ABCMdb

PMID: 22068586

Flamein F, Riffault L, Muselet-Charlier C, Pernelle J, Feldmann D, Jonard L, Durand-Schneider AM, Coulomb A, Maurice M, Nogee LM, Inagaki N, Amselem S, Dubus JC, Rigourd V, Bremont F, Marguet C, Brouard J, de Blic J, Clement A, Epaud R, Guillot L
Molecular and cellular characteristics of ABCA3 mutations associated with diffuse parenchymal lung diseases in children.
Hum Mol Genet. 2012 Feb 15;21(4):765-75. Epub 2011 Nov 7., [PubMed]

Sentences

No. Mutations Sentence Comment

7 ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Asp253His Functional studies of p.D253H and p.T1173R mutations revealed that p.D253H and p.T1173R induced abnormal lamellar bodies. Login to comment 8 ABCA3 p.Thr1173Arg Additionally, p.T1173R increased IL-8 secretion in vitro. Login to comment 27 ABCA3 p.Glu292Val ABCA3 p.Glu292Val For instance, the heterozygous c.875A.T (p.Glu292Val, p.E292V) ABCA3 mutation was identified in several older children and young adults with desquamative interstitial pneumonitis (1). Login to comment 29 ABCA3 p.Glu292Val ABCA3 p.Glu292Val For instance, the heterozygous c.875A.T (p.Glu292Val, p.E292V) ABCA3 mutation was identified in several older children and young adults with desquamative interstitial pneumonitis (1). Login to comment 39 ABCA3 p.Arg208Trp ABCA3 p.Gly210Val The two mutations p.G210V and p.R208W have been already identified (13,14). Login to comment 41 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Arg208Trp ABCA3 p.Arg208Trp ABCA3 p.Gly210Val ABCA3 p.Arg1521Trp ABCA3 p.Arg1583Trp Analysis of genomic DNA from the parents and kindred showed that the compound heterozygous p.R1583W/ p.S128Rfs (Fig. 1A) and p.R208W/p.R1521W (Fig. 1B) mutations were inherited, as well as the homozygous mutations p.T1173R (Fig. 1C) and p.D253H (Fig. 1D). Login to comment 43 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Arg208Trp ABCA3 p.Arg1521Trp ABCA3 p.Arg1583Trp Analysis of genomic DNA from the parents and kindred showed that the compound heterozygous p.R1583W/ p.S128Rfs (Fig. 1A) and p.R208W/p.R1521W (Fig. 1B) mutations were inherited, as well as the homozygous mutations p.T1173R (Fig. 1C) and p.D253H (Fig. 1D). Login to comment 54 ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Asp253His ABCA3 p.Arg208Trp ABCA3 p.Gly210Val ABCA3 p.Arg1521Trp ABCA3 p.Arg1583Trp ABCA3 p.Leu462Arg ABCA3 p.Ala1027Pro ABCA3 p.Gly202Arg ABCA3 p.Gly964Ser ABCA3 p.Leu303Val Genetic analysis results in the 10 children harboring homozygous and compound heterozygous (shaded) or heterozygous ABCA3 mutations Patient NRD Clinical outcome ABCA3 mutation ABCA3 SNPs ABCA3 variants cDNA level Protein level dbSNPs rs# cluster id Missense variants in conserved amino acid 1 Yes ILD c.[3518C.G] + [3518C.G] p.[T1173R] + [T1173R] rs149532, rs13332514 2 Yes ILD c.[757G.C] + [757G.C] p.[D253H] + [D253H] 3 Yes Death c.[1385T.G] + [2890G.A] p.[L462R] + [G964S] rs149532 4 Yes Death c.[4747C.T] + c.[384delC] p.[R1583W] + p.[S128Rfs] rs149532 c.[450G.A] (het) 5 No Death c.[629G.T] + [3079G.C] p.[G210V] + [A1027P] rs149532 6 Yes ILD c.[622C.T] + [4561C.T] p.[R208W] + [R1521W] rs149532, rs323043 7 Yes Death c.[604G.C] + [907C.G] p.[G202R] + [L303V] rs149532, rs323043 (het), rs13332514 8 Yes Death c.[2888A.G] + [?] Login to comment 55 ABCA3 p.Tyr963Cys p.[Y963C] + [?] Login to comment 56 ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Asp253His ABCA3 p.Arg208Trp ABCA3 p.Gly210Val ABCA3 p.Arg1521Trp ABCA3 p.Arg1583Trp ABCA3 p.Leu462Arg ABCA3 p.Ala1027Pro ABCA3 p.Gly202Arg ABCA3 p.Gly964Ser ABCA3 p.Leu303Val Genetic analysis results in the 10 children harboring homozygous and compound heterozygous (shaded) or heterozygous ABCA3 mutations Patient NRD Clinical outcome ABCA3 mutation ABCA3 SNPs ABCA3 variants cDNA level Protein level dbSNPs rs# cluster id Missense variants in conserved amino acid 1 Yes ILD c.[3518C.G] + [3518C.G] p.[T1173R] + [T1173R] rs149532, rs13332514 2 Yes ILD c.[757G.C] + [757G.C] p.[D253H] + [D253H] 3 Yes Death c.[1385T.G] + [2890G.A] p.[L462R] + [G964S] rs149532 4 Yes Death c.[4747C.T] + c.[384delC] p.[R1583W] + p.[S128Rfs] rs149532 c.[450G.A] (het) 5 No Death c.[629G.T] + [3079G.C] p.[G210V] + [A1027P] rs149532 6 Yes ILD c.[622C.T] + [4561C.T] p.[R208W] + [R1521W] rs149532, rs323043 7 Yes Death c.[604G.C] + [907C.G] p.[G202R] + [L303V] rs149532, rs323043 (het), rs13332514 8 Yes Death c.[2888A.G] + [?] Login to comment 57 ABCA3 p.Tyr963Cys ABCA3 p.Arg709Trp p.[R709W] + [?] Login to comment 59 ABCA3 p.Arg709Trp ABCA3 p.Ser872Gly p.[S872G] + [?] Login to comment 61 ABCA3 p.Ser872Gly p.[S872G] + [?] Login to comment 68 ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Arg208Trp ABCA3 p.Arg1521Trp ABCA3 p.Arg1583Trp Pedigree of the families with the ABCA3 mutations p.R1583W/p.S128Rfs (A), p.R1521W/R208W (B), p.T1173R/p.T1173R (C) and p.D253H/ p.T1173R (D). Login to comment 70 ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Arg208Trp ABCA3 p.Arg1521Trp ABCA3 p.Arg1583Trp Pedigree of the families with the ABCA3 mutations p.R1583W/p.S128Rfs (A), p.R1521W/R208W (B), p.T1173R/p.T1173R (C) and p.D253H/ p.T1173R (D). Login to comment 78 ABCA3 p.Asp253His Electron microscopy was performed on a lung biopsy from the patient harboring the p.D253H mutation and showed abnormal lamellar bodies with dense inclusions (Fig. 3C). Login to comment 79 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Arg1583Trp ABCA3 p.Leu462Arg ABCA3 p.Gly202Arg ABCA3 p.Gly964Ser ABCA3 p.Leu303Val ABCA3 p.Tyr963Cys ABCA3 p.Ser872Gly BALF analysis Western blot analysis of surfactant proteins (Fig. 4) was performed in seven patients, who had the following ABCA3 mutations: p.D253H (patient 2), p.T1173R (patient 1), p.L462R/ p.G964S (patient 3), p.G202R/p.L303V (patient 7), p.Y963C (patient 8), p.R1583W/p.S128Rfs (patient 4) and p.S872G (patient 10), respectively. Login to comment 80 ABCA3 p.Asp253His ABCA3 p.Gly202Arg ABCA3 p.Leu303Val SP-C (Fig. 4A) and SP-B (Fig. 4B) were detected at a size of 3.7 and 8 kDa in variable amounts, the smallest amount being found in the patient with the p.G202R/p.L303V ABCA3 mutation (patient 7) in whom SP-C and SP-B are only faintly visible. Login to comment 81 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Arg1583Trp ABCA3 p.Leu462Arg ABCA3 p.Gly202Arg ABCA3 p.Gly964Ser ABCA3 p.Leu303Val ABCA3 p.Tyr963Cys ABCA3 p.Ser872Gly BALF analysis Western blot analysis of surfactant proteins (Fig. 4) was performed in seven patients, who had the following ABCA3 mutations: p.D253H (patient 2), p.T1173R (patient 1), p.L462R/ p.G964S (patient 3), p.G202R/p.L303V (patient 7), p.Y963C (patient 8), p.R1583W/p.S128Rfs (patient 4) and p.S872G (patient 10), respectively. Login to comment 82 ABCA3 p.Gly202Arg ABCA3 p.Leu303Val SP-C (Fig. 4A) and SP-B (Fig. 4B) were detected at a size of 3.7 and 8 kDa in variable amounts, the smallest amount being found in the patient with the p.G202R/p.L303V ABCA3 mutation (patient 7) in whom SP-C and SP-B are only faintly visible. Login to comment 84 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Characterization of ABCA3 mutations The two mutations p.T1173R and p.D253H were deliberately chosen for subsequent functional studies because they were homozygous. Login to comment 86 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Characterization of ABCA3 mutations The two mutations p.T1173R and p.D253H were deliberately chosen for subsequent functional studies because they were homozygous. Login to comment 89 ABCA3 p.Asp253His (A) HRCT scan, (B) hematoxylin and eosin (HE) staining of lung tissue and (C) electron microscopy of type-II cells from the patient (patient 2) harboring the ABCA3 homozygous mutation p.D253H. Login to comment 91 ABCA3 p.Asp253His (A) HRCT scan, (B) hematoxylin and eosin (HE) staining of lung tissue and (C) electron microscopy of type-II cells from the patient (patient 2) harboring the ABCA3 homozygous mutation p.D253H. Login to comment 97 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Thus, p.D253H and p.T1173R mutants were not associated with a localization defect. Login to comment 99 ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Asp253His In WT, p.T1173R and p.D253H cells, anti-GFP antibody revealed two bands of 180 kDa (150 kDa ABCA3 + 30 kDa GFP) and 220 kDa (190 + 30 kDa GFP), respectively (Fig. 6). Login to comment 101 ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Asp253His Lamellar bodies in ABCA3 WT, p.D253H and p.T1173R cells. Login to comment 103 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Lamellar bodies in ABCA3 WT, p.D253H and p.T1173R cells. Login to comment 104 ABCA3 p.Asp253His The p.D253H mutation induced abnormal lamellar bodies with electron-dense inclusions (Fig. 7, dashed arrows), consistent with the results of the patient`s lung biopsy (Fig. 3C). Login to comment 105 ABCA3 p.Thr1173Arg In cells transfected with the p.T1173R mutation, abnormal lamellar bodies are the most frequently observed (irregularly arranged, phospholipid lamellae but eccentrically packed), even if some appeared almost normal. Login to comment 106 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Asp253His Cytokine production by ABCA3 WT, p.D253H and p.T1173R cells. Login to comment 107 ABCA3 p.Thr1173Arg In cells transfected with the p.T1173R mutation, abnormal lamellar bodies are the most frequently observed (irregularly arranged, phospholipid lamellae but eccentrically packed), even if some appeared almost normal. Login to comment 108 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Cytokine production by ABCA3 WT, p.D253H and p.T1173R cells. Login to comment 109 ABCA3 p.Thr1173Arg Using quantitative PCR (qPCR), we found that IL-8 mRNA levels were increased in p.T1173R cells (Fig. 8A). Login to comment 110 ABCA3 p.Asp253His A significant, faint increase in IL-8 mRNA expression was also observed between WT and p.D253H cells. Login to comment 111 ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 mRNA levels were similar in WT and mutated cells (data not shown), indicating that the increased IL-8 mRNA level in p.T1173R cells was not due to a transfection issue. Login to comment 112 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His At the protein level, ELISA results confirmed that A549 cells expressing the p.T1173R mutant produced more IL-8 than did WT cells (Fig. 8A). Login to comment 113 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His In contrast, IL-8 production by p.D253H cells was similar to that of WT cells (Fig. 8B). Login to comment 114 ABCA3 p.Thr1173Arg At the protein level, ELISA results confirmed that A549 cells expressing the p.T1173R mutant produced more IL-8 than did WT cells (Fig. 8A). Login to comment 115 ABCA3 p.Asp253His In contrast, IL-8 production by p.D253H cells was similar to that of WT cells (Fig. 8B). Login to comment 116 ABCA3 p.Thr1173Arg To determine whether these signaling pathways were involved in the observed IL-8 overproduction by p.T1173R cells, we used specific inhibitors. Login to comment 118 ABCA3 p.Thr1173Arg To determine whether these signaling pathways were involved in the observed IL-8 overproduction by p.T1173R cells, we used specific inhibitors. Login to comment 123 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Human Molecular Genetics, 2012, Vol. 21, No. 4 769 ERK1/2 inhibitor treatment, IL-8 production by p.T1173R cells remained increased compared with WT and p.D253H cells. Login to comment 124 ABCA3 p.Thr1173Arg These results suggest that, even if ERK1/2 signaling is involved in IL-8 production, another signaling pathway may be involved in the increased IL-8 production detected in p.T1173R cells. Login to comment 125 ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Western blot and relative quantification of ERK1/2 phosphorylation confirmed that the observed IL-8 overproduction in p.T1173R cells was independent of ERK1/2 signaling. Login to comment 126 ABCA3 p.Thr1173Arg These results suggest that, even if ERK1/2 signaling is involved in IL-8 production, another signaling pathway may be involved in the increased IL-8 production detected in p.T1173R cells. Login to comment 127 ABCA3 p.Thr1173Arg Western blot and relative quantification of ERK1/2 phosphorylation confirmed that the observed IL-8 overproduction in p.T1173R cells was independent of ERK1/2 signaling. Login to comment 132 ABCA3 p.Arg1521Trp ABCA3 p.Arg1583Trp ABCA3 p.Arg208Val Finally, parents heterozygous for the p.R1583W, p.S128Rfs, p.R1521W or p.R208V mutations were not affected. Login to comment 134 ABCA3 p.Arg1521Trp ABCA3 p.Arg1583Trp ABCA3 p.Arg208Val Finally, parents heterozygous for the p.R1583W, p.S128Rfs, p.R1521W or p.R208V mutations were not affected. Login to comment 137 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Intracellular localization of wild-type ABCA3 and of the p.D253H and p.T1173R mutants. Login to comment 138 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His A549 cells either non-transfected or transfected with mock vector, wild-type protein ABCA3-WT (A) or mutated proteins p.D253H (B) and p.T1173R (C) were analyzed using confocal microscopy. Login to comment 139 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Intracellular localization of wild-type ABCA3 and of the p.D253H and p.T1173R mutants. Login to comment 140 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His A549 cells either non-transfected or transfected with mock vector, wild-type protein ABCA3-WT (A) or mutated proteins p.D253H (B) and p.T1173R (C) were analyzed using confocal microscopy. Login to comment 141 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Western blot analysis of ABCA3 in cells transiently transfected with ABCA3-WT or the p.D253H or p.T1173R mutation. Login to comment 143 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Western blot analysis of ABCA3 in cells transiently transfected with ABCA3-WT or the p.D253H or p.T1173R mutation. Login to comment 155 ABCA3 p.Asp253His To confirm the hypothesis, it would have been interesting to compare the patterns of siblings harboring the p.D253H mutation. Login to comment 157 ABCA3 p.Asp253His To confirm the hypothesis, it would have been interesting to compare the patterns of siblings harboring the p.D253H mutation. Login to comment 161 ABCA3 p.Asp253His The presence of abnormal lamellar bodies in the patient harboring the p.D253H mutation supports a pathogenic effect of this mutation. Login to comment 162 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His These abnormalities were also observed in vitro in p.D253H- and p.T1173R-transfected cells, suggesting that ABCA3 abnormalities may consistently induce abnormal lamellar bodies. Login to comment 163 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His However, since we do not have the corresponding biopsy from the patient harboring the p.T1173R, we cannot draw a firm conclusion on this point. Login to comment 164 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His These abnormalities were also observed in vitro in p.D253H- and p.T1173R-transfected cells, suggesting that ABCA3 abnormalities may consistently induce abnormal lamellar bodies. Login to comment 165 ABCA3 p.Thr1173Arg However, since we do not have the corresponding biopsy from the patient harboring the p.T1173R, we cannot draw a firm conclusion on this point. Login to comment 166 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His A549 cells transfected with mock vector (pEGFP-N1), ABCA3-WT (A), or mutated ABCA3-D253H (B) or ABCA3-T1173R-GFP were analyzed using electron microscopy. Login to comment 167 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Human Molecular Genetics, 2012, Vol. 21, No. 4 771 We performed in vitro experiments to elucidate the pathophysiological effects of two mutations associated with progression towards ILD, p.D253H and T1173R. Login to comment 168 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His A549 cells transfected with mock vector (pEGFP-N1), ABCA3-WT (A), or mutated ABCA3-D253H (B) or ABCA3-T1173R-GFP were analyzed using electron microscopy. Login to comment 169 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His We performed in vitro experiments to elucidate the pathophysiological effects of two mutations associated with progression towards ILD, p.D253H and T1173R. Login to comment 172 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His The p.D253H and p.T1173R mutations induced abnormal lamellar bodies. Login to comment 174 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His The p.D253H and p.T1173R mutations induced abnormal lamellar bodies. Login to comment 175 ABCA3 p.Thr1173Arg The p.T1173R mutation was also associated with increased production of IL-8, a well-known chemotactic molecule for neutrophils. Login to comment 177 ABCA3 p.Thr1173Arg The p.T1173R mutation was also associated with increased production of IL-8, a well-known chemotactic molecule for neutrophils. Login to comment 192 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His (B) Role for MAPK and NF-kB-dependent signaling in IL-8 secretion by ABCA3-WT, D253H and T1173R cells. Login to comment 193 ABCA3 p.Thr1173Arg Cells were treated with 10 mM inhibitors of ERK1/2 (U0126), p38 (SB203580), JNK (SP600125) or NF-kB (BAY11-7082) for 24 h; * P ≤ 0.05: ABCA3 versus T1173R; #P ≤ 0.05: vehicle versus U0126. Login to comment 194 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His (B) Role for MAPK and NF-kB-dependent signaling in IL-8 secretion by ABCA3-WT, D253H and T1173R cells. Login to comment 195 ABCA3 p.Thr1173Arg Cells were treated with 10 mM inhibitors of ERK1/2 (U0126), p38 (SB203580), JNK (SP600125) or NF-kB (BAY11-7082) for 24 h; * P ࣘ 0.05: ABCA3 versus T1173R; #P ࣘ 0.05: vehicle versus U0126. Login to comment 227 ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Asp253His Mutagenesis primers (Sigma) were as follows: D253H-For-5' -ACCCGCCGTTCATCGCACACCCCTTCC-3' , D253H-Rev-5' -GGAAGGGGTGTGCGATGAACGGCGGGT- 3' ; T1173R-For-5' -ACGTGCGTGCCTTCAGGCGGGACG-3' , and T1173R-Rev-5' -CGTCCCGCCTGAAGGCACGCACGT- 3' . Login to comment 228 ABCA3 p.Thr1173Arg ABCA3 p.Thr1173Arg ABCA3 p.Asp253His ABCA3 p.Asp253His Mutagenesis primers (Sigma) were as follows: D253H-For-5' -ACCCGCCGTTCATCGCACACCCCTTCC-3' , D253H-Rev-5' -GGAAGGGGTGTGCGATGAACGGCGGGT- 3' ; T1173R-For-5' -ACGTGCGTGCCTTCAGGCGGGACG-3' , and T1173R-Rev-5' -CGTCCCGCCTGAAGGCACGCACGT- 3' . Login to comment 230 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Cells (1 × 106 ) were transfected with 1 mg of ABCA3-WT, ABCA3-D253H or ABCA3-T1173R plasmid using a nucleofector device (Lonza, Cologne, Germany) as recommended by the manufacturer. Login to comment 231 ABCA3 p.Thr1173Arg ABCA3 p.Asp253His Cells (1 &#d7; 106 ) were transfected with 1 mg of ABCA3-WT, ABCA3-D253H or ABCA3-T1173R plasmid using a nucleofector device (Lonza, Cologne, Germany) as recommended by the manufacturer. Login to comment