ABCA3 p.Gly964Asp
| Predicted by SNAP2: | A: D (53%), C: D (66%), D: D (66%), E: D (63%), F: D (80%), H: D (63%), I: D (75%), K: D (66%), L: D (80%), M: D (66%), N: N (53%), P: D (71%), Q: D (59%), R: D (66%), S: D (63%), T: N (53%), V: D (71%), W: D (71%), Y: D (75%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, S: D, T: D, V: D, W: D, Y: D, |
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[hide] A large kindred of pulmonary fibrosis associated w... Respir Res. 2014 Apr 15;15:43. doi: 10.1186/1465-9921-15-43. 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., [PMID:24730976]
Abstract [show]
BACKGROUND: Interstitial lung disease occurring in children is a condition characterized by high frequency of cases due to genetic aberrations of pulmonary surfactant homeostasis, that are also believed to be responsible of a fraction of familial pulmonary fibrosis. To our knowledge, ABCA3 gene was not previously reported as causative agent of fibrosis affecting both children and adults in the same kindred. METHODS: We investigated a large kindred in which two members, a girl whose interstitial lung disease was first recognized at age of 13, and an adult, showed a diffuse pulmonary fibrosis with marked differences in terms of morphology and imaging. An additional, asymptomatic family member was detected by genetic analysis. Surfactant abnormalities were investigated at biochemical, and genetic level, as well as by cell transfection experiments. RESULTS: Bronchoalveolar lavage fluid analysis of the patients revealed absence of surfactant protein C, whereas the gene sequence was normal. 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. Interestingly, the lung specimens from the girl displayed a morphologic usual interstitial pneumonitis-like pattern, whereas the specimens from one of the two adult patients showed rather a non specific interstitial pneumonitis-like pattern. CONCLUSIONS: We have detected a large kindred with a novel ABCA3 mutation likely causing interstitial lung fibrosis affecting either young and adult family members. We suggest that ABCA3 gene should be considered in genetic testing in the occurrence of familial pulmonary fibrosis.
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No. Sentence Comment
6 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.
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ABCA3 p.Gly964Asp 24730976:6:144
status: NEW43 After identification of the ABCA3 Gly964Asp mutation in the proband, genomic DNA of the parents and siblings was examined if available.
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ABCA3 p.Gly964Asp 24730976:43:34
status: NEW44 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.
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ABCA3 p.Gly964Asp 24730976:44:6
status: NEW75 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`.
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ABCA3 p.Gly964Asp 24730976:75:10
status: NEWX
ABCA3 p.Gly964Asp 24730976:75:170
status: NEWX
ABCA3 p.Gly964Asp 24730976:75:215
status: NEW114 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).
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ABCA3 p.Gly964Asp 24730976:114:198
status: NEW123 Interestingly two subjects (herein after referred as patients B and C) were homozygous for the ABCA3 Gly964Asp mutation.
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ABCA3 p.Gly964Asp 24730976:123:101
status: NEW168 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.
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ABCA3 p.Gly964Asp 24730976:168:165
status: NEW172 When consanguinity with patient A was disclosed, the ABCA3 gene was sequenced and homozygosity for the Gly964Asp mutation was identified.
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ABCA3 p.Gly964Asp 24730976:172:103
status: NEW195 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).
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ABCA3 p.Gly964Asp 24730976:195:192
status: NEW196 Protein concentrations of HA-tagged ABCA3 in ABCA3-WT and ABCA3-G964D transiently transfected A549 cells were similar (Figure 8b).
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ABCA3 p.Gly964Asp 24730976:196:64
status: NEW197 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).
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ABCA3 p.Gly964Asp 24730976:197:101
status: NEW198 The ratio of upper to lower bands was not significantly different in ABCA3-WT and p.G964D cells (Figure 8c).
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ABCA3 p.Gly964Asp 24730976:198:84
status: NEW199 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).
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ABCA3 p.Gly964Asp 24730976:199:183
status: NEW201 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).
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ABCA3 p.Gly964Asp 24730976:201:57
status: NEW202 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).
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ABCA3 p.Gly964Asp 24730976:202:20
status: NEW203 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).
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ABCA3 p.Gly964Asp 24730976:203:176
status: NEW204 The G964D mutation decreases ABCA3-induced intracellular accumulation of phospholipids and free cholesterol in HEK cells.
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ABCA3 p.Gly964Asp 24730976:204:4
status: NEW205 Phospholipids and cholesterol were determined in HEK cells stably transfected with vectors coding for ABCA3-WT and G964D.
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ABCA3 p.Gly964Asp 24730976:205:115
status: NEW213 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).
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ABCA3 p.Gly964Asp 24730976:213:209
status: NEW214 Electron microscopy of both stably transfected WT and G964D HEK cells possessed organelles which resemble lamellar bodies (Figure 10).
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ABCA3 p.Gly964Asp 24730976:214:54
status: NEW215 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.
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ABCA3 p.Gly964Asp 24730976:215:71
status: NEW216 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.
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ABCA3 p.Gly964Asp 24730976:216:80
status: NEW220 a. ABCA3 mRNA expression levels analyzed by quantitative real time PCR (P < 0.001 for WT/G964D vs. A549/Mock).
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ABCA3 p.Gly964Asp 24730976:220:89
status: NEW240 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.
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ABCA3 p.Gly964Asp 24730976:240:10
status: NEW241 Shown are cellular contents of phosphatidylcholine, phosphatidylglycerol, phosphatidylinositol, and free cholesterol in HEK cells expressing wild type ABCA3 and ABCA3 G964D, respectively.
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ABCA3 p.Gly964Asp 24730976:241:167
status: NEW244 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.
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ABCA3 p.Gly964Asp 24730976:244:10
status: NEW260 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.
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ABCA3 p.Gly964Asp 24730976:260:55
status: NEW266 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.
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ABCA3 p.Gly964Asp 24730976:266:14
status: NEW272 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.
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ABCA3 p.Gly964Asp 24730976:272:216
status: NEW276 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.
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ABCA3 p.Gly964Asp 24730976:276:179
status: NEW277 However G964D cells appeared to have a more variable and less mature organisation of these organelles than the WT.
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ABCA3 p.Gly964Asp 24730976:277:8
status: NEW278 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.
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ABCA3 p.Gly964Asp 24730976:278:188
status: NEW279 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.
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ABCA3 p.Gly964Asp 24730976:279:132
status: NEWX
ABCA3 p.Gly964Asp 24730976:279:376
status: NEW[hide] Sequencing of idiopathic pulmonary fibrosis-relate... BMJ Open Respir Res. 2014 Dec 10;1(1):e000057. doi: 10.1136/bmjresp-2014-000057. eCollection 2014. Coghlan MA, Shifren A, Huang HJ, Russell TD, Mitra RD, Zhang Q, Wegner DJ, Cole FS, Hamvas A
Sequencing of idiopathic pulmonary fibrosis-related genes reveals independent single gene associations.
BMJ Open Respir Res. 2014 Dec 10;1(1):e000057. doi: 10.1136/bmjresp-2014-000057. eCollection 2014., [PMID:25553246]
Abstract [show]
BACKGROUND: Previous studies investigating a genetic basis for idiopathic pulmonary fibrosis (IPF) have focused on resequencing single genes in IPF kindreds or cohorts to determine the genetic contributions to IPF. None has investigated interactions among the candidate genes. OBJECTIVE: To compare the frequencies and interactions of mutations in six IPF-associated genes in a cohort of 132 individuals with IPF with those of a disease-control cohort of 192 individuals with chronic obstructive pulmonary disease (COPD) and the population represented in the Exome Variant Server. METHODS: We resequenced the genes encoding surfactant proteins A2 (SFTPA2), and C (SFTPC), the ATP binding cassette member A3 (ABCA3), telomerase (TERT), thyroid transcription factor (NKX2-1) and mucin 5B (MUC5B) and compared the collapsed frequencies of rare (minor allele frequency <1%), computationally predicted deleterious variants in each cohort. We also genotyped a common MUC5B promoter variant that is over-represented in individuals with IPF. RESULTS: We found 15 mutations in 14 individuals (11%) in the IPF cohort: (SFTPA2 (n=1), SFTPC (n=5), ABCA3 (n=4) and TERT (n=5)). No individual with IPF had two different mutations, but one individual with IPF was homozygous for p.E292V, the most common ABCA3 disease-causing variant. We did not detect an interaction between any of the mutations and the MUC5B promoter variant. CONCLUSIONS: Rare mutations in SFTPA2, SFTPC and TERT are collectively over-represented in individuals with IPF. Genetic analysis and counselling should be considered as part of the IPF evaluation.
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No. Sentence Comment
93 Homozygous recessive or compound heterozygous ABCA3 mutations cause neonatal respiratory failure and childhood ILD, and single ABCA3 mutations (p.E292V and p.D123N) interacting with SFTPC p.I73T have been reported in two families, one with childhood ILD and one with IPF.10 34 Our identification of one individual who was homozygous for ABCA3 p.E292V adds to the recent identification of a kindred with pulmonary fibrosis due to a p.G964D thus further supporting the possibility that adult-onset fibrotic lung disease due to homozygous or compound heterozygous mutations in ABCA3 may occur.11 In contrast to our previous study that demonstrated an enrichment of single ABCA3 mutations in newborns with RDS, suggesting a developmental interaction that increased risk or severity of disease, we did not find the frequency of single ABCA3 mutations in the IPF cohort to be greater than the 3-5% in the general population.
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ABCA3 p.Gly964Asp 25553246:93:433
status: NEW