PMC9027814_01
Novel loss-of-function variant causing SASH3 deficiency. (A) Schematic structure of SASH3 protein. The location of the c.505C>T/p.Gln169* variant is indicated below in red and Sanger sequencing confirmation is shown. The three other variants reported by Delmonte et al. are indicated in gray in the upper part. (B) Pedigree and familial segregation of the SASH3 variant. The patient was hemizygous and his mother, a heterozygous carrier. (C) Western blot showing absence of SASH3 protein in patient PBMCs. The immunogen recognized by the antibody used (SASH3 polyclonal antibody #PA5-70305, Termofisher Scientific) is located at the N-terminal region of human SASH3 protein, before the amino acid 169 (where the STOP codon is located).
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To date only three SASH3 variants have been reported, two nonsense and one missense, with p.Gln169* being the most N-terminal one (Figure 1A). The variant was confirmed by Sanger and family study, which showed that the mother is a heterozygous carrier (Figure 1B). Western blot of the patient's PBMCs using a polyclonal antibody targeting the N-terminus of SASH3 showed complete absence of protein, thus confirming the LOF nature of the c.505C>T/p.Gln169* variant (Figure 1C).
Multicolor flow cytometry evaluation of advanced lymphocyte subpopulations and T cell activation. (A) Dot plots illustrating the main differences in B cell subpopulations between the patient and a healthy control show a dramatic reduction in pre-switch, isotype switching, and plasmablast populations in the patient. (B) Advanced T lymphocyte analysis shows decreased naive CD4 and CD8 populations, with a striking increase of lymphocytes with TEMRA phenotype. (C) Absence of cTfh cells in the patient vs. the control. (D) CD69 expression after activation with anti-CD3/CD28 in CD4 (upper) and CD8 (lower) lymphocytes, depicts decreased activation capacity in the SASH3-deficient patient.
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Most B cells were naive, with reductions in pre-switch and switch memory B cells (Figure 2A). T lymphocyte subset distributions demonstrated a marked decrease in CD4+ and CD8+ naive T cells (Figure 2B; Table 1). Conversely, CD4+ and CD8+ TEMRA cells were elevated, more markedly in the case of CD8+ T cells, and were accompanied by an increase in HLA-DR expression. There was a complete absence of circulating T follicular helper (cTfh) cells (Figure 2C). CD4+ and CD8+ T cells both showed low CD69 expression on stimulation with anti-CD3 and anti-CD28, indicating defective activation (Figure 2D).