Gene copy-number variation and associated polymorphisms of complement component C4 in human systemic lupus erythematosus (SLE): low copy number is a risk factor for and high copy number is a protective factor against SLE susceptibility in European Americans

Abstract

Interindividual gene copy-number variation (CNV) of complement component C4 and its associated polymorphisms in gene size (long and short) and protein isotypes (C4A and C4B) probably lead to different susceptibilities to autoimmune disease. We investigated the C4 gene CNV in 1,241 European Americans, including patients with systemic lupus erythematosus (SLE), their first-degree relatives, and unrelated healthy subjects, by definitive genotyping and phenotyping techniques. The gene copy number (GCN) varied from 2 to 6 for total C4, from 0 to 5 for C4A, and from 0 to 4 for C4B. Four copies of total C4, two copies of C4A, and two copies of C4B were the most common GCN counts, but each constituted only between one-half and three-quarters of the study populations. Long C4 genes were strongly correlated with C4A (R=0.695; P<.0001). Short C4 genes were correlated with C4B (R=0.437; P<.0001). In comparison with healthy subjects, patients with SLE clearly had the GCN of total C4 and C4A shifting to the lower side. The risk of SLE disease susceptibility significantly increased among subjects with only two copies of total C4 (patients 9.3%; unrelated controls 1.5%; odds ratio [OR] = 6.514; P=.00002) but decreased in those with > or =5 copies of C4 (patients 5.79%; controls 12%; OR=0.466; P=.016). Both zero copies (OR=5.267; P=.001) and one copy (OR=1.613; P=.022) of C4A were risk factors for SLE, whereas > or =3 copies of C4A appeared to be protective (OR=0.574; P=.012). Family-based association tests suggested that a specific haplotype with a single short C4B in tight linkage disequilibrium with the -308A allele of TNFA was more likely to be transmitted to patients with SLE. This work demonstrates how gene CNV and its related polymorphisms are associated with the susceptibility to a human complex disease.

Figure 1.

CNVs and size variations of complement C4 genes and RCCX modules in the human MHC. A, Map showing the gene organization of the MHC-complement gene cluster with a bimodular LL haplotype for RCCX. Horizontal arrows represent gene transcriptional orientations. The PmeI restriction sites flanking the RCCX modules in BF and TNXB are shown as dotted arrows. Vertical arrows with letters depict the locations and names of DNA probes employed for genomic Southern-blot analyses. B, Structures and configurations of RCCX length variants observed in the white study populations. The fragment sizes of TaqI restriction fragments for RP-C4, CYP21, and TNX are shown on the right.^–

Figure 2.

Different genetic mechanisms leading to lower expression of C4A protein than of C4B protein in four families with SLE. In each family, the RCCX modular structures were determined by PFGE of PmeI-digested genomic DNA, Southern blotting, and hybridization to a C4d-specific probe (A). TaqI genomic RFLP was applied to yield details of genomic structures for the RCCX constituents, including the dichotomies of RP1 and RP2, C4 long and C4 short linked to RP1 or RP2, CYP21B, CYP21A, TNXB, and TNXA (B). PshAI-PvuII RFLP was applied to determine the relative GCNs of C4A and C4B (C). The C4A and C4B protein polymorphisms were elucidated by immunofixation of EDTA plasma resolved by high-voltage agarose-gel electrophoresis (D). P = patient; M = mother; F = father; S = sibling (S1 = sibling 1; S2 = sibling 2).

Figure 3.

C4 gene CNVs in female patients with SLE, first-degree relatives, and unrelated healthy subjects. A–C, Distribution patterns of total C4, C4A and C4B GCN groups among white female patients with SLE (FP [red bars]), their female first-degree relatives (FF [yellow bars]), and unrelated female controls (FC [blue bars]). D and E, GCN-dependent variation in OR and SLE disease susceptibility for total C4 (D) and C4A (E).

Figure 4.

Distribution patterns of long and short C4 genes and RCCX haplotype structures among female patients with SLE (FP [red bars]), their female first-degree relatives (FF [yellow bars]), and unrelated female controls (FC [blue bars]). A, Gene CNV of long C4. B, Gene CNV of short C4. C, Haplotype variation of RCCX structures. T-Q = trimodular and quadrimodular.

Figure 5.

Significant increase in the frequency of monomodular S RCCX haplotype with C4A deficiency linked to the −308A allele of the TNFA gene (S-TNF2) in SLE. A, Number and frequency (Freq.) of each haplotype in unrelated female controls (FC), female first-degree relatives (FF), and female patients with SLE (FP). B, Differences between the three groups. NonS-TNF2 = TNF2 allele not linked to monomodular S haplotype of RCCX; NonS-TNF1 = −308G allele of the TNFA gene not linked to monomodular S haplotype of RCCX.