CD48 is a coactivating receptor within the signaling lymphocytic activation molecule (SLAM) family that is important for the regulation of lymphocyte cytotoxicity and T cell activation. Volkmer et al. previously described a patient case with recurrent inflammatory disease secondary to a de novo heterozygous S220Y mutation in the CD48 gene (1). We also previously reported a novel, de novo, heterozygous S220F mutation with a similar clinical phenotype. As CD48 is tolerant of loss-of-function variants (probability of loss of function intolerance [pLI] = 0), CD48 haploinsufficiency could not explain the cellular derangements seen with S220 CD48 variants and investigated whether S220 causes immune disease through S220, which is the omega amino acid to which the glycosylphosphatidylinositol (GPI) is covalently linked. Further testing has demonstrated that these S220 variants are dominant-negative inborn error of immunity caused by impaired GPI anchor linkage.
Combinations of hCD48 expression vectors—either wild type (WT), S220Y, S220F, or empty—were transfected into the HEK-293 cell line, and surface or intracellular CD48 levels were measured by flow cytometry. Phospholipase C (PLC) from Bacillus cereus was used to cleave surface GPI anchors prior to flow cytometry. In separate experiments, an unfolded protein response (UPR) reporter was stably transduced prior to transfection with hCD48 expression vectors. pLVX-ATF4 mScarlet NLS was a gift from David Andrews (Addgene plasmid # 115969; http://n2t.net/addgene:115969; RRID:Addgene_115969).
S220F and S220Y decreased surface CD48 when co-transfected with WT hCD48 (p < 0.01 compared to empty vector). PLC reduced surface CD48 by 73% for S220Y and 79% for S220F. UPR reporter activity was higher for S220F (p = 0.01) and S220Y (p = 0.08).
S220F/Y interacts with the GPI transamidase within the ER, but the efficiency of GPI linkage is severely reduced. This situation increases ER stress and induces the unfolded protein response. When monoallelic, S220 variants in CD48 dominantly impair surface expression of WT CD48, lower than levels seen with gene haploinsufficiency. Heterozygous S220 mutations are a new class of dominant-negative inborn error immunity with severely deficient surface expression and increased unfolded protein response as the mechanism of pathogenicity.
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