Determining the one, two, three, or four long and short loci of human complement C4 in a major histocompatibility complex haplotype encoding C4A or C4B proteins

Abstract

The complex genetics of human complement C4 with unusually frequent variations in the size and number of C4A and C4B, as well as their neighboring genes, in the major histocompatibility complex has been a hurdle for accurate epidemiological studies of diseases associated with C4. A comprehensive series of novel or improved techniques has been developed to determine the total gene number of C4 and the relative dosages of C4A and C4B in a diploid genome. These techniques include (1) definitive genomic restriction-fragment-length polymorphisms (RFLPs) based on the discrete duplication patterns of the RCCX (RP-C4-CYP21-TNX) modules and on the specific nucleotide changes for C4A and C4B isotypes; (2) module-specific PCR to give information on the total number of C4 genes by comparing the relative quantities of RP1- or TNXB-specific fragments with TNXA-RP2 fragments; (3) labeled-primer single-cycle DNA polymerization procedure of amplified C4d genomic DNA for diagnostic RFLP analysis of C4A and C4B; and (4) a highly reproducible long-range-mapping method that employs PmeI-digested genomic DNA for pulsed-field gel electrophoresis, to yield precise information on the number of long and short C4 genes in a haplotype. Applications of these vigorously tested techniques may clarify the roles that human C4A and C4B gene-dosage variations play in infectious and autoimmune diseases.

Figure 1

Schematic diagram of the human MHC complement gene cluster (MCGC) and eight unique modular variants of the RCCX. A, The gene organization of the MCGC and the location of the two PmeI restriction sites. A horizontal arrow represents the direction of transcription of a gene. A vertical arrow represents the position at which probes A, D, E, or F hybridizes to the DNA. The position of probe 22–25 is not shown, but its location is three exons upstream of probe D. B, The alignment of eight unique RCCX length variants at the telomeric end, which are grouped into either T, B, or M haplotypes. K(C4) = endogenous retrovirus HERV-K(C4); 21A = CYP21A; 21B = CYP21B; XA = TNXA; T = trimodular; B = bimodular; M = monomodular.

Figure 2

Defining the RCCX modular structure using different genomic RFLP Southern blot analyses. A, Restriction patterns of TaqI RFLP for the five selected individuals on simultaneous hybridization of probes specific for 3′ RP, CYP21, and 3′ TNX. B, PshAI RFLP hybridized to a 3′ RP probe. C, BamHI RFLP hybridized to a 3′ TNX probe. D, Phenotyping of C4A and C4B proteins by immunofixation of EDTA-blood plasma resolved by HVAGE. E, Immunoblot analysis of Rg1 and Ch1 antigenic determinants in plasma C4 proteins after HVAGE. F, Genomic Southern blot analysis of C4A and C4B genes by PshAI-RFLP (using Probe D) and by PshAI-PvuII RFLP (using probe 22–25). G, HLA class I and class II alleles of the five subjects.

Figure 3

Module-specific PCR analysis, to detect the modular variation of RCCX. A, Schematic diagram illustrating the approximate locations to which the primer sets a–c and b–d bind in the three modular haplotypes. B, Module-specific PCR for the RP1/TNXA-RP2 reaction, using primer set a–c (lanes 1–5, top), and for the TNXB/TNXA-RP2 reaction, using primer set b–d (lanes 6–10, top). The relative band intensities obtained from RP1 versus TNXA-RP2 or TNXB versus TNXA-RP2 were plotted in the graph and, except for lanes 1 and 6, are shown at bottom.

Figure 4

PCR techniques to determine C4A/C4B gene dosage. A, Scheme depicting the intron/exon structure between exons 21 and 31 (middle) and the basis of definitive RFLP for C4A/C4B (top) and for Rg1/Ch1 (bottom). Exons are in solid boxes. Primers E26.5 and E29.3 are indicated as horizontal arrows. B, PCR products of E26.5/E29.3. C, SSP-PCR assays for the presence and absence of C4A and C4B genes. D, LSP-PshAI or XcmI RFLP, to determine the C4A/C4B and C4 genes associated with Rg1 or Ch1, respectively. An asterisk indicates the fragment for Rg1 with the labeled E29.3 primer. E, Graphic comparisons between expected results for the relative gene dosage of C4A and C4B and results obtained from LSP-RFLPs and from ethidium bromide gels. Experiments with labeled DNA products digested with PshAI that distinguishes C4A and C4B are shown at left, and results of labeled DNA products digested with XcmI that distinguishes C4 genes associated with Rg1 and Ch1 are shown at right.

Figure 5

Elucidation of the number and size of RCCX modules present in MHC haplotypes by PmeI PFGE. A, Selected individuals, to show homozygous and heterozygous haplotypes of length variants of RCCX structures. B, PmeI RCCX patterns of the five individuals with two to six C4 genes, as characterized in figure 2.