67S mutation causes a defect in channel trafficking and corresponds to a class II mutation [16].
X
ABCC7 p.Pro67Ser 17495464:207:151
status: NEW211 Interestingly, the P67S mutation appeared to reduce channel surface expression even more than CFTR whole-cell conductance (fig. 3).
X
ABCC7 p.Pro67Ser 17495464:211:19
status: NEW213 However, from structural considerations it is unlikely that the P67S mutation affects the channel pore or the gating mechanism [3, 30].
X
ABCC7 p.Pro67Ser 17495464:213:64
status: NEW218 Thus, the P67S mutation is located distant from the channel pore or from domains likely to be involved in channel gating.
X
ABCC7 p.Pro67Ser 17495464:218:10
status: NEW219 Moreover, our finding that the P67S mutation does not affect the anion selectivity of the channel supports our conclusion that the mutation does not affect the channel pore.
X
ABCC7 p.Pro67Ser 17495464:219:31
status: NEW221 In conclusion it is unlikely that the P67S mutation has a major effect on channel properties like single channel conductance or open probability.
X
ABCC7 p.Pro67Ser 17495464:221:38
status: NEW227 In our study we found that the P67L mutant had a similar inhibitory effect on the CFTR whole-cell conductance as the P67S mutant.
X
ABCC7 p.Pro67Ser 17495464:227:117
status: NEW228 These findings confirm the functional relevance of the P67 residue and support our conclusion that the newly identified P67S mutation can lead to a mild CF phenotype.
X
ABCC7 p.Pro67Ser 17495464:228:120
status: NEW231 Thus, in the oocyte expression system the inhibitory effect of the ΔF508 mutation seems to be rather similar to that observed in the present study for the P67S and P67L mutant channels.
X
ABCC7 p.Pro67Ser 17495464:231:161
status: NEW236 Thus, the temperature sensitivity of the ΔF508-CFTR may explain its more severe phenotype compared to the P67S or the P67L mutations.
X
ABCC7 p.Pro67Ser 17495464:236:112
status: NEW237 In any case, the mild CF phenotype of patients with the P67S or the P67L mutations suggests that the residual function of these mutant channels is sufficient to prevent severe symptoms.
X
ABCC7 p.Pro67Ser 17495464:237:56
status: NEW239 The P67S mutation in our patient was found on the background of a M470V polymorphism of CFTR.
X
ABCC7 p.Pro67Ser 17495464:239:4
status: NEW241 It is conceivable that the combination of V470 and P67S leads to a mild phenotype of CF whereas the combination of M470 and P67S shows a normal phenotype.
X
ABCC7 p.Pro67Ser 17495464:241:51
status: NEWX
ABCC7 p.Pro67Ser 17495464:241:124
status: NEW242 However, to clarify this question it would be necessary to identify persons with a M470 P67S genotype and to test for symptoms of CF.
X
ABCC7 p.Pro67Ser 17495464:242:88
status: NEW243 In addition we have to consider that the phenotype of the P67S-CFTR mutation may be influenced by modifier genes or other factors [43].
X
ABCC7 p.Pro67Ser 17495464:243:58
status: NEW245 The P67S mutant channels can be stimulated by exposure to IBMX/Fsk and have normal anion selectivity.
X
ABCC7 p.Pro67Ser 17495464:245:4
status: NEW247 The residual function of the P67S mutant channel is consistent with the mild CF phenotype of the affected patient.
X
ABCC7 p.Pro67Ser 17495464:247:29
status: NEW[hide] Misfolding of the cystic fibrosis transmembrane co... Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15. Cheung JC, Deber CM
Misfolding of the cystic fibrosis transmembrane conductance regulator and disease.
Biochemistry. 2008 Feb 12;47(6):1465-73. Epub 2008 Jan 15., 2008-02-12 [PMID:18193900]
Abstract [show]
Understanding the structural basis for defects in protein function that underlie protein-based genetic diseases is the fundamental requirement for development of therapies. This situation is epitomized by the cystic fibrosis transmembrane conductance regulator (CFTR)-the gene product known to be defective in CF patients-that appears particularly susceptible to misfolding when its biogenesis is hampered by mutations at critical loci. While the primary CF-related defect in CFTR has been localized to deletion of nucleotide binding fold (NBD1) residue Phe508, an increasing number of mutations (now ca. 1,500) are being associated with CF disease of varying severity. Hundreds of these mutations occur in the CFTR transmembrane domain, the site of the protein's chloride channel. This report summarizes our current knowledge on how mutation-dependent misfolding of the CFTR protein is recognized on the cellular level; how specific types of mutations can contribute to the misfolding process; and describes experimental approaches to detecting and elucidating the structural consequences of CF-phenotypic mutations.
Comments [show]
None has been submitted yet.
No. Sentence Comment
92 The P67S mutation was FIGURE 3: The six "helical hairpins" and inclusive extracellular loops derived from the two portions (TMD1 and TMD2) of the transmembrane domain of wild type CFTR.
X
ABCC7 p.Pro67Ser 18193900:92:4
status: NEW