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Polymorphism of human complement component C6: an amino acid substitution (Glu/Ala) within the second thrombospondin
G Dewald1, M M Nöthen, S Cichon
1Institute of Human Genetics, University of Bonn, Germany.
Insights
Human complement component C6 (C6) shows genetic variation. A new PCR-based DNA typing method accurately identifies C6 allotypes, linking specific DNA sequences to C6 protein types.
Area of Science:
- Immunogenetics
- Molecular Biology
- Human Genetics
Background:
- The human complement system component C6 (C6) exhibits genetic polymorphism across populations.
- Isoelectric focusing has identified common allotypes (C6 A and C6 B) and rare variants.
Purpose of the Study:
- To investigate the genetic basis of C6 polymorphism at codon 98.
- To develop and validate a DNA-based typing method for C6 allotypes.
Main Methods:
- Polymerase chain reaction (PCR) amplification of the human C6 gene segment containing codon 98.
- Restriction fragment length polymorphism (RFLP) analysis using DdeI digestion.
- DNA sequencing to confirm nucleotide substitution.
- Comparison with C6 protein phenotyping.
Main Results:
- A nucleotide substitution (C-->A) in the second position of codon 98 was identified as the cause of polymorphism.
- Allele 1 (GCG) codes for Alanine (Ala), and Allele 2 (GAG) codes for Glutamic acid (Glu).
- PCR-based DNA typing showed absolute concordance with C6 protein typing results for 46 individuals.
Conclusions:
- The C6 A allotype is associated with Glutamic acid (Glu) at position 98.
- The C6 B allotype is associated with Alanine (Ala) at position 98.
- The developed PCR-based method is a reliable tool for C6 genetic typing.
Abstract:
Component C6 of the human complement system exhibits a genetic polymorphism in all populations tested so far. Using isoelectric focusing two common allotypes, C6 A ('acidic') and C6 B ('basic') and a number of rare variants have been described. A comparison of the two published cDNA sequences of C6 suggests a polymorphism in codon 98. Using polymerase chain reaction (PCR) we amplified a segment of the human C6 gene encompassing the presumably polymorphic codon. According to the restriction fragment patterns obtained after DdeI digestion of the PCR product, three genotypes were distinguished. The polymorphism is caused by a nucleotide substitution (C-->A) in the second position of codon 98; allele 1 (GCG) codes for Ala, allele 2 (GAG) for Glu. Sequencing of PCR products confirmed the mutation. For 46 unrelated individuals genotyped by this PCR-based method we also determined C6 protein phenotype. Three phenotypes were observed (C6 A, C6 AB, C6 B). There was an absolute concordance between C6 protein typing and DNA typing. We thus conclude that the C6 A allotype is characterized by a Glu and the C6 B allotype by an Ala residue in position 98 of the C6 polypeptide chain.