(A) Microarray analysis was used to compare levels of gene expression in the patient’s cells to those found in thymocytes or a panel of leukemias. DP1 and DP2 represent arrays performed on 2 different developmental stages of normal human CD4⁺CD8⁺ thymocytes. The y axis shows the fold change in expression, with values above 1 representing an increase and those below 1 a decrease. When compared with thymocytes and other leukemias, LMO2, NOTCH1, HES1, STIL, TAL1, and CMPK gene expression is upregulated. In contrast, expression levels of tumor suppressor genes p14(ARF1) and p16(INK4a) were reduced. γc mRNA was not overexpressed relative to thymocytes (consistent with surface expression data), although it was in comparison with the heterogeneous group of leukemias. (B) The vector insertion site is 35087 bp upstream of the LMO2 transcription start site in the opposite orientation (red X). LAM-PCR analysis (inset) of 10 ng and 1 ng of d717 PBMC DNA identified 1 dominant clone. M, 100-bp ladder; –C, 100 ng nontransduced DNA. When using limiting amounts of DNA, the internal control is out-competed by the LMO2 amplicon. The genomic locus and expression levels of genes surrounding LMO2 are shown in comparison with a dataset of arrays performed on other childhood leukemias or DP1 and DP2 human thymocytes. (C) Sequencing of denaturing HPLC analysis revealed a R1599P substitution in the HD-N domain of NOTCH1 in the leukemic cells (d717) but not before. No other NOTCH1 mutations were found.