Deficiencies of human complement component C4A and C4B and heterozygosity in length variants of RP-C4-CYP21-TNX (RCCX) modules in caucasians. The load of RCCX genetic diversity on major histocompatibility complex-associated disease

Abstract

The complement component C4 genes located in the major histocompatibility complex (MHC) class III region exhibit an unusually complex pattern of variations in gene number, gene size, and nucleotide polymorphism. Duplication or deletion of a C4 gene always concurs with its neighboring genes serine/threonine nuclear protein kinase RP, steroid 21-hydroxylase (CYP21), and tenascin (TNX), which together form a genetic unit termed the RCCX module. A detailed molecular genetic analysis of C4A and C4B and RCCX modular arrangements was correlated with immunochemical studies of C4A and C4B protein polymorphism in 150 normal Caucasians. The results show that bimodular RCCX has a frequency of 69%, whereas monomodular and trimodular RCCX structures account for 17.0 and 14.0%, respectively. Three quarters of C4 genes harbor the endogenous retrovirus HERV-K(C4). Partial deficiencies of C4A and C4B, primarily due to gene deletions and homoexpression of C4A proteins, have a combined frequency of 31.6%. This is probably the most common variation of gene dosage and gene size in human genomes. The seven RCCX physical variants create a great repertoire of haplotypes and diploid combinations, and a heterozygosity frequency of 69.4%. This phenomenon promotes the exchange of genetic information among RCCX constituents that is important in homogenizing the structural and functional diversities of C4A and C4B proteins. However, such length variants may cause unequal, interchromosomal crossovers leading to MHC-associated diseases. An analyses of the RCCX structures in 22 salt-losing, congenital adrenal hyperplasia patients revealed a significant increase in the monomodular structure with a long C4 gene linked to the pseudogene CYP21A, and bimodular structures with two CYP21A, which are likely generated by recombinations between heterozygous RCCX length variants.

Figure 1

A schematic diagram of the human MHC complement gene cluster. The functional genes of the RCCX modules are highlighted. A horizontal arrow represents the transcriptional orientation of a gene; vertical arrows represent locations of the DNA hybridized to probes A–F. K(C4), endogenous retrovirus HERV-K(C4); 21A, CYP21A; 21B, CYP21B; XA, TNXA.

Figure 2

Genotypic and phenotypic analyses of the RCCX modules in selected individuals. (I) TaqI and BamHI RFLPs, to define the RCCX modular structure of each chromosome. Probes A, E, and F were used for TaqI RFLP; probe E was used for BamHI RFLP. (II) Detection of the C4A and C4B genes and C4 genes associated with Rg1 or Ch1 by PshAI (A) and EcoO109 (B) RFLPs with probes C and D, respectively. (III) Detection of C4A and C4B allotypes by immunofixation (A) and immunoblot analysis (B), respectively. T, trimodular; C, CAH carrier.

Figure 4

Diploid combinations of the RCCX modules in the Caucasian population. (A) Frequencies of RCCX modular combinations based on the number of modules. (B) Variations in the number of C4 genes. (C) Frequencies of the RCCX modules based on the number of modules and size of C4 genes.

Figure 3

The frequencies and structures of RCCX modules in the normal Caucasian population. The most common haplotypes are in bold. L, long C4 gene; S, short C4 genes.

Figure 5

RCCX modular variations in classical CAH patients. (A) TaqI RFLP on seven selected CAH patients to show the RCCX diploid combinations. The 8-digit number above each lane represents the relative dosage of the 7.0-, 6.4-, 6.0-, and 5.4-kb fragments for RP-C4, and the 3.7-, 3.2-, 2.5-, and 2.4-kb fragments for CYP21B, CYP21A, TNXB, and TNXA, respectively, as shown in Table . (B) PshAI RFLP to determine the C4A and C4B gene dosage in the corresponding patients. (C) Interpretation of the RCCX structures and the relative gene dosage of CYP21A and CYP21B, and of C4A and C4B.

Figure 6

Alignments of the heterozygous RCCX length variants during meiosis to generate CAH disease haplotypes (A and B), or to facilitate appropriate pairings by the mono-S haplotype (C). For A, a pairing of a (trimodular) LLL structure with a mono-L structure may give a similar result. For C-3, a pairing at the telomeric end may not allow a correct alignment between the CYP21 genes from the homologous chromosomes and therefore may reduce the exchange of sequences.