ABCMdb

PMID: 19017867

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., [PubMed]

Sentences

No. Mutations Sentence Comment

5 ABCC7 p.Ala204Thr 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 9 ABCC7 p.Ala204Thr 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 ABCC7 p.Ala204Thr 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 ABCC7 p.Ala204Thr 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 23 ABCC7 p.Ala204Thr 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 25 ABCC7 p.Ala204Thr 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 ABCC7 p.Phe693Leu ABCC7 p.Ala204Thr 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 51 ABCC7 p.Phe693Leu ABCC7 p.Ala204Thr 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 57 ABCC7 p.Phe693Leu 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 diabetes mellitus, and PEM; and a 9-year-old girl (patient P038) with mild pulmonary symptoms, GI symptoms, and severe PEM. Login to comment 59 ABCC7 p.Ala204Thr ABCC7 p.Ala204Thr 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 61 ABCC7 p.Phe693Leu ABCC7 p.Ala204Thr ABCC7 p.Ala204Thr 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 63 ABCC7 p.Ala204Thr ABCC7 p.Ala204Thr ABCC7 p.Ala204Thr 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 65 ABCC7 p.Ala204Thr ABCC7 p.Ala204Thr 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 ABCC7 p.Ala204Thr 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 69 ABCC7 p.Phe693Leu The p.V573I ENaC variant was found in the patient who was heterozygous for the c.3120 ϩ 1GϾA CFTR mutation in combination with the TG12T7 variant, while the p.V348M ENaC mutation was observed in a patient with the p.F693L CFTR mutation. Login to comment 71 ABCC7 p.Phe693Leu ABCC7 p.Ala204Thr 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 73 ABCC7 p.Phe693Leu The p.V573I ENaC variant was found in the patient who was heterozygous for the c.3120 ϩ 1GϾA CFTR mutation in combination with the TG12T7 variant, while the p.V348M ENaC mutation was observed in a patient with the p.F693L CFTR mutation. Login to comment 75 ABCC7 p.Phe693Leu ABCC7 p.Ala204Thr 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 ABCC7 p.Phe693Leu ABCC7 p.Phe693Leu ABCC7 p.Ala204Thr 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 91 ABCC7 p.Phe693Leu ABCC7 p.Phe693Leu ABCC7 p.Phe693Leu ABCC7 p.Ala204Thr 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 95 ABCC7 p.Phe693Leu CFTR Allele Variants, % (n ϭ 120) p.F693L TϾG at 2211 13 PheϾLeu at 693 2 1.67 c.3120 ϩ 1GϾA GϾA at 3120 ϩ 1 Intron 16 Splicing mutation 1 0.83 p.A204T* GϾA at 742 6a AlaϾThr at 742 1 0.83 c.4575 ϩ 2GϾA GϾA at 4575 ϩ 2 3Ј UTR 1 0.83 p.T854T TϾG at 2694 14a Sequence variation 52 43.33 p.Q1463Q GϾA at 4521 24 Sequence variation 14 11.7 p.M470V AϾG at 1540 10 Sequence variation 13 10.83 c.1898 ϩ 152TϾA TϾA at 1898 ϩ 152 12 Sequence variation 9 7.5 p.P1290P AϾG at 4002 20 Sequence variation 6 5 c.1001 ϩ 11CϾT CϾT at 1001 ϩ 11 Intron 6b Sequence variation 5 4.17 p.E527E AϾG at 1713 10 Sequence variation 3 2.5 c.2752 - 15CϾG CϾG at 2752 - 15 Intron 14a Sequence variation 3 2.5 c.3041 - 71AϾG GϾC at 3041 - 71 Intron 15 Sequence variation 2 1.67 c.3272 - 32TϾC TϾC at 3072 - 32 Intron17a 1 0.83 *Identified novel missense mutation. Login to comment 97 ABCC7 p.Phe693Leu ABCC7 p.Ala204Thr 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 ABCC7 p.Ala204Thr 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 ABCC7 p.Ala204Thr Left, A: the electrophoregram shows the wt and A204T DNA sequence analyses of exon 6a. Login to comment 101 ABCC7 p.Phe693Leu ABCC7 p.Ala204Thr 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 ABCC7 p.Ala204Thr 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 103 ABCC7 p.Ala204Thr Left, A: the electrophoregram shows the wt and A204T DNA sequence analyses of exon 6a. Login to comment 105 ABCC7 p.Ala204Thr Bottom right, C: Western blotting analysis of HeLa cells transiently expressing CFTR-wt, F508del, or A204T. Login to comment 109 ABCC7 p.Ala204Thr ABCC7 p.Ala204Thr Bottom right, C: Western blotting analysis of HeLa cells transiently expressing CFTR-wt, F508del, or A204T. Login to comment 113 ABCC7 p.Ala204Thr 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 125 ABCC7 p.Phe693Leu For instance, the two patients with a p.F693L CFTR mutation carried the p.V348M or p.T577T SCNN1B mutation, which were not detected in the control group. Login to comment 129 ABCC7 p.Phe693Leu For instance, the two patients with a p.F693L CFTR mutation carried the p.V348M or p.T577T SCNN1B mutation, which were not detected in the control group. Login to comment