Quantitative sequence analysis of FBN1 premature termination codons provides evidence for incomplete NMD in leukocytes.

Details

Serval ID
serval:BIB_4ECB5518460B
Type
Article: article from journal or magazin.
Collection
Publications
Institution
Title
Quantitative sequence analysis of FBN1 premature termination codons provides evidence for incomplete NMD in leukocytes.
Journal
Human Mutation
Author(s)
Magyar I., Colman D., Arnold E., Baumgartner D., Bottani A., Fokstuen S., Addor M.C., Berger W., Carrel T., Steinmann B., Mátyás G.
ISSN
1098-1004[electronic]
Publication state
Published
Issued date
2009
Volume
30
Number
9
Pages
1355-1364
Language
english
Abstract
We improved, evaluated, and used Sanger sequencing for quantification of single nucleotide polymorphism (SNP) variants in transcripts and gDNA samples. This improved assay resulted in highly reproducible relative allele frequencies (e.g., for a heterozygous gDNA 50.0+/-1.4%, and for a missense mutation-bearing transcript 46.9+/-3.7%) with a lower detection limit of 3-9%. It provided excellent accuracy and linear correlation between expected and observed relative allele frequencies. This sequencing assay, which can also be used for the quantification of copy number variations (CNVs), methylations, mosaicisms, and DNA pools, enabled us to analyze transcripts of the FBN1 gene in fibroblasts and blood samples of patients with suspected Marfan syndrome not only qualitatively but also quantitatively. We report a total of 18 novel and 19 known FBN1 sequence variants leading to a premature termination codon (PTC), 26 of which we analyzed by quantitative sequencing both at gDNA and cDNA levels. The relative amounts of PTC-containing FBN1 transcripts in fresh and PAXgene-stabilized blood samples were significantly higher (33.0+/-3.9% to 80.0+/-7.2%) than those detected in affected fibroblasts with inhibition of nonsense-mediated mRNA decay (NMD) (11.0+/-2.1% to 25.0+/-1.8%), whereas in fibroblasts without NMD inhibition no mutant alleles could be detected. These results provide evidence for incomplete NMD in leukocytes and have particular importance for RNA-based analyses not only in FBN1 but also in other genes.
Pubmed
Web of science
Create date
08/10/2009 13:29
Last modification date
20/08/2019 14:04
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