|Year : 2018 | Volume
| Issue : 6 | Page : 643-647
Variant of EOMES Associated with Increasing Risk in Chinese Patients with Relapsing-remitting Multiple Sclerosis
Sheng Chen1, Juan Zhang2, Qi-Bing Liu3, Jing-Cong Zhuang3, Lei Wu2, Yong-Feng Xu2, Hong-Fu Li2, Zhi-Ying Wu2, Bao-Gou Xiao1
1 Department of Neurology, Huashan Hospital, Fudan University, Shanghai 200040, China
2 Department of Neurology and Research Center of Neurology, Second Affiliated Hospital, Key Laboratory of Medical Neurobiology of Zhejiang Province, Zhejiang University School of Medicine, Hangzhou 310009, Zhejiang, China
3 Department of Neurology and Institute of Neurology, First Affiliated Hospital, Fujian Medical University, Fuzhou 350005, Fujian, China
|Date of Submission||12-Nov-2017|
|Date of Web Publication||9-Mar-2018|
Dr. Bao-Gou Xiao
Department of Neurology, Huashan Hospital, Fudan University, Shanghai 200040
Source of Support: None, Conflict of Interest: None
Background: Multiple sclerosis (MS) is a common central nervous system autoimmune disorder. Increasing number of genome-wide association study (GWAS) analyses hint that MS is strongly associated with genetics. Unfortunately, almost all the GWAS analyses were Caucasian population based. Numbers of risk loci might not be replicated in Chinese MS patients. Hence, we performed a MassArray Assay to genotype the previously reported variants located in the transcription regulation genes in order to elucidate their role in the Chinese MS patients.
Methods: One hundred and forty-two relapsing-remitting MS (RRMS) patients and 301 healthy controls were consecutively collected from September 2, 2008, to June 7, 2013, as stage 1 subjects. Eight reported transcription regulation-related single-nucleotide polymorphisms (SNPs) were genotyped using the Sequenom MassArray system. In stage 2, another 44 RRMS patients and 200 healthy controls were consecutively collected and Sanger sequenced from April 7, 2015, to June 29, 2017, for the validation of positive results in stage 1. Differences in allele and genotype frequencies between patients and healthy controls, odds ratios, and 95% confidence intervals were calculated with the Chi-square test or Fisher's exact test. Hardy-Weinberg equilibrium was tested also using the Chi-square test.
Results: In stage 1 analysis, we confirmed only one previously reported risk variant, rs11129295 in EOMES gene. We found that the frequency of T/T genotype was much higher in MS group (χ2 = 10.251, P = 0.005) and the T allele of rs11129295 increased the risk of MS (χ2 = 10.022, P = 0.002). In stage 2 and combined analyses, the T allele of rs11129295 still increased the risk of MS (χ2 = 4.586, P = 0.030 and χ2 = 16.378, P = 5.19 × 10−5, respectively).
Conclusions: This study enhances the knowledge that the variant of EOMES is associated with increasing risk in Chinese RRMS patients and provides a potential therapeutic target in RRMS.
背景：多发性硬化（Multiple Sclerosis, MS）是一种常见的中枢神经系统自身免疫性疾病。越来越多的全基因组关联分析（genome-wide association study, GWAS）表明多发性硬化可能与遗传因素密切相关。遗憾的是, 几乎所有的GWAS都是基于高加索人群， 实验中得到的很多风险位点并不能在中国MS病人身上得到验证。
方法：自2008年9月2日至2013年6月7日，我们连续收集了142例复发缓解型MS（relapsing-remitting MS, RRMS）患者及301名健康志愿者作为第一阶段的研究对象，通过Sequenom MassArray 技术对8个GWAS研究发现的转录调节相关风险位点进行单核苷酸多态性（single-nucleotide polymorphisms, SNPs）分型。为了验证第一阶段所得到的阳性结果，我们自2015年4月7日至2017年6月29日又连续收集了44例RRMS患者及200名健康志愿者，通过Sanger测序进行SNP位点检测。患者与健康志愿者基因型以及等位基因之间的差异、风险比及95%置信区间通过χ2或Fisher精确概率法进行检验。Hardy-Weinberg平衡亦通过χ2检验进行计算。
结果：在第一阶段分析中，我们发现仅有EOMES基因rs11129295位点这一个风险位点可能与MS发病风险相关，患者中携带T/T基因型的频率显著高于健康志愿者 （χ2=10.251, P=0.005），T等位基因提高MS发病风险（χ2=10.022, P= 0.002）。在第二阶段及联合分析中，rs11129295位点T等位基因依然能提高MS发病风险（χ2 = 4.586, P= 0.030 以及χ2 =16.378, P = 5.19×10-5）。
Keywords: Genetic Association Studies; Multiple Sclerosis; Risk Factors; Single-nucleotide Polymorphism
|How to cite this article:|
Chen S, Zhang J, Liu QB, Zhuang JC, Wu L, Xu YF, Li HF, Wu ZY, Xiao BG. Variant of EOMES Associated with Increasing Risk in Chinese Patients with Relapsing-remitting Multiple Sclerosis. Chin Med J 2018;131:643-7
|How to cite this URL:|
Chen S, Zhang J, Liu QB, Zhuang JC, Wu L, Xu YF, Li HF, Wu ZY, Xiao BG. Variant of EOMES Associated with Increasing Risk in Chinese Patients with Relapsing-remitting Multiple Sclerosis. Chin Med J [serial online] 2018 [cited 2018 Mar 18];131:643-7. Available from: http://www.cmj.org/text.asp?2018/131/6/643/226892
| Introduction|| |
Multiple sclerosis (MS; MIM 126200) is a common, partially heritable central nervous system (CNS) autoimmune disorder being considered as one of the most common causes of neurological disability in young adults., Relapsing-remitting MS (RRMS) is the most common form of MS which has a biphasic disease course characterized by alternating episodes of acute neurological deficits, followed by a complete or partial recovery. Although the exact pathogenesis of MS is still not well classified, it is no doubt that genetic factors are primarily responsible for the substantially increased frequency of the disease, especially in the relatives of affected individuals.,, For years, the major histocompatibility complex (MHC) was the only known MS susceptibility region. However, hundreds of variants outside MHC region had now been identified by several genome-wide association studies (GWASs).,
The major pathogenic process of MS mainly starts with the activation of autoreactive lymphocytes and their migration across the blood-brain barrier. Transcription factors (TFs) are a group of key regulators involving in controlling lineage differentiation and cell survival. Certainly, TFs are indispensable for the T helper (Th) cell fate determination and cytokine production. A number of previous studies have demonstrated that the dysregulation of TFs is deeply involved in the pathogenesis of MS.,, In addition, a series of GWAS analyses had discovered several risk loci within transcription regulation genes. In 2011, a Caucasian population-based multicenter GWAS analysis reported at least eight transcription regulator-related risk loci among 29 novel MS susceptibility loci. Later on, another GWAS analysis replicated these results in a larger cohort. In addition, it has been indicated that the mRNA expression of one TF, EOMES, was significantly and consistently lower in MS patients comparing to healthy controls (HCs).
However, to date, few analyses were conducted to identify the variants located in transcription regulation genes in Asian MS populations. Here, we performed a MassArray Assay to genotype the previously reported variants located in the transcription regulation genes in order to elucidate their role in the Chinese MS patients.
| Methods|| |
The written informed consent was signed by each participant before inclusion in the study. This study was approved by the Ethics Committee of Huashan Hospital of Fudan University, the Second Affiliated Hospital of Zhejiang University School of Medicine, and the First Affiliated Hospital of Fujian Medical University.
In the first stage, a total of 142 MS patients (59 males, 83 females; mean age 39.8 ± 12.3 years; range: 13.0–66.0 years) were consecutively collected from September 2, 2008, to June 7, 2013. All patients underwent detailed neurological examinations, laboratory tests, and magnetic resonance imaging scans for the brain and/or spinal cord. Patients were then diagnosed according to the revised McDonald criteria., In addition, 301 healthy individuals (175 males, 126 females; mean age 37.0 ± 15.6 years; range 16.0–85.0 years) with no history of autoimmune diseases were recruited as HCs matched for case ethnicity and region. To validate the positive result from the first stage, another 44 RRMS patients and 200 HCs were consecutively collected from April 7, 2015, to June 29, 2017.
Genomic DNA was extracted from peripheral blood mononuclear cell using a QIAamp DNA Blood Minikit (QIAGEN, Hilden, Germany). Eight previously reported TF-related variants were genotyped using the Sequenom MassArray system. MassArray Assay Design 3.1 software (Sequenom, San Diego, USA) was used to design the polymerase chain reaction primers used in the genotyping [Supplementary Table 1 [Additional file 1]]. Alleles were detected using a matrix-assisted laser desorption/ionization time of flight mass spectrometry platform (MassArray™, Sequenom Inc., San Diego, CA, USA) according to a previously described method. In validation of the first stage, rs11129295 was further Sanger sequenced in subjects recruited for the second stage using a pair of in-house designed primer (Forward: 5'-TCTTGTTTTCTGGAGAGGAGC-3'; Reverse: 5'-ACCCACCTTCAGGAATTTCAAT-3').
Quantitative measures were summarized with descriptive statistics, such as mean ± standard deviation (SD) and 95% confidence interval (CI) of mean. Difference in ages was measured using the Student's t-test. Differences in genders, allele and genotype frequencies between MS and HC, odds ratios (OR s), and 95% CI s were calculated using the Chi-square test or Fisher's exact test. Hardy-Weinberg equilibrium (HWE) was tested using the Chi-squaretest. Statistical analyses were performed using SPSS 19.0 software (SPSS Inc., Chicago, IL, USA) and GraphPad Prism 6.02 (GraphPad Inc, San Diego, CA, USA). The criterion for a signifcant difference was a value of P< 0.05.
| Results|| |
The demographic data are summarized in [Table 1]. In stage 1, the average ages of the MS patients and HCs were 39.8 ± 12.3 and 37.0 ± 15.6 years, respectively (t = 1.840, P = 0.070). In stage 2, the average ages of the MS patients and HCs were 39.0 ± 13.8 and 33.3 ± 11.3 years, respectively (t = 2.740, P = 0.007). In total, the average ages of the MS patients and HCs were 39.6 ± 12.5 and 35.5 ± 14.2 years, respectively (t = 3.310, P = 0.001).
|Table 1: Demographic and clinical characteristics of RRMS patients and healthy controls|
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EOMES variant rs11129295 associated with increasing risk in Chinese multiple sclerosis patients
The average genotyping success rate across the single-nucleotide polymorphisms (SNPs) was 97.5% using the Sequenom MassArray system. As shown in [Supplementary Table 2 [Additional file 2]], most of the SNPs in each group were under the HWE except STAT3 variant rs744166 in MS patients (P = 0.020). Eventually, as summarized in [Table 2], we found that the frequency of T/T genotype in EOMES variant rs11129295 was much higher in MS group with a significant difference (χ2 = 10.251, P = 0.005). In addition, the T allele of rs11129295 increased the risk of MS (OR = 1.764, P = 0.002). Besides, when analyzed in a dominant model, the genotype T/T and C/T together can increase the risk of MS (OR = 4.076, P = 0.019). Hence, it was a recessive model (OR = 1.776, P = 0.007). Furthermore, we replicated these findings in another group of population using Sanger sequencing. In this stage, we still found that the frequency of T/T genotype in rs11129295 was much higher in MS group with a significant difference (P = 0.020). Besides, the T allele increased the risk of MS (OR = 1.830, P = 0.030). However, we did not find significance in recessive model in stage 2 [Table 2].
|Table 2: Allele and genotype distributions of rs11129295 between MS and healthy controls|
Click here to view
In combined analyses of the two stages, we found that the frequency of T/T genotype in rs11129295 was much higher in MS group with a significant difference (P = 4.17 × 10−5), as well as the T allele increased the risk of MS (OR = 1.834, P = 5.19 × 10−5) and the higher frequency of T/T plus C/T in dominant model (OR = 6.615, P = 8.12 × 10−5) and T/T genotype in recessive model (OR = 1.750, P = 0.002; [Table 2]). As shown in [Table 3], we did not find any other significant differences between MS and HC groups among other previously reported variants within transcription-related genes.
|Table 3: Other variants involved in transcription regulation between MS and healthy controls|
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| Discussion|| |
MS is an autoimmune demyelinating disease of CNS. Increasing number of evidences hint that MS is strongly associated with genetics. The MHC loci made the early success in demonstrating the important role of genetic factors. To date, 13 MHC loci had been identified. Nevertheless, little progress was made in unraveling the non-MHC genes underlying susceptibility to MS until the advent of GWAS technology. Over the last decade, over 200 non-MHC genes had been identified using GWAS analyses in large cohorts.,, Unfortunately, almost all the GWAS analyses were Caucasian population based. A number of risk loci might not be replicated in Chinese MS patients. In our previous studies, Liu et al. found that the variants of interferon regulatory factor 5 were not associated with MS in the southeastern Han Chinese population. Moreover, Cai et al. found the association between autophagy-related gene 5 (ATG5) and neuromyelitis optica, but failed in MS. Fortunately, Zhuang et al., identified that the variants in interleukin 7 (IL7) and CYP27B1 were associated with MS. Similarly, in the present study, we confirmed only one previously reported risk variant, rs11129295 in EOMES gene. We found that the frequency of T/T genotype was much higher in the MS group and the T allele of rs11129295 increased the risk of MS.
TFs are playing critical roles in the differentiation of Th cell. As was demonstrated previously, T-bet is a major factor for Th1 cell differentiation and IFN-γ production. Similarly, Foxp3 and RORγt are the master TFs of nTreg cell and Th17 cell, respectively.,EOMES, also termed as TBR2, encodes a TF which is crucial for embryonic development of the CNS in vertebrates. Besides, multiple lines of evidences have demonstrated that EOMES deeply involves in defense against viral infections., Its function in CD4+ Th cell differentiation was remarkably noted recently. A functional experiment revealed that EOMES expression directly suppresses the Rorc and IL-17a expressions through binding to the promoter regions of these genes, which results in suppression of Th17 cell differentiation.
Meanwhile, EOMES expression itself is suppressed by transforming growth factor beta via a Smad-independent pathway in autoimmune disorders. In addition, a Caucasian population-based mRNA sequencing study further indicated the negative role of EOMES dysregulation played in MS progression. However, more recent functional studies were questioning the role of EOMES in some specific conditions. One study even revealed that the higher expression of EOMES in Th cell could result in the occurrence of secondary-progressive MS. Another study conducted by Lupar et al. revealed that the expression of EOMES limits the Foxp3 induction in a cell-intrinsic way. However, more recently, Zhang et al. reported that EOMES promotes the development of type 1 regulatory T cells, a Foxp3-negative, IL-10-producing T cell subset, which has potent immunosuppressive functions in autoimmunity. Thus, the role of EOMES playing in the pathogenesis of MS seems rather complicated and might be divided into two phases, acute phase and chronic phase. The more specific mechanisms of EOMES in these two phases remain to be clarified further.
In summary, we have identified a transcription regulation-related variant in Chinese MS patients. This variant is associated with increasing risk. The findings in the current study, together with previous studies, enhanced the knowledge that EOMES low expression in the acute phase of RRMS could promote the disease progression. As well, it possibly hints that overexpression of EOMES in the acute phase of RRMS might be a potential therapeutic target in RRMS.
The authors sincerely thank the participants for their willingness to take part in this study.
Supplementary information is linked to the online version of the paper on the Chinese Medical Journal website.
Financial support and sponsorship
This study was supported by the grants from the National Natural Science Foundation of China (No. 81371414 and No. 81125009).
Conflicts of interest
There are no conflicts of interest.
| References|| |
Reich DS, Lucchinetti CF, Calabresi PA. Multiple sclerosis. N Engl J Med 2018;378:169-80. doi: 10.1056/NEJMra1401483.
Chen H, Liu SM, Zhang XX, Liu YO, Li SZ, Liu Z, et al.
Clinical features of patients with multiple sclerosis and neuromyelitis optica spectrum disorders. Chin Med J 2016;129:2079-84. doi: 10.4103/0366-6999.189046.
] [Full text]
Willer CJ, Dyment DA, Risch NJ, Sadovnick AD, Ebers GC, Canadian Collaborative Study Group. et al.
Twin concordance and sibling recurrence rates in multiple sclerosis. Proc Natl Acad Sci U S A 2003;100:12877-82. doi: 10.1073/pnas.1932604100.
Mumford CJ, Wood NW, Kellar-Wood H, Thorpe JW, Miller DH, Compston DA, et al
. The british isles survey of multiple sclerosis in twins. Neurology 1994;44:11-5.
Dyment DA, Yee IM, Ebers GC, Sadovnick AD, Canadian Collaborative Study Group. Multiple sclerosis in stepsiblings: Recurrence risk and ascertainment. J Neurol Neurosurg Psychiatry 2006;77:258-9. doi: 10.1136/jnnp.2005.063008.
Moutsianas L, Jostins L, Beecham AH, Dilthey AT, Xifara DK, Ban M, et al.
Class II HLA interactions modulate genetic risk for multiple sclerosis. Nat Genet 2015;47:1107-13. doi: 10.1038/ng.3395.
International Multiple Sclerosis Genetics Consortium, Wellcome Trust Case Control Consortium 2, Sawcer S, Hellenthal G, Pirinen M, Spencer CC, et al.
Genetic risk and a primary role for cell-mediated immune mechanisms in multiple sclerosis. Nature 2011;476:214-9. doi: 10.1038/nature10251.
International Multiple Sclerosis Genetics Consortium (IMSGC), Beecham AH, Patsopoulos NA, Xifara DK, Davis MF, Kemppinen A, et al.
Analysis of immune-related loci identifies 48 new susceptibility variants for multiple sclerosis. Nat Genet 2013;45:1353-60. doi: 10.1038/ng.2770.
Ivanov II, McKenzie BS, Zhou L, Tadokoro CE, Lepelley A, Lafaille JJ, et al.
The orphan nuclear receptor RORgammat directs the differentiation program of proinflammatory IL-17+ T helper cells. Cell 2006;126:1121-33. doi: 10.1016/j.cell.2006.07.035.
Schraml BU, Hildner K, Ise W, Lee WL, Smith WA, Solomon B, et al.
The AP-1 transcription factor batf controls T(H)17 differentiation. Nature 2009;460:405-9. doi: 10.1038/nature08114.
Zhu J, Yamane H, Paul WE. Differentiation of effector CD4 T cell populations (*). Annu Rev Immunol 2010;28:445-89. doi: 10.1146/annurev-immunol-030409-101212.
Parnell GP, Gatt PN, Krupa M, Nickles D, McKay FC, Schibeci SD, et al.
The autoimmune disease-associated transcription factors EOMES and TBX21 are dysregulated in multiple sclerosis and define a molecular subtype of disease. Clin Immunol 2014;151:16-24. doi: 10.1016/j.clim.2014.01.003.
Polman CH, Reingold SC, Edan G, Filippi M, Hartung HP, Kappos L, et al.
Diagnostic criteria for multiple sclerosis: 2005 revisions to the “McDonald criteria”. Ann Neurol 2005;58:840-6. doi: 10.1002/ana.20703.
Polman CH, Reingold SC, Banwell B, Clanet M, Cohen JA, Filippi M, et al.
Diagnostic criteria for multiple sclerosis: 2010 revisions to the mcDonald criteria. Ann Neurol 2011;69:292-302. doi: 10.1002/ana.22366.
Buetow KH, Edmonson M, MacDonald R, Clifford R, Yip P, Kelley J, et al.
High-throughput development and characterization of a genomewide collection of gene-based single nucleotide polymorphism markers by chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Proc Natl Acad Sci U S A 2001;98:581-4. doi: 10.1073/pnas.021506298.
De Jager PL, Jia X, Wang J, de Bakker PI, Ottoboni L, Aggarwal NT, et al.
Meta-analysis of genome scans and replication identify CD6, IRF8 and TNFRSF1A as new multiple sclerosis susceptibility loci. Nat Genet 2009;41:776-82. doi: 10.1038/ng.401.
Liu QB, Wu L, Zhao GX, Cai PP, Li ZX, Wu ZY, et al.
Variants of interferon regulatory factor 5 are associated with neither neuromyelitis optica nor multiple sclerosis in the Southeastern Han Chinese population. Chin Med J 2015;128:1743-7. doi: 10.4103/0366-6999.159347.
] [Full text]
Cai PP, Wang HX, Zhuang JC, Liu QB, Zhao GX, Li ZX, et al.
Variants of autophagy-related gene 5 are associated with neuromyelitis optica in the Southern Han Chinese population. Autoimmunity 2014;47:563-6. doi: 10.3109/08916934.2014.929668.
Zhuang JC, Huang ZY, Zhao GX, Yu H, Li ZX, Wu ZY, et al.
Variants of CYP27B1 are associated with both multiple sclerosis and neuromyelitis optica patients in Han Chinese population. Gene 2015;557:236-9. doi: 10.1016/j.gene.2014.12.045.
Zhuang JC, Wu L, Qian MZ, Cai PP, Liu QB, Zhao GX, et al.
Variants of interleukin-7/Interleukin-7 receptor alpha are associated with both neuromyelitis optica and multiple sclerosis among Chinese han population in Southeastern China. Chin Med J 2015;128:3062-8. doi: 10.4103/0366-6999.169093.
] [Full text]
Szabo SJ, Kim ST, Costa GL, Zhang X, Fathman CG, Glimcher LH, et al.
A novel transcription factor, T-bet, directs th1 lineage commitment. Cell 2000;100:655-69.
Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor foxp3. Science 2003;299:1057-61. doi: 10.1126/science.1079490.
Sessa A, Mao CA, Hadjantonakis AK, Klein WH, Broccoli V. Tbr2 directs conversion of radial glia into basal precursors and guides neuronal amplification by indirect neurogenesis in the developing neocortex. Neuron 2008;60:56-69. doi: 10.1016/j.neuron.2008.09.028.
Intlekofer AM, Takemoto N, Wherry EJ, Longworth SA, Northrup JT, Palanivel VR, et al.
Effector and memory CD8+ T cell fate coupled by T-bet and eomesodermin. Nat Immunol 2005;6:1236-44. doi: 10.1038/ni1268.
Cui N, Su LX, Wang H, Xiao M, Yang F, Zheng M, et al.
MTOR modulates lymphocyte differentiation through T-bet and eomesodermin in response to invasive pulmonary aspergillosis in rats. Chin Med J 2016;129:1704-10. doi: 10.4103/0366-6999.185858.
] [Full text]
Ichiyama K, Sekiya T, Inoue N, Tamiya T, Kashiwagi I, Kimura A, et al.
Transcription factor smad-independent T helper 17 cell induction by transforming-growth factor-β is mediated by suppression of eomesodermin. Immunity 2011;34:741-54. doi: 10.1016/j.immuni.2011.02.021.
Raveney BJ, Oki S, Hohjoh H, Nakamura M, Sato W, Murata M, et al.
Eomesodermin-expressing T-helper cells are essential for chronic neuroinflammation. Nat Commun 2015;6:8437. doi: 10.1038/ncomms9437.
Lupar E, Brack M, Garnier L, Laffont S, Rauch KS, Schachtrup K, et al.
Eomesodermin expression in CD4+ T cells restricts peripheral foxp3 induction. J Immunol 2015;195:4742-52. doi: 10.4049/jimmunol.1501159.
Zhang P, Lee JS, Gartlan KH, Schuster IS, Comerford I, Varelias A, et al.
Eomesodermin promotes the development of type 1 regulatory T (TR1) cells. Sci Immunol 2017;2. pii: eaah7152. doi: 10.1126/sciimmunol.aah7152.
[Table 1], [Table 2], [Table 3]