Figure 1.
Identification and functional characterization of PSTPIP1 mutation N236K in a patient with PAMI syndrome. (A) Laboratory testing results for markers of inflammation and for leukocyte subset frequencies. Results were obtained on at least two occasions for erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), ferritin, and zinc, and the indicated leukocyte subsets. Repeated measures obtained on separate dates are separated by semicolons. Values above the age-matched normal range are bolded. Values below the normal range are in red font. Data were obtained while the patient was between 5 and 22 mo of age. ■measurement taken while on anakinra; ⊕while on ruxolitinib. NK, natural killer. (B) Timeline of patient’s clinical course. WES, whole-exome sequencing. (C) Co-immunoprecipitation (co-IP) experiments assessing the ability of WT and mutant forms of PSTPIP1 (pEZY-FLAG-PSTPIP1) to bind pyrin (pCMV6-MEFV-Myc). EV, empty vector control; IB, immunoblot. (D) Quantitation of PSTPIP1 protein, relative to pyrin protein, in co-IP samples from three independent experiments performed as in B. *P < 0.05, paired two-tailed T test. (E) ASC speck assays using HEK293-ASCYFP cells transfected with pEZY-FLAG-PSTPIP1 plasmids (EV, WT, N236K, or E250K), with or without pyrin (pCMV6-MEFV-Myc). ASC is visualized in green, and DAPI-stained nuclei are visualized in blue. (F) Quantitation of ASC specs, normalized to DAPI-stained nuclei, from fluorescence microscopy images using automated counting in ImageJ. Each data point is the average of at least 10 fields per condition in a single experiment. Graph shows results from n = 4 independent experiments. *P < 0.05, **P < 0.01, paired two-tailed T test. (G) A partial crystal structure of WT PSTPIP1 surrounding the N236 residue is shown, from Protein Data Bank entry 7AAN. The N236 amino acid is polar and undergoes Van der Waals interactions (VDW) with neighboring amino acids. Modeling of the N236K mutation shows loss of VDW interactions and polarity. (H) Patterns of gene expression in whole blood samples from PAMI patients (n = 2), healthy pediatric controls (HP, n = 4), and healthy adult controls (HC, n = 4). All genes included on heatmap showed P < 0.05 in comparisons of patients vs. healthy adult controls and patients vs. healthy pediatric controls, and P > 0.05 in comparisons of healthy adult controls vs. healthy pediatric controls (two-tailed unpaired T test). Source data are available for this figure: SourceData F1.

Identification and functional characterization of PSTPIP1 mutation N236K in a patient with PAMI syndrome. (A) Laboratory testing results for markers of inflammation and for leukocyte subset frequencies. Results were obtained on at least two occasions for erythrocyte sedimentation rate (ESR), C-reactive protein (CRP), ferritin, and zinc, and the indicated leukocyte subsets. Repeated measures obtained on separate dates are separated by semicolons. Values above the age-matched normal range are bolded. Values below the normal range are in red font. Data were obtained while the patient was between 5 and 22 mo of age. ■measurement taken while on anakinra; ⊕while on ruxolitinib. NK, natural killer. (B) Timeline of patient’s clinical course. WES, whole-exome sequencing. (C) Co-immunoprecipitation (co-IP) experiments assessing the ability of WT and mutant forms of PSTPIP1 (pEZY-FLAG-PSTPIP1) to bind pyrin (pCMV6-MEFV-Myc). EV, empty vector control; IB, immunoblot. (D) Quantitation of PSTPIP1 protein, relative to pyrin protein, in co-IP samples from three independent experiments performed as in B. *P < 0.05, paired two-tailed T test. (E) ASC speck assays using HEK293-ASCYFP cells transfected with pEZY-FLAG-PSTPIP1 plasmids (EV, WT, N236K, or E250K), with or without pyrin (pCMV6-MEFV-Myc). ASC is visualized in green, and DAPI-stained nuclei are visualized in blue. (F) Quantitation of ASC specs, normalized to DAPI-stained nuclei, from fluorescence microscopy images using automated counting in ImageJ. Each data point is the average of at least 10 fields per condition in a single experiment. Graph shows results from n = 4 independent experiments. *P < 0.05, **P < 0.01, paired two-tailed T test. (G) A partial crystal structure of WT PSTPIP1 surrounding the N236 residue is shown, from Protein Data Bank entry 7AAN. The N236 amino acid is polar and undergoes Van der Waals interactions (VDW) with neighboring amino acids. Modeling of the N236K mutation shows loss of VDW interactions and polarity. (H) Patterns of gene expression in whole blood samples from PAMI patients (n = 2), healthy pediatric controls (HP, n = 4), and healthy adult controls (HC, n = 4). All genes included on heatmap showed P < 0.05 in comparisons of patients vs. healthy adult controls and patients vs. healthy pediatric controls, and P > 0.05 in comparisons of healthy adult controls vs. healthy pediatric controls (two-tailed unpaired T test). Source data are available for this figure: SourceData F1.

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