PMID: 10516113

Illek B, Zhang L, Lewis NC, Moss RB, Dong JY, Fischer H
Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein.
Am J Physiol. 1999 Oct;277(4 Pt 1):C833-9., [PubMed]
Sentences
No. Mutations Sentence Comment
6 ABCC7 p.Gly551Asp
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ABCC7 p.Gly551Asp 10516113:6:137
status: NEW
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It is published 12 times aAJP - Cell Physiology rapid communication Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein BEATE ILLEK,1 LEI ZHANG,2 NANCY C. LEWIS,1 RICHARD B. MOSS,3 JIAN-YUN DONG,2 AND HORST FISCHER1 1Research Institute and Pulmonary Center, Children`s Hospital Oakland, Oakland 94609; 2Laboratory Medicine, University of California at San Francisco, San Francisco 94143; and 3Pediatric Pulmonary Medicine, Stanford University Medical Center, Stanford, California 94305 Illek, Beate, Lei Zhang, Nancy C. Lewis, Richard B. Moss, Jian-Yun Dong, and Horst Fischer. Login to comment
7 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:7:68
status: NEW
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Defective function of the cystic fibrosis-causing missense mutation G551D is recovered by genistein. Login to comment
8 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:8:177
status: NEW
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Am. J. Physiol. 277 (Cell Physiol. 46): C833-C839, 1999.-The patch-clamp technique was used to investigate the effects of the isoflavone genistein on disease-causing mutations (G551D and ⌬F508) of the cystic fibrosis transmembrane conductance regulator (CFTR). Login to comment
9 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:9:65
status: NEW
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:9:158
status: NEW
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In HeLa cells recombinantly expressing the trafficking-competent G551D-CFTR, the forskolin-stimulated Cl currents were small, and average open probability of G551D-CFTR was Po ϭ 0.047 Ϯ 0.019. Login to comment
10 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:10:74
status: NEW
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Addition of genistein activated Cl currents ϳ10-fold, and the Po of G551D-CFTR increased to 0.49 Ϯ 0.12, which is a Po similar to wild-type CFTR. Login to comment
13 ABCC7 p.Gly551Asp
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ABCC7 p.Gly551Asp 10516113:13:70
status: NEW
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Therefore, we tested the effects of genistein in CF patients with the G551D mutation in nasal potential difference (PD) measurements in vivo. Login to comment
14 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:14:37
status: NEW
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The perfusion of the nasal mucosa of G551D CF patients with isoproterenol had no effect; however, genistein stimulated Cl-dependent nasal PD by, on average, -2.4 Ϯ 0.6 mV, which corresponds to 16.9% of the responses (to beta-adrenergic stimulation) found in healthy subjects. Login to comment
18 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:18:62
status: NEW
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The glycine-to- aspartic acid missense mutation at codon 551 (G551D) is the third commonest CF mutation, with a worldwide frequency of ϳ2% on CF chromosomes (25, 27). Login to comment
19 ABCC7 p.Gly551Asp
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ABCC7 p.Gly551Asp 10516113:19:22
status: NEW
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Higher frequencies of G551D are found in populations of Celtic descent, with incidences of 6.9% and 5.5% in the Irish and Australian CF populations, respectively (3, 12, 25). Login to comment
22 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:22:4
status: NEW
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The G551D-CFTR protein traffics to the apical membrane, but Cl channel function is markedly reduced (class III mutation) (27). Login to comment
23 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:23:90
status: NEW
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Compared with CF patients homozygous for the ⌬F508 mutation, patients carrying the G551D mutation show a threefold reduction in the incidence of meconium ileus (neonatal intestinal blockage), a trend toward later age at diagnosis of pancreatic insufficiency (12), and milder disease in homozygotes (22). Login to comment
24 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:24:54
status: NEW
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:24:197
status: NEW
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Interestingly, both the ⌬F508 mutation and the G551D mutation are in NBD1 of CFTR; however, ATP binding of NBD1 is not affected by the ⌬F508 mutation but is significantly impaired in G551D-CFTR (21). Login to comment
28 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:28:35
status: NEW
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We therefore tested its effects on G551D-CFTR, which is present in the cell membrane, and on ⌬F508-CFTR, once it was translocated in sufficient quantity to the membrane after 4-PB treatment. Login to comment
29 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:29:72
status: NEW
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MATERIALS AND METHODS Generation of recombinant adenovirus carrying the G551D gene. Login to comment
30 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:30:41
status: NEW
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Recombinant adenovirus carrying the CFTR G551D mutation was generated by a modified ligation procedure. Login to comment
31 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:31:45
status: NEW
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With the use of PCR-mediated point mutation, G551D was generated by converting the codon for glycine (GGT) at position 551 to an aspartic acid codon (GAT). Login to comment
35 ABCC7 p.Gly551Asp
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ABCC7 p.Gly551Asp 10516113:35:4
status: NEW
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The G551D cDNA was inserted into a shuttle vector, pLadC, which contains the left-hand terminal of adenovirus sequence and a cytomegalovirus promoter. Login to comment
36 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:36:88
status: NEW
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The recombinant adenovirus, AdcG551D, was generated by ligation of the linearized pLadC-G551D to the right-hand portion of the adenovirus sub360, which contains the deletion in the E1a and E3 regions. Login to comment
40 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:40:13
status: NEW
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The inserted G551D cDNA was confirmed using PCR protocols. Login to comment
45 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:45:80
status: NEW
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Twenty-four hours before use, cells were infected with viral vectors containing G551D-CFTR, wtCFTR, or LacZ as control at a multiplicity of infection of ϳ100. Login to comment
63 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:63:27
status: NEW
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Patients with at least one G551D-CFTR allele were recruited from Children`s Hospital Oakland and from the Stanford CF Center at Lucile Salter Packard Children`s Hospital. Login to comment
70 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:70:0
status: NEW
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G551D-CFTR-mediated current is stimulated by genistein. Login to comment
71 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:71:52
status: NEW
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A: whole cell recording from a HeLa cell expressing G551D-CFTR. Login to comment
89 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:89:162
status: NEW
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F: average calculated Po values during forskolin treatment (fsk, open bars) and forskolin ϩ genistein treatment (geni, solid bars) in HeLa cells expressing G551D or wild-type (wt) CFTR. Login to comment
90 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:90:40
status: NEW
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Genistein significantly increased Po of G551D-CFTR (P ϭ 0.01, n ϭ 4) and wtCFTR (P ϭ 0.02, n ϭ 4, paired t-tests). Login to comment
94 ABCC7 p.Gly551Asp
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ABCC7 p.Gly551Asp 10516113:94:32
status: NEW
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RESULTS Effects of genistein on G551D-CFTR in patch-clamp recordings. Login to comment
95 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:95:29
status: NEW
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HeLa cells infected with the G551D-CFTR-containing adenovirus were patch clamped in the whole cell and the cell-attached mode. Login to comment
96 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:96:45
status: NEW
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Figure 1 shows a whole cell recording from a G551D-CFTR-expressing cell. Login to comment
101 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:101:79
status: NEW
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Current variance to mean current plots was used to calculate the average Po of G551D-CFTR-mediated whole cell current. Login to comment
106 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:106:37
status: NEW
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Figure 1F shows average Po values of G551D-CFTR compared with wtCFTR. Login to comment
107 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:107:19
status: NEW
view ABCC7 p.Gly551Asp details
Note the low Po of G551D-CFTR during forskolin stimulation (0.047 Ϯ 0.19, n ϭ 4), which was increased by genistein to 0.49 Ϯ 0.12, a value not different from wtCFTR. Login to comment
109 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:109:47
status: NEW
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Figure 2 shows the effects of genistein on the G551D-CFTR channel in a cell-attached patch-clamp recording. Login to comment
114 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:114:60
status: NEW
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Figure 2B shows the average single channel I-V relation for G551D-CFTR recorded in the cell-attached mode. Single channel I-V relations were typically outwardly rectifying in the cell-attached mode (7), with a conductance in the positive voltage range of g ϭ 8.7 Ϯ 1.0 pS (n ϭ 4, forskolin) and 9.6 Ϯ 0.8 pS (forskolin ϩ genistein), which were similar to wtCFTR (g ϭ 8.6 Ϯ 0.91 pS, n ϭ 3) recorded under the same conditions. Login to comment
116 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:116:143
status: NEW
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Genistein showed no effects on Cl current, indicating that 1) HeLa cells had no endogenous genistein-activated Cl current, and 2) unstimulated G551D-CFTR could not be activated by genistein alone. Login to comment
118 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:118:65
status: NEW
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:118:153
status: NEW
view ABCC7 p.Gly551Asp details
This indicated that genistein was not a direct channel opener of G551D-CFTR but relied on regulation by the cAMP/protein kinase A (PKA) pathway and that G551D-CFTR was only active when both genistein was present and PKA was active. Login to comment
119 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:119:45
status: NEW
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In summary, these patch-clamp data show that G551D-CFTR is a channel with markedly reduced open probability. Login to comment
120 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:120:13
status: NEW
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Treatment of G551D-CFTR with genistein effectively restored regulation of Po by the cAMP pathway similar to wtCFTR. Login to comment
127 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:127:27
status: NEW
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Cell-attached recording of G551D-CFTR. Login to comment
138 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:138:85
status: NEW
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We performed nasal potential difference (PD) measurements in CF patients bearing the G551D mutation on at least one allele and in healthy human subjects. Login to comment
141 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:141:81
status: NEW
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Figure 5 shows measurements of nasal PD from a healthy volunteer (Fig. 5A) and a G551D CF patient (Fig. 5B). Login to comment
143 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:143:154
status: NEW
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Perfusion with Cl-free solution and subsequent addition of isoproterenol hyperpolarized nasal PD in healthy subjects, whereas nasal PD was depolarized in G551D CF patients, indicating a lack of a beta-adrenergic- stimulated Cl conductance in CF patients. Login to comment
144 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:144:100
status: NEW
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However, perfusion of the nasal mucosa with genistein significantly hyperpolarized nasal PD both in G551D CF patients and in healthy subjects, indicative of a genistein-induced Cl conductance in both groups. Login to comment
146 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:146:3
status: NEW
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In G551D CF patients, genistein caused a hyperpolarization of, on average, -2.4 Ϯ 0.6 mV (n ϭ 5), which corresponds to 16.9% of the average response of normal subjects to treatment with Cl-free solution and isoproterenol (-14.0 Ϯ 2.1 mV, n ϭ 8). Login to comment
147 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:147:111
status: NEW
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Another tested flavonoid, quercetin, was also active but less potent than genistein in stimulating nasal PD in G551D CF patients (by -0.9 Ϯ 0.3 mV, n ϭ 4, significantly different from 0, one sample t-test). Login to comment
148 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:148:45
status: NEW
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DISCUSSION Genistein restores cAMP-dependent G551D-CFTR activity. Login to comment
149 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:149:31
status: NEW
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ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:149:58
status: NEW
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For this study we selected the G551D mutation because the G551D-CFTR protein was described as a trafficking-competent mutant (23). Login to comment
150 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:150:95
status: NEW
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We found that acute treatment of cells with genistein in the presence of forskolin resulted in G551D-CFTR activity that was functionally similar to wtCFTR during stimulation with forskolin. Login to comment
151 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:151:25
status: NEW
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The genistein-stimulated G551D-CFTR had a single channel conductance of 9.6 Ϯ 0.8 pS, showed time-and voltage-independent currents, and Po was 0.49 Ϯ 0.12, which are typical biophysical characteristics of wtCFTR. Login to comment
152 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:152:28
status: NEW
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In addition, stimulation of G551D-CFTR by genistein required intrinsic regulation of the channel by the cAMP/PKA system. Login to comment
155 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:155:27
status: NEW
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Genistein has no effect on G551D-CFTR-mediated Cl currents in unstimulated cells. Login to comment
156 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:156:47
status: NEW
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Whole cell recording of a HeLa cell expressing G551D-CFTR. Login to comment
171 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:171:24
status: NEW
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Effects of genistein in G551D CF patients. Login to comment
172 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:172:29
status: NEW
view ABCC7 p.Gly551Asp details
Our nasal PD measurements in G551D CF patients showed the typical abnormalities present in other CF patients, which are: a hyperpolarized basal nasal PD, an increased amiloride-sensitive PD, a depolarization of nasal PD with Cl-free solutions, and a lack of response to the beta-adrenergic agonist isoproterenol (20). Login to comment
173 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:173:50
status: NEW
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In contrast, perfusion of the nasal epithelium of G551D CF patients with genistein resulted in a hyperpolarization of nasal PD in all patients, which under the experimental conditions corresponds to an activation of a Cl conductance. Login to comment
183 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:183:47
status: NEW
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Thus the responses to genistein of nasal PD in G551D CF patients correspond to 13.6% of normal in terms of Cl current, which may have therapeutic significance. Login to comment
189 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:189:64
status: NEW
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Genistein activates Cl-selective potentials in CF patients with G551D mutation. Login to comment
194 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:194:32
status: NEW
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B: measurement of nasal PD in a G551D CF patient. Login to comment
195 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:195:57
status: NEW
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Patient was a 14-year-old Caucasian female with genotype G551D/⌬F508. Login to comment
200 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:200:24
status: NEW
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C: average responses of G551D CF patients (n ϭ 5) to treatment with amiloride (amil, 50 µM), Cl-free solution (exchanged for gluconate) and 10 µM isoproterenol (Cl free/iso), and 30 µM genistein (geni), in comparison to measurements in healthy subjects (n ϭ 8). Login to comment
202 ABCC7 p.Gly551Asp
X
ABCC7 p.Gly551Asp 10516113:202:69
status: NEW
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ABCC7 p.Gly551Asp
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ABCC7 p.Gly551Asp 10516113:202:119
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Asp
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ABCC7 p.Gly551Asp 10516113:202:125
status: NEW
view ABCC7 p.Gly551Asp details
ABCC7 p.Gly551Asp
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ABCC7 p.Gly551Asp 10516113:202:150
status: NEW
view ABCC7 p.Gly551Asp details
Genotypes (and responses of PD to genistein) of patients were: three G551D/⌬F508 (-0.8, -2.4, and -2.3 mV), one G551D/G551D (-2.0 mV), and one G551D/unknown (-4.3 mV). Login to comment
205 ABCC7 p.Gly551Asp
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ABCC7 p.Gly551Asp 10516113:205:3
status: NEW
view ABCC7 p.Gly551Asp details
In G551D CF patients average nasal PD values were: NaCl, -33.8 Ϯ 3.2 mV; amiloride, -11.7 Ϯ 0.9 mV; isoproterenol/Cl free, -6.4 Ϯ 1.7 mV; and genistein, -8.7 Ϯ 1.6 mV. Login to comment