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C6 haplotypes: associations of a Dde I site polymorphism to complement deficiency genes and the Msp I restriction
B A Fernie1, M J Hobart, G Delbridge
1Molecular Immunopathology Unit, MRC Centre, Cambridge, UK.
Insights
Researchers developed a genotyping method to study complement C6 deficiency. This method identified distinct genetic backgrounds for complete (C6Q0) and sub-total (C6SD) C6 deficiency, indicating independent origins.
Area of Science:
- Immunogenetics
- Molecular Biology
- Human Genetics
Background:
- Complement C6 exhibits a common charge polymorphism (C6A and C6B) with known gene frequencies.
- The molecular basis of this polymorphism is likely an amino acid substitution at position 98.
- Previous studies have established methods for detecting C6 polymorphisms.
Purpose of the Study:
- To apply a novel Dde I restriction fragment length polymorphism (RFLP) genotyping method to investigate complement C6 deficiency.
- To determine the genetic associations of complete (C6Q0) and sub-total (C6SD) C6 deficiency phenotypes.
- To elucidate the allelic backgrounds and origins of different C6 deficiency forms.
Main Methods:
- Polymerase chain reaction (PCR) amplification of genomic DNA fragments.
- Restriction enzyme digestion (Dde I and Msp I) to detect RFLPs.
- Genotyping analysis of individuals with C6 deficiency and their families.
Main Results:
- The Dde I A/B polymorphism, detected via PCR and restriction digestion, was successfully applied to C6-deficient individuals.
- Sixteen of seventeen unrelated C6Q0 subjects were Dde I B/Msp I-negative, suggesting a specific genetic background.
- The C6SD phenotype was associated with the Dde I A/Msp I-positive genotype in families with C6/C7 deficiency.
Conclusions:
- Complete (C6Q0) and sub-total (C6SD) C6 deficiencies appear to have arisen independently on distinct C6 allelic backgrounds.
- The developed genotyping method allows for the characterization of rare families with combined C6 deficiency types.
- Normal C6 Dde I/Msp I haplotypes were defined in Caucasian and Cape Coloured populations.
Abstract:
Complement C6 has a common charge polymorphism designated A and B with gene frequencies of 0.65 and 0.35. The probable molecular basis for this is a Glu (C6A) for Ala (C6B) substitution at amino acid position 98, and is detected by digestion with the restriction enzyme Dde I of a polymerase chain reaction (PCR)-amplified fragment of genomic DNA. C6A was found to be Dde I-positive and C6B corresponds to Dde I-negative. We have applied our Dde I A/B polymorphism genotyping method to the investigation of C6-deficient individuals with complete (C6Q0) and sub-total deficiency (C6SD) protein phenotypes, including members of four families. We have also investigated the RFLP detected by digestion of genomic DNA with the enzyme Msp I, which is due to a polymorphic site located in the 5' section of the gene, the variable sequence of which has yet to be determined. Sixteen out of seventeen unrelated C6Q0 subjects were found to be genotypically Dde I B/Msp I-negative; the remaining subject was heterozygous at both the loci under investigation. The C6SD phenotype was found to be associated with the Dde I A/Msp I-positive genotype in two families with combined C6/C7 subtotal deficiency and two with C6SD. It can be concluded that the two forms of C6 deficiency, C6Q0 and C6SD, arose independently on two different C6 allelic backgrounds. These associations have allowed the genotyping of the rare families that contain both types of deficiency. We have also defined a number of normal C6 Dde I/Msp I haplotypes in Caucasians and Cape Coloured populations.