PMC7338719_01
Family pedigree and PNP gene sequencing of patients II.1 and II.2. (A) Family pedigree demonstrating the three siblings (II.1, II.2, and II.3) that are homozygous for the PNP gene mutation putatively causing p.His257Asp (depicted as H257D), while the parents (I.1 and I.2) and an additional sibling (II.4) were heterozygous for the mutated and for the wild-type (depicted as WT) alleles. (B) Sequencing of the PNP gene Sanger sequencing of the PNP gene from patients II.1 (upper panel) and II.2 (middle panel) demonstrating a G nucleotide at position 769, while a healthy control (bottom panel) has a C nucleotide at this position. Sequencing was performed with the forward primer TTACAGGTGTGAACCACTGC and the reverse primer GAAGAAAGTGGGAAAGGTGA.
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F2
Patient II.1 (family pedigree depicted in Figure 2A), a 21-year-old female university student, was referred for an evaluation of recurrent sino-pulmonary infections and persistent lymphopenia. The presence of the mutation was subsequently confirmed by Sanger Sequencing (Figure 2B, upper panel). The homozygous mutation was also identified in siblings II.2 (Figure 2B middle panel) and confirmed by Sanger sequencing.
PMC7338719_02
Immune evaluations in patients with partial PNP deficiency. (A) CD8+ T cells diversity in patient II.1. T cell diversity was determined by flow cytometry as the percentage of CD3+CD8+ T cells expressing one of 24 TCR V-beta chains. Diversity of patient II.1 is depicted by black bars, while the mean and 1 standard deviation of healthy donors are represented in the open bars. (B) Proliferation of CD4+ T cells of patients with partial PNP deficiency. The response of CD4+ T cells from patient II.1 (light violet) and II.2 (light pink) after stimulation with antiCD3 and soluble antiCD28 for 5 days was measured by flow cytometry dilution of intracellular Violet dye and compared with same day healthy control (yellow).
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F3
T cell receptor excision circles were not detected. The percentages of the CD3+CD4+CD25+CD127Low regulatory T cells were normal. The diversity of patient II.1 T cells was restricted, particularly for CD8+ T cells, where 8 of 24 V-beta families were absent (Figure 3A). PHA. Similarly, patients II.1 and II.2 CD4+ T cells demonstrated normal proliferation, as determined by flow cytometry analysis of violet dye dilution (Figure 3B).
PNP protein expression in cells from a patient with partial PNP deficiency. (A) Western blotting of EBV transformed lymphoblastoid cells from patient II.1, a patient with complete PNP deficiency, a PNP-proficient healthy control, and an ADA-deficient patient. TATPNP and PNP that have molecular weights of 34 and 32 KDa, respectively, and molecular weight markers were also blotted. (B) Semi-quantitative assessment of PNP protein in EBV transformed lymphoblastoid cells from the patients and controls described above were calculated by comparison to beta-actin band intensity and TATPNP using the IMAGEJ open platform software.
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F4
Western blotting was performed on EBV transformed lymphoblastoid cell lysates to evaluate the effects of the PNP gene mutation on its protein expression (Figure 4A). Semi-quantitative assessment, by comparison of PNP to beta-actin ratio and band intensity for TATPNP, suggested that the amount of PNP in the cells of patient II.1 was ~50 ng and similar to that in PNP-proficient cells (Figure 4B).
Sensitivity of cells from patients with partial PNP deficiency to irradiation. Viability of lymphoblastoid cells from the patients II.1 and II.2, a patient with complete absence of PNP activity, a patient with ataxia telangiectasia, and two unrelated PNP-proficient controls was determined 3 days after irradiation with 10 Gy. The Survival Fraction (SF) was calculated by dividing the number of viable cells following irradiation by the number of viable cells in non-irradiated cultures. Statistical differences in SF were determined by 2-way Anova.
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F5
Accordingly, the viability of lymphoblastoid cells was determined 3 days after irradiation with 10 Gy in comparison with the initial number of cells before irradiation (Figure 5).