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Molecular analysis of glycophorin C deficiency in human erythrocytes
1Lawrence Berkeley Laboratory, University of California, Berkeley 94720.
Insights
The Leach blood group phenotype is linked to a deficiency in erythrocyte glycophorin C and D. This is caused by a deletion in the glycophorin C gene, affecting exons 3 and 4.
Area of Science:
- Genetics
- Hematology
- Molecular Biology
Background:
- Human erythrocyte glycophorin C is crucial for red blood cell shape and membrane stability.
- The Leach phenotype is characterized by a deficiency of glycophorins C and D in erythrocytes.
Purpose of the Study:
- To characterize the genetic basis of glycophorin C and D deficiency in the Leach phenotype.
- To investigate the molecular mechanisms underlying the absence of these proteins in erythrocytes.
Main Methods:
- Polymerase chain reaction (PCR) amplification of reticulocyte cDNA and genomic DNA.
- Southern hybridization analysis.
- Analysis of glycophorin C gene structure (4 exons) and mRNA expression.
Main Results:
- PCR analysis revealed a missing or altered 3' end of the glycophorin C mRNA in Leach phenotype individuals.
- Exon 4 of the glycophorin C gene was found to be absent in Leach genomic DNA.
- A deletion or significant alteration of exons 3 and 4 of the glycophorin C gene causes the deficiency.
- A stable, albeit truncated, mRNA transcript is produced by the mutant gene.
Conclusions:
- The absence of glycophorin C and D in Leach phenotype erythrocytes results from a mutation in the glycophorin C gene affecting exons 3 and 4.
- The mutant mRNA, though detectable, cannot produce functional membrane-bound glycophorin C due to the lack of transmembrane and cytoplasmic domains.
Abstract:
Human erythrocyte glycophorin C plays a functionally important role in maintaining erythrocyte shape and regulating membrane mechanical stability. We report here the characterization of the glycophorins C and D deficiency in erythrocytes of the Leach phenotype. Glycophorin C gene is encoded by 4 exons. Amplification of reticulocyte cDNA from Leach phenotype and normal individuals generated a 140-bp fragment when using primers spanning exons 1 and 2. However, no polymerase chain reaction (PCR) products were detected in the Leach phenotype using primers flanking either exons 1 and 3 or exons 1 and 4, suggesting that the 3' end of the mRNA was missing or altered. Exon 4 also appeared to be missing from Leach genomic DNA, based on both Southern hybridization and PCR. These results indicate that an absence of glycophorin C and glycophorin D in erythrocytes from these Leach phenotype individuals is a consequence of a deletion or marked alteration of exon 3 and exon 4 of their glycophorin C gene. Surprisingly, the mutant gene encodes an mRNA stable enough to be detected in circulating reticulocytes. Although this mRNA could encode an N-terminal fragment of glycophorin C, these protein isoform(s) would not be expressed in the membrane because they lack the transmembrane and cytoplasmic domains.