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An Allele-specific Gene Expression Assay to Test the Functional Basis of Genetic Associations
Published on: November 3, 2010
Dr(a-) polymorphism of decay accelerating factor. Biochemical, functional, and molecular characterization and
D M Lublin1, E S Thompson, A M Green
1Department of Pathology, Washington University School of Medicine, St. Louis, Missouri 63110.
Insights
Researchers identified a rare blood group phenotype, Dr(a-), linked to a mutation in the decay accelerating factor (DAF) gene. This discovery defines a new DAF allele, Drb, and offers potential for improved immunohematology diagnostics.
Area of Science:
- Immunology
- Genetics
- Biochemistry
Background:
- The Dra antigen is part of the Cromer-related blood group system, found on decay accelerating factor (DAF).
- DAF is a glycosyl-phosphatidylinositol-anchored protein protecting cells from complement-mediated damage.
- The rare inherited Dr(a-) phenotype presents an opportunity to study DAF polymorphism.
Purpose of the Study:
- To investigate the biochemical and functional changes in DAF associated with the Dr(a-) phenotype.
- To characterize the genetic basis of the Dr(a-) polymorphism.
Main Methods:
- Flow cytometry and radioimmunoassay to assess DAF surface expression on erythrocytes.
- Western blotting to analyze DAF protein characteristics.
- Polymerase chain reaction (PCR) to identify genetic mutations.
- Creation of transfected cell lines for serological testing.
Main Results:
- Dr(a-) erythrocytes showed 40% of normal DAF surface expression, distinct from paroxysmal nocturnal hemoglobinuria.
- Western blots revealed reduced DAF expression and slightly faster mobility.
- A C to T point mutation (nucleotide 649) causing Ser165 to Leu substitution was identified as the cause of the Dr(a-) phenotype, defining the Drb allele.
- Transfected cell lines confirmed the specificity of anti-Dra alloantisera.
Conclusions:
- The Dr(a-) phenotype is caused by a specific mutation in the DAF gene, defining the Drb allele.
- This allele-specific characterization of DAF provides a foundation for novel immunohematology approaches.
- Understanding DAF polymorphism is crucial for transfusion medicine and understanding complement regulation.
Abstract:
The Dra antigen belongs to the Cromer-related blood group system, a series of antigens on decay accelerating factor (DAF), a glycosyl-phosphatidylinositol-anchored membrane protein that protects host cells from complement-mediated damage. We studied the rare inherited Dr(a-) phenotype to ascertain the associated biochemical and functional changes in DAF and to characterize the basis for this polymorphism. Radioimmunoassay assay and flow cytometric analysis of Dr(a-) erythrocytes demonstrated 40% of normal surface expression of DAF but normal levels of several other glycosyl-phosphatidylinositol-anchored proteins, distinguishing this phenotype from that of paroxysmal nocturnal hemoglobinuria. Western blots confirmed this reduced DAF expression and indicated a slightly faster mobility of the molecule on SDS-PAGE. Despite the reduced DAF expression, Dr(a-) erythrocytes functioned normally in the complement lysis sensitivity assay. Utilization of the polymerase chain reaction to amplify mononuclear cell genomic DNA from three unrelated Dr(a-) individuals demonstrated that a point mutation underlies the Dr(a-) phenotype: a C to T change in nucleotide 649 resulting in a serine165 to leucine change. This defines the Drb allele of DAF, which can be distinguished from Dra by a Taq I restriction fragment length polymorphism. We created transfected Chinese hamster ovary cell lines expressing either the Dra or the Drb allelic form of DAF. These allele-specific transfectants were tested by inhibition of hemagglutination or flow cytometry and confirmed the specificity of anti-Dra alloantisera. The allele-specific transfectants could form the basis of a new serological approach to immunohematology.
