The table presents data on lymphocyte counts pre- and post-transplant, percent donor chimerism post-transplant, and sensitivity of the patient's cells to low-dose radiation. It includes measurements for various cell types such as C D 3, C D 4, C D 8, C D 4 slash C D 45 R A, C D 19, and C D 3 slash C D 56. The table is divided into sections showing age at testing, lymphocyte counts pre- and post-transplant, percent donor chimerism post-transplant, and sensitivity of the patient's cells to low-dose radiation. Each section provides specific cell counts and percentages at different time points. In Panel a For Cluster of Differentiation 3 cells per microliter, the reference range is 1400 to 7000. Values are 28 at 10 days old, 52 at 1 month, 71 at 2 months, and 60 at 2.5 months pre-transplant. Post-transplant values are 247 at 6.5 months (2 weeks post-transplant), 315 at 7 months (1 month post-transplant), 509 at 8 months (2 months post-transplant), 1051 at 9 months (3 months post-transplant), and 2633 at 17 months (11 months post-transplant). For Cluster of Differentiation 4 cells per microliter, the reference range is 1000 to 5300. Values are 24, 49, 67, and 54 pre-transplant at the same ages, and 165, 222, 194, 549, and 1872 post-transplant. For Cluster of Differentiation 8 cells per microliter, the reference range is 400 to 2300. Values are 1, 3, 3, and 3 pre-transplant, and 51, 67, 239, 415, and 696 post-transplant. For Cluster of Differentiation 4 positive slash Cluster of Differentiation 45 R A positive cells per microliter, the reference range is 41 to 1121. Values are no data at 10 days, then 31, 41, and 34 pre-transplant, and 93, no data, no data, 406, and no data post-transplant. For Cluster of Differentiation 19 cells per microliter, the reference range is 600 to 2700. Values are 310, 160, 229, and 642 pre-transplant, and 353, 424, 282, 340, and 314 post-transplant. For Cluster of Differentiation 3 negative slash Cluster of Differentiation 56 positive and/or Cluster of Differentiation 16 positive cells per microliter, the reference range is 200 to 1400. Values are 6, 23, 4, and 10 pre-transplant, and 177, 69, 238, 128, and 72 post-transplant. For percent donor chimerism post-transplant, the categories include non-enriched cells, T-cell, B-cell, and myeloid. For non-enriched cells, values are 9 at 7 months old (1 month post-transplant), 24 at 8 months old (2 months post-transplant), 29 at 9 months old (3 months post-transplant), and 40 at 17 months old (11 months post-transplant). For T-cells, values are 54 at 7 months old, 83 at 8 months old, 90 at 9 months old, and 88.4 at 17 months old. For B-cells, values are 1 at 7 months old, 6 at 8 months old, 7 at 9 months old, and 4 at 17 months old. For myeloid cells, values are 2 at 7 months old, 3 at 8 months old, 1 at 9 months old, and 2.4 at 17 months old. For sensitivity of the patient’s cells to low dose radiation at 1 hour post irradiation, in T 2 cells the delta percent for phosphorylated ataxia telangiectasia mutated is 80.2 (low), phosphorylated structural maintenance of chromosomes 1 is 80.9, and phosphorylated histone H 2 A X (gamma H 2 A X) is 67.2 (low), with reference ranges 86.9 to 97.0, 78.0 to 96.7, and 93.4 to 99.1 respectively. The ratio (mean fluorescence intensity) values are 4.8, 2.8, and 18.6, with reference ranges 3.3 to 7.8, 2.3 to 6.4, and 7.4 to 84.1. In B 2 cells at 1 hour post-irradiation, delta percent values are 54.4 (low) for phosphorylated ataxia telangiectasia mutated, 48.6 (low) for phosphorylated structural maintenance of chromosomes 1, and 48.5 (low) for phosphorylated histone H 2 A X, with reference ranges 70.3 to 100.0, 73.7 to 95.8, and 62.5 to 96.9. The ratio values are 6.7, 2.8, and 26.0, with reference ranges 0.2 to 8.1, 2.5 to 6.7, and 8.0 to 256.3. At 24 hours post-irradiation, in T 2 cells the ratio values are 1.7 for phosphorylated ataxia telangiectasia mutated, 1.4 for phosphorylated structural maintenance of chromosomes 1, and 2.1 for phosphorylated histone H 2 A X, with reference ranges 1.0 to 3.0, 1.2 to 2.9, and 0.6 to 2.9. In B 2 cells, the ratio values are 1.0, 1.0, and 1.0, with reference ranges 0.7 to 2.2, 1.5 to 4.2, and 0.6 to 3.3. Initial viability percent is 65.7 (low) with a reference range greater than or equal to 74.6. For apoptosis at 24 hours (fold increase), values are 1.7 in T 2 cells and 1.2 in B 2 cells, with reference ranges 1.0 to 7.8 and 0.8 to 3.3. For cell death at 24 hours (fold increase), values are 3.6 in T 2 cells and 1.6 in B 2 cells, with reference ranges 1.7 to 22.3 and 1.1 to 6.4. Panel c and panel d show two dimensional U M A P projections of single cell populations, with labeled clusters representing distinct cell types. In panel c, the clusters are color coded and clearly separated. The largest regions include D N (double negative), D P (double positive), and S P (single positive) thymocyte populations. Nearby are Innate T and Innate lymphoid clusters. Additional labeled clusters include E T P (early T progenitor), N M P, Mast (mast cells), M g k (megakaryocytes), Ery (erythroid cells), Mac slash Mono (macrophage/monocyte), D C (dendritic cells), B (B cells), T E C (thymic epithelial cells), F b (fibroblasts), V S M C (vascular smooth muscle cells), and Endo (endothelial cells). Each population forms a distinct island of points. In panel d, the same U M A P structure is shown but with a different coloring scheme. Most cells appear in light blue, while scattered multicolored points are overlaid across clusters, particularly within D N and D P regions. The overall spatial arrangement of clusters remains identical to panel c, indicating the same cellular landscape with an alternate data overlay. Panel e presents flow cytometry density plots comparing control (top row) and patient (bottom row) samples across three marker combinations. In the left column, C D 1 a is plotted on the vertical axis and C D 7 on the horizontal axis. In the control sample, the major population is C D 1 a positive C D 7 negative at 55.1 percent, with C D 1 a positive C D 7 positive at 12.9 percent, C D 7 positive C D 1 a negative at 29.4 percent, and C D 1 a negative C D 7 negative at 2.74 percent. In the patient sample, C D 1 a-positive C D 7-positive cells are markedly increased to 77.3 percent, while C D 1 a positive C D 7 negative are 2.00 percent, C D 7 positive C D 1 a negative are 17.4 percent, and double negative cells are 3.21 percent. In the middle column, C D 4 is plotted vertically and C D 8 beta horizontally. In the control sample, quadrant distributions are Q 5 1.87 percent, Q 6 52.5 percent, Q 7 12.5 percent, and Q 8 33.2 percent. In the patient sample, Q 5 is 27.1 percent, Q 6 15.1 percent, Q 7 3.57 percent, and Q 8 54.2 percent, indicating a shift in C D 4 and C D 8 beta distribution. In the right column, T cell receptor alpha beta is plotted on the vertical axis and C D 3 on the horizontal axis. In the control sample, T cell receptor alpha beta positive C D 3 positive cells are 46.3 percent, and T cell receptor alpha beta negative C D 3 positive cells are 0.83 percent. In the patient sample, T cell receptor alpha beta positive CD3 positive cells are markedly reduced to 1.34 percent, and T cell receptor alpha beta negative C D 3 positive cells are 0.40 percent.
RECQL4 deficiency is associated with DNA radiosensitivity and a hematopoietic-intrinsic T cell defect than can be rescued with HSCT. (a) Lymphocyte cell counts pre- and post-transplant and percent donor chimerism after transplant. (b) Defective induction and completion of DNA double-stranded break repair in the patient’s T and B cells as measured after exposure to low-dose irradiation in a commercial assay. Too few NK cells were present in the patient sample for analysis. 1 and 24 h after irradiation, the percentage of cells in which each marker (pATM, pSMC, and γ H2AX) was phosphorylated was measured by flow cytometry. The percent of cells phosphorylated is expressed as the difference (delta) between irradiated and unirradiated cells. The degree of phosphorylation is expressed as the ratio of the mean fluorescence intensity of irradiated/unirradiated cells. The viability of total lymphocytes was low at the start of the assay, but there was no significant increase in cell death or apoptosis at 24 h after irradiation, though there may be increased cell death over time, resulting in lymphopenia. (c) Uniform Manifold Approximation and Projection visualization showing the cellular composition of the human thymus obtained from the collection of single-cell datasets available on the UCSC Cell Browser (https://fetal-thymus.cells.ucsc.edu). (d) Expression of RECQL4 in the human thymus atlas. RECQL4 is expressed in the human thymus in CD4− CD8− double negative (DN), CD4+ CD8+ double positive (DP) thymocytes, and in thymic epithelial cells (TECs). (e) Defective T cell differentiation from CD34+ stem cells in the patient after 7 wk in an ATO system. The ATOs are made by aggregating a human DLL4-expressing stromal cell line (MS5-hDLL4) with CD34+ cells isolated from peripheral blood of either the patient or a healthy control. Plots shown are gated on CD45+CD56− cells. Data shown are representative of two independent experiments. ETP, early T cell progenitor; MFI, mean fluorescence intensity. tx, transplant; IR = irradiation; DC, dendritic cell; SP, single positive thymocyte; NMP, neutrophil-myeloid progenitor; VSCM, vascular smooth muscle cell.