In several carcinomas, the SET Domain Containing 3, Actin Histidine Methyltransferase (SETD3)is associated with oncogenesis. However, there is little knowledge about the role of SETD3 in the progression and prognosis of breast cancer. In this study, we first analyzed the prognostic value of SETD3 in breast cancer patients using the database of the public Kaplan-Meier plotter. Moreover, in vitro assays were performed to assess the role of SETD3 in the viability and capacity of invasion of human breast cancer cell lines. We observed that the high expression of SETD3 was associated with better relapse-free survival (RFS) of the whole collective of 3,951 patients, of Estrogen Receptorpositive, and of Luminal A-type breast cancer patients. However, in patients lacking expression of estrogen-, progesterone-and HER2-receptor, and those affected by a p53-mutation, SETD3 was associated with poor RFS. In vitro analysis showed that SETD3 siRNA depletion affects the viability of triple-negative cells as well as the cytoskeletal function and capacity of invasion of highly invasive MDA-MB-231 cells. Interestingly, SETD3 regulates the expression of other genes associated with cancer such as β-actin, FOXM1, FBXW7, Fascin, eNOS, and MMP-2. Our study suggests that SETD3 expression can act as a subtype-specific biomarker for breast cancer progression and prognosis.MTT proliferation assay. SETD3 depleted cells or controls (5 × 10 3 ) were plated in 96-well plates with DMEM medium (without phenol red) (Gibco ® , cat. No. 31053028, Germany) with FCS and incubated for 96 hours. After the incubation time, the medium was removed completely and cells were incubated with 20 µl/well of 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) (cat. No. M2128-1G), at 5 mg/ml for 4 hours, at 37 °C. Subsequently, 100 µl of Stopping buffer, pH 4.7 composed of 10% (w/v) sodium dodecyl sulfate (SDS) (cat. No. 3599286) and 50% (v/v) N,) was added to stop the reaction and dissolve the formazan crystals. The absorbance was measured in a VersaMax ® Microplate Reader (Molecular Devices, Sunnyvale, CA, USA) at a wavelength of 595 nm. The proliferation of the control cells was defined as 100%. Results were derived from three independent sets of triplicate experiments. MTT, SDS, and N,N-Dimethyl formamide were from Sigma-

OBJECTIVE Multiple genome-wide association studies have identified a strong genetic linkage between the SKAP2 locus and type 1 diabetes (T1D), but how this leads to disease remains obscure. Here, we characterized the functional consequence of a novel SKAP2 coding mutation in a patient with T1D to gain further insight into how this impacts immune tolerance. RESEARCH DESIGN AND METHODS We identified a 24-year-old individual with T1D and other autoimmune and inflammatory conditions. The proband and first-degree relatives were recruited for whole-exome sequencing. Functional studies of the protein variant were performed using a cell line and primary myeloid immune cells collected from family members. RESULTS Sequencing identified a de novo SKAP2 variant (c.457G>A, p.Gly153Arg) in the proband. Assays using monocyte-derived macrophages from the individual revealed enhanced activity of integrin pathways and a migratory phenotype in the absence of chemokine stimulation, consistent with SKAP2 p.Gly153Arg being constitutively active. The p.Gly153Arg variant, located in the well-conserved lipid-binding loop, induced similar phenotypes when expressed in a human macrophage cell line. SKAP2 p.Gly153Arg is a gain-of-function, pathogenic mutation that disrupts myeloid immune cell function, likely resulting in a break in immune tolerance and T1D. CONCLUSIONS SKAP2 plays a key role in myeloid cell activation and migration. This particular mutation in a patient with T1D and multiple autoimmune conditions implicates a role for activating SKAP2 variants in autoimmune T1D.

<b>OBJECTIVE: </b>Multiple genome-wide association studies (GWAS) have identified a strong genetic linkage between the <i>SKAP2</i> locus and type 1 diabetes (T1D) but how this leads to disease remains obscure. Here, we characterized the functional consequence of a novel <i>SKAP2</i> coding mutation in a T1D patient to gain further insight into how this impacts immune tolerance. <p><b> </b></p> <p><b>RESEARCH DESIGN AND METHODS: </b>We identified a 24-year-old individual with T1D and other autoimmune and inflammatory conditions. The proband and first-degree relatives were recruited for whole exome sequencing. Functional studies of the protein variant were performed using a cell line and primary myeloid immune cells collected from family members.</p> <p><b> </b></p> <p><b>RESULTS: </b>Sequencing identified a <i>de novo</i> <i>SKAP2</i> variant (c.457G>A, p.Gly153Arg) in the proband. Assays using monocyte-derived macrophages from the individual revealed enhanced activity of integrin pathways and a migratory phenotype in the absence of chemokine stimulation, consistent with SKAP2 p.Gly153Arg being constitutively active. The p.Gly153Arg variant, located in the well-conserved lipid-binding loop, induced similar phenotypes when expressed in a human macrophage cell line. SKAP2 p.Gly153Arg is a gain-of-function, pathogenic mutation that disrupts myeloid immune cell function, likely resulting in a break in immune tolerance and T1D.</p> <p><b> </b></p> <p><b>CONCLUSIONS: </b>SKAP2 plays a key role in myeloid cell activation and migration. This particular mutation in a patient with T1D and multiple autoimmunity implicates a role for activating <i>SKAP2</i> variants in autoimmune T1D. <br> </p>