Rare SH2B3 variants were identified in SLE patients and HCs. (A) Pedigrees of families with rare single nucleotide variants (SNV) in SH2B3 associated with autoimmunity. Black arrows indicate the proband of each cohort. Individuals colored black are affected by SLE, and individuals colored gray are affected by RA. The amino acid changes in SH2B3 protein are shown next to sequenced individuals, with “+” sign indicating WT. (B) Location of patient-specific and HC SNVs in the schematic diagram of SH2B3 amino acid sequence. DD: dimerization domain, SH2: Src homology 2 domain, PH: Pleckstrin homology domain. (C) Summary of variant information, in silico deleteriousness prediction, and MAF of each SNV from HC and SLE patients. PP2: PolyPhen-2. (D) Relative activity of STAT1-GAS in HEK293 cells overexpressing empty vector, WT, or mutant human SH2B3 in the presence of IFN-γ (50 ng/ml) stimulation. Relative activity was calculated by normalizing all STAT1-GAS-Firefly/CMV-Renilla ratios to the average ratios in the unstimulated cells transfected with empty vectors. Results were pooled from seven independent experiments. Sample numbers for each condition are listed as follows: empty (n = 21), WT (n = 21), R43C (n = 9), C133Y (n = 12), E208Q (n = 21), E400K (n = 15), A536T (n = 18), Q540X (n = 18), and R566Q (n = 21). Means are shown as bars, and all conditions were compared with cells transfected with WT SH2B3. lmer with emmeans using the experiment as the blocking factor was used for the statistical analyses. ***: P < 0.001, ****: P < 0.0001. (E) Hydrogen bonding network between Arg369-Glu372-Arg387 (left) that helps position the BC loop relative to the central β-sheet and disrupted hydrogen bond formation (right) when Glu372 is replaced with Lys. (F) Percentage of binding of murine WT or E372K SH2B3 to IL6ST pY757 peptide chip after preincubation with varying concentrations of mutant JAK pY813 peptides (left; WT n = 8, E372K: n = 6) or phenylphosphate (right; WT: n = 4, E372K: n = 2). Data are shown as the mean ± SD of technical replicates from at least three independent experiments. (G) Melting temperatures of WT (left) and E400K (right) SH2B3 protein at apo state (unbound) or bound to pY813 JAK2. Data are displayed as the mean ± SD of technical replicates from at least three independent experiments. WT Apo, WT + pY813, and E400K Apo: n = 3; E400K pY813: n = 7.
Figure 1.

Rare SH2B3 variants were identified in SLE patients and HCs. (A) Pedigrees of families with rare single nucleotide variants (SNV) in SH2B3 associated with autoimmunity. Black arrows indicate the proband of each cohort. Individuals colored black are affected by SLE, and individuals colored gray are affected by RA. The amino acid changes in SH2B3 protein are shown next to sequenced individuals, with “+” sign indicating WT. (B) Location of patient-specific and HC SNVs in the schematic diagram of SH2B3 amino acid sequence. DD: dimerization domain, SH2: Src homology 2 domain, PH: Pleckstrin homology domain. (C) Summary of variant information, in silico deleteriousness prediction, and MAF of each SNV from HC and SLE patients. PP2: PolyPhen-2. (D) Relative activity of STAT1-GAS in HEK293 cells overexpressing empty vector, WT, or mutant human SH2B3 in the presence of IFN-γ (50 ng/ml) stimulation. Relative activity was calculated by normalizing all STAT1-GAS-Firefly/CMV-Renilla ratios to the average ratios in the unstimulated cells transfected with empty vectors. Results were pooled from seven independent experiments. Sample numbers for each condition are listed as follows: empty (n = 21), WT (n = 21), R43C (n = 9), C133Y (n = 12), E208Q (n = 21), E400K (n = 15), A536T (n = 18), Q540X (n = 18), and R566Q (n = 21). Means are shown as bars, and all conditions were compared with cells transfected with WT SH2B3. lmer with emmeans using the experiment as the blocking factor was used for the statistical analyses. ***: P < 0.001, ****: P < 0.0001. (E) Hydrogen bonding network between Arg369-Glu372-Arg387 (left) that helps position the BC loop relative to the central β-sheet and disrupted hydrogen bond formation (right) when Glu372 is replaced with Lys. (F) Percentage of binding of murine WT or E372K SH2B3 to IL6ST pY757 peptide chip after preincubation with varying concentrations of mutant JAK pY813 peptides (left; WT n = 8, E372K: n = 6) or phenylphosphate (right; WT: n = 4, E372K: n = 2). Data are shown as the mean ± SD of technical replicates from at least three independent experiments. (G) Melting temperatures of WT (left) and E400K (right) SH2B3 protein at apo state (unbound) or bound to pY813 JAK2. Data are displayed as the mean ± SD of technical replicates from at least three independent experiments. WT Apo, WT + pY813, and E400K Apo: n = 3; E400K pY813: n = 7.

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