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Molecular cloning and characterization of decay-accelerating factor deficiency in Cromer blood group Inab phenotype
L Wang1, M Uchikawa, H Tsuneyama
1Japanese Red Cross Central Blood Center, Tokyo, Japan.
Insights
A novel mutation in the decay-accelerating factor (DAF) gene causes the Inab blood group phenotype. This genetic change prevents DAF from attaching to red blood cells, impacting complement regulation.
Area of Science:
- Hematology
- Immunology
- Genetics
Background:
- The decay-accelerating factor (DAF) is crucial for regulating complement activation on cell surfaces.
- The Inab phenotype is characterized by the absence of Cromer blood group system antigens and DAF on red blood cells.
Purpose of the Study:
- To investigate the molecular basis of a newly identified Inab phenotype in a Japanese individual.
- To understand the impact of the identified mutation on DAF protein expression and function.
Main Methods:
- Immunoblotting using a monoclonal antibody to DAF to confirm DAF deficiency.
- Molecular analysis, including DNA sequencing, to identify the genetic mutation.
- Analysis of mRNA splicing and predicted protein translation.
Main Results:
- The patient (H.A.) was homozygous for a C1579-->A nucleotide substitution in the DAF gene.
- This mutation activated a cryptic splice site, leading to a 26 bp deletion in the DAF mRNA.
- The deletion caused a frameshift and premature stop codon, resulting in a truncated DAF protein lacking functional domains and GPI anchoring signals.
- No DAF protein was detected on the patient's red blood cell surface.
Conclusions:
- The identified C1579-->A mutation is responsible for the Inab phenotype by disrupting DAF mRNA splicing and protein synthesis.
- This molecular defect leads to a complete absence of cell surface DAF, potentially affecting complement regulation.
- Further studies are warranted to explore the clinical implications of absent cell surface DAF in the context of the Inab phenotype.
Abstract:
An additional decay-accelerating factor (DAF) mutation, designated as Inab phenotype in the Cromer blood group system, was recently identified in a 28-year-old Japanese woman (H.A.). The red blood cells of H.A., like those of other Inab phenotype individuals, were negative for Cromer system antigens, Cra, Tca, Dra, UMC, and IFC. The deficiency of DAF on the red blood cells of H.A. has been shown by immunoblotting with a murine monoclonal antibody to DAF. Molecular analysis has shown that H.A. is homozygous for a single nucleotide substitution, C1579-->A, at the position 24 bp upstream of the 3'-end of exon 2 of the DAF gene. This substitution causes the activation of a novel cryptic splice site and results in the production of mRNA with a 26 bp deletion. The deletion introduces a reading frame shift and creates a stop codon immediately downstream of the deletion. Translation of mRNA would be terminated at the first amino acid residue of the second short consensus repeat (SCR2) domain (exon 3) of DAF. The functional domains of DAF's complement regulatory activity and the carboxy-terminal signal domains for glycosylphosphatidylinositol (GPI) anchoring are predicted to be lacking in H.A. Thus, there would be no DAF present on the cell surface.