ABCMdb

ABCC7 p.Pro574Ser

ClinVar:	c.1721C>A , p.Pro574His D , Pathogenic c.1720C>T , p.Pro574Ser ? , not provided
CF databases:	c.1720C>T , p.Pro574Ser (CFTR1) D , The P574S mutation was simultaneously detected in two families with no apparent relation but living in the center part of France (Auvergne). The P574S mutation was detected in CFTR gene by DGGE and identified by sequencing. In one family this mutation was associated with N1303K, in the other family it was associated with the F508del. c.1721C>A , p.Pro574His (CFTR1) ? , Although the amino acid pro at this position is not highly conserved across different ATP-binding folds, his seems to be a drastic substitution. This change is not detected in 52 other CF chromosomes nor 15 normal chromosomes, 4 of which have the same group IV haplotype.
Predicted by SNAP2:	A: D (91%), C: D (95%), D: D (95%), E: D (95%), F: D (95%), G: D (95%), H: D (53%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (95%), Q: D (95%), R: D (95%), S: D (95%), T: D (95%), V: D (95%), W: D (95%), Y: D (95%),
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,

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Publications
[hide] Diminished self-chaperoning activity of the DeltaF... PLoS Comput Biol. 2008 Feb 29;4(2):e1000008. Serohijos AW, Hegedus T, Riordan JR, Dokholyan NV
Diminished self-chaperoning activity of the DeltaF508 mutant of CFTR results in protein misfolding.
PLoS Comput Biol. 2008 Feb 29;4(2):e1000008., [PMID:18463704]

Abstract [show]
The absence of a functional ATP Binding Cassette (ABC) protein called the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) from apical membranes of epithelial cells is responsible for cystic fibrosis (CF). Over 90% of CF patients carry at least one mutant allele with deletion of phenylalanine at position 508 located in the N-terminal nucleotide binding domain (NBD1). Biochemical and cell biological studies show that the DeltaF508 mutant exhibits inefficient biosynthetic maturation and susceptibility to degradation probably due to misfolding of NBD1 and the resultant misassembly of other domains. However, little is known about the direct effect of the Phe508 deletion on the NBD1 folding, which is essential for rational design strategies of cystic fibrosis treatment. Here we show that the deletion of Phe508 alters the folding dynamics and kinetics of NBD1, thus possibly affecting the assembly of the complete CFTR. Using molecular dynamics simulations, we find that meta-stable intermediate states appearing on wild type and mutant folding pathways are populated differently and that their kinetic accessibilities are distinct. The structural basis of the increased misfolding propensity of the DeltaF508 NBD1 mutant is the perturbation of interactions in residue pairs Q493/P574 and F575/F578 found in loop S7-H6. As a proof-of-principle that the S7-H6 loop conformation can modulate the folding kinetics of NBD1, we virtually design rescue mutations in the identified critical interactions to force the S7-H6 loop into the wild type conformation. Two redesigned NBD1-DeltaF508 variants exhibited significantly higher folding probabilities than the original NBD1-DeltaF508, thereby partially rescuing folding ability of the NBD1-DeltaF508 mutant. We propose that these observed defects in folding kinetics of mutant NBD1 may also be modulated by structures separate from the 508 site. The identified structural determinants of increased misfolding propensity of NBD1-DeltaF508 are essential information in correcting this pathogenic mutant.

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No. Sentence Comment
167 Interestingly, the P574S mutation has been observed in a CF family also possessing the DF508 mutation but without significant pulmonary or pancreatic disease. Login to comment
169 The mutations P574S and F594N may promote contact formation between Q493 and P574 during NBD1DF508 folding, thus rescuing NBD1DF508 from the misfolding defect. Login to comment

[hide] Do common in silico tools predict the clinical con... Clin Genet. 2010 May;77(5):464-73. Epub 2009 Jan 6. Dorfman R, Nalpathamkalam T, Taylor C, Gonska T, Keenan K, Yuan XW, Corey M, Tsui LC, Zielenski J, Durie P
Do common in silico tools predict the clinical consequences of amino-acid substitutions in the CFTR gene?
Clin Genet. 2010 May;77(5):464-73. Epub 2009 Jan 6., [PMID:20059485]

Abstract [show]
Computational methods are used to predict the molecular consequences of amino-acid substitutions on the basis of evolutionary conservation or protein structure, but their utility in clinical diagnosis or prediction of disease outcome has not been well validated. We evaluated three popular computer programs, namely, PANTHER, SIFT and PolyPhen, by comparing the predicted clinical outcomes for a group of known CFTR missense mutations against the diagnosis of cystic fibrosis (CF) and clinical manifestations in cohorts of subjects with CF-disease and CFTR-related disorders carrying these mutations. Owing to poor specificity, none of tools reliably distinguished between individual mutations that confer CF disease from mutations found in subjects with a CFTR-related disorder or no disease. Prediction scores for CFTR mutations derived from PANTHER showed a significant overall statistical correlation with the spectrum of disease severity associated with mutations in the CFTR gene. In contrast, PolyPhen- and SIFT-derived scores only showed significant differences between CF-causing and non-CF variants. Current computational methods are not recommended for establishing or excluding a CF diagnosis, notably as a newborn screening strategy or in patients with equivocal test results.

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No. Sentence Comment
64 Mutations in the CFTR gene grouped by clinical category Cystic fibrosis CFTR-related disease No disease T338I D614G L320V V920L L90S M470V H199R S1251N I203M G550R P111A I148T Q1291H R560K L1388Q L183I R170H I1027T S549R D443Y P499A L1414S T908N R668C S549N A455E E1401K Q151K G27E I1234L Y563N R347P C866R S1118C P1290S R75Q A559T V520F P841R M469V E1401G P67L G85E S50Y E1409K R933G G458V G178R Y1032C R248T I980K G85V V392G L973P L137H T351S R334W I444S V938G R792G R560T R555G L1339F D1305E P574H V1240G T1053I D58G G551D L1335P I918M F994C S945L L558S F1337V R810G D1152H G1247R P574S R766M D579G W1098R H949R F200I R352Q L1077P K1351E M244K L206W M1101K D1154G L375F N1303K R1066C E528D D110Y R347H R1070Q A800G P1021S S549K A1364V V392A damaging` (is supposed to affect protein function or structure) and 'probably damaging` (high confidence of affecting protein function or structure). Login to comment

[hide] Orphan missense mutations in the cystic fibrosis t... J Mol Diagn. 2011 Sep;13(5):520-7. Epub 2011 Jun 25. Fresquet F, Clement R, Norez C, Sterlin A, Melin P, Becq F, Kitzis A, Thoreau V, Bilan F
Orphan missense mutations in the cystic fibrosis transmembrane conductance regulator a three-step biological approach to establishing a correlation between genotype and phenotype.
J Mol Diagn. 2011 Sep;13(5):520-7. Epub 2011 Jun 25., [PMID:21708286]

Abstract [show]
More than 1860 mutations have been found within the human cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence. These mutations can be classified according to their degree of severity in CF disease. Although the most common mutations are well characterized, few data are available for rare mutations. Thus, genetic counseling is particularly difficult when fetuses or patients with CF present these orphan variations. We describe a three-step in vitro assay that can evaluate rare missense CFTR mutation consequences to establish a correlation between genotype and phenotype. By using a green fluorescent protein-tagged CFTR construct, we expressed mutated proteins in COS-7 cells. CFTR trafficking was visualized by confocal microscopy, and the cellular localization of CFTR was determined using intracellular markers. We studied the CFTR maturation process using Western blot analysis and evaluated CFTR channel activity by automated iodide efflux assays. Of six rare mutations that we studied, five have been isolated in our laboratory. The cellular and functional impact that we observed in each case was compared with the clinical data concerning the patients in whom we encountered these mutations. In conclusion, we propose that performing this type of analysis for orphan CFTR missense mutations can improve CF genetic counseling.

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No. Sentence Comment
50 The genotype was as follows: p.[Asn1303Lys] ϩ [Pro574Ser]. Login to comment
97 Sequences of Site-Directed Mutagenesis Primers Mutation Sense oligonucleotide sequences L102P 5=-GTACAGCCTCTCTTACCGGGAAGAATCATAGCTTCC-3= L167R 5=-AAGAAGACTTTAAAGCGGTCAAGCCGTGTTCTAG-3= P574S 5=-GCTGATTTGTATTTATTAGACTCTTCTTTTGGATACCTAGATG-3= V562I 5=-AGAATTTCTTTAGCAAGAGCAATATACAAAGATGCTGATTTG-3= K696R 5=-CAGACTGGAGAGTTTGGGGAAAGAAGGAAGAATTCTATTCTC-3= P841R 5=-GATATGGAGAGCATACGAGCAGTGACTACATGG-3= CFTR Missense Mutation Biological Assay 3 JMD Month 2011, Vol. xx, No. Login to comment
109 Mixed Phenotype with P574S CFTR Variant The missense substitution p.[Pro574Ser] (P574S) lies within nucleotide-binding domain 1. Login to comment
112 Moreover, the P574S mutant does not fully colocalize with the ER marker and seems to escape this cellular compartment because a strong green stain can be observed, which clearly differs from the calreticulin red signal. Login to comment
113 After immunoblotting (Figure 2B), we quantify the ratio of immature core-glycosylated CFTR/total CFTR [band B/(band B ϩ band C)]: attributing 100% to WT-CFTR, this ratio increases to 349.5% Ϯ 88% (n ϭ 3) for P574S. Login to comment
115 Surprisingly, the iodide efflux assay exhibits a dramatic inhibition of CFTR channel activity: when 100% activation is attributed to the maximal activation peak of WT-CFTR, activation is reduced to 13.07% Ϯ 5.23% (n ϭ 4) for P574S (Figure 2C). Login to comment
116 The lowered maturation of P574S suggests an attenuated class II mutation. Login to comment
118 These data should be confronted to the patient genotype to evaluate the pathogenicity of the P574S mutation. Login to comment
122 Summary of the Patients` Data, Concerning Genotype, Phenotype, and Protein Dysfunction Patient no./sex/age at molecular diagnostics (years) Genotype Phenotype Protein dysfunctions Channel activity* Maturation† Intracellular localization‡ 1/F/3 p.[Leu102Pro] ϩ [Arg553X] Positive sweat test result, bacterial lung colonization, no pancreatitis ϩϩ ϩϩ ϩϩ 2/F/newborn p.[Phe508del] ϩ [Leu167Arg] Positive sweat test result, recurrent pancreatitis, no lung infection ϩϩ ϩϩ ϩϩ 3/F/3 p.[Asn1303Lys] ϩ [Pro574Ser] Normal sweat test result, asymptomatic ϩϩ ϩ ϩ 4/M/31 p.[Arg74Trp;Val201Met; Asp1270Asn] ϩ [Pro841Arg]; c. Login to comment
145 This confirms that V562I substitution should be a polymorphism with no structural or functional impact on the CFTR protein. Login to comment
151 In our model, two missense mutations, L102P and A calreticulin egremP201L calreticulin egremP201L L167R calreticulin mergeL167R calreticulin merge CB F508del L102P L167R WT F508del L102P L167R *** *** ***WT + inh172 L102P L167R *** *** ***WT + inh172 L102P L167R B C CFTR B C CFTR % of maximal activation C. Login to comment
154 L102P and L167R amino acid substitutions impair CFTR protein maturation. Login to comment
161 B A calreticulin egremS475P calreticulin egremS475P WT F508del P574S CFTR C WT F508del P574S CFTR C B CFTR NaKATPase B CFTR NaKATPase C WT P574S *** WT P574S ***** 00 WT P574S 0 00 00 00 00 **** 00 WT P574S 0 00 00 00 00 0 50 100 WT % of maximal activation 0 50 100 WT % of maximal activationB/(B+C) (%) 100 0 200 300 400 500 B/(B+C) (%) 100 0 200 300 400 500 Figure 2. Login to comment
162 P574S amino acid substitution decreases CFTR protein maturation. Login to comment
181 The results obtained for P574S are more complex to interpret. Login to comment
183 Contrary to L102P and L167R, P574S is not fully retained in the ER compartment because we can see the presence of the fully glycosylated mature form of CFTR by using Western blot analysis. Login to comment
184 This observation is sustained by confocal microscopic imaging clearly showing different staining patterns for P574S- CFTR and the ER resident calreticulin. Login to comment
185 Taken together, these results strengthen the idea that a small part of P574S could reach post-Golgi compartments and, thus, the plasma membrane. Login to comment
187 His genotype was p.[Asn1303Lys] ϩ [Pro574Ser], and the allele in cis is considered a CF-causing mutation.21 Five sweat test results were negative, and this patient is now a healthy 3-year-old girl, asymptomatic of CF disease. Login to comment
189 Their genotype was p.[Phe508del] ϩ [Pro574Ser]. Login to comment
190 These data clearly indicate that P574S induces a CF-related disorder phenotype (CBAVD) for males, consistent with a normal sweat test result and no symptoms of CF disease for the girl. Login to comment
191 However, if the results of our Western blot analysis indicate a mild phenotype, P574S-CFTR-impaired channel activation would suggest a severe CF phenotype. Login to comment
193 K696R and P841R amino acid substitutions affect CFTR channel activity. Login to comment
199 A B C V562I actin mergeV562I actin merge % of maximal activation ns 0 50 100 WT V562I % of maximal activation ns 0 50 100 WT V562I % of maximal activation ns 0 50 100 WT V562I B CFTR NaKATPase C WT F508del V562I B CFTR NaKATPase C WT F508del V562I Figure 4. Login to comment
200 The V562I amino acid substitution does not alter CFTR phenotype. Login to comment
207 Obviously, patch-clamp analysis would be useful to measure the real impact of P574S in CFTR channel gating. Login to comment
224 Our results are concordant with those previously obtained in baby hamster kidney cells overexpressing CFTR23 (by using Western blot analysis, iodide effluxes, and inside-out patch-clamp experiments in which the V562I mutant behaves like WT CFTR). Login to comment
228 Moreover, it appears that P574S, an allele exhibiting a lower severity for two of three criteria, is not a CF-causing mutation, despite strongly decreased estimated channel activity. Login to comment
53 The two boys had the same genotype: p.[Phe508del] ϩ [Pro574Ser]); they presented with congenital bilateral aplasia of the vas deferens (CBAVD). Login to comment
60 Sequences of Site-Directed Mutagenesis Primers Mutation Sense oligonucleotide sequences L102P 5=-GTACAGCCTCTCTTACCGGGAAGAATCATAGCTTCC-3= L167R 5=-AAGAAGACTTTAAAGCGGTCAAGCCGTGTTCTAG-3= P574S 5=-GCTGATTTGTATTTATTAGACTCTTCTTTTGGATACCTAGATG-3= V562I 5=-AGAATTTCTTTAGCAAGAGCAATATACAAAGATGCTGATTTG-3= K696R 5=-CAGACTGGAGAGTTTGGGGAAAGAAGGAAGAATTCTATTCTC-3= P841R 5=-GATATGGAGAGCATACGAGCAGTGACTACATGG-3= was presented. Login to comment
81 Summary of the Patients` Data, Concerning Genotype, Phenotype, and Protein Dysfunction Patient no./sex/age at molecular diagnostics (years) Genotype Phenotype Protein dysfunctions Channel activity* Maturation† Intracellular localization‡ 1/F/3 p.[Leu102Pro] ϩ [Arg553X] Positive sweat test result, bacterial lung colonization, no pancreatitis ϩϩ ϩϩ ϩϩ 2/F/newborn p.[Phe508del] ϩ [Leu167Arg] Positive sweat test result, recurrent pancreatitis, no lung infection ϩϩ ϩϩ ϩϩ 3/F/3 p.[Asn1303Lys] ϩ [Pro574Ser] Normal sweat test result, asymptomatic ϩϩ ϩ ϩ 4/M/31 p.[Arg74Trp;Val201Met; Asp1270Asn] ϩ [Pro841Arg]; c. Login to comment
102 Fluorescence was examined with a spectral confocal station FV-1000 in- B A calreticulin egremS475P calreticulin egremS475P WT F508del P574S CFTR C WT F508del P574S CFTR C B CFTR NaKATPase B CFTR NaKATPase C WT P574S *** WT P574S ***** 00 WT P574S 0 00 00 00 00 **** 00 WT P574S 0 00 00 00 00 0 50 100 WT % of maximal activation 0 50 100 WT % of maximal activationB/(B+C) (%) 100 0 200 300 400 500 B/(B+C) (%) 100 0 200 300 400 500 Figure 2. Login to comment
103 P574S amino acid substitution decreases CFTR protein maturation. Login to comment
152 After immunoblotting (Figure 2B), we quantify the ratio of immature core-glycosylated CFTR/total CFTR [band B/(band B ϩ band C)]: attributing 100% to WT-CFTR, this ratio increases to 349.5% Ϯ 88% (n ϭ 3) for P574S. Login to comment
155 The lowered maturation of P574S suggests an attenuated class II mutation. Login to comment
157 These data should be compared to the patient genotype to evaluate the pathogenicity of the P574S mutation. Login to comment
194 This observation is sustained by confocal microscopic imaging clearly showing different staining patterns for P574S- CFTR and the ER resident calreticulin. Login to comment
195 Taken together, these results strengthen the idea that a small part of P574S could reach post-Golgi compartments and, thus, the plasma membrane. Login to comment
197 His genotype was p.[Asn1303Lys] ϩ [Pro574Ser], and the allele in cis is considered a CF-causing mutation.21 Five sweat test results were negative, and this patient is now a healthy 3-year-old girl, asymptomatic of CF disease. Login to comment
201 However, if the results of our Western blot analysis indicate a mild phenotype, P574S-CFTR-impaired channel activation would suggest a severe CF phenotype. Login to comment
203 Obviously, patch-clamp analysis would be useful to measure the real impact of P574S in CFTR channel gating. Login to comment