ABCMdb

PMID: 24730976

Campo I, Zorzetto M, Mariani F, Kadija Z, Morbini P, Dore R, Kaltenborn E, Frixel S, Zarbock R, Liebisch G, Hegermann J, Wrede C, Griese M, Luisetti M
A large kindred of pulmonary fibrosis associated with a novel ABCA3 gene variant.
Respir Res. 2014 Apr 15;15:43. doi: 10.1186/1465-9921-15-43., [PubMed]

Sentences

No. Mutations Sentence Comment

6 ABCA3 p.Gly964Asp By contrast, sequence of the ABCA3 gene showed a novel homozygous G > A transition at nucleotide 2891, localized within exon 21, resulting in a glycine to aspartic acid change at codon 964. Login to comment 43 ABCA3 p.Gly964Asp After identification of the ABCA3 Gly964Asp mutation in the proband, genomic DNA of the parents and siblings was examined if available. Login to comment 44 ABCA3 p.Gly964Asp ABCA3 Gly964Asp SNP detection was performed with the Taqman Assay on a Light cycler 480 (Roche), with the following primers and probe: forward primer: AGGTCCCGGGA ACTGAGAA, reverse primer: CCATGCTGAGGCTGAC CTT, reporter 1: VIC-CGAGTACGGCAGAACC, reporter 2: FAM-CGAGTACGACAGAACC, quencher: NFQ. Login to comment 75 ABCA3 p.Gly964Asp ABCA3 p.Gly964Asp ABCA3 p.Gly964Asp The ABCA3-G964D point mutation was introduced into the pUB6-ABCA3-WT vector using the Quick Change Site-directed mutagenesis kit (Stratagene) with the following primers: G964D-for 5`-GCGAGTAC GACAGAACCGTCGTG-3` and G964D-rev 5`-CACGAC GGTTCTGTCGTACTCGC-3`. Login to comment 114 ABCA3 p.Gly964Asp Genetic analysis Sequence analysis of all 30 coding exons of the ABCA3 gene revealed the presence of a novel homozygous G > A transition at nucleotide 2891, localized within exon 21, resulting in a glycine to aspartic acid change at codon 964 (Figure 4). Login to comment 123 ABCA3 p.Gly964Asp Interestingly two subjects (herein after referred as patients B and C) were homozygous for the ABCA3 Gly964Asp mutation. Login to comment 168 ABCA3 p.Gly964Asp The family genetic screening by the Taqman assay resulted in the identification of two other paternal members (black arrows), who were homozygous for the same ABCA3 Gly964Asp mutation. Login to comment 172 ABCA3 p.Gly964Asp When consanguinity with patient A was disclosed, the ABCA3 gene was sequenced and homozygosity for the Gly964Asp mutation was identified. Login to comment 195 ABCA3 p.Gly964Asp Expression, intracellular processing, and cellular effects resulting from ABCA3 G > A transition at nucleotide 2891 Transient transfection of A549 cells with DNA coding for ABCA3-WT and ABCA3-G964D induced ABCA3-mRNA transcription 500 to 600-fold compared to non-transfected cells (Figure 8a). Login to comment 196 ABCA3 p.Gly964Asp Protein concentrations of HA-tagged ABCA3 in ABCA3-WT and ABCA3-G964D transiently transfected A549 cells were similar (Figure 8b). Login to comment 197 ABCA3 p.Gly964Asp Intracellular processing as assessed by western immunoblotting did not differ between ABCA3-WT and p.G964D, both proteins were found to have a 150 kDa as well as a larger 190 kDa processing form (Figure 8b). Login to comment 198 ABCA3 p.Gly964Asp The ratio of upper to lower bands was not significantly different in ABCA3-WT and p.G964D cells (Figure 8c). Login to comment 199 ABCA3 p.Gly964Asp Immunofluorescence staining of transfected A549 cells revealed a mostly vesicular ABCA3 pattern, which colocalized with the lamellar-body marker LAMP3 for both HA-tagged ABCA3-WT and G964D proteins (Figure 8d). Login to comment 201 ABCA3 p.Gly964Asp Almost no colocalization was observed for ABCA3-WT and p.G964D with the ER-resident chaperone calnexin, so ER-retention due to protein-misfolding can be excluded (Figure 8e). Login to comment 202 ABCA3 p.Gly964Asp Expression of the p.G964D mutation did not induce the early-endosomal marker (EEA1), the ER chaperone calnexin or the ER-resident stress marker BiP- when compared with the ABCA3-WT protein (Figure 8f). Login to comment 203 ABCA3 p.Gly964Asp Similar results for the intra-cellular processing and the lack of induction of ER stress were obtained with stably transfected HEK cells with DNA coding for ABCA3-WT and ABCA3-G964D (Additional file 1: Figure S1). Login to comment 204 ABCA3 p.Gly964Asp The G964D mutation decreases ABCA3-induced intracellular accumulation of phospholipids and free cholesterol in HEK cells. Login to comment 205 ABCA3 p.Gly964Asp Phospholipids and cholesterol were determined in HEK cells stably transfected with vectors coding for ABCA3-WT and G964D. Login to comment 213 ABCA3 p.Gly964Asp The involvement of the upper lobes is very similar to that of the brother, patient B. of PC, phosphatidylglycerol (PG) and PI were significantly increased in cells expressing wild type ABCA3 when compared to G964D stably transfected HEK cells (Figure 9). Login to comment 214 ABCA3 p.Gly964Asp Electron microscopy of both stably transfected WT and G964D HEK cells possessed organelles which resemble lamellar bodies (Figure 10). Login to comment 215 ABCA3 p.Gly964Asp Whereas these organelles appeared in different states of organisation, G964D transfected HEK had less mature and more variable appearing organisation of the organelles than the WT. Login to comment 216 ABCA3 p.Gly964Asp Taken together, these results suggest that transport function of ABCA3 with the G964D mutation is severely impaired especially with regard to the sorting of important surfactant phospholipids and FC, as well as quality of lamellar body formation. Login to comment 220 ABCA3 p.Gly964Asp a. ABCA3 mRNA expression levels analyzed by quantitative real time PCR (P < 0.001 for WT/G964D vs. A549/Mock). Login to comment 233 ABCA3 p.Glu292Val ABCA3 p.Glu292Val A compound heterozygous mutation involving a substitution of valine for glutamic acid in codon 292 (E292V) on at least one allele has been identified in children with chronic ILD. Login to comment 239 ABCA3 p.Trp308Arg Sequence analysis revealed that the patient was a homozygous carrier of the loss-of-function mutation p.Trp308Arg (c.922 T > C) at exon 9 of the ABCA3 gene, without any additional abnormalities in SFTPB, SFTPC and CSF2RA genes. Login to comment 240 ABCA3 p.Gly964Asp The ABCA3 G964D mutation identified in our patients has never been described previously and it shows an autosomal recessive pattern of inheritance, as suggested by evidence that lung disease develops only in Figure 9 Levels of phospholipids and free cholesterol in HEK cells. Login to comment 241 ABCA3 p.Gly964Asp Shown are cellular contents of phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, and free cholesterol in HEK cells expressing wild type ABCA3 and ABCA3 G964D, respectively. Login to comment 244 ABCA3 p.Gly964Asp The ABCA3 G964D mutation was not detected in a large control group of healthy subjects, thus it is unlikely that it is a polymorphism, but rather a rare variant. Login to comment 246 ABCA3 p.Leu462Arg ABCA3 p.Gly964Ser [25] reported a case where an infant died from neonatal respiratory distress, which carried a ABCA3 G964S mutation in compound heterozygosity with a L462R mutation. Login to comment 260 ABCA3 p.Gly964Asp Transient cellular expression of wild type and mutated G964D ABCA3 in alveolar epithelial cells resulted in enhanced expression of normal and mutated protein which did not differ in size and molecular form, intracellular processing or localization Figure 10 Transmission electron microscopy of stable transfected HEK293-cells. Login to comment 266 ABCA3 p.Gly964Asp In the strain G964D these organelles appear less organized: lamellae are often not arranged perfectly parallel (see inset), and the concentric organisation is not as distinct as in the WT. Login to comment 272 ABCA3 p.Gly964Asp We further demonstrated reduced level of the lipids characteristic for pulmonary surfactant, i.e. phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and free cholesterol, in those cells that carried the G964D - ABCA3. Login to comment 273 ABCA3 p.Asn568Asp In accordance with these results it was also shown previously that phosphatidylcholine accumulation in cells expressing ABCA3 with the walker A motif mutant N568D was diminished [27]. Login to comment 276 ABCA3 p.Gly964Asp By electron microscopy lamellar bodies, as characteristic in type II alveolar epithelial cells in the lungs, were clearly induced and visualized in both stably transfected WT and G964D HEK cells. Login to comment 277 ABCA3 p.Gly964Asp However G964D cells appeared to have a more variable and less mature organisation of these organelles than the WT. Login to comment 278 ABCA3 p.Gly964Asp This finding is concordant with the functional transport defect for phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol and free cholesterol observed in the cells carrying the G964D - ABCA3 mutations and described above. Login to comment 279 ABCA3 p.Gly964Asp ABCA3 p.Gly964Asp Therefore, based on these cell culture data, disordered lipid composition, reduced and faulty lamellar body organization due to the G964D - ABCA3 mutation may represent mechanisms involved in the pathogenesis on ILD in this family, whereas misfolding, aberrant processing, mislocalization and ER-stress can be excluded as factors contributing to the development of ILD by the G964D mutation. Login to comment