Genetic sophistication of human complement components C4A and C4B and RP-C4-CYP21-TNX (RCCX) modules in the major histocompatibility complex

Abstract

Human populations are endowed with a sophisticated genetic diversity of complement C4 and its flanking genes RP, CYP21, and TNX in the RCCX modules of the major histocompatibility complex class III region. We applied definitive techniques to elucidate (a) the complement C4 polymorphisms in gene sizes, gene numbers, and protein isotypes and (b) their gene orders. Several intriguing features are unraveled, including (1) a trimodular RCCX haplotype with three long C4 genes expressing C4A protein only, (2) two trimodular haplotypes with two long (L) and one short (S) C4 genes organized in LSL configurations, (3) a quadrimodular haplotype with four C4 genes organized in a SLSL configuration, and (4) another quadrimodular structure, with four long C4 genes (LLLL), that has the human leukocyte antigen haplotype that is identical to ancestral haplotype 7.2 in the Japanese population. Long-range PCR and PshAI-RFLP analyses conclusively revealed that the short genes from the LSL and SLSL haplotypes are C4A. In four informative families, an astonishingly complex pattern of genetic diversity for RCCX haplotypes with one, two, three and four C4 genes is demonstrated; each C4 gene may be long or short, encoding a C4A or C4B protein. Such diversity may be related to different intrinsic strengths among humans to defend against infections and susceptibilities to autoimmune diseases.

Figure 1

The 1-2-3–locus concept of human C4 and RCCX modular variations. This map of the human MHC class III region shows the physical length variants among the monomodular, bimodular, and trimodular RCCX structures. The MHC class I genes are located at the telomeric end; the MHC class II genes are located at the centromeric end.

Figure 2

Homoexpression of C4A in a trimodular LLL haplotype from a white family. A, Pedigree of the family C008. B, Genomic TaqI RFLP, to show the RCCX modular variations. C, Genomic PshAI-RFLP, to show the number of RCCX modules by determining the relative dosages of RP2 and RP1. D, PmeI-PFGE analysis, to show the length variants of RCCX haplotypes. E, Immunofixation, to show the relative quantities of C4A and C4B allotypes. F, PshAI-PvuII RFLP, to show the relative dosage of C4A and C4B genes in each individual. G, LSP-RFLP analysis, to determine the relative dosage of C4A and C4B. The dark-background panels are ethidium bromide–stained pictures that contain artifact results because of heteroduplexes. The light-background pictures are results of autoradiography corresponding to LSP-RFLP images free of heteroduplex effects.

Figure 3

A family with a trimodular RCCX haplotype consisting of two long and one short C4 genes. A, Genomic TaqI-RFLP analysis of RCCX modules. B, PmeI-PFGE analysis, to show haplotypes of RCCX length variants. C, Immunofixation of EDTA-plasma, to show the qualitative and quantitative variations of C4A and C4B proteins. D, Genomic PshAI-PvuII RFLP, to determine the relative dosage of C4A and C4B.

Figure 4

A patient with JRA who had the quadrimodular RCCX haplotype consisting of two short C4 and two long C4 genes. A, Genomic TaqI-RFLP analysis of RCCX modules. B, PshAI RFLP, to show the relative dosage of RP2 and RP1. C, Immunofixation, to determine the C4A and C4B allotypes. D, Genomic PshAI-PvuII RFLP, to determine the relative gene dosage of C4A and C4B. E, PmeI PFGE, to show the quadrimodular RCCX in J20 (lane 2). Lanes 1 and 3 are references for LLL/LL and LS/L RCCX structures, respectively.

Figure 5

Quadrimodular and trimodular RCCX structures in an Asian family with a pediatric patient with SLE. A, Genomic TaqI-RFLP analysis of RCCX modules. B, PmeI PFGE of the length variants in RCCX haplotypes. C, Immunofixation of human complement C3 (anti-C3) and C4 (anti-C4). The sample from lane 1A was taken from the pediatric patient with SLE (CS-P4) while her disease was in remission; the sample from lane 1 was taken from the patient in disease flare. Lanes 2 and 3 were samples from CS-M4 and CS-F4, respectively. D, Genomic PshAI-PvuII RFLP, to determine the relative dosage of C4A and C4B.

Figure 6

Determining the configurations of long and short C4A and C4B genes in multi-modular RCCX structures. A, PacI-PFGE analysis of long or short C4 genes linked to TNXA and TNXB. B, A scheme illustrating the locations (upward arrows) and size of the PacI restriction fragments containing the C4 and TNX in three different length variants. C, Exon-intron structure of a short C4 gene and PCR strategy to amplify the genomic region between exons 9 and 31. E9.5 and E31.3 are primers used for amplification of the 7.0-kb genomic fragment for the short C4 gene. The PshAI restriction maps for the C4A and C4B genes are shown in red and blue, respectively. D, Restriction analysis of the PCR fragments from four different individuals with short C4. Lanes 1–4 are undigested PCR products of E9.5-E31.3. Lanes 5–8 are digested with PshAI. Lanes 1 and 5 are from an individual with homozygous, monomodular short C4B genes. E, Southern blot analysis of the genomic fragments in panel D, using a C4d exons 22–25 probe.

Figure 7

Mechanistic models to generate LSL (A) and SLSL (B) modules of RCCX by unequal crossovers