Abstract
SASH3 deficiency was recently reported as a novel cause of X-linked combined immunodeficiency in four unrelated patients (Delmonte OM et al. Blood, 2021). Three of them carried loss-of-function variants leading to lack of SASH3 protein expression, whereas a hypomorphic variant was detected in one patient. Clinical and laboratory manifestations of SASH3 deficiency included refractory autoimmune cytopenias, recurrent infections, and multilineage immunodeficiency involving naïve CD4+ T-cells, B-cells and NK-cells. SASH3 deficiency leads to abnormal TCR signaling, decreased diversity of the T-cell repertoire, and prominent clonotypic expansions. We report bone marrow features in these patients including marked expansion of abnormal T-cells with features overlapping with T-cell lymphoproliferative disorders. In addition, we report relevant features of the SASH3 R347C knock-in (KI) mice developed using CRISPR-Cas9 technology. Unlike the other 3 SASH3 mutations, R347C results in a protein-positive phenotype.
Bone marrow biopsies were performed for evaluation of refractory cytopenias in the four patients at 22, 49, 28, and 34 years of age, respectively. Three of four patients had a known history of cytopenias and sinopulmonary infections since childhood. All patients had leukopenia with neutropenia, thrombocytopenia, and/or autoimmune hemolytic anemia. Marrow cellularity ranged from mildly hypocellular to normocellular with inverted M:E ratios reflecting myeloid hypoplasia. No marrow showed increased blasts or dysplasia. One patient had AFB-negative non-necrotizing granulomas. The most striking findings included extensive interstitial CD8+ and TIA1+ T-cell lymphocytosis with T-cell aggregates in the marrow. In contrast, the peripheral blood showed lymphopenia or normal ALC. Flow cytometry (FC) was performed in 3 cases and demonstrated expansion of cytotoxic T-cells expressing CD3, CD8, CD57, dim CD5, and CD7, and negative for CD4 and CD56. CD4:CD8 ratios were inverted in all cases ranging from 0.11 to 0.74. One patient also had an abnormal NK/T-cell population that was CD3+, CD56+, CD8+ (partial), CD7+, CD57-, CD5-, and CD4-. A clonal TCR gamma gene rearrangement was detected in two of three patients tested raising concern for clonal T-cell large granular lymphocyte (LGL) proliferation or T-LGL leukemia. All had severe NK-cell and B-cell lymphopenia. Plasma cells were polytypic and demonstrated atypical expression of CD56. Myeloid cells, while reduced, showed progressive maturation.There was no evidence of hemophagocytosis. One patient had a prior bone marrow biopsy after G-CSF treatment which showed post treatment myeloid hyperplasia, but the neutropenia was not corrected. Erythroid maturation was complete in all cases. As expected in immune-mediated thrombocytopenia, there was megakaryocytic hyperplasia with increased immature megakaryocytes.
We developed SASH3 R347C knock-in mice using CRISPR-Cas9 technology. The mice were obtained by breeding females heterozygous for SASH3 knockin (SASH3 KI) with wild type (WT) males. The male pups obtained were either WT or Hemizygous for SASH3 KI. The mice did not show a difference in marrow cellularity, progenitor numbers or lineage subsets with the exception of NK cells. SASH3 KI R347C mice showed significantly decreased NK and T/NK cells, but no evidence of neutropenia at the age of 6-9 weeks, unlike the patient. Interestingly, the CD4:CD8 ratio of T-cells in the SASH3 KI mice were altered with a significant reduction in the proportion of CD8+ T-cells.
In summary, SASH3 deficiency causes an X-linked combined immunodeficiency that presents with multiple cytopenias and infections, although a milder phenotype, characterized by infections but not autoimmunity, has been recently reported in another patient (Labrador-Horrillo et al., Front Immunol 2022). Bone marrow may demonstrate extensive infiltration by CD8+ T-cells that may be clonal and have features overlapping with T-LGL leukemia. The mouse model recapitulates predominantly the NK lymphopenia at early time points. A longer follow up in SASH3 KO mice and the SASH3 R347C KI mouse model may be informative and lead to insight into the mechanisms of cytopenias and other immune system dysfunction in SASH3 deficiency. SASH3 deficiency should be considered in male patients presenting with cytopenias, immunodeficiency, and immune dysregulation.
Disclosures
Cortese:Keires, AG; Nouscom, AG; and PDC*line Pharma: Other: shareholder.
Author notes
Asterisk with author names denotes non-ASH members.
