Evaluation of a densitometric method for detecting null genes of the fourth component of complement, C4

Boisseau, Deirdre-Ann (1989) Evaluation of a densitometric method for detecting null genes of the fourth component of complement, C4. Masters thesis, Memorial University of Newfoundland.

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Abstract

The fourth component of complement (C4) is encoded by tandem MHC-linkcd genes, C4A and C4B. C4A and C4B have multiple alleles, including nulls, at each locus. Individuals who are homozygous null for C4A or C4B are identified by the absence of C4A or C4B in scrum. The complete absence of C4A is strongly associated with systemic lupus crythematosus (SLE). Although the C4A or C4B heterozygous-null genotype can only be assigned with certainty when informative family data are available, many investigators use relative intensity of C4A/C4B bands after serum electrophoresis to assign null alleles. These assignments are based on unproven assumptions regarding gene-dose and relative C4A/B expression, and pose a serious problem in population studies that attempt to show associations between null C4 alleles and disease, as for example in SLE. In this study the usefulness of C4A:C4B ratio measured by densitometry of typing bands as a method for detecting null alleles was examined. -- C4A:C4B densitometric ratios were determined for 108 individuals genotyped by family studies. Discriminant analysis was used to compare genotype derived from family data with genotype predicted from A:B ratio. Mean ratios for various C4 genotypes and total C4 concentrations by single radial immunodiffusion were also evaluated. C4A:C4B ratios were then determined for six populations of unrelated individuals including SLE and rheumatoid arthritis (RA) patients and control subjects. Observed frequencies for genotypes predicted by A:B ratio were compared to expected frequencies, assuming Hardy-Weinberg equilibrium. MHC data were evaluated for individuals mis-classified by densitometry, and those with very high and very low ratios. -- The results of this study showed that the overall accuracy of the densitometric method was approximately 80%. The main error in the method was the false identification of subjects as heterozygous null for C4A. The overall mis-classification trend toward the heterozygous null C4A group suggested that many individuals may have more C4B than C4A. Using densitometric measures of C4A:C4B ratio to estimate population frequencies of carriers of null C4 genes tended to overestimate the frequency of carriers of null C4A. The overestimation was pronounced in populations in which the actual frequency of the null C4A gene was low. The assumptions upon which the densitometric method is based may not be valid because the MHC haplotype HLA-B17, C4A6C4B1, HLA-DR7 may carry a C4A6 that is expressed at low levels, and the haplotype HLA-B44, C4A3C4BQ0 may carry two copies of C4A3 or a single A3 that is expressed at high levels. -- In summary, the ratio of C4A to C4B in an individual's serum is an inaccurate predictor of his/her genotype.

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