title: Quantitative sequence analysis of FBN1 premature termination codons provides evidence for incomplete NMD in leukocytes.
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.
authors.affiliation: Division of Medical Molecular Genetics and Gene Diagnostics, Institute of Medical Genetics, University of Zurich, Zurich, Switzerland.
authors.forename: István
authors.initials: I
authors.lastname: Magyar
authors.forename: Dvora
authors.initials: D
authors.lastname: Colman
authors.forename: Eliane
authors.initials: E
authors.lastname: Arnold
authors.forename: Daniela
authors.initials: D
authors.lastname: Baumgartner
authors.forename: Armand
authors.initials: A
authors.lastname: Bottani
authors.forename: Siv
authors.initials: S
authors.lastname: Fokstuen
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Addor
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Berger
authors.forename: Thierry
authors.initials: T
authors.lastname: Carrel
authors.forename: Beat
authors.initials: B
authors.lastname: Steinmann
authors.forename: Gábor
authors.initials: G
authors.lastname: Mátyás
mesh_terms: D000483:Alleles; D001483:Base Sequence; D018389:Codon, Nonsense / Q000235:genetics* / Q000378:metabolism; D004252:DNA Mutational Analysis; D000071838:Fibrillin-1; D000071837:Fibrillins; D014644:Genetic Variation; D006801:Humans; D007962:Leukocytes / Q000378:metabolism*; D008382:Marfan Syndrome / Q000235:genetics*; D008840:Microfilament Proteins / Q000235:genetics*; D020871:RNA Stability / Q000235:genetics*; D012333:RNA, Messenger / Q000378:metabolism*; D054392:Adipokines
chemical_list: D018389:Codon, Nonsense; D000071838:Fibrillin-1; D000071837:Fibrillins; D008840:Microfilament Proteins; D012333:RNA, Messenger; C000605722:FBN1 protein, human; D054392:Adipokines