ABCD1 p.Ser213Cys
| Predicted by SNAP2: | A: D (85%), C: D (85%), D: D (95%), E: D (95%), F: D (95%), G: D (85%), H: D (95%), I: D (95%), K: D (95%), L: D (95%), M: D (95%), N: D (91%), P: D (95%), Q: D (91%), R: D (95%), T: D (91%), V: D (95%), W: D (95%), Y: D (95%), |
| Predicted by PROVEAN: | A: D, C: D, D: D, E: D, F: D, G: D, H: D, I: D, K: D, L: D, M: D, N: D, P: D, Q: D, R: D, T: D, V: D, W: D, Y: D, |
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[hide] Simultaneous quantitative and allele-specific expr... BMC Genet. 2004 May 5;5:8. Ding C, Maier E, Roscher AA, Braun A, Cantor CR
Simultaneous quantitative and allele-specific expression analysis with real competitive PCR.
BMC Genet. 2004 May 5;5:8., [PMID:15128429]
Abstract [show]
BACKGROUND: For a diploid organism such as human, the two alleles of a particular gene can be expressed at different levels due to X chromosome inactivation, gene imprinting, different local promoter activity, or mRNA stability. Recently, imbalanced allelic expression was found to be common in human and can follow Mendelian inheritance. Here we present a method that employs real competitive PCR for allele-specific expression analysis. RESULTS: A transcribed mutation such as a single nucleotide polymorphism (SNP) is used as the marker for allele-specific expression analysis. A synthetic mutation created in the competitor is close to a natural mutation site in the cDNA sequence. PCR is used to amplify the two cDNA sequences from the two alleles and the competitor. A base extension reaction with a mixture of ddNTPs/dNTP is used to generate three oligonucleotides for the two cDNAs and the competitor. The three products are identified and their ratios are calculated based on their peak areas in the MALDI-TOF mass spectrum. Several examples are given to illustrate how allele-specific gene expression can be applied in different biological studies. CONCLUSIONS: This technique can quantify the absolute expression level of each individual allele of a gene with high precision and throughput.
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No. Sentence Comment
132 The ddNTPs/dNTP mixtures are: ddATP/ddCTP/ddGTP/dTTP for interleukin 6 and ABCD1 Q672X, ddTTP/ddCTP/ddGTP/dATP for lexA, and ddATP/ddCTP/ddTTP/dGTP for ABCD-1 S108W and S213C.
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ABCD1 p.Ser213Cys 15128429:132:169
status: NEW[hide] Co-expression of mutated and normal adrenoleukodys... Hum Mol Genet. 2000 Nov 1;9(18):2609-16. Unterrainer G, Molzer B, Forss-Petter S, Berger J
Co-expression of mutated and normal adrenoleukodystrophy protein reduces protein function: implications for gene therapy of X-linked adrenoleukodystrophy.
Hum Mol Genet. 2000 Nov 1;9(18):2609-16., [PMID:11063720]
Abstract [show]
Inherited defects in the X-chromosomal adrenoleukodystrophy (ALD; ABCD1) gene are the genetic cause of the severe neurodegenerative disorder X-linked adrenoleukodystrophy (X-ALD). Biochemically the accumulation of very long-chain fatty acids, caused by impaired peroxisomal beta-oxidation, is the pathognomonic characteristic of the disease. Due to the X-chromosomal inheritance of X-ALD no data are available to clarify the question whether mutated adrenoleukodystrophy proteins (ALDPs) can negatively influence normal ALDP function. Here we show that restoration of beta-oxidation in X-ALD fibroblasts following transient transfection with normal ALD cDNA is more effective in ALDP-deficient fibroblasts compared with fibroblasts expressing normal amounts of mutated ALDP. Furthermore, we utilized the HeLa Tet-on system to construct a stable HeLa cell line expressing a constant level of endogenous ALDP and doxycycline-inducible levels of mutated ALDP. The induction was doxycycline dosage-dependent and the ALDP correctly localized. Interestingly, although mutated ALDP increased >6-fold in a dosage-dependent manner the total amount of ALDP (mutated and normal) remained approximately even as demonstrated by western blot and flow cytometric analyses. Thus, apparently mutated and normal ALDP compete for integration into a limited number of sites in the peroxisomal membrane. Consequently, increased amounts of mutated ALDP resulted in decreased peroxisomal beta-oxidation and accumulation of very long-chain fatty acids. These findings have direct implications on future gene therapy approaches for treatment of X-ALD, since in some patients a non-functional endogenous protein could act in a dominant negative way or displace the introduced, normal protein.
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49 Average transfection efficiency was determined in three independent experiments and was 14% for the ALDP-deficient cell line (range 10-17%), 20% for the D194H cell line (range 15-23%), 26% for the S213C cells and 24% for the N148S cell line (range 22-30%).
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ABCD1 p.Ser213Cys 11063720:49:197
status: NEW149 Of the three mutations analysed in transient transfection assays, N148S and S213C are located in transmembrane domains, whereas D194H is in the first cytosolic loop of the ALDP (Fig. 6).
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ABCD1 p.Ser213Cys 11063720:149:76
status: NEW150 In yeast two-hybrid experiments performed with ALDP lacking the N-terminal 361 amino acids, homodimers still formed (13), arguing against the possibility that the three mutations N148S, D194H and S213C interfere with dimerization of ALDP.
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ABCD1 p.Ser213Cys 11063720:150:196
status: NEW155 Missense mutations N148S and S213C are both located within putative transmembrane domains and D194H is located in the first cytosolic loop.
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ABCD1 p.Ser213Cys 11063720:155:29
status: NEW161 12 in ref. 30) were provided by Dr Wanders (Amsterdam) and cell line S213C (31) was obtained from Drs Holzinger and Roscher (Munich).
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ABCD1 p.Ser213Cys 11063720:161:69
status: NEW