Analysis of the genetic variability of complement component C4 by real-time PCR

Recent findings revealed that the repetitions of long DNA sequences comprising the sequence of different genes (CNV - copy-number variations) are very common in the human genome. A well-known example for this type of variations is the "RCCX-module" that implies the tandem duplication of four genes - RP, 21-hydroxylase, complement component C4 and tenascin X - in a haplotype block. Only the C4 gene is active in each module, besides, each module may contain C4A or C4B gene encoding the two isoforms of complement C4. Copy number variation of C4 genes has functional relevance; specific combinations could be risk factors for autoimmune or other immunological diseases. We developed a new, real-time PCR based method for the quantification of C4A and C4B genes. This assay applies specific TaqMan probes, therefore combines the advantages of quantitative measurement (copy number determination) and SNP genotyping (for distinguishing the C4A and C4B isoforms) techniques. The developed real-time PCR methodology was used to determine C4A and C4B gene dosages in a healthy Hungarian population (N=118). The obtained data were compared to the results of an earlier study of the same population analyzed by different techniques. The novel method was demonstrated to be a simple, fast and reliable choice for studies of the complement C4 system, especially in large-scale populations screening.