Nambiar M P, Enyedy E J, Warke V G, Krishnan S, Dennis G, Wong H K, Kammer G M, Tsokos G C
Walter Reed Army Institute of Research, Silver Spring, Maryland 20910-7500, USA.
Arthritis Rheum. 2001 Jun;44(6):1336-50. doi: 10.1002/1529-0131(200106)44:6<1336::AID-ART226>3.0.CO;2-8.
T cells from patients with systemic lupus erythematosus (SLE) display antigen receptor-mediated signaling aberrations associated with defective T cell receptor (TCR) zeta chain protein and messenger RNA (mRNA) expression. This study was undertaken to explore the possibility that coding-region mutations/polymorphisms of the TCR zeta chain could account for its decreased expression and altered signaling in SLE T cells.
TCR zeta chain mRNA from 48 SLE patients, 18 disease controls, and 21 healthy volunteers was reverse transcribed, amplified by polymerase chain reaction, and cloned, and complementary DNA (cDNA) was sequenced. DNA sequences from multiple clones were analyzed for silent single-nucleotide polymorphisms, mutations, and splice variations, to promote the identification of heterozygosity.
DNA sequence analysis revealed several widely distributed missense mutations and silent polymorphisms in the coding region of the TCR zeta chain, which were more frequent in SLE patients than in patients with other rheumatic diseases or healthy controls (P < 0.0001). Several of the missense mutations were located in the 3 immunoreceptor tyrosine activation motifs or the GTP binding domain, and this could lead to functional alterations in the TCR zeta chain. A splice variant of the TCR zeta chain with a codon CAG (glutamine) insertion between exons IV and V was found in half of the SLE and control samples. Two larger spliced isoforms of the TCR zeta chain, with an insertion of 145 bases and 93 bases between exons I and II, were found only in SLE T cells. We also identified various alternatively spliced forms of the TCR zeta chain resulting from the deletion of individual exons II, VI, or VII, or a combined deletion of exons V and VI; VI and VII; II, III, and IV; or V, VI, and VII in SLE T cells. The frequency of the deletion splice variants was significantly higher in SLE than in control samples (P = 0.004). These variations were observed in cDNA and may not reflect the status of the genomic DNA.
These findings demonstrate that heterogeneous mutations/polymorphisms and alternative splicing of TCR zeta chain cDNA are more frequent in SLE T cells than in T cells from non-SLE subjects and may underlie the molecular basis of known T cell signaling abnormalities in this disease.
系统性红斑狼疮（SLE）患者的T细胞表现出与T细胞受体（TCR）ζ链蛋白及信使核糖核酸（mRNA）表达缺陷相关的抗原受体介导的信号转导异常。本研究旨在探讨TCRζ链编码区突变/多态性是否可解释其在SLE T细胞中表达降低及信号转导改变的可能性。
对48例SLE患者、18例疾病对照者及21名健康志愿者的TCRζ链mRNA进行逆转录，通过聚合酶链反应扩增并克隆，然后对互补DNA（cDNA）进行测序。分析多个克隆的DNA序列，以确定沉默单核苷酸多态性、突变及剪接变异，从而促进杂合性的鉴定。
DNA序列分析显示，TCRζ链编码区存在多个广泛分布的错义突变和沉默多态性，在SLE患者中比在其他风湿性疾病患者或健康对照中更常见（P<0.0001）。其中一些错义突变位于3个免疫受体酪氨酸激活基序或GTP结合域中，这可能导致TCRζ链功能改变。在一半的SLE及对照样本中发现了一种TCRζ链剪接变异体，其在外显子IV和V之间插入了密码子CAG（谷氨酰胺）。仅在SLE T细胞中发现了两种较大的TCRζ链剪接异构体，分别在外显子I和II之间插入了145个碱基和93个碱基。我们还在SLE T细胞中鉴定出了TCRζ链的各种选择性剪接形式，这些形式是由于外显子II、VI或VII的单个缺失，或外显子V和VI、VI和VII、II、III和IV或V、VI和VII的联合缺失所致。缺失剪接变异体的频率在SLE中显著高于对照样本（P = 0.004）。这些变异在cDNA中观察到，可能无法反映基因组DNA的状态。
这些发现表明，TCRζ链cDNA的异质性突变/多态性和选择性剪接在SLE T细胞中比在非SLE受试者的T细胞中更常见，可能是该疾病中已知T细胞信号异常的分子基础。
