ABCMdb

ABCC7 p.Glu725Lys

ClinVar:	c.2173G>A , p.Glu725Lys ? , not provided
CF databases:	c.2173G>A , p.Glu725Lys (CFTR1) ? , This mutation was detected by DGGE and direct sequencing. E725K was found in one female CF patient of Greek origin (1/500chromosomes); her other mutation is unknown.
Predicted by SNAP2:	A: D (63%), C: D (59%), D: N (61%), F: D (66%), G: D (71%), H: D (53%), I: D (71%), K: D (80%), L: D (66%), M: D (66%), N: D (71%), P: D (75%), Q: D (71%), R: D (80%), S: D (71%), T: D (66%), V: D (66%), W: D (80%), Y: D (71%),
Predicted by PROVEAN:	A: N, C: D, D: N, F: D, G: D, H: N, I: N, K: N, L: N, M: N, N: N, P: N, Q: N, R: N, S: N, T: N, V: N, W: D, Y: D,

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Publications
[hide] A short segment of the R domain of cystic fibrosis... J Biol Chem. 2002 Jun 21;277(25):23019-27. Epub 2002 Apr 11. Xie J, Adams LM, Zhao J, Gerken TA, Davis PB, Ma J
A short segment of the R domain of cystic fibrosis transmembrane conductance regulator contains channel stimulatory and inhibitory activities that are separable by sequence modification.
J Biol Chem. 2002 Jun 21;277(25):23019-27. Epub 2002 Apr 11., 2002-06-21 [PMID:11950844]

Abstract [show]
The regulatory (R) domain of the cystic fibrosis transmembrane conductance regulator (CFTR) contains consensus phosphorylation sites for cAMP-dependent protein kinase (PKA) that are the basis for physiological regulation of the CFTR chloride channel. A short peptide segment in the R domain with a net negative charge of B9 (amino acids 817-838, NEG2) and predicted helical tendency is shown to play a critical role in CFTR chloride channel function. Deletion of NEG2 from CFTR completely eliminates the PKA dependence of channel activity. Exogenous NEG2 peptide interacts with CFTR to exert both stimulatory and inhibitory effects on the channel function. The NEG2 peptide with sequence scrambled to remove helical tendencies also inhibits channel function, but does not stimulate. Similar results are found for a NEG2 peptide whose helical structure is disrupted by a proline residue. When six of the negatively charged carboxylic acid residues are replaced by their cognate amides, reducing net negative charge to B3, but increasing helical propensity as assessed by circular dichroism, the peptide stimulates CFTR channel function, but does not inhibit. We speculate that the NEG2 region interacts with other cytosolic domains of CFTR to control opening and closing transitions of the chloride channel.

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No. Sentence Comment
231 One amino acid substitution, noted in a patient with CF, is reported in the CF Mutation Consortium data base in the NEG1 region (E725K). Login to comment

[hide] The PEST sequence does not contribute to the stabi... BMC Biochem. 2002 Oct 2;3:29. Epub 2002 Oct 2. Chen EY, Clarke DM
The PEST sequence does not contribute to the stability of the cystic fibrosis transmembrane conductance regulator.
BMC Biochem. 2002 Oct 2;3:29. Epub 2002 Oct 2., 2002-10-02 [PMID:12361483]

Abstract [show]
BACKGROUND: Endoplasmic reticulum retention of misfolded cystic fibrosis transmembrane conductance regulator (CFTR) mutants and their rapid degradation is the major cause of cystic fibrosis (CF). An important goal is to understand the mechanism of how the misfolded proteins are recognized, retained, and targeted for degradation. RESULTS: Using a web-based algorithm, PESTFind, we found a PEST sequence in the regulatory (R) domain of CFTR. The PEST sequence is found in many short-lived eukaryotic proteins and plays a role in their degradation. To determine its role in the stability and degradation of misprocessed CFTR, we introduced a number of site-directed mutations into the PEST sequence in the cDNA of DeltaF508 CFTR, the most prevalent misprocessed mutation found in CF patients. Analysis of these mutants showed that the disruption of the PEST sequence plays a minor role in the degradation of the CFTR mutants. Multiple mutations to the PEST sequence within the R domain of CFTR inhibit maturation of CFTR and prevent the formation of a 100 kDa degradation product. The mutations, however, do not improve the stability of the mutant DeltaF508 CFTR. CONCLUSION: These observations show that disruption of the structure of the R domain of CFTR can inhibit maturation of the protein and that the predicted PEST sequence plays no significant role in the degradation of CFTR.

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48 The E725K/E726K mutant was included to test if alteration of charges would affect the function of the PEST sequence. Login to comment
51 However, since both S728A and T717A mutant have a high PEST score (T717A mutant alone has PEST score of +3.87), we added an additional E725K mutation to the T717A mutant just to further disrupt the PEST sequence. Login to comment
109 Misfolded or misassembled proteins, such as processing defective ∆F508 mutant CFTR, are recognized and retained in the ER by the quality control system in the ER; although the exact mechanism for recognition is yet to be elucidated, evidence indicate that the prolonged association with mo- Table 1: Mutations introduced into the predicted PEST sequence of CFTR Mutant Sequence PEST Score Wild-type (WT) 716 - KTPLQMNGIEEDSDEPLER - 734 +6.91 Poly-Valine 716 - KTPLQMNGIVVVVVVPLER - 734 -26.19 E725K/E726K 716 - KTPLQMNGIKKDSDEPLER - 734 N/A S728A 716 - KTPLQMNGIEEDADEPLER - 734 +4.07 T717A/E725K 716 - KAPLQMNGIKEDSDEPLER - 734 N/A * Residues in bold are the mutations introduced. Login to comment
134 Mature Immature WT CFTR Mature Immature ∆F508 CFTR WT/∆F508 Poly-Valine S728A E725K/E726K T717A/E725K A. B. Login to comment
135 205 130 90 kDa 100 kD 81 kD Immature ∆F508 Poly-Valine ∆F508/Poly-Valine ∆F508/S728A ∆F508/E725K/E726K ∆F508/T717A/E725K volved in protein-protein interactions: direct interaction with ubc9 [33] and ligand recognition [34,35]. Login to comment
153 A) Pulse-chase radiograph for non-processing defective constructs: WT, WT/E725K/E726K, and WT/T717A/E725K; results for WT/S728A not shown, but is similar to that of WT. Login to comment
154 B) Pulse-chase radiograph for processing defective constructs: ∆F508, ∆F508/poly-valine, ∆F508/E725K/E726K, and ∆F508/T717/E725K; results for WT/Poly-valine and ∆F508/S728A not shown but they show similar results as ∆F508. Login to comment
156 Hours 0 2 4 6 8 12 24 E725K/E726K Mature Immature T717A/E725KMature Immature WT Mature Immature A. Login to comment
157 Hours 0 2 4 6 8 12 24 ∆∆∆∆F508Mature Immature ∆∆∆∆F508/ E725K/E726K Mature Immature ∆∆∆∆F508/ T717A/E725K Mature Immature Mature Immature ∆∆∆∆F508/ Poly-Valine B. mutant protein to not mature suggests otherwise. Login to comment
177 0 0.1 1 10 100 1000 [Trypsin] (µg/ml) T717A/E725KMature Immature E725K/E726KMature Immature S728AMature Immature WTMature Immature Poly-ValineMature Immature ∆F508Mature Immature 0 0.1 1 10 100 1000 [Trypsin] (µg/ml) ∆F508/ Poly-Valine Mature Immature ∆F508/ S728A Mature Immature ∆F508/ E725K/E726K Mature Immature ∆F508/ T717A/E725K Mature Immature the PEST region of another nuclear SUMO-1 target protein, HIPK2 [48]. Login to comment
202 WT/∆F508Mature Immature Poly-ValineMature Immature S728AMature Immature E725K/E726KMature Immature T717A/E725KMature Immature - + - + WT CFTR ∆∆∆∆F508 CFTR 2µµµµM MG-132 tion at 44,000 × g for 45 min. at 4°C and resuspended with 300 µl of TBS (Tris-buffered saline; 10 mM Tris-HCl, pH 7.5, 150 mM NaCl). Login to comment

[hide] Primary sclerosing cholangitis in childhood is ass... J Pediatr. 2007 Sep;151(3):255-9. Epub 2007 Jul 24. Pall H, Zielenski J, Jonas MM, DaSilva DA, Potvin KM, Yuan XW, Huang Q, Freedman SD
Primary sclerosing cholangitis in childhood is associated with abnormalities in cystic fibrosis-mediated chloride channel function.
J Pediatr. 2007 Sep;151(3):255-9. Epub 2007 Jul 24., [PMID:17719933]

Abstract [show]
OBJECTIVE: To determine whether primary sclerosing cholangitis (PSC) in childhood is associated with abnormalities in cystic fibrosis transmembrane conductance regulator (CFTR). STUDY DESIGN: Subjects with PSC diagnosed in childhood (n = 20) were recruited from Children's Hospital. Subjects had testing with sweat chloride concentration, nasal transmembrane potential difference, and extensive genetic analysis of the CFTR gene. Disease control subjects consisted of 14 patients with inflammatory bowel disease alone and no liver disease. t tests were performed to determine statistical significance. RESULTS: In the PSC group, CFTR chloride channel function (deltaChloride free + isoproterenol) was markedly diminished at -8.6 +/- 8.2 mV (reference range: -24.6 +/- 10.4 mV). In contrast, disease control subjects had normal function, at -17.8 +/- 9.7 mV (P = .008). Sweat chloride concentration in subjects with PSC was greater than in disease control subjects (20.8 +/- 3.4 mmol/L vs 12.0 +/- 1.6 mmol/L, P = .045). Comprehensive CFTR genotyping revealed that 5 of 19 (26.3%) subjects with PSC had a CFTR mutation or variant, compared with 6 of 14 (42.9%) disease control subjects. CONCLUSIONS: There is a high prevalence of CFTR-mediated ion transport dysfunction in subjects with childhood PSC.

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No. Sentence Comment
90 Mutations/Variants Polymorphism 1540 locus T Tract Sweat chloride (mmol/L) ⌬Cl ؉ Iso (mV)* PSC 1 GG 7/7 20.2 -1.2 2 GG 7/7 19.7 -17.3 3 R75Q AA 7/7 14.8 -3.9 4 4521G/A(8T/9T) AA 7/7 19.4 -13.3 5 2694T/G AG 7/7 NP NP 6 GG 7/7 5.1 -12.5 7 NP 5.8 -17.8 8 AA 7/7 9.9 -12.8 9 E725K AG 7/7 56.3 -5.1 10 R75Q AA 7/7 30.3 -5.5 11 4006 - 200G/A, 1233A/T AA 7/9 32.1 -29 12 2694T/G AG 7/7 8.7 -2.4 13 4521G/A, 4700T8/9 AG 7/7 4.8 -3.8 14 1525 - 61A/G AG 7/9 45.3 -2.2 15 3030G/A GG 7/7 18.6 -0.45 16 AA 7/7 7.6 -4.5 17 R75Q AG 7/9 21.6 -1.8 18 1001 ϩ 11C/T AG 7/9 45 1.2 19 AA 7/7 11 -11.5 20 1716G ¡ A AG 7/7 18.9 -20.1 IBD 21 1001 ϩ 11C/T AG 7/9 8.9 -13 22 S1235R/2752 - 26A ¡ G 185 ϩ 324C/T AG 7/7 15 -15 23 AG 7/7 17.8 -20 24 IVS8T5 AA 5/7 4.8 -10.4 25 875 ϩ 40A/G, 125G/C GG 7/7 10.8 -30 26 AG 7/7 22.9 -10.5 27 IVS8T5 AG 5/7 15.1 NP 28 AG 7/7 20.1 -8 29 R75Q GG 7/7 10.1 -17 30 AA 7/7 14 NP 31 Q1352H 4521A(hom), 4700T8/8 AG 7/7 3.5 -35 32 AA 7/7 11.8 -8 33 125G/C AG 7/7 9.4 -33 34 R75Q/IVS8T5 1898 ϩ 152T/A AG 5/7 4 -14 Subjects with IBD were disease control subjects. Login to comment
91 CFTR mutations shown in bold are E725K, S1235R, and 2752 - 26A ¡ G. Login to comment

[hide] N-terminal CFTR missense variants severely affect ... Hum Mutat. 2008 May;29(5):738-49. Gene GG, Llobet A, Larriba S, de Semir D, Martinez I, Escalada A, Solsona C, Casals T, Aran JM
N-terminal CFTR missense variants severely affect the behavior of the CFTR chloride channel.
Hum Mutat. 2008 May;29(5):738-49., [PMID:18306312]

Abstract [show]
Over 1,500 cystic fibrosis transmembrane conductance regulator (CFTR) gene sequence variations have been identified in patients with cystic fibrosis (CF) and related disorders involving an impaired function of the CFTR chloride channel. However, detailed structure-function analyses have only been established for a few of them. This study aimed evaluating the impact of eight N-terminus CFTR natural missense changes on channel behavior. By site-directed mutagenesis, we generated four CFTR variants in the N-terminal cytoplasmic tail (p.P5L, p.S50P, p.E60K, and p.R75Q) and four in the first transmembrane segment of membrane-spanning domain 1 (p.G85E/V, p.Y89C, and p.E92K). Immunoblot analysis revealed that p.S50P, p.E60K, p.G85E/V, and p.E92K produced only core-glycosylated proteins. Immunofluorescence and whole cell patch-clamp confirmed intracellular retention, thus reflecting a defect of CFTR folding and/or trafficking. In contrast, both p.R75Q and p.Y89C had a glycosylation pattern and a subcellular distribution comparable to the wild-type CFTR, while the percentage of mature p.P5L was considerably reduced, suggesting a major biogenesis flaw on this channel. Nevertheless, whole-cell chloride currents were recorded for all three variants. Single-channel patch-clamp analyses revealed that the channel activity of p.R75Q appeared similar to that of the wild-type CFTR, while both p.P5L and p.Y89C channels displayed abnormal gating. Overall, our results predict a major impact of the CFTR missense variants analyzed, except p.R75Q, on the CF phenotype and highlight the importance of the CFTR N-terminus on channel physiology.

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133 Genotype^Phenotype Correlation in the N-Terminal CFTR MissenseVariants Under StudyÃ Missense varianta Phenotype Second allele (number of patients)b p.P5L CF p.F508del (1), p.P205S (1) p.S50P CBAVD p.F508del (1), p.E115del (1) p.E60K CF p.G542X (1), p.I507del (1) p.R75Q HT p.F508del (3), p.E725K (1) B p.R347H (1), p.R75Q (1), n.i. (4) Br c.1584G4A (2), c.1210-7_1210-6delTT (1), n.i.(3) NT p.F508del (1) CP c.1584G4A (1), n.i. (3) MI n.i. (1) CUAVD n.i. (2) OZ n.i. (2) Normal p.R75Q (1), c.2052_2053insA (1), n.i. (1) p.G85E CF p.F508del (8), p.G542X (2), p.I507del (1), c.580-1G4T (1), p.G85E (1), c.1477_ 1478delCA (1) CBAVD p.G576A (1) HT p.L997F (1),WT (1) p.G85V CF p.F508del (2), p.G542X (2), p.Y1092X (1), c.265715G4A (1), p.A1006E, c.1210-7_1210- 6delTT (1), n.i. (1) p.Y89C CF n.i. (1)c p.E92K CF p.F508del (2), p.Q890X (1), p.L206W (1) CBAVD c.1210-7_1210-6delTT (1) ÃThe recommendations for mutation nomenclature (www.hgvs.org/mutnomen/) were used to name CFTR gene sequence variations at both the nucleotide level and the protein level. Login to comment

[hide] Molecular evaluation of CFTR sequence variants in ... Int J Androl. 2005 Oct;28(5):284-90. Larriba S, Bonache S, Sarquella J, Ramos MD, Gimenez J, Bassas L, Casals T
Molecular evaluation of CFTR sequence variants in male infertility of testicular origin.
Int J Androl. 2005 Oct;28(5):284-90., [PMID:16128988]

Abstract [show]
Although the involvement of the CFTR gene has been well established in congenital agenesia of vas deferens, its role in non-obstructive (NOb) infertility is still a matter of debate. In order to definitively define the involvement of the CFTR gene in spermatogenic impairment and a potential synergistic contribution to known genetic and clinical factors, genetic variants in the entire coding sequence and the immediately flanking regions of the CFTR gene, along with a thorough clinical evaluation, were analysed in 83 NOb infertile patients and 87 clinically well-defined fertile individuals as controls. The results of our study showed no statistical difference between CFTR carrier frequency in the infertile and fertile population. Specifically, the IVS8-6(5T) allele carrier frequency was similar in NOb infertile patients when compared with fertile men, but it is noteworthy that, when fertile men were classified into having optimal and suboptimal fertility, no 5T allele was found among the 35 men with optimal fertility parameters. In conclusion, extensive CFTR analysis in infertile individuals and fertile population as adequate control definitively excludes the involvement of the CFTR gene variants in sperm production and stresses the importance of carefully identifying those individuals with obstructive defects, in whom CFTR screening will be beneficial.

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53 Thirteen CFTR gene sequence variants [p.R75Q, p.I148T, p.T351S, p.F508del, p.G576A, p.R668C, p.E725K, p.V754M, p.D836Y, p.L997F, p.S1235R, IVS8-6(5T) and c.1716G>A] were determined in 11 F1 and 15 F2 individuals (Table 1) giving a frequency of 29.9%. Login to comment
85 Continued No. Phenotype CFTR genotype Associated factors Testicular histologya b c 13 F2 p.I148T p.R75Q No nd 14 F2 p.T351S No nd 15 F2 p.F508del No nd 16 F2 p.E725K No nd 17 F2 p.V754M No nd 18 F2 p.L997F No nd 19 F2 (T)5-(TG)12 No nd 20 F2 (T)5-(TG)12 No nd 21 F2 (T)5-(TG)11 No nd 22 F2 (T)5-(TG)11 No nd 23 F2 c.1716 G>A No nd 24 F2 c.1716 G>A No nd 25 F2 c.1716 G>A No nd 26 F2 c.1716 G>A No nd Phenotype: NOb (SO), non-obstructive severe oligozoospermia; NOb (A), non-obstructive azoospermia; F1, optimal fertility; F2, suboptimal fertility. Login to comment

[hide] Definition of a "functional R domain" of the cysti... Mol Genet Metab. 2000 Sep-Oct;71(1-2):245-9. Chen JM, Scotet V, Ferec C
Definition of a "functional R domain" of the cystic fibrosis transmembrane conductance regulator.
Mol Genet Metab. 2000 Sep-Oct;71(1-2):245-9., [PMID:11001817]

Abstract [show]
The R domain of the cystic fibrosis transmembrane conductance regulator (CFTR) was originally defined as 241 amino acids, encoded by exon 13. Such exon/intron boundaries provide a convenient way to define the R domain, but do not necessarily reflect the corresponding functional domain within CFTR. A two-domain model was later proposed based on a comparison of the R-domain sequences from 10 species. While RD1, the N-terminal third of the R domain is highly conserved, RD2, the large central region of the R domain has less rigid structural requirements. Although this two-domain model was given strong support by recent functional analysis data, the simple observation that two of the four main phosphorylation sites are excluded from RD2 clearly indicates that RD2 still does not satisfy the requirements of a "functional R domain." Nevertheless, knowledge of the CFTR structure and function accumulated over the past decade and reevaluated in the context of a comprehensive sequence comparison of 15 CFTR homologues made it possible to define such a "functional R domain," i.e., amino acids C647 to D836. This definition is validated primarily because it contains all of the important potential consensus phosphorylation sequences. In addition, it includes the highly charged motif from E822 to D836. Finally, it includes all of the deletions/insertions in this region. This definition also aids in understanding the effects of missense mutations occurring within this domain.

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49 Similarly, E725K and D836Y both occur in well-conserved, FIG. 1. Login to comment