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. Author manuscript; available in PMC: 2017 Jun 1.
Published in final edited form as: Arthritis Rheumatol. 2016 Jun;68(6):1442–1453. doi: 10.1002/art.39589

Effects of Complement C4 Gene Copy Number Variations, Size Dichotomy, and C4A Deficiency on Genetic Risk and Clinical Presentation of Systemic Lupus Erythematosus in East Asian Populations

Ji Yih Chen 1,#, Yee Ling Wu 2,#, Mo Yin Mok 3, Yeong-Jian Jan Wu 1, Katherine E Lintner 2, Chin-Man Wang 4, Erwin K Chung 2, Yan Yang 2, Bi Zhou 2, Huanyu Wang 2, Dennis Yu 2, Alaaedin Alhomosh 2, Karla Jones 2, Charles H Spencer 2, Haikady N Nagaraja 5, Yu Lung Lau 6, Chak-Sing Lau 3, C Yung Yu 2,
PMCID: PMC5114127  NIHMSID: NIHMS398178  PMID: 26814708

Abstract

Objectives

Human complement C4 is sophisticatedly complex with multiple layers of diversity. This study aims to elucidate the CNVs of C4A and C4B in disease risk of SLE, and compare the basis of race-specific C4A-deficiency in East-Asians (EA) and Europeans.

Patients and Methods

Our EA study-population included 999 SLE patients and 1,347 healthy subjects. Variations in gene copy-numbers (GCNs) for total C4, C4A, C4B, long and short genes were determined and validated rigorously by independent genotyping technologies. Genomic regions with C4B96 were investigated to determine the basis of the most basic C4B protein that is concurrent with C4A-deficiency.

Results

In EA, strong protective effects of high GCNs for total C4 and C4A against SLE were notable; low and medium GCNs for total C4 and C4A, and the absence of short genes were risk factors of SLE. Homozygous C4A-deficiency was infrequent but had an odds-ratio (OR) of 12.4 (p=0.0015). Patients who experienced very-low serum complement were associated with low GCNs of total C4 (OR=3.27, p=7.0×10−7) and C4B (OR=2.55, p=2.5×10−5). Patients with low complement had high frequencies of anti-dsDNA (OR=4.96, p=9.7×10−17), hemolytic anemia (OR=3.89, p=3.6×10−10) and renal disease (OR=2.18, p=8.5×10−6). The monomodular-short haplotype with C4A-deficiency and in linkage-disequilibrium with HLA-DRB1*0301 prevalent in European was scarce in EA. Instead, most EA-subjects with C4A-deficiency shared a recombinant haplotype with bimodular-LS encoding C4B1 and C4B96, which was linked to HLA-DRB1*1501. DNA sequencing revealed the E920K polymorphism for C4B96.

Conclusion

C4 CNVs and C4A-deficiency are important in the risk and manifestations of East-Asian and European SLE.

INTRODUCTION

Systemic lupus erythematosus (SLE) is a female dominant autoimmune disease characterized by the generation of autoantibodies against nuclear antigens including double-stranded DNA (dsDNA) (1-3). The formation of immune complexes between autoantibodies and self-antigens activates complement, causing systemic tissue injuries and organ damages (4,5). Reduced serum levels or depressed hemolytic activities of complement are common clinical laboratory findings in patients with SLE and glomerulonephritis (4,6-9). Such phenomenon is generally interpreted as a result of consumption due to SLE disease activities. However, low serum complement can both be a cause and an effect of SLE. Human subjects with a homozygous genetic deficiency in anyone of the early components for the classical pathway of complement activation (Supporting Information: Figure S1), C1q, C1r, C1s and C4 were almost always inflicted with SLE irrespective of race, gender and HLA haplotypes (10-14). Under susceptible backgrounds, mice with genetic knockout of complement C4 or C1q manifested lupus-like phenotypes with high titers of antinuclear antibodies (ANA) and anti-dsDNA and high frequency of glomerulonephritis (15-18). Cumulated information suggests that complement deficiency contributes to pathogenesis of SLE through impaired handling and clearance of immune complexes, deficient scavenging of apoptotic cells, aberrant induction of peripheral tolerance because of inefficient elimination of self-reactive B cells to produce high affinity autoantibodies with class switching, and defective control of cytokine production including type I interferons (5,19,20).

While a homozygous genetic deficiency for complement C1 or C4 can be a causal factor of SLE, their prevalence is extremely rare as only 122 cases have ever been documented (4). Genome-wide association studies led to the identification of numerous single nucleotide polymorphisms (SNPs) associated with increased risk of SLE, but most of those SNPs are of low effect-size with odds ratios (OR) between 1.1 and 1.5 (1-3,21).

SLE affects all racial groups but patients of Asian, African and Hispanic ancestries/ethnicities often have severe disease with renal involvement (22,23). While SLE risk factors identified in Asians are also germane to Europeans, there are remarkable differences between races with respect to effect-sizes and frequencies of risk alleles (24). In a case-control study of C4 gene copy-number variations (CNVs; online Figure S1) in SLE of European ancestry, we found that low GCNs of total C4 and C4A were risk factors for, and high GCNs of total C4 and C4A were protective factors against, SLE disease susceptibility (25). A common major histocompatibility (MHC, or HLA in human) haplotype with a single short C4B gene (mono-S) and the absence of C4A is prevalent among European SLE patients. This mono-S haplotype is in strong linkage disequilibrium (LD) with the HLA class II gene DRB1 allele *0301 (or DR3) (26,27). Although the increased risk for C4A-deficiency in Asians had been observed in earlier studies (28-30), the mechanism of C4A-deficiency and diversity associated with C4-CNVs have not been investigated meticulously. Here, we report an in-depth study to elucidate patterns of CNVs for complement C4 and associated variants in large cohorts of East-Asian (Chinese) SLE and race-matched healthy controls. This study unravels the molecular basis of C4A-deficiency and fine details pertinent to C4-CNVs in East-Asian and European SLE disease susceptibility and pathogenesis.

STUDY POPULATIONS AND METHODS

Study populations

Our study populations included 999 Chinese SLE patients and 1,347 race-matched healthy controls (Table 1). All patients fulfilled at least four of the eleven revised 1982-ACR diagnostic criteria for human SLE (31,32), The patients’ female to male sex ratio was 11.6 to 1. Their mean age (±SD) at disease diagnosis was 30.61±11.45 years old. Besides the presence of ANA that was almost universal among SLE patients, the frequencies for SLE diagnostic disorders with details on hematologic and immunologic disorders are tabulated in panel B of Table 1. None of the race-match healthy controls reported to have an autoimmune disease. The IRB of the Nationwide Children's Hospital (USA), the ethics committees from the University of Hong Kong (Hong Kong) and the Chang Gung Memorial Hospital (Taiwan) approved the study. All blood donors provided written consent.

Table 1.

Clinical characteristics of East-Asian SLE patients

Hong Kong Taiwan Combined
Age of onset (yr) 30.27±10.70 30.68±11.62 30.61±11.45
Female/Male (N) 172 / 8 748 / 71 920 / 79
N (yes/no) frequency N (yes/no) frequency N (yes/no) frequency
Malar rash 113 / 67 0.628 467 / 352 0.570 580 / 419 0.581
Discoid rash 39 / 141 0.217 158 / 661 0.193 197 / 802 0.197
Photosensitivity 62 / 118 0.344 189 / 630 0.231 251 / 748 0.251
Oral ulcer 23 / 157 0.128 224 / 595 0.274 247 / 752 0.247
Arthritis 147 / 33 0.817 520 / 299 0.635 667 / 332 0.668
Serositis 35 / 145 0.194 229 / 590 0.280 264 / 735 0.264
Renal Disease 52 / 128 0.289 464 / 355 0.567 516 / 483 0.517
Neurologic disease 11 / 169 0.061 137 / 682 0.167 148 / 851 0.148
Immunologic disorder 150 / 30 0.833 600 / 219 0.733 750 / 249 0.751
Hematologic disease 118 / 62 0.656 598 / 221 0.730 716 / 283 0.717
    leukopenia <3.5K 471 /348 0.575
    hemolytic anemia 263 / 556 0.321
    thrombocytopenia 216 / 602 0.264
    anti-dsDNA 624 / 179 0.771
    anti-RNP-1 295 / 374 0.441
    anti-Sm 262 / 408 0.391
    anti-Ro (Ssa) 362 / 189 0.657
    anti-La (SSb) 148 / 404 0.268
    ACA-IgG 188 / 461 0.290
    ACA-IgM 55 / 544 0.092
    Low C3/C4 641 / 166 0.794
    Pericarditis 101 / 718 0.123
    Pleuralitis 159 / 660 0.194
    Ascites 43 / 776 0.053
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Healthy controls included 765 subjects (396 females and 369 males) from Taiwan, 371 subjects (204 females, 162 males, 5 unknown sex) from Hong Kong, and 211 subjects (122 females and 89 males) from central Ohio, USA.

Determination of C4 gene CNVs

Processing of blood samples for plasma, peripheral mononuclear cells (PBMC) and genomic DNA were as described previously (33). The copy-numbers of total C4, C4A and C4B genes, and long and short C4 genes were determined by TaqMan based, quantitative realtime PCR, and/or “hot-stop PCR” (33-35). Validation for CNV calls were achieved when GCNs of total C4=C4A+C4B. The RCCX haplotypes for the Ohio cohort were further determined by long-range mapping of PmeI and PacI-digested genomic DNA and resolved by pulsed-field gel electrophoresis (PFGE), and TaqI, PshAI/PvuII restriction fragment length polymorphisms (RFLPs).

Phenotyping of complement C4

Protein polymorphisms of C4A and C4B were determined by immunofixation of EDTA-plasma resolved by high-voltage agarose gel-electrophoresis (33,36,37). Ambiguous and intermediate allotypes migrating between C4B and C4A were further resolved by immunoblot analyses using monoclonal antibodies against Rodgers or Chido antigens (38,39). Interpretation of C4A and C4B allotypes were substantiated by their corresponding genotypes.

Autoantibody assays

Autoantibody titers were determined by ELISA. ANA was considered as positive when serum titers were >1:80 by Hep-2 cell assay. Anti-ENA (Ro/SSA, La/SSB, Sm, and RNP) and anti-cardiolipin were assessed by ELISA according to vendor's instructions (Pharmacia Diagnostics).

Statistics

Statistical analyses were performed using JMP Genomics version 6.0 (SAS Institute, Cary, NC) and Prism6 (GraphPad, San Diego, CA) software. Descriptive statistics were displayed as mean ± standard deviation (SD) for normally distributed data, and simple comparisons were made using Student's t-test for continuous data, or by χ2 analysis for categorical data. P-values were derived from Likelihood ratios. Odds ratios (OR) and 95% confidence levels were calculated by analysis of 2×2 tables through Fisher's exact test. For all analyses, p≤0.05 was considered significant.

RESULTS

CNVs of C4A, C4B, long genes, short genes and total C4

CNV of C4A

The number of C4A varied from 0 to 6 copies among different individuals (panel A, Fig. 1; Supporting Information: Table S1). There was a great difference in the distribution of GCN groups for C4A between SLE and controls (p=8.0×10−9). Homozygous deficiency of C4A (GCN=0) was found in nine patients and only one control, which translated into an OR of 12.4 (95% confidence interval, 1.57-97.9) and p=0.0015. The prevalence of heterozygous deficiency for C4A (GCN=1) was greater, 13.8% in SLE and 11.1% in controls, but its effect-size was modest: OR=1.28 (1.00-1.65), p=0.049. The most prevalent GCN group for C4A was two, which had a frequency of 67.8% in SLE and 61.9% in controls. Three and four copies of C4A had frequencies of 13.1% and 1.7%, respectively, in SLE; compared to 20.2% and 4.3%, respectively, in controls. For very high copy-number of C4A (GCN=5 and 6), the frequencies were low but similar between SLE and controls. The mean GCN (±SD) of C4A was 2.09±0.79 in SLE and 2.25±0.82 in controls (p=3.4×10−6, t-test).

Figure 1.

Figure 1

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Variations of complement C4 gene copy-numbers (GCNs) in SLE patients (red) and controls (blue) of East-Asian ancestry. A-E, Distributions of GCN groups for C4A (A), C4B (B), long genes (C), short genes (D), total C4 (E). F. A summary of C4 genetic factors associated with SLE in East-Asians. High GCNs of total C4 and C4A were strong protective factors and deficiency of short genes is a risk factor for SLE.

CNV of C4B

The GCN of C4B varies from 0 to 5 copies among different individuals (panel B, Fig. 1). There was only slight difference in distribution of GCN groups for C4B between SLE and controls (p=0.045). The mean GCN of C4B was 1.85±0.68 in SLE and 1.88±0.71 in controls.

CNV of long-C4 genes

One to six copies of long-C4 genes were present in a diploid genome among different individuals but the distribution patterns of long C4 were similar between SLE and controls (panel C, Fig. 1). It is notable that no subject had an absence and only nine had one copy of long gene in the entire study population. The mean GCN of long-C4 was 2.88±0.79 in SLE and 2.94±0.82 in controls.

CNV of short-C4 genes

Zero to six copies of short-C4 genes was present and the distribution was different between SLE and controls (p=0.0008, χ2 analysis; panel D, Fig. 1). The absence of short gene existed in 29.7% of patients and 22.6% of controls; OR=1.48 (1.22-1.80), p=8.3×10−5. The mean copy-number of short genes was 1.05±0.97 in SLE and 1.17±0.94 in controls (p=0.005, t-test).

CNV of total C4

Two to eight copies of total C4 in a diploid genome were detectable (panel E, Fig. 1; Supporting Information: Table S1). The most prevalent GCN group for total C4 was four. Similar to C4A, there was a consistent shift for increased frequencies of the low and median copy-number groups, and reduced frequencies of the high copy-number groups in SLE (p=3.7×10−7, χ2 analyses). The mean GCN of total C4 was 3.95±0.87 in SLE and 4.14±0.92 in controls (p=8.9×10−8, t-test).

Among the C4 genes, the proportions for C4A and C4B were 53.2%/46.8% in SLE and 54.8%/45.2% in controls (p=0.011). For long and short genes, the proportions were 75.0%/25.0% in SLE; and 73.0%/27.0% in controls (p=0.017). SLE patients had consistent reduction in mean GCNs of total-C4, C4A and short-C4 (Fig 1F, Supporting Information: Table S1). The reduction of GCNs for total C4 or C4A was attributable to a reduction of short genes. The protective effects for high copy-numbers of total-C4 and C4A against SLE were highly significant.

Complement C4 gene CNVs as risk factors for SLE diagnostic disorders

Intra-group analyses of C4 CNVs were performed to investigate their associations with diagnostic disorders of SLE. Three different associations emerged (Table 2).

Table 2.

CNVs of complement C4 as risk factors for SLE diagnostic disorders

Yes No
Disorders Group N (freq.) / GCI N (freq.) / GCI OR (95% CI) p
a. Potentially causal relationship
    Malar Rash C4A = 0 9 (.016) 0 (0) na1 0.0016
C4A ≥ 1 547 (.984) 407 (1.0)
C4A GCI 2.05±.82 2.16±.75 0.034
    Thrombocytopenia C4B = 0 0 (0) 11 (.019) na2 0.0093
C4B ≥ 1 205 (1.0) 564 (.981)
C4B GCI 1.83±.63 1.82±.69 ns
b. Reduced GCN as a risk factor
    Low C3/C4 C4-total = 2-3 183 (.306) 18 (.118) 3.27 (1.94-5.52) 7.0×10−7
C4-total ≥ 4 416 (.695) 134 (.882)
C4-total GCI 3.88±.89 4.16±.83 0.0003
C4B = 0-1 194 (.324) 24 (.158) 2.55 (1.60-4.08) 2.5×10−5
C4B = 2-5 405 (.676) 128 (.842)
C4B GCI 1.77±.67 2.03±.65 1.3×10−5
C4-Long = 1-3 490 (.801) 107 (.686) 1.84 (1.24-2.72) 0.0028
C4-Long = 4-6 122 (.199) 49 .314)
C4-Long GCI 2.87±.78 3.06±.83 0.0084
    Anticardiolipin-IgM C4B = 0-1 23 (.426) 145 (.282) 1.89 (1.07-3.36) 0.032
C4B = 2-5 31 (.574) 370 (.718)
C4B GCI 1.63±.73 1.84±.68 0.029
    Discoid rash C4-Short = 0-1 124 (.821) 475 (.736) 1.64 (1.05-2.58) 0.026
C4-Short = 2-6 27 (.179) 170 (.264)
C4-Short GCI 0.83±.87 1.08±1.03 0.0060
    Arthritis C4-Short = 0 174 (.344) 76 (.261) 1.48 (1.07-2.03) 0.016
C4-Short ≥ 1 326 (.656) 214 (.738)
C4-Short GCI 0.97±.96 1.15±1.06 0.017
c. Increased GCN as a risk factor
    Pericarditis C4A = 3-6 28 (.292) 106 (.154) 2.26 (1.39-3.67) 0.0017
C4A = 0-2 68 (.708) 581 (.846)
C4A GCI 2.27±.79 2.08±.78 0.027
    Hemolytic anemia C4A = 3-6 58 (.234) 76 (.142) 1.84 (1.26-2.70) 0.0019
C4A = 0-2 190 (.766) 459 (.858)
C4A GCI 2.22±.88 2.05±.74 0.0061
    Thrombocytopenia C4-Short = 2-6 67 (.328) 124 (.216) 1.77 (1.24-2.52) 0.0018
C4-Short = 0-1 137 (.672) 449 (.784)
C4-Short GCI 1.27±1.17 0.94±.93 5.8×10−5
C4A = 3-6 46 (.224) 88 (.153) 1.61 (1.08-2.40) 0.022
C4A = 0-2 159 (.776) 489 (.848)
C4A GCI 2.26±.98 2.05±.70 0.0012
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GCI, gene copy-index – the mean of gene copy-number in a population with standard deviation; OR, odds ratio p-values for continuous data were calculated by t-tests; p-values and odds ratios of categorical data were derived by χ2 analyses.

na1, odds ratio not applicable; all nine SLE patients with homozygous C4A deficiency had malar rash; na2, odds ratio not applicable; no subjects with thrombocytopenia had a homozygous C4B-deficiency; ns, not significant

a. Potentially causal relationship

Nine SLE patients had a homozygous C4A-deficiency and all of them had malar rash (p=0.0016). Also, eleven SLE patients had homozygous C4B-deficiency and none of them had thrombocytopenia (p=0.0093). Thus, the absence of C4A could be a causal factor for malar rash, and the presence of C4B might be required for thrombocytopenia.

b. Risks associated with low GCNs

The ever presence of very-low serum complement levels were recorded in 79.4% of SLE patients. Very-low serum complement was strongly correlated with low GCNs of total C4, C4B and long-C4. Low GCN of total C4 had the largest effect-size [OR=3.27 (1.94-5.52), p=7.0×10−7]. The OR for C4B was 2.55 (1.60-4.08), p=2.5×10−5, and for long-C4 was 1.84 (1.24-2.72), p=0.0028. Low GCNs of total C4 leading to very-low serum complement were attributable to low GCNs of C4B, which were likely long genes.

Low GCN of C4B was also associated with the presence of anticardiolipin-IgM [C4B=0 or 1, OR=1.89 (1.07-3.36), p=0.032]. The absence or low copy-numbers of short-C4 were associated with discoid rash and arthritis with moderate effect-sizes [discoid rash: OR=1.64 (1.05-2.58), p=0.026; arthritis: OR=1.48 (1.07-2.03), p=0.016].

c. Risks associated with high GCNs

High GCN of C4A (GCN=3-6) appeared to be a risk factor for pericarditis [OR=2.26 (1.39-3.67), p=0.0017], hemolytic anemia [OR=1.84 (1.26-2.700, p=0.0019] and thrombocytopenia [OR=1.61 (2.08-2.40), p=0.022]. High GCN of short-C4 genes (≥2) appeared as a risk factor of thrombocytopenia [OR=1.77 (1.24-2.52), p=0.0018].

Associations of low serum C3/C4 levels with SLE diagnostic disorders

Besides low GCNs of total C4, C4B or long genes, low serum C3 and C4 levels could also reflect systemic complement consumption or SLE disease states (Table 3). Patients with very-low C3/C4 had an earlier age of disease-onset (mean age ± SD: 30.0±11.0 with low C3/C4 versus 33.3±13.8 without low C3/C4; p=0.0011). Very strong associations with large effect-sizes were found between very-low C3/C4 and (a) immunologic disorders such as the presence of anti-dsDNA [OR: 4.96 (3.41-7.22), p=9.7×10−17], anti-Sm [OR: 2.37 (1.54-3.64), p=3.6×10−5], and IgG-anticardiolipin [OR: 2.33 (1.41-3.87), p=0.0004]; (b) hematologic disorders such as hemolytic anemia [OR: 3.89 (2.42-6.27), p=3.6×10−10] and leukopenia [OR:1.92 (1.36-2.71), p=0.0002]; (c) renal disease [OR: 2.32 (1.63-3.31), p=2.2×10−6]; and (d) serositis including ascites [OR: 3.62 (1.10-11.8), p=0.011] and pericarditis [OR: 2.55 (1.29-5.01), p=0.0027]. Very-low C3/C4 also significantly associated with increased risks of serositis, thrombocytopenia, anti-RNP1, and anti-La/SSb, although their effect-sizes were modest.

Table 3.

Associations of low serum complement protein levels (C4/C3)* with clinical and immunologic features of EA-SLE.

Yes (N=641) frequency No (N=166) frequency OR (95% CI) p
Malar rash 0.577 0.530 ns
Discoid rash 0.184 0.217 ns
Photosensitivity 0.225 0.259 ns
Oral ulcers 0.270 0.295 ns
Arthritis 0.619 0.693 0.08
Neurologic disorder 0.181 0.120 0.055
Immunologic disorders**
    Anti-dsDNA 0.846 0.524 4.96 (3.41-7.22) 9.7×10–17
    Anti-Sm 0.429 0.241 2.37 (1.54-3.64) 3.6×10–5
    Anti-RNP-1 0.474 0.314 1.97 (1.32-2.94) 0.0007
    Anti-Ro/SSa 0.676 0.600 ns
    Anti-La/SSb 0.843 0.748 1.80 (1.10-2.98) 0.016
    Aca-IgG 0.320 0.168 2.33 (1.41-3.87) 0.0004
    Aca-IgM 0.097 0.068 ns
Hematologic disorders
    Hemolytic anemia 0.373 0.133 3.89 (2.42-6.27) 3.6×10–10
    Leukopenia ≤3.5k 0.607 0.446 1.92 (1.36-2.71) 0.0002
    Thrombocytopenia 0.281 0.206 1.50 (0.99-2.27) 0.048
Renal disease 0.608 0.416 2.18 (1.54-3.09) 8.5×10–6
Serositis
    Ascites 0.062 0.018 3.62 (1.10-11.8) 0.011
    Pericarditis 0.140 0.060 2.55 (1.29-5.01) 0.0027
Age of onset (yrs±SD) 29.98±10.95 33.29±13.75 0.0011
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*

Low C3 was defined as a documentation of serum C3 concentration <700 mg/L; low C4 was defined as a documentation of serum C4 concentration <100 mg/L.

**

As proposed by SLICC in 2012, low C4/C3 is one of the diagnostic factors for an immunologic disorder in SLE.

ns, not significant

A specific Asian haplotype with C4B96 and C4A-deficiency

Immunochemical analyses of EDTA-plasma from four SLE patients with homozygous C4A deficiency and one patient with both C4A and C4B present were shown in panels A and B of Figure 2. Immunofixation experiment revealed that three out of four C4A-deficient patients possessed the slowest-migrating C4 allotype, C4B96, in addition to C4B1. The fourth C4A-deficient patient had C4B1 only (panel A). Immunoblot experiment revealed that C4B96 and C4B1 (and C4A1) but not C4A3 were associated with Chido blood-group antigens (panel B) (38,39). C4A-deficient patients had two to four copies of C4 genes in a diploid genome and they all coded for C4B protein. Those C4 genes either existed in (a) monomodular RCCX haplotype with a single long gene coding for C4B1, or (b) bimodular RCCX coding for C4B1-B96 or C4B1-B1. The limited accessibility of patient samples restricted further characterization of homozygous C4A deficiency.

Figure 2.

Figure 2

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C4A-deficiency in East-Asians. A. Immunofixation of C4 allotypes showing the homozygous deficiency of C4A protein in four SLE patients (H097, H440, H288 and H288) and one control (H411). B. Immunoblot analysis of C4 plasma protein from the same subjects shown in panel A using monoclonal antibodies against Chido antigens that is generally associated with C4B protein. CH, Characterization of C4B96 protein by immunofixation (C), RCCX haplotypes by long range mapping of PmeI-digested genomic DNA (D), TaqI RFLP of RP-C4-CYP21-TNX modules (E), PshAI-PvuII RFLP for relative dosage of C4B and C4A genes (F) and rearrangement of RCCX in Subject-89 (G). A red-arrow indicates the presence of a TNXA-XB recombinant characterized by a 3.5 kb PshAI restriction fragment after Southern blot analysis using a TNX 3’ probe. H. DNA and amino acid sequences specific for C4B96 highlighting the E920K polymorphism with EcoRI RFLP. I. EcoRI RFLP of genomic PCR fragments from six subjects with C4B96 (lanes 1 and 3-7) and one subject without C4B96 (lane 2). J. The location of E920K in 3-dimensional structure of native C4 protein (indicated by a red arrow) (44). X-ray crystal structure of C4 was downloaded from RCSB PDB (http://www.rcsb.org/pdb/home/home.do; entry “4FXK”, and visualized by PyMOL).

Seven subjects in our American EA study-cohort contained C4B96 and Subject-89 was chosen for detailed analysis because of its relative simplicity (panels C-G, Fig. 2). PmeI-PFGE and TaqI-RFLP revealed that Subject-89 had heterozygous RCCX haplotypes with bimodular-LS and monomodular-L (panels D and E), and a total of two C4B and one C4A (panel F). The LS haplotype coding for C4B1-B96 was a recombinant between tenascin TNXB and TNXA, as characterized by the absence of pseudogene steroid 21-hydroxylase CYP21A, and the presence of an 120-bp insertion in TNXA, or XA+120, as documented previously (Supporting Information: Fig. S2) (40-42). Examinations of other Asian subjects with C4B96 revealed that they all shared the same bimodular-LS haplotype encoding C4B1-B96, with two CYP21B (no CYP21A) and the presence of an XA+120 recombinant. The coding sequences of C4 genes in Subject-89 were amplified, sequenced to completion and compared with known C4 sequences (Supporting Information: Figs. S2-S4; Table S2) (39,43). We identified a novel, non-synonymous G→A nucleotide change at exon 21 from the short gene coding for C4B96, which attributed to the E920K polymorphism. The negatively-charged glutamic acid-920 was changed to the positively-charged lysine-920 in C4B96. This basic residue is located at the MG7 domain of the C4 protein structure (panel J, Fig. 2) (44). The DNA sequence for K920 ablated a restriction site for EcoRI (GAATTC→AAATTC; panel H, Fig. 2). Thus, 1.7 kb PCR fragments spanning from intron 20 to exon 26 of C4B were generated from six subjects with C4B96 plus one control without this allotype. The PCR products were digested with EcoRI resolved by electrophoresis. All subjects with B96 displayed the 1.7 kb fragment in addition to the 1.5 kb from C4B1 gene (lanes 1 and 3-7, panel I). C4 protein allotyping and/or EcoRI RFLP of PCR-amplified DNA revealed that 75% of EA-SLE with homozygous C4A-deficiency contained C4B96. HLA-DRB1 genotyping revealed that all subjects with C4B96 had DRB1*1501 (DR2).

DISCUSSIONS

The phenomenon of common CNVs is gaining appreciation but their impacts on rheumatic diseases among different racial groups await accurate and in-depth investigations. Here we report the continuous variation in GCN of human complement C4 and polymorphisms for C4A, C4B, long genes and short genes in association with SLE disease susceptibility and clinical manifestations, and compare them to those of European Americans.

In East-Asians, homozygous deficiency of C4A was present only in ~1% of SLE patients but it had a very large effect-size on disease susceptibility (OR=12.4). Heterozygous C4A-deficiency and low GCNs for total C4 were more prevalent but their effects on SLE disease risk were modest (OR=1.28 and 1.45, respectively). On the other end, the protective effects for high copy-numbers C4A or total C4 against SLE were conspicuous and highly significant. Over ¼ of healthy controls had high copy-numbers of total C4 or C4A, and their frequencies were reduced in SLE (p=8.8×10−8 and 4.0×10−9). Overall, there were reductions in SLE on the mean copy-numbers of total C4 by 0.19, C4A by 0.15 and short genes by 0.12.

Compared with subjects of European ancestry, East-Asians have significantly higher GCNs of total C4 and its associated variants (Table 4, Supporting Information: Fig. S5). In European-SLE, homozygous and heterozygous deficiencies of C4A, low GCNs of total C4 and long genes were common and medium-to-high effect-size risk factors of SLE (25). Notably, 6.5% of had a homozygous C4A deficiency (OR=8.57), 27.6% had a heterozygous C4A deficiency (OR=1.97), 42.2% had low GCNs of total C4 (OR=1.77). The variability of total C4 or C4A in Europeans was driven by changes in copy-number of long genes instead of short genes as in East-Asians. Remarkably, 13.9% of European SLE patients had zero or one copy of long genes, which were infrequent in Asians (p=2.9×10−19, Supporting Information: Fig. S5, panel H).

Table 4.

CNVs of complement C4 as a risk or protective factor in human SLE: a comparison between East-Asian and European-American.

East-Asian European-American*
Total C4
    SLE - GCI: 3.95 ± 0.87 3.56 ± 0.78
    Controls - GCI: 4.14 ± 0.92 3.83 ± 0.69
        difference −0.19 −0.27
        p 3.7×10–7 3.6×10–6
        C4T=2+3, OR: 1.45 (1.20-1.77) 1.77 (1.28-2.45)
        C4T=5-8, OR: 0.55 (0.45-0.68) 0.53 (0.30-0.94)
C4A
    SLE - GCI: 2.09 ± 0.79 1.80 ± 0.90
    Controls - GCI: 2.25 ± 0.82 2.09 ± 0.75
        difference −0.16 −0.30
        p 3.4×10–6 3.4×10–6
        C4A=0, OR: 12.4 (1.57-97.9) 8.57 (2.81-26.1)
        C4A=1, OR: 1.28 (1.00-1.65) 1.97 (1.36-2.85)
        C4A≥3, OR: 0.58 (0.47-0.71) 0.55 (0.37-0.83)
C4B
    SLE - GCI: 1.85 ± 0.68 1.78 ± 0.59
    Controls - GCI: 1.88 ± 0.71 1.73 ± 0.63
        difference −0.03 −0.04
        p ns ns
Long C4
    SLE - GCI: 2.88 ± 0.79 2.63 ± 1.16
    Controls - GCI: 2.94 ± 0.82 2.95 ± 0.98
        difference −0.06 −0.31
        p ns 0.0002
        C4L≤2, OR: na 1.66 (1.26-2.18)
Short C4
    SLE - GCI: 1.05 ± 0.97 0.98 ± 0.85
    Controls - GCI: 1.17 ± 0.94 0.89 ± 0.79
        difference −0.12 0.09
        p 0.005 ns
        C4S=0, OR: 1.48 (1.22-1.80) na
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*

European-American data derived from White Ohio healthy subjects (N=500) and 232 White Ohio SLE patients (25).

na, not applicable; ns, not significant. GCI: gene copy-index - the mean of gene copy-number (± standard deviation).

The main cause for C4A-deficiency among Europeans is the presence of mono-S coding for C4B1 and the absence of a long C4A gene in haplotypes with HLA-DRB1*0301. This mono-S haplotype has a frequency of 0.113 in healthy subjects and 0.169 in SLE (25). Strikingly, in our study cohort >2000 Asian subjects, none had a homozygous mono-S (which is a homozygous deficiency of long genes). A new and predominant mechanism for C4A-deficiency in Asians unraveled in this study is a bimodular-LS haplotype coding for C4B1-B96 with markers characteristic of an ancient recombination and is linked to DRB1*1501 (or DR2).

Among SLE patients who ever experienced very-low serum levels of C4 and C3, low GCNs of total C4, C4B and long genes were major risk factors. Low GCN of total C4 would result in lower rate of biosynthesis and therefore a lower reservoir of C4 protein. During an active disease, high reactivity and high turnover of activated C4B protein would lead to fast depletion and therefore very low levels complement. Very-low serum complement levels were also strongly correlated with the presence of dsDNA autoantibodies, IgG-anticadiolipin, hemolytic anemia, renal disease and younger age of disease onset. The association between low C3/C4 and the presence of anti-dsDNA was remarkably strong, with OR=4.96 and p=9.7×10−17. Renal disease occurred in greater than half of East-Asian SLE patients. Among SLE patients with low GCN of total C4 (GCN=2 or 3), low C3/C4 protein levels had greater effects on the occurrence of renal disease [OR=3.95 (1.37-11.4), p=0.0067] than those with medium and high GCNs of total C4 [OR=1.45 (0.98-2.16), p=0.064]. Associations of low complement with renal disease and/or hematologic disease of SLE were also observed earlier in US patients (9,45). Other serologic factors that correlate with renal disease of SLE are anti-dsDNA and anti-C1q. In a recent multicenter study, it was found that simultaneous positivity of anti-C1q, anti-dsDNA and low complement associated with renal SLE with a combined odds ratio of 14.9 (5.8-38.4) (46). Measuring serum levels of C3 and C4 and anti-C1q, cell-bound levels of processed complement (e.g., erythrocyte-C4d) (47), and CNV genotyping of C4A and C4B are desirable laboratory tests to facilitate precision management of SLE and minimize renal disease.

The co-existence of C4A-deficiency with HLA risk alleles DRB1*0301 in Europeans and DRB1*1501 in East-Asians in SLE and other autoimmune diseases continue to be a fascinating topic on whether C4-CNVs or C4A-deficiency and DRB1 genetic variants are independent, additive or confounding risk factors (4,48-50). The pathologic effects for genetic and/or acquired deficiencies of C4, C1q, C1r and C1s on SLE have been overwhelming (4). Thus, it would be appropriate to investigate the feasibility of complement-guided therapeutics for SLE.

Supplementary Material

Supplementary information

ACKNOWLEDGMENTS

We are indebted to volunteer blood donors and SLE patients for participation in this study. We are grateful to Jeannie Shaw (Columbus OH) for helps in recruiting healthy subjects and the Hong Kong Red Cross for sharing expired blood samples. This work was supported in part by the National Institute of Arthritis and Musculoskeletal and Skin Disease of the National Institutes of Health grant 1R01 AR054459 (CYY), Institutional grant from the Research Institute of the Nationwide Children's Hospital, Columbus Ohio (CYY), Ministry of Science and Technology grant 101-2314-B-182A-062-MY3 (JYC), and Chang Gung Memorial Hospital Grant CMRPG3E0531 (JYC).

Footnotes

AUTHOR CONTRIBUTIONS

JYC, YLW, EKC, YY, CSL and CYY designed project. JYC, CSL, YLL, CHS, YJJW, YY and MYM performed diagnosis and clinical studies. JYC, YLW, MYM, YY, YJJW, CMW, EKC, KEL, BZ, KJ, CHS, YLL, CSL and CYY recruited study subjects. YLW, YJJW, KEL, CMW, EKC, YY, BZ, HW, DY, AA, HNN, JYC and CYY performed experiments and data analyses. CYY, JYC, YLW, EKC and YY drafted the manuscript. All authors read and approved the manuscript.

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