T cell signaling abnormalities in systemic lupus erythematosus are associated with increased mutations/polymorphisms and splice variants of T cell receptor zeta chain messenger RNA

Objective. T cells from patients with systemic lupus erythematosus (SLE) di splay antigen receptor-mediated signaling aberrations associated with defec tive T cell receptor (TCR) zeta chain protein and messenger RNA (mRNA) expr ession. This study was undertaken to explore the possibility that coding-re gion mutations/polymorphisms of the TCR zeta chain could account for its de creased expression and altered signaling in SLE T cells. Methods. TCR zeta chain mRNA from 48 SLE patients, 18 disease controls, and 21 healthy volunteers was reverse transcribed, amplified by polymerase cha in reaction, and cloned, and complementary DNA (cDNA) was sequenced. DNA se quences from multiple clones were analyzed for silent single-nucleotide pol ymorphisms, mutations, and splice variations, to promote the identification of heterozygosity. Results. DNA sequence analysis revealed several widely distributed missense mutations and silent polymorphisms in the coding region of the TCR zeta ch ain, which were more frequent in SLE patients than in patients with other r heumatic 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 S LE 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 fo und only in SLE T cells. We also identified various alternatively spliced f orms 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, I II, 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 s tatus of the genomic DNA. Conclusion. These findings demonstrate that heterogeneous mutations/polymor phisms and alternative splicing of TCR chain cDNA are more frequent in SLE T cells than in T cells from non-SLE subjects and may underlie the molecula r basis of known T cell signaling abnormalities in this disease.