Congenital afibrinogenemia: mutations leading to premature termination codons in fibrinogen Aα-chain gene are not associated with the decay of the mutant.

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Congenital afibrinogenemia: mutations leading to premature termination codons in fibrinogen Aα-chain gene are not associated with the decay of the mutant mRNAs by Rosanna Asselta, Stefano Duga, Silvia Spena, Elena Santagostino, Flora Peyvandi, Gavino Piseddu, Roberto Targhetta, Massimo Malcovati, Pier Mannuccio Mannucci, and Maria Luisa Tenchini Blood Volume 98(13):3685-3692 December 15, 2001 ©2001 by American Society of Hematology

Mutations in the fibrinogen Aα-chain gene identified in the analyzed patients with congenital afibrinogenemia.(A) Schematic representation of the fibrinogen Aα-chain gene showing the positions of the identified mutations. Mutations in the fibrinogen Aα-chain gene identified in the analyzed patients with congenital afibrinogenemia.(A) Schematic representation of the fibrinogen Aα-chain gene showing the positions of the identified mutations. Heterozygous mutations are shown in italics; homozygous mutations are shown in bold. Exons (numbered) and introns are indicated by boxes and narrow lines, respectively, and are drawn to scale. The shaded portions of exons represent the regions where a premature termination codon should not induce nonsense-mediated mRNA decay. Exon 6 of Aα-chain gene, which is present only in 2% of transcripts, has been omitted. (B) Predicted effects of the identified mutations at the protein level. Schematic representations of the fibrinogen Aα-chain (omitting the signal peptide encoded by exon 1) and of the predicted mutant truncated polypeptides are shown. Portions of Aα-chain encoded by different exons are drawn to scale and shaded in different grays. Numbers above the schematic drawing of the mature Aα-chain refer to amino acid positions corresponding to exon/exon boundaries; numbers beside the truncated polypeptides refer to their amino acid lengths. Rosanna Asselta et al. Blood 2001;98:3685-3692 ©2001 by American Society of Hematology

Fluorescent hot-stop PCR Fluorescent hot-stop PCR.Accuracy of the quantitation of the relative abundance of 2 DNA species by the newly developed fluorescent hot-stop PCR was verified by performing a number of PCR amplifications using different molar ratios of pT-Aα-wt and pT-Aα-Arg... Fluorescent hot-stop PCR.Accuracy of the quantitation of the relative abundance of 2 DNA species by the newly developed fluorescent hot-stop PCR was verified by performing a number of PCR amplifications using different molar ratios of pT-Aα-wt and pT-Aα-Arg149stop plasmids as template. Labeled PCR products were subjected to allele-specific digestion withMaeIII and quantitated on an Abi-310 Genetic Analyzer. Areas of fluorescence peaks corresponding to the mutant and wild-type restriction fragments were measured by GeneScan Analysis software 3.1. (A) GeneScan Analysis windows showing fluorescence peaks corresponding to wild-type (280 nt) and Arg149stop mutant (309 nt) single-stranded fragments. The x-axis represents GeneScan data points and the y-axis represents fluorescence units (FUs). (B) Results of the semiquantitative analysis. The reported values correspond to the peak areas, setting the more abundant mRNA species of each experiment equal to 100%. Rosanna Asselta et al. Blood 2001;98:3685-3692 ©2001 by American Society of Hematology

Analysis of the relative abundance of wild-type and mutant mRNAs Analysis of the relative abundance of wild-type and mutant mRNAs.(A) Example of GeneScan analysis window showing fluorescence peaks corresponding to wild-type and mutant fragments for Arg110stop mutation. Analysis of the relative abundance of wild-type and mutant mRNAs.(A) Example of GeneScan analysis window showing fluorescence peaks corresponding to wild-type and mutant fragments for Arg110stop mutation. These fragments, whose lengths are indicated below each peak, were obtained by a suitable allele-specific digestion subsequent to an RT-PCR performed on RNA extracted from transfected COS-1 cells, coexpressing wild type and each mutant Aα-chain (see “Materials and methods”). The x-axis represents GeneScan data points, and the y-axis represents FUs. (B) Comparison of wild-type and mutant mRNAs levels. The reported values correspond to the peak area measured by means of GeneScan analysis software, with the wild-type mRNA level set equal to 100%. Rosanna Asselta et al. Blood 2001;98:3685-3692 ©2001 by American Society of Hematology

Expression of wild-type and mutant fibrinogens Expression of wild-type and mutant fibrinogens.Fibrinogens secreted from COS-1 cells, transfected with wild-type Bβ- and γ-chains, together with each mutant (Gly13stop, FS51stop, Ser100stop, Arg110stop, and Arg149stop) Aα-chain were analyzed as shown. Expression of wild-type and mutant fibrinogens.Fibrinogens secreted from COS-1 cells, transfected with wild-type Bβ- and γ-chains, together with each mutant (Gly13stop, FS51stop, Ser100stop, Arg110stop, and Arg149stop) Aα-chain were analyzed as shown. Transfections, untransfected HepG2 controls, SDS-PAGE, and Western blot were carried out as described in “Materials and methods.” Proteins were separated on 4% SDS-PAGE under nonreducing conditions. The arrowhead indicates the 340-kd normal fibrinogen. Rosanna Asselta et al. Blood 2001;98:3685-3692 ©2001 by American Society of Hematology