PMC8264782_01
(A) Family pedigree of the patient. (B) Sanger sequencing confirmed the presence of the mutation in exon 1 (c.115G>A; p.Asp39Asn). The heterozygous (mother) and WT states. (C) Scarring alopecia, hypotrichosis with ringworm of the scalp. (D) Schematic diagram of the IL-2RG protein, with its extracellular (EC), transmembrane (TM), intracellular (IC) domains. In red the mutation carried by the patient and in black the positions affected by the mutations already described in the literature.
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Investigation of his family history ( Figure 1A ) revealed that two of his maternal uncles displayed similar clinical manifestations, with one of them who has been treated for a celiac disease and who died of infectious complications at 22 years of age, while the other one, who was treated for bronchiectasis and severe refractory eczema, died at the age 28 years. The patient who was underweight exhibited during the first physical examination, at the age of 6 years, a ringworm of the scalp accompanied by hair loss ( Figure 1C ), and an eczema present on his ears, elbow folds, and bursae. A flow cytometry analysis of the different lymphocyte subsets ( Supplementary Figure S1 ) revealed normal numbers and frequencies of CD3+ T cells 84% (absolute count 2434/mm3), CD3+CD8+ T cells 61.5% (1782/mm3), CD19+ B cells 9.5% (275/mm3), and CD56+NK cells 6.5% (188/mm3). The presence of the missense mutation was further confirmed by Sanger sequencing ( Figure 1B ).
Immune functional and proliferation assays in subsets of lymphocytes. (A) Flow cytometry analysis of CD132 (gammac) expression on T, B and NK cells of the patient, his mother, and healthy control, and screening of siblings for CD132 expression on T cells. (B) Histogram of CD4+ T cell and monocyte response with STAT phosphorylation to several gammac dependent (IL-2, IL-4, IL-7) and independent (IL-6, IFN-gamma) cytokines in whole blood of patient. (C) Analysis of proliferation of healthy control (HC) and patient CFSE labeled cells with the indicated stimuli: 4 days of culture with Phytohemaglutinin (PHA) and 6 days with anti-CD3/anti-CD28 monoclonal antibodies. (D) The percentages of IL-17A, IFNgamma and IL-4-producing CD4+ T cells from patient and age-matched control using PMA/Ionomycin stimulation in the presence of monensin for 5 hours. *Gamma chain dependent cytokines; the others are gamma chain independent.
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Further characterization of the lymphocyte subpopulations ( Table 1 ) showed a substantial shift towards a memory phenotype ( Supplementary Figure S2 ) with an increased percentage of CD45RO+ CD4+ and CD45RO+ CD8+T cells, low percentages of naive T cells (CCR7+CD45RA+), and high percentages of CD4+ and CD8+ effector memory T cells (TEM) (CCR7-CD45RA-) ( Supplementary Figure S3 ). No difference for pSTAT3 expression was detected between patient and healthy control ( Figure 2B ). An immunostaining with anti-CD132 and flow cytometry analysis showed a significant reduction of gammac surface expressions on T, B and NK cells of the patient when compared to his parents, siblings, or healthy control ( Figure 2A ). However, gammac independent signaling in monocytes (IFN-gamma induced pSTAT1) was not affected ( Figure 2B ). T cells of the patient with anti-CD3 and anti-CD28-coated beads led to a reduced proliferation compared to healthy control (67% vs. 85%) ( Figure 2C ). Taken together, these data suggest that the patient has a "leaky" form of SCID. CD4+ T cells (Th1) and normal CD4+IL4+(Th2) and CD4+IL17A+(Th17) frequencies when compared to an age-matched control subject ( Figure 2D ). CD4+CD25highCD127low/-FoxP3+ Regulatory T-cells represent 11,52% of CD4+ T-cells.