Aims Considering the phenotypical consequences and association of C4 copy number variation (CNV) with various autoimmune diseases, we aimed to examine C4 CNVs for 1027 patients with Vogt-Koyanagi-Harada (VKH) syndrome and 2083 controls.
Methods C4 CNVs were examined by real-time PCR for 1027 patients with VKH and 2083 controls. Peripheral blood mononuclear cells (PBMC) were prepared from venous blood by Ficoll-Hypaque density-gradient centrifugation for cell culture. Cytokine production was examined by ELISA.
Results The expression of total C4 in serum was significantly decreased in patients with VKH as compared with controls (p=0.0010). A significant positive association between C4 expression with C4 CNVs was found (p=0.0023, r2=0.92). CNV analysis identified significantly decreased frequencies of more than two copies of C4A or more than four copies of total C4 in patients with VKH (Pc=1.42×10−3 to 3.56×10−4, OR=0.67 to 0.70). Linkage analysis showed the independent association of C4 with VKH syndrome from human leucocyte antigen (HLA)-DR4. No significant association was observed concerning type 1 T helper cell (Th1) cytokines and Th17 cytokine production by stimulated PBMCs and C4A copy number.
Conclusions Our findings indicate a decreased expression of serum C4 and a decreased frequency of high C4 gene copy number in patients with VKH.
Trial registration number Chinese Clinical Trial Registration Number: ChiCTR-CCC-12002184.
Statistics from Altmetric.com
Vogt-Koyanagi-Harada (VKH) syndrome is an autoimmune disorder characterised by bilateral granulomatous panuveitis frequently associated with systemic involvement including vitiligo, alopecia, poliosis, auditory and central nervous system.1 It primarily affects certain ethnic populations, such as Native Americans and Asians, and is rare in Caucasians.1 ,2 Although its exact aetiology and pathogenesis remain unclear, VKH syndrome is believed to be an autoimmune disease directed against melanocytes.3
The complement system has been shown to be involved in the development of certain ocular inflammatory diseases.4 Previous studies have shown that certain genetic variants of the complement system are associated with intraocular inflammatory diseases, such as uveitis5 and age-related macular degeneration.6 Wakefield et al5 were the first to observe a significantly increased frequency of the C4B2 allotype in patients with anterior uveitis. Complement component C4 (C4) deficiency has been shown to be associated with systemic lupus erythematosis (SLE),7 rheumatoid arthritis (RA)8 and juvenile idiopathic arthritis.9 The genes encoding C4 are located on chromosome 6q21.3 and include a C4A (Mendelian Inheritance in Man (MIM) 120810) and C4B (MIM 120820) isoform.10 Both isoforms show gene duplication, and individuals can inherit between 0 and 5 copies.11 C4 gene copy number variants confer risk for various autoimmune disorders including SLE,12 RA13 and Grave's disease.14 Whether C4 copy number variations (CNV) are also associated with VKH syndrome has not yet been reported and was therefore the subject of the study presented here. We analysed C4 CNVs in VKH syndrome in two Han Chinese populations. Our findings provide further support for a role of C4 in the pathogenesis of intraocular inflammation.
Materials and methods
Recruitment of patients and normal controls
A total of 500 patients with VKH and 647 normal controls were recruited from the Zhongshan Ophthalmic Center, Sun Yat-sen University (Guangzhou, China) (Guangdong cohort; table 1). The replication study included a total of 527 patients with VKH and 1436 healthy individuals recruited from the First Affiliated Hospital of Chongqing Medical University (Chongqing, China) (Chongqing cohort; table 1). The diagnosis of VKH syndrome was based on the revised diagnostic criteria 2001 for VKH syndrome.15 If the diagnosis was in doubt, the patients were excluded from the present study. The normal controls enrolled in this study were unrelated healthy individuals without any autoimmune disorder and intraocular disease. They were age-matched and ethnicity-matched with the patients with VKH. All study participants were Han Chinese and gave written informed consent. The present study adhered to the tenets of the Declaration of Helsinki and was approved by the Ethics Committee of our hospitals (Permit Number: 2009-201008).
Measurement of serum complement component C4 by ELISA
Blood was collected, allowed to clot, centrifuged and serum was stored at −80°C until used. The C4 level was measured using a human ELISA kit (Cusabio Biotech, Wuhan City, Hubei province, China) according to the manufacturer's instructions. Fifty normal controls were enrolled in the present study to evaluate the relationship of serum C4 levels and C4 CNV (see online supplementary table S1).
Genomic DNA extraction
Genomic DNA was extracted from peripheral blood using the QIAGEN QIAamp DNA Mini Blood Kit (Hilden, Germany) according to the manufacturer's instructions.
Analysis of complement C4 gene CNVs
C4 gene copy number was determined by TaqMan real-time PCR and performed in 96-well optical plates on a 7500 real-time PCR system following the manufacturer's protocols (Applied Biosystems, Foster City, California, USA). TaqMan assays labelled with fluorescein amidite were used to detect C4A (Hs07226349-cn, Overlaps Exon 26-Intron 26) or C4B (Hs07226350-cn, Overlaps Exon 26-Intron 26), respectively (Applied Biosystems, Foster City, California, USA). The TaqMan Copy Number assay was normalised using an endogenous reference gene (RNAseP) known to be present in two copies (Applied Biosystems, Foster City, California, USA). Three replicates were performed per sample to measure the C4 gene copy number.
Cells isolation and culture
Peripheral blood mononuclear cells (PBMC) were prepared from venous blood by Ficoll-Hypaque density-gradient centrifugation. Isolated PBMCs were resuspended at a concentration of 1×106 cells/mL in Roswell Park Memorial Institute 1640 complete medium supplemented with 10% fetal calf serum, 2 mM L-glutamine and 100 U/mL penicillin/streptomycin (Invitrogen, Carlsbad, California, USA). PBMCs were treated with lipopolysaccharide (100 ng/mL, Sigma, Missouri, USA) for 24 h to stimulate tumour necrosis factor (TNF)-α secretion in the culture supernatants. To stimulate interleukin (IL)-17A, IL17F and interferon (IFN)-γ production, PBMCs were treated with a combination of anti-CD3 (OKT3, 0.5 μg/mL) and anti-CD28 antibodies (15E8, 0.1 μg/mL) (Miltenyi Biotec, Palo Alto, California, USA) for 72 h.
The real-time PCR data were analysed by manufacturer supplied software, V2.0.6 (Applied Biosystems, Foster City, California, USA). Relative gene copy numbers were examined by the comparative CT method using CopyCaller, V.2.0 (Applied Biosystems, Foster City, California, USA). The number of total C4 genes equals the number of C4A plus C4B gene copies in an individual.16 The differences in total C4, C4A, C4B were compared between patients and controls by the χ2 test or Fisher's exact test using SPSS (V.17.0; SPSS, Chicago, Illinois, USA).The serum C4 concentration between groups was analysed by independent samples t test using SPSS (V.17.0; SPSS, Chicago, Illinois, USA). To account for multiple testing, the Bonferroni correction was applied.
Clinical features of patients with VKH syndrome
The distribution of demographic characteristics and clinical characteristics of the enrolled patients with VKH were assessed at the time of diagnosis and are summarised in table 1.
Downregulated serum C4 levels in patients with VKH syndrome
We investigated serum C4 levels in patients with VKH. Serum was collected from 29 patients with VKH and 30 healthy controls, and the concentration of C4 in serum was assessed by ELISA. C4 was significantly decreased in patients with VKH as compared with normal controls (patients vs controls (mean±SD): (120.9±42.9 vs 180.3±82.3) (p=0.0010); figure 1).
The relationship between serum C4 levels and C4 CNV
We also analysed the relationship between serum C4 levels and C4 CNV, in a group of healthy controls in whom we determined the C4 CNV status. Serum from 50 healthy controls was randomly collected and serum C4 was assessed by ELISA according to the manufacturer's instructions (see online supplementary table S1). Subdivision of these groups showed a significant positive association between serum C4 levels with the C4 gene copy numbers (p=0.0023, r2=0.92; figure 2).
C4 gene copy numbers in patients with VKH syndrome
C4A and C4B are encoded by different genes whereby each may show gene duplication. A null gene is also possible. We assessed the number of gene copies for each individual for either C4A or C4B and also added the gene copies for these two genes per individual. This total C4 copy number can be due to various combinations of either C4A or C4B gene copy numbers. The results showed that the frequency of patients with more than two copies of C4A or more than four copies of both the C4A and C4B genes were significantly decreased in patients with VKH syndrome as compared with normal controls in the Guangdong cohort (p=5.64×10−4 to 2.31×10−4, OR=0.59 to 0.61; table 2). A decreased frequency of carriers with more than two copies of C4A was found in VKH in the Chongqing cohort (p=1.70×10−3, OR=1.43; table 2). Combining the data of the aforementioned two VKH cohorts (Guangdong and Chongqing cohorts) showed a decreased frequency of carriers with more than two copies of C4A, or more than four copies of both C4A and C4B (Pc=1.42×10−3 to 3.56×10−4, OR=0.67–0.70; table 2). Furthermore, the frequency of carriers having two copies of C4A, C4B or four copies of both C4A and C4B was increased in patients with VKH syndrome (Pc=5.51×10−3 to 8.07×10−5, OR=1.36–1.47).
Previous reports showed that human leucocyte antigen (HLA)-DR4 was strongly associated with VKH in Han Chinese.17 ,18 Since HLA-DR4 and C4 are located on the same chromosome, we also constructed a linkage block for single nucleotide polymorphisms in C4 gene and HLA-DR4 using the 1000 genome data from the Chinese Han population. The result showed that the association of C4 with VKH syndrome is independent with its relation to HLA-DR4.
The influence of copy number variants of C4A on cytokine production
A number of studies conducted in recent years have indicated that type 1 T helper cell (Th1) cytokines such as TNF-α, IFN-γ and Th17 cytokines including IL-17A and IL-17F play important roles in the pathogenesis of VKH syndrome.19 Further experiments were therefore designed to investigate whether the different CNVs of C4A affected the production of the aforementioned cytokines (figure 3). The results revealed no significant association between TNF-α, IFN-γ, IL-17A and IL-17F production by stimulated PBMCs and C4A copy number (figure 3A–D).
The present study shows that serum C4 levels were decreased in patients with VKH syndrome compared with normal controls and that having more than two copies of C4A or more than four copies of both the C4A and C4B gene, significantly decreased the risk of VKH syndrome. Furthermore, we found a positive relationship between serum C4 concentration and C4 copy number variants. These results suggest that a genetically controlled high C4 level protects an individual from developing VKH syndrome, whereby the actual manifestation of the disease may depend on as yet unknown environmental triggers. We did not detect an effect of C4 CNVs on the production of cytokines including TNF-α, IFN-γ, IL-17A and IL-17F.
Complement component C4 serves important roles in the clearance of infectious microorganisms, apoptotic cells, cellular debris and immune complexes, as well as in the regulation of T and B lymphocyte activation.20 Our findings are in agreement with earlier observations concerning C4 CNV in other autoimmune diseases such as SLE,11 despite an inconsistent result reported by Boteva and colleagues.12 Animal models have shown that C4 deficiency leads to spontaneous SLE in mice21 but not in guinea pigs.22 SLE is a typical immune complex mediated disease, whereas a role of immune complexes in VKH has not yet been shown. Previous studies have shown evidence for autoantibody involvement in both VKH syndrome and SLE, such as antibodies directed against uveal antigens or against outer segments of photoreceptors and Müller cells23–25 in VKH syndrome and anti-nuclear antibodies and anti-dsDNA antibodies in SLE.26 ,27 As mentioned above, C4 may also be involved in T and B lymphocyte activation, and its exact role in VKH remains to be determined. Collectively, these data suggest that C4 deficiency may act as a common risk factor for autoimmune diseases including VKH syndrome and SLE although the precise mechanisms may differ between these diseases. Previous results from our group showed an increased expression of C4 and an association between high copy number variants of C4A and the occurrence of ocular Behcet's disease, another common uveitis entity seen in China.28 The discrepancy between C4 CNV and susceptibility to these two uveitis entities may be due to the fact that VKH syndrome is mediated by an autoantibody response against melanocytes and is thus a good example of an autoimmune disease,29 whereas Behcet's disease is considered an autoinflammatory disease due to an aberrant response against invading viruses or microbes.30 In our study, we also examined the C4 concentration in Chinese normal controls. The range of the serum C4 concentration in the present study (50–400 μg/mL) is similar to that reported by other groups.31 Additionally, a positive relationship between serum C4 concentration and C4 CNV was also found. This finding is in accordance with earlier studies that showed a positive relationship between C4 CNV and the serum level of this protein.31 We are aware of the fact that our study has some limitations. The patients were recruited from Chinese Han populations and may represent a selected population of patients. Although we have a very high number of patients included in our study, the sample size was not yet large enough to study whether stratification of our patients according to their disease manifestations might reveal a special association of a subgroup of patients with C4 CNV. Furthermore, confirmation of our data is needed by investigating patients with VKH from other ethnic cohorts. C4 genes are part of an extended haplotype involving several genes that may contribute to, or be responsible for, the increased frequency of VKH syndrome. Linkage analysis studies involving HLA-DR4 showed that the observed association with C4 CNV was independent of this gene that also resides on chromosome 6. Further studies are needed to elucidate the exact role of C4 and the possible contribution of adjacent genes in the development of VKH syndrome.
In conclusion, our results indicate that a high copy number of C4 protects against susceptibility to VKH syndrome in a Han Chinese population and suggests that C4 may play a role in the pathogenesis of this disease.
The authors would like to thank all donors enrolled in the present study. This work was supported by Natural Science Foundation Major International (Regional) Joint Research Project (81320108009), Key Project of Natural Science Foundation (81130019), National Natural Science Foundation Project (31370893, 81270990), Basic Research program of Chongqing (cstc2013jcyjC10001), Chongqing Key Laboratory of Ophthalmology (CSTC, 2008CA5003), National Key Clinical Specialties Construction Program of China, Fund for PAR-EU Scholars Program and The Youth Talent Support Plan of Chongqing.
This web only file has been produced by the BMJ Publishing Group from an electronic file supplied by the author(s) and has not been edited for content.
Files in this Data Supplement:
- Data supplement 1 - Online supplement
Contributors Experimental design: SH and PY. Sample collection: SH, JQ and DL, Experiments: SH, JQ, DL and LC. Data analysis: JQ, DL and JF. Paper writing: SH, AK and PY.
Competing interests None.
Patient consent Obtained.
Ethics approval Ethics approval was provided by the Ethics Committee of The First Affiliated Hospital of Chongqing Medical University (Permit Number: 2009-201008).
Provenance and peer review Not commissioned; externally peer reviewed.
If you wish to reuse any or all of this article please use the link below which will take you to the Copyright Clearance Center’s RightsLink service. You will be able to get a quick price and instant permission to reuse the content in many different ways.