ABCMdb

ABCC7 p.Ala204Thr

Predicted by SNAP2:	C: D (59%), D: D (80%), E: D (80%), F: D (80%), G: N (87%), H: D (85%), I: D (71%), K: D (75%), L: D (66%), M: D (71%), N: D (59%), P: D (71%), Q: D (75%), R: D (85%), S: N (78%), T: N (82%), V: N (57%), W: D (85%), Y: D (71%),
Predicted by PROVEAN:	C: D, D: D, E: D, F: D, G: N, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: N, T: N, V: N, W: D, Y: D,

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[hide] Genetic analysis of Rwandan patients with cystic f... Chest. 2009 May;135(5):1233-42. Epub 2008 Nov 18. Mutesa L, Azad AK, Verhaeghe C, Segers K, Vanbellinghen JF, Ngendahayo L, Rusingiza EK, Mutwa PR, Rulisa S, Koulischer L, Cassiman JJ, Cuppens H, Bours V
Genetic analysis of Rwandan patients with cystic fibrosis-like symptoms: identification of novel cystic fibrosis transmembrane conductance regulator and epithelial sodium channel gene variants.
Chest. 2009 May;135(5):1233-42. Epub 2008 Nov 18., [PMID:19017867]

Abstract [show]
BACKGROUND: The defect in chloride and sodium transport in cystic fibrosis (CF) patients is a consequence of CF transmembrane conductance regulator (CFTR) loss of function and an abnormal interaction between CFTR and the epithelial sodium channel (ENaC). A few patients were described with CF-like symptoms, a single CFTR mutation, and an ENaC mutation. METHODS: To study African patients with CF-like symptoms and to relate the disease to gene mutations of both CFTR and ENaC genes, we collected clinical data and DNA samples from 60 African patients with a CF phenotype. The CFTR gene was first analyzed in all patients by denaturing high-performance liquid chromatography followed by direct sequencing; whereas, the sodium channel non-voltage-gated 1 alpha (SCNN1A), sodium channel non-voltage-gated 1 beta (SCNN1B), and sodium channel non-voltage-gated 1 gamma (SCNN1G) subunits of the ENaC gene were analyzed by sequencing in the five patients who carried only one CF mutation. The frequency of all identified ENaC variants was established in a control group of 200 healthy individuals and in the 55 CF-like patients without any CFTR mutation. RESULTS: Three CFTR mutants, including one previously undescribed missense mutation (p.A204T), and a 5T/7T variant were identified in five patients. ENaC gene sequencing in these five patients detected the following eight ENaC variants: c.72T>C and p.V573I in SCNN1A; p.V348M, p.G442V, c.1473 + 28C>T, and p.T577T in SCNN1B; and p.S212S and c.1176 + 30G>C in SCNN1G. In the 55 CF-like patients without any CFTR mutation, we identified five of these eight ENaC variants, including the frequent p.G442V polymorphism, but we did not detect the presence of the p.V348M, p.T577T, and c.1176 + 30G>C ENaC variants. Moreover, these last three ENaC variants, p.V348M, p.T577T, and c.1176 + 30G>C, were not found in the control group. CONCLUSION: Our data suggest that CF-like syndrome in Africa could be associated with CFTR and ENaC mutations.

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25 Cell Transfection HeLa cells (7.105 cells per 100-mm dish) were transfected with 5 ␮g of pcDNA3, wild-type (wt)-CFTR, F508del, or A204T plasmid (Transfectin; Bio-Rad Laboratories; Gent, Belgium). Login to comment
63 A 4-year-old girl (patient P041) with mild pulmonary disease associated with severe PEM status and a positive sweat chloride concentration (113 mmol/L) had a novel GϾA mutation at nucleotide position 742 (Fig 1, 2 left, A), substituting a threonine for an alanine at amino acid position 204 (p.A204T). Login to comment
103 Left, A: the electrophoregram shows the wt and A204T DNA sequence analyses of exon 6a. Login to comment
109 Bottom right, C: Western blotting analysis of HeLa cells transiently expressing CFTR-wt, F508del, or A204T. Login to comment
9 Results: Three CFTR mutants, including one previously undescribed missense mutation (p.A204T), and a 5T/7T variant were identified in five patients. Login to comment
23 Site-Directed Mutagenesis and Expression of CFTR Mutant The p.A204T mutation was introduced in the pcDNA3-CFTR expression plasmid (QuikChange XL Site-Directed Mutagenesis kit; Stratagene; La Jolla, CA). Login to comment
51 We found 14 CFTR variants (Table 2), as follows: two known mutations (p.F693L and c.3120 ϩ 1GϾA); a novel p.A204T missense mutation; and nine sequence polymorphisms; and two previously uncharacterized intronic nucleotide changes, c.3272-32T Ͼ C in the intron 17a and c.4575 ϩ 2GϾA in the 3Ј-untranslated region (UTR). Login to comment
61 The p.F693L mutation was identified in the following two patients: a 13-year-old boy (patient P004) with recurrent respiratory infections, lung colonization by P aeruginosa, GI symptoms, failure to thrive, Table 1-Characteristics of 60 Patients With CF-Like Symptoms Variables Patients (n ϭ 60) % Age, yr Range 2-14 Mean Ϯ SD 5.8 Ϯ 1.7 Sex Male 33 55 Female 27 45 Phenotype Chronic lung disease 39 65 GI symptoms 41 68 Pancreatic insufficiency 19 32 Failure to thrive 23 38 PEM 52 87 Diabetes mellitus 4 7 Nasal polyps 3 5 Sweat chloride test results Positive (Ͼ 60 mmol/L) 37 62 Borderline (40-60 mol/L) 11 18 Normal (Ͻ 40 mmol/L) 9 15 Test not performed 3 5 www.chestjournal.org CHEST / 135 / 5 / MAY, 2009 1235 (c) 2009 American College of Chest Physicians at University of North Carolina on August 8, diabetes mellitus, and PEM; and a 9-year-old girl (patient P038) with mild pulmonary symptoms, GI symptoms, and severe PEM. Login to comment
65 To investigate its causative role in the CF phenotype, the expression of the p.A204T CFTR was studied after transient transfection of a p.A204T-CFTR-pcDNA3 expression vector in HeLa cells. Login to comment
67 Expression of the p.A204T CFTR allowed the expression of the mature glycosylated CFTR isoform at a level higher than that observed with the p.F508del mutant but reproducibly lower than the wt-CFTR level. Login to comment
75 The silent polymorphism p.T577T was found in a patient with a p.F693L CFTR mutation, whereas the c.72TϾC was detected in the patient with the novel p.A204T CFTR mutation. Login to comment
91 Table 3-Comparison of Clinical Findings in Five Patients With Identified CFTR and ENaC Mutants* Patient/Sex/Age, yr Phenotype Sweat Test Value, mmol/L CFTR Genotype (͓TG͔mTn) Genotype ENaC Genotype (␣, beta, and ␥ Subunits) P004/M/13 LD, PA, GI, PEM 94 p.F693L/- (TG)10T7/(TG)11T9 p.V348M (beta) p.S212S† (␥) P038/F/9 LD, SA, GI, PEM 124 p.F693L/- (TG)10T7/(TG)10T9 p.T577T (beta) p.G442V† (beta) P007/F/7 LD, PA, GI, PEM 85 c.3120 ϩ 1GϾA/- (TG)11T7/(TG)12T7 p.V573I (␣) p.G442V† (beta) P029/M/2 GI, PI, FT, PEM 77 c.4575 ϩ 2GϾA‡/- (TG)10T7/(TG)11T5 c.1473 ϩ 28CϾT (beta) c.1176 ϩ 30GϾC (␥) P041/F/4 LD, PEM 113 p.A204T/- (TG)10T7/(TG)10T7 c.72 TϾC (␣ 5Ј UTR) p.G442V† (beta) *Tn ϭ poly-T tract; TGm ϭ poly-TG loci; LD ϭ lung disease; PA ϭ P aeruginosa lung colonization; FT ϭ failure to thrive; DM ϭ diabetes mellitus; PI ϭ pancreatic insufficiency; SA ϭ Staphylococcus aureus lung colonization; F ϭ female; M ϭ male. Login to comment
101 The identified mutations were as follows: (1) a c.3120 ϩ 1GϾA CFTR mutation, frequently observed in African patients and responsible for a splicing defect4; and (2) a p.F693L CFTR missense mutation in two patients with CF-like severe symptoms including recurrent lung disease (the same amino acid change at the same residue has been previously reported23 in an Italian CF patient with severe symptoms; moreover, these two patients carried SCNN1B mutations) [Table 3]; (3) a TG11T5 variant, which can be associated with mild CF signs; and (4) a novel p.A204T CFTR mutation. Login to comment
102 This 204 residue is in the first membrane-spanning domain (MSD1), and almost all of the mutations within this region are associated with a mild phenotype, as was observed in our patient.24,25 This missense mutation Figure 2. p.A204T missense mutation in the CFTR gene. Login to comment
113 Exogenous expression of a p.A204T-CFTR protein in cells led to a weak expression and an altered protein glycosylation, but functional studies are required in order to assess precisely the consequences of this variant. Login to comment
5 Results: Three CFTR mutants, including one previously undescribed missense mutation (p.A204T), and a 5T/7T variant were identified in five patients. Login to comment
19 Site-Directed Mutagenesis and Expression of CFTR Mutant The p.A204T mutation was introduced in the pcDNA3-CFTR expression plasmid (QuikChange XL Site-Directed Mutagenesis kit; Stratagene; La Jolla, CA). Login to comment
21 Cell Transfection HeLa cells (7.105 cells per 100-mm dish) were transfected with 5 ␮g of pcDNA3, wild-type (wt)-CFTR, F508del, or A204T plasmid (Transfectin; Bio-Rad Laboratories; Gent, Belgium). Login to comment
47 We found 14 CFTR variants (Table 2), as follows: two known mutations (p.F693L and c.3120 ϩ 1GϾA); a novel p.A204T missense mutation; and nine sequence polymorphisms; and two previously uncharacterized intronic nucleotide changes, c.3272-32T Ͼ C in the intron 17a and c.4575 ϩ 2GϾA in the 3Ј-untranslated region (UTR). Login to comment
59 A 4-year-old girl (patient P041) with mild pulmonary disease associated with severe PEM status and a positive sweat chloride concentration (113 mmol/L) had a novel GϾA mutation at nucleotide position 742 (Fig 1, 2 left, A), substituting a threonine for an alanine at amino acid position 204 (p.A204T). Login to comment
71 The silent polymorphism p.T577T was found in a patient with a p.F693L CFTR mutation, whereas the c.72TϾC was detected in the patient with the novel p.A204T CFTR mutation. Login to comment
87 Table 3-Comparison of Clinical Findings in Five Patients With Identified CFTR and ENaC Mutants* Patient/Sex/Age, yr Phenotype Sweat Test Value, mmol/L CFTR Genotype (͓TG͔mTn) Genotype ENaC Genotype (␣, beta, and ␥ Subunits) P004/M/13 LD, PA, GI, PEM 94 p.F693L/- (TG)10T7/(TG)11T9 p.V348M (beta) p.S212S† (␥) P038/F/9 LD, SA, GI, PEM 124 p.F693L/- (TG)10T7/(TG)10T9 p.T577T (beta) p.G442V† (beta) P007/F/7 LD, PA, GI, PEM 85 c.3120 ϩ 1GϾA/- (TG)11T7/(TG)12T7 p.V573I (␣) p.G442V† (beta) P029/M/2 GI, PI, FT, PEM 77 c.4575 ϩ 2GϾA‡/- (TG)10T7/(TG)11T5 c.1473 ϩ 28CϾT (beta) c.1176 ϩ 30GϾC (␥) P041/F/4 LD, PEM 113 p.A204T/- (TG)10T7/(TG)10T7 c.72 TϾC (␣ 5Ј UTR) p.G442V† (beta) *Tn ϭ poly-T tract; TGm ϭ poly-TG loci; LD ϭ lung disease; PA ϭ P aeruginosa lung colonization; FT ϭ failure to thrive; DM ϭ diabetes mellitus; PI ϭ pancreatic insufficiency; SA ϭ Staphylococcus aureus lung colonization; F ϭ female; M ϭ male. Login to comment
97 The identified mutations were as follows: (1) a c.3120 ϩ 1GϾA CFTR mutation, frequently observed in African patients and responsible for a splicing defect4; and (2) a p.F693L CFTR missense mutation in two patients with CF-like severe symptoms including recurrent lung disease (the same amino acid change at the same residue has been previously reported23 in an Italian CF patient with severe symptoms; moreover, these two patients carried SCNN1B mutations) [Table 3]; (3) a TG11T5 variant, which can be associated with mild CF signs; and (4) a novel p.A204T CFTR mutation. Login to comment
98 This 204 residue is in the first membrane-spanning domain (MSD1), and almost all of the mutations within this region are associated with a mild phenotype, as was observed in our patient.24,25 This missense mutation Figure 2. p.A204T missense mutation in the CFTR gene. Login to comment
99 Left, A: the electrophoregram shows the wt and A204T DNA sequence analyses of exon 6a. Login to comment
105 Bottom right, C: Western blotting analysis of HeLa cells transiently expressing CFTR-wt, F508del, or A204T. Login to comment