Small ubiquitin-like modifier 4 (SUMO4) polymorphisms and Vogt-Koyanagi-Harada (VKH) syndrome in the Chinese Han population.

Purpose To examine whether small ubiquitin-like modifier 4 (SUMO4) polymorphisms were associated with Vogt-Koyanagi-Harada (VKH) syndrome in the Chinese Han population. Methods Genotyping for SUMO4 polymorphisms at G-847A, A-504G, A+163G, and C+438T loci was performed on 231 VKH patients and 302 controls using polymerase chain reaction restriction fragment length polymorphism. Results A decreased frequency of SUMO4 +438 TT genotype was found in VKH patients compared with healthy controls (p=0.009). However, the significance was lost after Bonferroni correction. Human leukocyte antigens (HLA)-DR4 and HLA-DRw53 were significantly associated with susceptibility to VKH syndrome (p=3.21×10−16 and 7.08×10−5, respectively). Stratification analysis based on HLA-DR4 and HLA-DRw53 did not show any associations between SUMO4 polymorphisms and VKH syndrome, although there was a big difference in the percentage of certain allele and genotype frequencies between HLA-DRw53 negative patients and controls. There was no significance in clinical findings and gender stratification analysis. Conclusions HLA-DR4 and HLA-DRw53 are strongly associated with the susceptibility to VKH syndrome in the Chinese Han population. However, none of the currently known single nucleotide polymorphisms (SNPs) of SUMO4 are associated with this syndrome.

Guangzhou, P.R. China approved this study, and informed consent was obtained from all tested subjects. DNA extraction: Genomic DNA samples were extracted and isolated from ethylene diamine tetraacetic acid (EDTA) anticoagulated peripheral blood mononuclear cells (PBMCs) of VKH patients and healthy controls by a conventional salting out method. These DNA samples were diluted in PCR grade water and stored at −70 °C until used.
Statistical analysis: The χ 2 test was applied to analyze the Hardy-Weinberg equilibrium (HWE). The χ 2 test or Fisher's exact test was performed to compare the allelic, genotypic, and haplotypic distribution between VKH patients and healthy controls using version 12.0 of SPSS for Windows (SPSS Inc., Chicago, IL). Analysis of linkage disequilibrium (LD) of each SNP and haplotype was performed using the Haploview v3.32 program [28,29]. The p values were corrected using the Bonferroni correction to account for multiple testing. Sample sizes were estimated by Quanto 1.2 software (Department of Preventive Medicine, University of Southern California, Los Angles, CA).

Descriptive data of the tested patients and controls:
Detailed clinical findings of the enrolled VKH patients are shown in Table1. The average age of the VKH patients was 33.6±12.4 years and that of healthy controls was 35.4±12.0 years. No statistical difference was observed between VKH patients and controls in the distribution of age and gender (p>0.05).
Single nucleotide polymorphism and haplotype analyses between polymorphisms of SUMO4 and Vogt-Koyanagi-Harada syndrome: The distribution of genotype for each SNP including G-847A, A-504G, A+163G, and C+438T did not deviate from the HWE in VKH patients and healthy controls (p>0.05). A decreased frequency of SUMO4 +438 TT genotype was observed in VKH patients compared with healthy controls (p=0.009, χ 2 =9.36). However, it did not remain significant after Bonferroni correction ( Table 2).
Stratification analysis of SUMO4 polymorphisms with the status of HLA-DR4, HLA-DRw53, the clinical findings, and gender: Our study showed that the frequency of HLA-DR4  Table 5).
Stratification analysis was also performed according to clinical findings including neck stiffness, tinnitus, alopecia, poliosis, dysacusis, scalp hypersensitivity, and vitiligo. No association was found between the four SNPs and any extraocular findings. The analysis of gender stratification also showed no association of SUMO4 polymorphisms with VKH syndrome.

DISCUSSION
In this study, we examined the association of SUMO4 polymorphisms with VKH syndrome in the Chinese Han population. Our results failed to find an association between SUMO4 polymorphisms and VKH syndrome even after stratification for HLA-DR4, HLA-DRw53, clinical features, and gender.  SUMO4 has been shown to be involved in the regulation of NF-кB, an important transcription factor in autoimmune diseases. It has been reported that the SUMO4 A+163G (M55V) polymorphism is an essential polymorphism involved in regulating its own sumoylation, and it has been shown to be associated with certain autoimmune diseases such as type 1 diabetes, autoimmune thyroid disease, and rheumatoid arthritis without amyloidosis [19]. These results suggest that this polymorphism could be a susceptibility gene shared by certain autoimmune diseases, although conflicting data have been reported in Sjögren's syndrome [19]. The identification of a general susceptibility gene for several autoimmune diseases could make an important contribution to the understanding of the pathogenesis and modulation of these diseases. The question was therefore raised whether the SUMO4 A+163G polymorphism was also associated with VKH syndrome. This study was designed to clarify this issue. We strictly chose the patients who were definitely diagnosed with VKH syndrome according to the revised criteria [30] to exclude the influence of misdiagnosis. As ethnic confounding could also influence the association results, only VKH patients with Chinese Han nationality as well as age-and sexmatched controls with the same nationality were enrolled in this study. The frequency of the +163G allele in the control population presented in our study is similar with that in the Chinese population reported by Li et al. [31] and in the Japanese population reported by Noso et al. [20]. Meanwhile, a power analysis of the study population showed that our sample size was large enough to detect a possible association. Unexpectedly, we failed to find an association of the SUMO4 A+163G polymorphism with VKH syndrome. This suggests that this polymorphism may not be involved in the development of susceptibility to VKH syndrome.
Others SNPs including G-847A, A-504G, and C+438T polymorphisms have been identified by direct sequencing of the whole SUMO4 gene in the Japanese population [20,32]. Our previous results showed an association of SUMO4 C +438T polymorphism with Behcet's disease [22], another common uveitis entity observed in China. The present study also failed to show any association of the SUMO4 G-847A, A-504G, and C+438T polymorphisms with VKH syndrome. This difference may result from the different features of these two uveitis entities. One of the striking features of Behcet's disease is its characteristic non-granulomatous inflammation while VKH syndrome is in fact a granulomatous inflammation [33].
Like HLA-DR4, HLA-DRw53 have been demonstrated to be strongly associated with VKH syndrome. Therefore, genotyping of HLA-DR4 and HLA-DRw53 was performed. The association of HLA-DR4 and HLA-DRw53 with VKH syndrome was extremely strong in this study. The results were generally consistent with those previously reported in Chinese [4,8,34] and Spanish patients [35]. Furthermore, stratification analysis according to HLA-DR4 and HLA-DRw53 did not show any association of SUMO4 with VKH syndrome in our study. This result is consistent with the previous studies that

SNPs
Genotype allele

HLA-DR4+
It is worthy to point out that there was a big difference in the percentages of certain alleles and genotypes between HLA-DRw53 negative patients and controls, although the difference did not reach statistical significance. As the sample size of HLA-DRw53 negative is small (28 patients), it is necessary to further test the association of SUMO4 polymorphisms with HLA-DRw53 negative patients using larger samples.
In conclusion, we failed to detect an association of SUMO4 polymorphisms with VKH syndrome in Chinese Han population. In agreement with earlier studies, we found a strong association of HLA-DR4 and HLA-DRw53 with susceptibility to VKH syndrome. A big but insignificant difference of allele and genotype frequency was noted in HLA-DRw53 negative patients when compared with HLA-DRw53 negative controls. Further studies are necessary to elucidate the association of SUMO4 polymorphisms with VKH syndrome in a HLA-DRw53 negative population using larger samples.