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Characterization of two hybrid C4 allotypes (C4A*12 and C4B*3) by electrophoretic, serological and restriction
R H McLean1, W B Bias, C Giles
1Department of Pediatrics, Johns Hopkins University School of Medicine, Baltimore, Maryland.
Insights
Pedigree analysis reveals rare C4 allotypes, including a C4A-deficient individual with a hybrid C4B*3 gene and another with a Rodgers-negative C4A*12 allotype. These findings advance understanding of complement component 4 (C4) gene variations.
Area of Science:
- Immunogenetics
- Molecular Biology
Background:
- The complement component 4 (C4) gene system is highly polymorphic.
- Understanding rare C4 allotypes is crucial for genetic studies and disease association.
Purpose of the Study:
- To report informative pedigree analyses of two rare C4 allotypes.
- To characterize the genetic basis of observed C4 variations.
Main Methods:
- Pedigree analysis
- SDS-PAGE
- Hemagglutination inhibition (HAI)
- Western blotting
- Restriction Fragment Length Polymorphism (RFLP) analysis
Main Results:
- Characterization of a C4A-deficient proband with a hybrid C4B*3 gene exhibiting normal hemolytic activity and specific serological/blotting profiles.
- Identification of a Rodgers-negative C4A*12 allotype associated with a unique TaqI fragment and specific RFLP patterns.
- RFLP analysis confirmed serologic and electrophoretic findings, suggesting a gene conversion event for C4B*3.
Conclusions:
- Gene conversion is a plausible mechanism for the formation of the hybrid C4B*3 gene.
- Detailed characterization of rare C4 allotypes aids in understanding C4 gene regulation and evolution.
- RFLP analysis is a powerful tool for confirming C4 allotype variations.
Abstract:
Informative pedigree analysis of two rare C4 allotypes is reported. One proband was C4A deficient as a consequence of having one haplotype with a deleted C4A gene, and the second haplotype with two C4B genes--one encoding the common C4B*1 and one encoding a unique hybrid gene product C4B*3. C4B*3 had approximately normal C4B hemolytic activity, a single alpha-chain of MR 94,000 by SDS-PAGE but was positive for Rg:1,2 by hemagglutination inhibition (HAI) and for Rg:1 by Western blotting. The hybrid nature was confirmed by RFLP analysis with a Rg:1-associated fragment by Eco0109 digestion but no C4A-associated fragments by N1aIV digestion were identified. A gene conversion at Locus I which included just the C4 isotype region could explain the structure of C4B*3. The second pedigree had a Rodgers negative C4A*12 allotype. This C4A gene, which segregated with a single 7.0 kb TaqI fragment, encoded a C4A alpha-chain, which was negative for Rg:1 epitope. The affected haplotype lacked the Rg:1-associated fragment by Eco0109 digestion yet had the C4A specific N1aIV digestion fragment. These studies successfully employed RFLP analyses to confirm serologic and electrophoretic observations.