台灣華人第二補體成分基因多型性與全身性紅斑性狼瘡的關聯分析

Association of complement component 2 polymorphism with clinical features of systemic lupus erythematosus in a Taiwanese population

陳詩欣(Chen, Hsih Hsin)

指導教授 ： 李寬容 ； 陳得源

國立清華大學/生命科學院/分子醫學研究所/博士(2015年)

摘要

補體系統屬先天性免疫(innate immunity)，由一群未被激活的血漿蛋白組成，主要參與由免疫複合體媒介的免疫反應。補體系統的活化有三條路徑：古典路徑(classical pathway)由抗原與抗體先行結合所引起；甘露糖結合凝集素路徑(mannose-binding lectin pathway)由補體成分與微生物表面特定醣分子結合引起；替代路徑(alternative pathway)為補體成分直接與微生物細胞壁成分結合所致；補體系統的生理作用如下：（一）清除免疫複合體（二）移除凋亡細胞（三）防禦病原菌感染（四）引起局部發炎反應（五）調節適應性免疫系統(adaptive immunity)。
當補體反應不足或補體成分缺乏時，會使得凋亡細胞碎片無法順利及時清除，造成自體抗原過度暴露進而導致自體免疫疾病的發生，如果缺乏反應路徑上較早期的補體成分則疾病發生機率會上升、病況嚴重程度也會加劇。依據先前紅斑性狼瘡致病機轉的研究，針對長期性發炎症狀、大量自體抗體形成、免疫複合體的沉積現象、顯著相關的染色體區域及老鼠遺傳模式，挑選出十一個候選基因，包括IL1B、IL1RN、LTA、TNF-α、LTB、C2、C4、PDCD1、FCGR2A、FCGR3A及FASLG。第二補體成分歸屬於古典路徑，經由酵素激活後能與第四補體成分(C4)組成第三補體成分(C3)的轉化酶(convertase)主導後續的連鎖反應。另有研究顯示當第二補體成分蛋白缺乏時，病患會表現出全身性紅斑性狼瘡的症狀，以上結果使我們確信第二補體成分對於紅斑性狼瘡臨床病症應有其相關的重要性。
本研究探討的主題：第二補體成分基因多型性與紅斑性狼瘡臨床表徵的關聯性分析；第二補體成分基因的單一核苷酸多型性挑選主要依據美國國家生物技術與資訊中心(NCBI)的資料庫，在啟動子區域及可轉譯出蛋白質區域作搜尋，著重於轉錄因子結合或氨基酸序列改變的候選單一核苷酸多型性，選擇三個內含子區域的單一核苷酸多型性(intronic SNPs：rs2844455、rs3130681、rs2734335)及三個外顯子區域的單一核苷酸多型性(exonic SNPs：rs1042663、rs1042664、rs4151648)；利用連鎖不平衡檢定並挑選小等位基因频率在病患組具有意義者(minor allele frequency > 5%)，最後決定了位於內含子區域(GC-rich region)的單一核苷酸多型性(SNP rs2844455)依基因型與病徵、基因表現情形作後續分析。
首先完成病患資料建檔，包括臨床症狀、腎臟病徵、全血液檢查、補體成分濃度及活性分析、自體抗體種類檢測，接著為基因型之鑑定及基因表現之定量。結果顯示與正常人相互比較下，有較高頻率的AA基因型會出現在病患組(22.1% vs. 9.5%，P<0.05)；A對偶基因在病患組的頻率則更高(40.5% vs. 28.4%，P<0.05)。
針對紅斑性狼瘡病人，A對偶基因與毛髮脫落、皮膚光敏感性、抗心磷脂質抗體之存在均有顯著相關；反之，G對偶基因與這些病症則為顯著不相關。基因表現的定量分析(qPCR)，可發現第二補體成分基因在病患組的相對表現情形(relative mRNA level)明顯高於正常人；再依個別基因型與相對表現量進一步分析可以發現，若與GG基因型病人(中位數：35.76，四分位距：19.33-49.71)相比，AA基因型病人(中位數：18.86，四分位距：11.36-22.43，P=0.002)或AG基因型病人(中位數：18.38，四分位距：13.13-28.82，P=0.004)其第二補體基因轉錄情形皆為明顯降低；在RNA轉錄異構體(transcript isoforms)的定性分析(RT-PCR)中，能看到所檢測的異構體種類在病人與正常人的表現不論在型式或比例上都有差異；另外分析了第二補體成分的血清濃度，結果顯示病人的血清濃度明顯低於正常人，依基因型分組則發現血清中補體濃度與基因型沒有顯著相關。
根據以上的結果，我們知道位於第二補體成分基因的單一核苷酸多型性(SNP rs2844455)是一個在基因轉錄表現上具調控功能的位點，不僅影響基因整體的表現量與異構體種類，也明顯與臨床病徵相關如毛髮脫落、皮膚光敏感症狀及抗心磷脂質抗體的產生。
本研究以台灣華人族群遺傳背景為基礎探討第二補體成分基因多型性與全身性紅斑性狼瘡病徵之間的關聯性，我們將A對偶基因歸納為引發紅斑性狼瘡的危險因子，而G對偶基因較常出現於正常人及沒有明顯症狀的病人，相對於A對偶基因的高風險性，G對偶基因應該是一個能對抗疾病的保護因子。

關鍵字

抗心磷脂質抗體 ； 第二補體成分 ； 光敏感性 ； 單一核苷酸多型性 ； 全身性紅斑性狼瘡

並列摘要

The complement system comprises a large number of plasma proteins participating in pathogen defense, apoptotic cell removal, immune-complex clearance and inflammatory response of innate immunity; it also has regulatory potencies affecting B cell maturation and altering T cell function.
Impaired complement function or complement deficiency is believed to induce defective clearance of apoptotic cells to drive the autoimmunity in systemic lupus erythematosus (SLE). Complement component 2 (C2), an early member of the classical complement pathway, is correlated with systemic manifestations of SLE. We hypothesize that C2 polymorphism may confer genetic susceptibility to complement dysfunction in SLE so we aim to investigate the clinical and serological associations of C2 variants in Taiwanese patients with SLE.
The single-nucleotide polymorphisms (SNPs) were selected according to the NCBI dbSNP database and analyzed using the Genomatix software suite; we also performed computational calculations to estimate the linkage disequilibrium (LD) relationships for effective selection including three intronic SNPs (rs2844455, rs3130681 and rs2734335) and three exonic SNPs (rs1042663, rs1042664 and rs4151648). These SNP allele frequencies were tested in normal control subjects; upon comparison with the allele frequency in SLE patients, if the minor allele frequency (MAF) exceeded 5%, the SNPs were chosen for further study.
The rs2844455 located in the GC-rich region of C2 gene was genotyped by direct sequencing in 95 SLE patients and 95 matched normal control subjects. The gene expression profiles were generated by quantitative real-time PCR (qPCR) and reverse transcription PCR (RT-PCR); the serum C2 levels were determined using commercial ELISA tests.
Our results showed that SLE patients had significantly higher frequencies of the AA genotype (22.1% vs. 9.5%, P<0.05) and the A allele (40.5% vs. 28.4%, P<0.05) when compared with normal control subjects. The A allele was strongly associated with the occurrence of hair loss, photosensitivity and anticardiolipin antibodies; whereas the G allele was associated with lower frequencies of these clinical presentations.
The relative expression levels of C2 mRNA were significantly lower in patients with the AA genotype [median: 18.86, interquartile range (IQR): 11.36-22.43, P=0.002] and the AG genotype (18.38, IQR: 13.13-28.82, P=0.004) than in those with the GG genotype (35.76, IQR: 19.33-49.71). Differential expression patterns of C2 gene were demonstrated while the gel showed that the banding patterns and their intensity were obviously related to the genetic variants in patients; however, serum protein assays showed no significant connection existed between rs2844455 genotypes and serum C2 level in SLE patients (n=10 for each genotype; ANOVA F =0.071, P>0.05).
As expected, we confirmed the A allele as a risk factor for SLE development in a Taiwanese population, in contrast, the G allele might be a protective factor against the pathogenic autoantibody formation and cutaneous manifestations in SLE patients.

並列關鍵字

anticardiolipin antibody ； complement component 2 ； photosensitivity ； single-nucleotide polymorphism ； systemic lupus erythematosus

參考文獻

1. Marshall E. Lupus: mysterious disease holds its secrets tight. Science. 2002;296:689-91.

2. Kyttaris VC. Systemic lupus erythematosus: from genes to organ damage. Methods Mol Biol. 2010;662:265-83.

3. Popescu A, Kao AH. Neuropsychiatric systemic lupus erythematosus. Current neuropharmacology. 2011;9:449-57.

4. Nowling TK, Gilkeson GS. Mechanisms of tissue injury in lupus nephritis. Arthritis research & therapy. 2011;13:250.

5. Mayadas TN, Tsokos GC, Tsuboi N. Mechanisms of immune complex-mediated neutrophil recruitment and tissue injury. Circulation. 2009;120:2012-24.