Characterization of RAG1 gene mutations and T-cell receptor (TCR) Vβ repertoire. (A) The RAG1 gene was amplified from DNA extracted from normal PBMCs, the patient's granulocytes and PBMCs, and the parents' PBMCs. Direct sequencing was performed using an automated sequencer. A thin bar shows the position of the delC mutation. Pt indicates patient. (B) Sequence analysis of the same genomic region in subcloned PCR products obtained from the patient's T cells. A thick bar highlights the position of the second-site mutations. (C) Predicted structures of mutated RAG1 molecules. HD indicates homeodomain. (D) Expression profile of TCRVβ subfamilies. Peripheral blood samples were stained with monoclonal antibodies (mAbs) for individual TCRVβ together with anti-CD4 and anti-CD8 mAbs. The percentage of each TCRVβ expression within CD4⁺ or CD8⁺ T cells was analyzed by a flow cytometry. (E) CDR3 spectratyping. Each TCRVβ fragment was amplified from cDNA with one of the Vβ-specific primers. The size distribution of PCR products was determined by an automated sequencer and GeneScan software.