Identification of a novel non-coding mutation in C1qB in a Dutch child with C1q deficiency associated with recurrent infections.

INTRODUCTION: C1q deficiency is a rare genetic disorder that is strongly associated with development of systemic lupus erythematosus (SLE). Several mutations in the coding regions of the C1q genes have been described that result in stop-codons or other genetic abnormalities ultimately leading to C1q deficiency. Here we report on a Dutch boy suffering from recurrent infections with a complete C1q deficiency, without any SLE symptoms. METHODS: The presence of C1q in serum was assessed using ELISA and hemolytic assay. By western blot we examined the different C1q chains in cell lysates. We identified the mutation using deep-sequencing. By qPCR we studied the mRNA expression of C1qA, C1qB and C1qC in the PBMCs of the patient. RESULTS: Deep-sequencing revealed a homozygous mutation in the non-coding region of C1qB in the patient, whereas both parents were heterozygous. The mutation is located two nucleotides before the splice site of the second exon. In-silico analyses predict a complete abrogation of this natural splice site. Analyses of in vitro cultured cells from the patient revealed a lack of production of C1q and intracellular absence of C1qB in the presence of C1qA and C1qC peptides. Quantitative PCR analysis revealed total absence of C1qB mRNA, a reduced level of C1qA mRNA and normal levels of C1qC mRNA. CONCLUSION: In this study we report a new mutation in the non-coding region of C1qB that is associated with C1q deficiency.

Effect of redox balance alterations on cellular localization of LAT and downstream T-cell receptor signaling pathways.

The integral membrane protein linker for activation of T cells (LAT) is a central adapter protein in the T-cell receptor (TCR)-mediated signaling pathways. The cellular localization of LAT is extremely sensitive to intracellular redox balance alterations. Reduced intracellular levels of the antioxidant glutathione (GSH), a hallmark of chronic oxidative stress, resulted in the membrane displacement of LAT, abrogated TCR-mediated signaling and consequently hyporesponsiveness of T lymphocytes. The membrane displacement of LAT is accompanied by a considerable difference in the mobility of LAT upon native and nonreducing denaturing polyacrylamide gel electrophoresis analysis, a finding indicative of a conformational change. Targeted mutation of redox-sensitive cysteine residues within LAT created LAT mutants which remain membrane anchored under conditions of chronic oxidative stress. The expression of redox-insensitive LAT mutants allows for restoration of TCR-mediated signal transduction, whereas CD28-mediated signaling pathways remained impaired. These results are indicative that the membrane displacement of LAT as a result of redox balance alterations is a consequence of a conformational change interfering with the insertion of LAT into the plasma membrane. Conclusively, the data suggest a role for LAT as a crucial intermediate in the sensitivity of TCR signaling and hence T lymphocytes toward chronic oxidative stress.