Phenotypes, genotypes and disease susceptibility associated with gene copy number variations: complement C4 CNVs in European American healthy subjects and those with systemic lupus erythematosus

Abstract

A new paradigm in human genetics is high frequencies of inter-individual variations in copy numbers of specific genomic DNA segments. Such common copy number variation (CNV) loci often contain genes engaged in host-environment interaction including those involved in immune effector functions. DNA sequences within a CNV locus often share a high degree of identity but beneficial or deleterious polymorphic variants are present among different individuals. Thus, common gene CNVs can contribute, both qualitatively and quantitatively, to a spectrum of phenotypic variants. In this review we describe the phenotypic and genotypic diversities of complement C4 created by copy number variations of RCCX modules (RP-C4-CYP21-TNX) and size dichotomy of C4 genes. A direct outcome of C4 CNV is the generation of two classes of polymorphic proteins, C4A and C4B, with differential chemical reactivities towards peptide or carbohydrate antigens, and a range of C4 plasma protein concentrations (from 15 to 70 mg/dl) among healthy subjects. Deliberate molecular genetic studies enabled development of definitive techniques to determine exact patterns of RCCX modular variations, copy numbers of long and short C4A and C4B genes by Southern blot analyses or by real-time quantitative PCR. It is found that in healthy European Americans, the total C4 gene copy number per diploid genome ranges from 2 to 6: 60.8% of people with four copies of C4 genes, 27.2% with less than four copies, and 12% with more than four copies. Such a distribution is skewed towards the low copy number side in patients with systemic lupus erythematosus (SLE), a prototypic autoimmune disease with complex etiology. In SLE, the frequency of individuals with less than four copies of C4 is significantly increased (42.2%), while the frequency of those with more than four copies is decreased (6%). This decrease in total C4 gene copy number in SLE is due to increases in homozygous and heterozygous deficiencies of C4A but not C4B. Therefore, it is concluded that lower copy number of C4 is a risk factor for and higher gene copy number of C4 is a protective factor against SLE disease susceptibility.

Fig. 1

Polymorphism of complement C4A and C4B proteins. Human plasma proteins were digested with neuraminidase and carboxyl peptidase B to remove heterogeneities caused by glycosylations and incomplete processing of C-termini at the beta and alpha chains of C4 proteins. The proteins were resolved by high voltage agarose gel electrophoresis based on gross differences in electric charge, immunofixed by antisera against human C4, blotted to remove diffusible proteins and then stained. Plasma samples from 15 subjects are shown. The basic C4B proteins migrate slower than the acidic C4A proteins (Blanchong et al., 2000).

Fig. 2

Dichotomous size variation of human C4 genes. Each human C4 gene consists of 41 exons. The long C4 gene contains the endogenous retrovirus HERV-K(C4) in intron 9. The thioester bond is encoded by exon 24, the C4A and C4B isotypic residues by exon 26, and the major Rodgers and Chido blood group antigenic determinants by exon 28. Among the European Americans, 76% of the C4 genes belong to the long form and 24% belong to the short form (Yu, 1991).

Fig. 3

Modular variation of RP-C4-CYP21-TNX (RCCX) in the central region of the human major histocompatibility complex (MHC) on chromosome 6. (A) MHC haplotypes with 1, 2, 3 or 4 RCCX modules. (B) Details of a duplicated RCCX module (Yu and Whitacre, 2004).

Fig. 4

Demonstration of RCCX modular variations by pulsed-field gel electrophoresis (PFGE) of PmeI digested genomic DNA. (A) Genetic map of the MHC complement gene cluster. Horizontal arrows show the transcriptional orientations of genes. Upward arrows show the locations of the PmeI restriction sites, or the locations of DNA probes for hybridization. (B) Demonstration of quadrimodular (Q), trimodular (T), bimodular (B) and monomodular (M) RCCX haplotypes by PmeI PFGE of genomic DNA from 12 human subjects. Lanes 1–4 are from subjects with homozygous trimodular LLL, bimodular LL, monomodular L, and monomodular S, respectively. Lanes 5–12 are from subjects who are heterozygous in RCCX with different combinations of haplotypes.

Fig. 5

Genotypic and phenotypic variations of human C4 from five human subjects with 2, 3, 4, 5 or 6 copies of C4 genes. (A) TaqI RFLP to demonstrate the configurations of RP1 linked to a long C4 or a short C4, the presence and relative quantities of RP2 linked to a long C4 or a short C4, the presence and relative quantities of CYP21B and CYP21A, and the presence and relative quantities of TNXB and TNXA. (B) Demonstration of RCCX haplotypes by PmeI PFGE. Lane 1 is homozygous S/S; lane 2 is heterozygous LS/S; lane 3 is homozygous LL/LL; lane 4 is heterozygous LSL/LL; lane 5 is homozygous LLL/LLL. (C) PshAI RFLP demonstrating the relative quantities of RP1 and RP2. RP2 is not present in monomodular RCCX haplotypes. The number of RP1 genes is constant (i.e., 2 copies per diploid genome) among all human subjects. The relative intensities of RP2 to RP1 restriction fragments give information about the number of duplicated RCCX modules present in an individual. (D) PshAI-PvuII RFLP to determine the presence and relative quantities of C4A and C4B genes. (E) Immunofixation of EDTA-plasma resolved by high voltage agarose gel electrophoresis to demonstrate C4A and C4B protein polymorphisms, using polyclonal serum against human C4. (F) Immunoblot experiments to show C4 proteins associated with Chido or with Rodgers blood group antigens. A monoclonal antibody against Ch1 was used in the upper panel; a monoclonal antibody against Rg1 was used in the lower panel (Chung et al., 2002b).

Fig. 6

Frequencies of gene copy number (GCN) groups for total C4, C4A, C4B, long C4 (C4L) and short C4 (C4S) in healthy subjects. The GCN frequencies were calculated from data based on >500 female and male healthy subjects of European ancestry from central Ohio (Yang et al., 2007).

Fig. 7

Copy number variations of total C4, C4A and C4B in female SLE patients and female controls of European ancestry (Yang et al., 2007).