ABCMdb

ABCC7 p.Asp58Asn

ClinVar:	c.172G>A , p.Asp58Asn ? , not provided c.173A>G , p.Asp58Gly ? , not provided
CF databases:	c.172G>A , p.Asp58Asn (CFTR1) D , Missense mutations D58N was found in a CBAVD patient with an yet unidentified mutation on his other allele. c.173A>G , p.Asp58Gly (CFTR1) D , This putative mutation was detected by DGGE and identified by DNA fluorescent sequencing in a CBAVD patient from Tunisia, who is heterozygous for the mutation N1303K.
Predicted by SNAP2:	A: N (53%), C: N (53%), E: N (72%), F: D (63%), G: D (53%), H: N (53%), I: D (63%), K: D (71%), L: D (66%), M: D (63%), N: N (72%), P: D (71%), Q: D (53%), R: D (66%), S: N (53%), T: N (66%), V: D (63%), W: D (66%), Y: D (63%),
Predicted by PROVEAN:	A: N, C: D, E: N, F: D, G: D, H: D, I: D, K: N, L: D, M: D, N: N, P: N, Q: N, R: N, S: N, T: N, V: D, W: D, Y: D,

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[hide] Cysteine substitutions reveal dual functions of th... J Biol Chem. 2001 Sep 21;276(38):35660-8. Epub 2001 Jul 23. Fu J, Kirk KL
Cysteine substitutions reveal dual functions of the amino-terminal tail in cystic fibrosis transmembrane conductance regulator channel gating.
J Biol Chem. 2001 Sep 21;276(38):35660-8. Epub 2001 Jul 23., 2001-09-21 [PMID:11468285]

Abstract [show]
Previously, we observed that the cystic fibrosis transmembrane conductance regulator (CFTR) channel openings are destabilized by replacing several acidic residues in the amino-terminal tail with alanines (Naren, A. P., Cormet-Boyaka, E., Fu, J., Villain, M., Blalock, J. E., Quick, M. W., and Kirk, K. L. (1999) Science 286, 544-548). Here we determined whether this effect is due to the loss of negative charge at these sites and whether the amino-terminal tail also modulates other aspects of channel gating. We introduced cysteines at two of these positions (E54C/D58C) and tested a series of methanethiosulfonate (MTS) reagents for their effects on the gating properties of these cysteine mutants in intact Xenopus oocytes and excised membrane patches. Covalent modification of these sites with either neutral (MMTS) or charged (2-carboxyethylmethanethiosulfonate (MTSCE) and 2-(trimethylammonium)ethylmethanethiosulfonate (MTSET)) reagents markedly inhibited channel open probability primarily by reducing the rate of channel opening. The MTS reagents had negligible effects on the gating of the wild type channel or a corresponding double alanine mutant (E54A/D58A) under the same conditions. The inhibition of the opening rate of the E54C/D58C mutant channel by MMTS could be reversed by the reducing agent dithiothreitol (200 microm) or by elevating the bath ATP concentration above that required to activate maximally the wild type channel (>1 mm). Interestingly, the three MTS reagents had qualitatively different effects on the duration of channel openings (i.e. channel closing rate), namely the duration of openings was negligibly changed by the neutral MMTS, decreased by the positively charged MTSET, and increased by the negatively charged MTSCE. Our results indicate that the CFTR amino tail modulates both the rates of channel opening and channel closing and that the negative charges at residues 54 and 58 are important for controlling the duration of channel openings.

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16 A disease-associated mutant that maps to one of these sites (D58N CFTR) exhibited similar alterations in macroscopic current kinetics and open channel burst duration (14). Login to comment
32 Although we failed to observe a dramatic effect of mutations in this region on channel opening rate, we did observe a modest increase in interburst duration (i.e. closed time) for D58N CFTR (14) as if this mutation also affected the ability of the channel to open. Login to comment
182 We also observed that a disease-associated mutant (D58N) that maps to this region exhibited unstable openings (i.e. shortened open channel burst duration) in excised patches (14). Login to comment
239 These data are consistent with the shorter channel openings that were previously observed for the disease-associated D58N mutant (14), which harbors a structurally subtle substitution with the exception of the loss of negative charge at this position (asparagine for aspartate). Login to comment

[hide] A cluster of negative charges at the amino termina... J Physiol. 2001 Oct 15;536(Pt 2):459-70. Fu J, Ji HL, Naren AP, Kirk KL
A cluster of negative charges at the amino terminal tail of CFTR regulates ATP-dependent channel gating.
J Physiol. 2001 Oct 15;536(Pt 2):459-70., 2001-10-15 [PMID:11600681]

Abstract [show]
1. The cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel is activated by protein kinase A (PKA) phosphorylation of its R domain and by ATP binding at its nucleotide-binding domains (NBDs). Here we investigated the functional role of a cluster of acidic residues in the amino terminal tail (N-tail) that also modulate CFTR channel gating by an unknown mechanism. 2. A disease-associated mutant that lacks one of these acidic residues (D58N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP -activating cocktail than wild-type CFTR. 3. In excised membrane patches D58N CFTR exhibited a two-fold reduction in single channel open probability due primarily to shortened open channel bursts. 4. Replacing this and two nearby acidic residues with alanines (D47A, E54A, D58A) also reduced channel activity, but had negligible effects on bulk PKA phosphorylation or on the ATP dependence of channel activation. 5. Conversely, the N-tail triple mutant exhibited a markedly inhibited response to AMP-PNP, a poorly hydrolysable ATP analogue that can nearly lock open the wild-type channel. The N-tail mutant had both a slower response to AMP-PNP (activation half-time of 140 +/- 20 s vs. 21 +/- 4 s for wild type) and a lower steady-state open probability following AMP-PNP addition (0.68 +/- 0.08 vs. 0.92 +/- 0.03 for wild type). 6. Introducing the N-tail mutations into K1250A CFTR, an NBD2 hydrolysis mutant that normally exhibits very long open channel bursts, destabilized the activity of this mutant as evidenced by decreased macroscopic currents and shortened open channel bursts. 7. We propose that this cluster of acidic residues modulates the stability of CFTR channel openings at a step that is downstream of ATP binding and upstream of ATP hydrolysis, probably at NBD2.

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14 A disease-associated mutant that lacks one of these acidic residues (D58N CFTR) exhibited lower macroscopic currents in Xenopus oocytes and faster deactivation following washout of a cAMP -activating cocktail than wild-type CFTR. Login to comment
16 In excised membrane patches D58N CFTR exhibited a two-fold reduction in single channel open probability due primarily to shortened open channel bursts. Login to comment
45 A disease-associated mutant, D58N, maps to one of these acidic residues (Cystic Fibrosis Genetic Analysis Consortium:www.genet.sickkids.on.ca./cftr).Thismutation was found on a patient with CBAVD (Congenital Bilateral Absence of Vas Deferens), a disease that typically associates with mild CF alleles (De Braekeleer & Ferec, 1996). Login to comment
48 Here we report that the D58N CFTR mutant exhibits reduced macroscopic currents and accelerated deactivation kinetics in intact oocytes, and briefer channel openings in excised membrane patches as compared with the wild-type channel. Login to comment
49 The reduced chloride channel activity of D58N CFTR may explain how this mutation causes disease. Login to comment
59 D58N CFTR was constructed by PCR mutagenesis. Login to comment
112 RESULTS D58N CFTR exhibits reduced channel activity The disease-associated D58N CFTR mutant exhibited lower macroscopic currents upon activation with a cocktail containing cAMP, forskolin and IBMX than wild-type CFTR when equivalent cRNA amounts were injected into Xenopus oocytes (Fig. 1). Login to comment
113 The currents mediated by D58N CFTR were somewhat greater than CFTR channel regulation by the amino tailJ. Physiol. 536.2 those observed for a triple mutant in which three acidic residues were replaced with alanines (D47A, E54A, D58A). Login to comment
114 The currents mediated by D58N CFTR and the N-tail triple mutant also deactivated faster than those of wild-type CFTR after removal of the cAMP-containing cocktail (Fig. 1C). Login to comment
117 This finding, coupled with our observation that each of these N-tail mutants can be expressed as the mature form of CFTR with no obvious defect in biosynthetic processing (Naren et al. 1999; Fig. 3 and, for D58N CFTR, unpublished data), suggested to us the possibility that the D58N mutation influences the gating properties of the CFTR channel. Login to comment
119 D58N CFTR and N-tail triple mutant (D47A, E54A, D58A) exhibit lower macroscopic currents and faster deactivation than wild-type CFTR A, schematic diagram of CFTR topology (left) and helical wheel plot of N-tail region of interest (right) showing locations of the mutations analysed in this study. Login to comment
120 B, macroscopic currents for wild-type (WT) CFTR, D58N CFTR and the triple mutant. Login to comment
122 Equal amounts of wild-type, triple mutant or D58N CFTR cRNAs (1 ng) were injected into oocytes. Login to comment
126 Shown are representative current traces and mean estimates of the time to half-maximal deactivation (t1/2; n = 4 for wild-type CFTR, n = 4 for triple mutant CFTR, n = 5 for D58N CFTR). Login to comment
128 Since smaller currents tend to have faster deactivation rates (authors` unpublished observations), higher amounts of mutant CFTR cRNAs were injected (1 ng, 20 ng and 10 ng for wild-type, N-tail triple and D58N mutant CFTR, respectively) in order to achieve comparable macroscopic currents. Login to comment
131 To test the effects of the D58N mutation on CFTR gating we examined the properties of D58N CFTR channels in excised membrane patches. Login to comment
132 D58N CFTR exhibited a lower single channel open probability (Po) under conditions that maximally activate the wild-type channel (Fig. 2). Login to comment
133 Although the Po of D58N CFTR was substantially reduced as compared with the wild-type channel, it was not as markedly reduced as the macroscopic currents in intact oocytes (see Fig. 1B). Login to comment
134 We cannot rule out the possibility that this quantitative difference is due to some effect of the D58N mutation on channel density in the intact oocyte, although it is just as likely that this difference is due to the different activating conditions used in these two assays. Login to comment
136 The reduction in Po exhibited by D58N CFTR was due in large part to a reduction in the duration of channel openings (i.e. to briefer open channel bursts; see Methods for details). Login to comment
141 Mutants in which multiple acidic residues in the N-tail were replaced with alanine were chosen for these and the following experiments, since these mutants exhibit a greater degree of chloride channel dysfunction than the D58N CFTR mutant (Fig. 1 and Naren et al. 1999). Login to comment
153 D58N CFTR exhibits decreased channel activity in excised membrane patches due in part to shorter channel openings A, representative patch clamp records from inside-out excised patches that contained at least two active wild-type (WT) or D58N CFTR channels. Login to comment
154 B, C and D, mean single channel open probabilities, open channel burst durations and interburst durations, respectively, for wild-type CFTR and D58N CFTR. Login to comment
157 The principal effect of the D58N mutation was to reduce the open channel burst duration. Login to comment
161 The results of this analysis of single channel records are consistent with our previous estimates of mean open channel burst duration for this mutant (Naren et al. 1999) and D58N CFTR (Fig. 2) in which multichannel patches were analysed using the cycle time method (see Methods for details). Login to comment
214 In this study we examined the single channel properties and macroscopic current kinetics of a disease-associated mutant that maps to this region of the N-tail (D58N CFTR). Login to comment
217 Our results indicate that: (i) the negative charge at this position is likely to be an important determinant of the involvement of the N-tail in regulating the duration of channel openings and (ii) the mild disease that associates with the D58N mutation could well be due to the partial loss of channel activity that is exhibited by this mutant. Login to comment
255 In this regard, the longer interburst duration exhibited by D58N CFTR (Fig. 2D) implies that this mutation may also inhibit channel opening. Login to comment
260 In addition, our analysis of the D58N CFTR mutant has revealed that a disease-associated mutation that maps to this cluster of acidic residues also inhibits channel activity. Login to comment

[hide] Distinct spectrum of CFTR gene mutations in congen... Hum Genet. 1997 Sep;100(3-4):365-77. Dork T, Dworniczak B, Aulehla-Scholz C, Wieczorek D, Bohm I, Mayerova A, Seydewitz HH, Nieschlag E, Meschede D, Horst J, Pander HJ, Sperling H, Ratjen F, Passarge E, Schmidtke J, Stuhrmann M
Distinct spectrum of CFTR gene mutations in congenital absence of vas deferens.
Hum Genet. 1997 Sep;100(3-4):365-77., [PMID:9272157]

Abstract [show]
Congenital absence of the vas deferens (CAVD) is a frequent cause for obstructive azoospermia and accounts for 1%-2% of male infertility. A high incidence of mutations of the cystic fibrosis transmembrane conductance regulator (CFTR) gene has recently been reported in males with CAVD. We have investigated a cohort of 106 German patients with congenital bilateral or unilateral absence of the vas deferens for mutations in the coding region, flanking intron regions and promotor sequences of the CFTR gene. Of the CAVD patients, 75% carried CFTR mutations or disease-associated CFTR variants, such as the "5T" allele, on both chromosomes. The distribution of mutation genotypes clearly differed from that observed in cystic fibrosis. None of the CAVD patients was homozygous for delta F508 and none was compound heterozygous for delta F508 and a nonsense or frameshift mutation. Instead, homozygosity was found for a few mild missense or splicing mutations, and the majority of CAVD mutations were missense substitutions. Twenty-one German CAVD patients were compound heterozygous for delta F508 and R117H, which was the most frequent CAVD genotype in our study group. Haplotype analysis indicated a common origin for R117H in our population, whereas another frequent CAVD mutation, viz. the "5T allele" was a recurrent mutation on different intragenic haplotypes and multiple ethnic backgrounds. We identified a total of 46 different mutations and variants, of which 15 mutations have not previously been reported. Thirteen novel missense mutations and one unique amino-acid insertion may be confined to the CAVD phenotype. A few splice or missense variants, such as F508C or 1716 G-->A, are proposed here as possible candidate CAVD mutations with an apparently reduced penetrance. Clinical examination of patients with CFTR mutations on both chromosomes revealed elevated sweat chloride concentrations and discrete symptoms of respiratory disease in a subset of patients. Thus, our collaborative study shows that CAVD without renal malformation is a primary genital form of cystic fibrosis in the vast majority of German patients and links the particular expression of clinical symptoms in CAVD with a distinct subset of CFTR mutation genotypes.

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No. Sentence Comment
80 The D58N mutation was uncovered in a Lebanese CBAVD patient who had inherited D58N from his father and a "5T" allele from his mother. Login to comment
86 The V938G substitution was identified in two unrelated patients, one homozygote with unilateral ab- 368 Table 1A Frequency distribution and haplotypes of CFTR mutations in 106 CAVD patients Mutationa Nucleotide changesb Locationc Frequencyd Haplotypee Referencef 174delA deletion of A at 174 exon 1 1 D3 This study E56K G→A at 298 exon 3 1 B3 This study D58N G→A at 304 exon 3 1 C2 This study D110H G→A at 460 exon 4 2 C2 Dean et al. (1990) R117H G→A at 482 exon 4 24 B6 Dean et al. (1990) A120T G→A at 490 exon 4 1 n.p. Chillón et al. (1994) ̃L138 insertion of CTA after 546 exon 4 1 A2 This study L206W T→G at 749 exon 6a 1 B8 Claustres et al. (1993) M265R T→G at 926 exon 6b 1 A2 Schwarz et al. (pers. comm.) R297W C→T at 1021 exon 7 1 C2 This study 1078delT deletion of T at 1078 exon 7 1 C2 Claustres et al. (1992) R334W C→T at 1132 exon 7 1 B1 Gasparini et al. (1991) R334L G→T at 1133 exon 7 1 D3 This study I336K T→A at 1139 exon 7 1 A2 Cuppens et al. (1993) R347H G→A at 1172 exon 7 3 D1 Cremonesi et al. (1992) L375F A→C at 1257 exon 8 1 B3 Jézéquel et al. (1996) ∆F508 deletion of 3 bp between 1652-1655 exon 10 57 B1 Kerem et al. (1989) G542X G→T at 1756 exon 11 2 B1 Kerem et al. (1990) R553X C→T at 1789 exon 11 1 A4 Cutting et al. (1990) L568F G→T at 1836 exon 12 1 B3 This study 2184insA insertion of A at 2184 exon 13 1 D3 Dörk et al. (1994b) 2789+5 G→A G→A at 2789+5 intron 14b 4 D3 Highsmith et al. (1997) R933S A→T at 2931 exon 15 1 n.p. Login to comment
137 Complex alleles are indicated a One CF allele with R75X and 125G→C b One CBAVD allele with R75Q and R933S c One CBAVD allele with 5T and Q1352H d Two CF alleles with F508C and S1251N e One CF allele with 1716G→A and L619S f G576A and R668C were linked on two CBAVD and three CF alleles, whereas two additional CF alleles carried R668C together with the 3849+10kB C→T mutation (Dörk and Stuhrmann 1995) 371 Table 3 CFTR mutation genotypes in 106 males with CAVD Genotype PolyT Frequency Ethnic descent Diagnosis ∆F508/R117H 9/7 21 German, Austrian 20 CBAVD, 1 CUAVD ∆F508/5T 9/5 9 German, Austrian 8 CBAVD, 1 CUAVD ∆F508/F508C 9/7 3 German CBAVD ∆F508/R347H 9/9 2 German CBAVD ∆F508/1716 G→A 9/7 2 German CBAVD ∆F508/3272-26 A→G 9/7 2 German CBAVD ∆F508/E56K 9/7 1 German CBAVD ∆F508/M265R 9/7 1 German-Portuguese CBAVD ∆F508/R334W 9/9 1 German CBAVD ∆F508/T351S 9/9 1 German CBAVD ∆F508/L375F 9/7 1 Volga German CBAVD ∆F508/G576A & R668C 9/7 1 German CBAVD ∆F508/R933S 9/7 1 German CBAVD ∆F508/L997F 9/9 1 German CBAVD ∆F508/Y1032C 9/7 1 German CBAVD ∆F508/D1152H 9/7 1 German CBAVD ∆F508/K1351E 9/7 1 German CBAVD ∆F508/D1377H 9/7 1 Portuguese CBAVD ∆F508/L1388Q 9/7 1 German CBAVD ∆F508/unknown 9/7 4 German 3 CBAVD, 1 CUAVD 5T/5T 5/5 2 German CBAVD 5T/G542X 5/9 2 German, Turkish CBAVD 5T/D58N 5/7 1 Lebanese CBAVD 5T/̃L138 5/7 1 German-Polish CBAVD 5T/1078delT 5/7 1 German CBAVD 5T/R553X 5/7 1 German CBAVD 5T/2184insA 5/7 1 Turkish CBAVD 5T/D979A 5/7 1 Vietnamese CBAVD 5T/D1152H 5/7 1 Turkish CBAVD 5T/3659delC 5/7 1 German CBAVD 5T/S1235R 5/7 1 Greek CBAVD 5T/W1282X 5/7 1 German CBAVD 5T & Q1352H/ R297W & Q1352H 5/7 1 Vietnamese CBAVD 5T/unknown 5/7 1 German CBAVD R117H/L206W 7/9 1 German CBAVD R117H/2789+5 G→A 7/7 1 German CBAVD R117H/unknown 7/7 1 German CBAVD 2789+5 G→A/2789+5 G→A 7/7 1 Lebanese CBAVD 2789+5 G→A/L973F 7/7 1 German CBAVD V938G/V938G 7/7 1 Greek CBAVD V938G/174delA 7/7 1 German CBAVD D110H/D110H 7/7 1 Turkish CBAVD R334L/I336K 7/7 1 German CBAVD R347H/N1303K 9/9 1 German CBAVD L568F/D1152H 7/7 1 Turkish CBAVD 3272-26 A→G/V1153E 7/7 1 German CBAVD R75Q/unknown 7/7 1 German CBAVD A120T/unknown 9/7 1 German CBAVD 1716G→A/unknown 7/7 1 German CBAVD G576A & R668C/unknown 7/7 1 German CBAVD 2752-15 C→G/unknown 7/7 1 Iranian CBAVD Unknown/unknown 17 German, Turkish 7 CBAVD and 1 CUAVD without observed renal agenesis, 9 CBAVD with renal agenesis allele and the R297W mutation on a homozygous Q1352H background may then reduce CFTR function to a disease-causing level. Login to comment
145 Maldigestion 13 25 5T/D58N 184 99 55 - 14 34 5T/̃L138 177 80 53 - 15 33 5T/1078delT 187 87 56 Recurrent bronchitis 16 31 5T/G542X 181 85 79 - 17 31 5T/2184insA n.d. n.d. 60 Borderline pancreatic sufficiency 18 31 5T/D979A n.d. n.d. 55 Recurrent infections, FEVI 76% 19 29 5T/D1152H n.d. n.d. 57 - 20 32 5T/W1282X 180 76 n.d. Recurrent infections, nasal polyposis 21 37 5T/unknown 180 74 n.d. Nasal polyposis 22 28 D110H/D110H 175 80 n.d Asthma bronchiale, obstipation 23 33 R334L/I336K 170 65 n.d. Recurrent infections, nasal polyposis, maldigestion, salt depletion episodes 24 35 N1303K/R347H 167 77 93 - 25 30 V938G/174delA n.d. n.d. 42 - 26 29 V938G/V938G 197 115 n.d. Asthma bronchiale Fig.2 Spectrum of CFTR mutation genotypes in CF patients (left) and in patients with congenital absence of the vas deferens (right). Login to comment