Nature Immunology: doi: /ni.3575
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2 Supplementary Figure 1 Histology of lymphoma and segregation of the mutation with the disease phenotype. (a) Histology of adenoid biopsy showing low grade B-cell lymphoma compatible with marginal zone lymphoma. Top left to right: Hematoxilin/Eosin staining, CD20 staining, CD3 staining. Bottom left to right: EBER, CD23, Ki67. The described findings suggest a low grade EBV-related B-cell lymphoma developed likely associated with the underlying immunodeficiency. (b) Segregation of the detected mutation among the core family (circle - female; square - male; line - dead; black filling - affected index patient).
3 Supplementary Figure 2 Functional characterization of T cells. (a) Invariant natural killer T (inkt) cells. A prominent TCRv 24-expressing cell population was detectable in patient peripheral blood, however these cells expressed CD8 and CD45RA surface markers, suggesting that they belong to oligoclonally expanded exhausted memory CD8 T cells. HD healthy donor; SC Shipment control. (b) Proliferative response of T cells determined by [ 3 H]-thymidine incorporation assay after stimulation with various stimuli after 3 days (OKT3, anti-cd3 antibody (clone OKT3)); PMA, phorbol 12-myristate 13-acetate; SEA: Staphylococcal enterotoxin A; SEB: Staphylococcal enterotoxin B; PHA: phytohaemagglutinin). (c) Proliferation of Jurkat T cells upon shrna-mediated knockdown of RASGRP1. shrna against Renilla luciferase was used as control. Cells were labeled with violet proliferation dye and analyzed by flow cytometry over the period of 5 days. (d) TCRV spectratyping of patient and control T cells indicating oligoclonality of the TCR repertoire of the patient. (e) Calcium flux of Jurkat T cells upon inducible shrna-mediated knockdown of RASGRP-1. shrna against Renilla luciferase was used as control. (f) Fluorescence microscopy of Ca 2+ -flux of one representative cell is displayed below the graph. Scale bar represents 10 µm. HD healthy donor; DIC differential interference contrast. (g) ICAM Ring formation and csmac exclusion of htert immortalized patient and control T cell lines on a lipid bilayer following OKT3 stimulation. (h) Cropped immunoblot showing downstream TCR signaling in expanded patient and shipment T cells upon CD3/CD28 stimulation. Cells were starved and restimulated with anti-cd3/anti-cd28 antibodies and subsequently analyzed for ERK1/2 phosphorylation. Data is representative of three (h), two (a,c,d,g) or one (b,f,e) independent experiment. Full gating strategy is shown in Figure 3.
4 Supplementary Figure 3 Gating Strategy Gating strategy for Figures 2 and 3 is presented in this figure.
5 Supplementary Figure 4 NK-cell studies. (a) Flow cytometric analysis of intracellular accumulation of IL-5, IL13 and IFN in patient and control NK cells. (b) Immune synapse formation in primary patient NK cells (c) Co-immunoprecipitation of Strep-HA tagged RASGRP1 with endogenous dynein light chain 1 (DYNLL1) (RASGRP1wt) wildtype Strep-HA-tagged RASGRP1 and (RASGRP1mut) mutant Strep-HA-tagged RASGRP1. Strep-HAtagged GFP was used as control. (d) Video microscopy of granule convergence in CRISPR-edited NK-92 cells lines HD, healthy donor. (d) Video microscopy of control or sgrasgrp1 NK-92 cells for granule convergence (Granules-red, Microtubuli-green). Data are representative of two biological replicates (a-d).(e) Gating strategy of FACS plots in Figure 4.
6 Supplementary Figure 5 T-cell activation using CXCL12. (a-c) Immunofluorescence (a) and quantification (b,c) of patient and healthy donor expanded CD8 T cells following CXCL12 stimulation either stained for total RhoA (green) or active RhoA (pink) and DAPI (blue). Data is representative of two (a-c) independent experiments.
7 Supplementary Table 1. Immunophenotyping results on several occasions of the index patient bearing the homozygous RASGRP1 stop gain mutation. Patient/ Age 7 years old 8 years old 12 years old 12.5 years old ALC (cells/mm³) (normal range) 4064 ( ) 3100 ( ) 2700 ( ) 2700 ( ) Immunoglobulins Ig A (g/dl) 660 (70-303) 680 (70-303) 640 (67-433) 766 (67-433) Ig G (g/dl) 2030 ( ) 3400 ( ) 1720 ( ) 1600 ( ) Ig M (g/dl) 188 (69-387) 346 (69-387) 218 (47-484) 306 (47-484) Lymphocyte subsets CD3 (%) 64 (55-78) (cells/mm³) 2600 ( ) 1891 ( ) 1125 ( ) 2187 ( ) CD4 (%) 13 (27-53) 10 (27-53) 13 (25-48) 8 (25-48) (cells/mm³) 528 ( ) 310 ( ) 195 ( ) 216 ( ) CD8 (%) 55 (19-34) 52 (19-34) 58 (9-35) 70 (9-35) (cells/mm³) 2235 ( ) 1612 ( ) 870 ( ) 1890 ( ) CD19 (%) 10 (10-31) 11 (10-31) 9 (8-24) 3 (8-24) (cells/mm³) 406 ( ) 341 ( ) 135 ( ) 81 ( ) CD16+56 (%) 22 (4-26) 26 (4-26) 11 (6-27) 13 (6-27) (cells/mm³) 894 (90-900) 806 (90-900) 165 ( ) 351 ( ) CD45RA 55 CD45RO 51 TCR αβ 62 TCR γδ 17 ALC, absolute lymphocyte count; TCR, T cell receptor.
8 Supplementary Table 2. Immunophenotyping of the healthy mother. Value Mother WBC (x10 3 /µl) 5.9 ( ) ALC (x10 3 /µl) 2.3 ( ) ANC (x10 3 /µl) 2.3 ( ) Monocytes (x10 3 /µl) 0.5 ( ) Eosinophils (x10 3 /µl) 0.4 ( ) Basophils (x10 3 /µl) 0 (0-0.2) Immunoglobulins Ig A (g/dl) Ig G (g/dl) 5.42 ( ) 18.5 (8-18) Ig M (g/dl) 3.29 ( ) Lymphocyte subsets CD3 (%) 68 (55-83) CD3 (1000/µl) 1.5 ( ) CD4 (%) 42 (28-57) CD4 (1000/µl) 1.0 ( ) CD8 (%) 25 (10-39) CD8 (1000/µl) 0.7 ( ) CD19 (%) 9 (6-19) CD19 (1000/µl) 0.2 ( ) CD16+56 (%) 14 (7-31) CD16+56 (1000/µl) 0.3 ( )
9 Supplementary Table 3. Relative immunophenotyping values of patient, mother brother and a healthy donor. Subset (rel. proportions in %) Healthy Donor Mother Brother Patient CD CD3+CD CD3+CD CD Gated on CD3+: inkt cells (TCRvα24+TCRvβ11+) recent thymic emigrants (CD3+CD4+CD45RA+CD31+) TCRvα7.2+CD161+ (MAIT) Gated on CD3+CD4+: CD45RA+CCR CD45RA+CCR CD45RA-CCR CD45RA-CCR CD3+CD4+FOXP n.d. n.d CD45RA+CD n.d. n.d CD45RA+CD n.d. n.d CD45RA-CD n.d. n.d CD45RA-CD n.d. n.d CD45RA+CD n.d. n.d. 0.6 CD45RA+CD n.d. n.d CD45RA-CD n.d. n.d CD45RA-CD n.d. n.d Gated on CD3+CD8+: CD45RA+CCR CD45RA+CCR CD45RA-CCR CD45RA-CCR CD45RA+CD n.d. n.d CD45RA+CD n.d. n.d CD45RA-CD n.d. n.d CD45RA-CD n.d. n.d. 62 CD45RA+CD n.d. n.d CD45RA+CD n.d. n.d. 1.3 CD45RA-CD n.d. n.d. 2.9 CD45RA-CD n.d. n.d Gated on CD19+: IgD+CD27- (naïve) IgD+CD27+(memory non-switched) IgD-CD27+(memory) IgD-CD CD21 low CD CD21 low CD
10 Supplementary Table 4. Evaluation of the detected variant in RASGRP1 (Chr15: : G>A; c. C726T, p. Arg246*) in exome variant databases (last accessed July, 13, 2016). Source dbsnp v142 1 no 60,706 Exomes from the Exome Aggregation Consortium 2 no 6503 Exomes from NHLBI Exome Sequencing Project 3 no CBio Portal 4 no 108 Exomes Hacettepe Exome Project no In-house collection of 500 individuals no (219 Exomes including 96 PID patients, 12 Genomes and 269 target enrichment screens on 229 PID patients and 40 SCID patients) Found
11 Supplementary Table 5a. Evaluation of the estimated population frequency severe homozygous RASGRP1 variants. To date, no severe (stopgain, frameshift, splice-site) homozygous variants have been observed in RASGRP1. This table lists rare heterozygous variants in RASGRP1 which have been identified and indicates an estimated population frequency of homozygous RASGRP1 loss-of-function individuals based on heterozygous carrier frequency, as obtained from ExAC browser (accessed April 29, 2016). Homo zygous Alleles Consequence Annotation African East Asian European (Non- Finnish) Heterozygous Allele count Finnish Latino Other South Asian p.gln793ter stop gained p.leu788phefs frameshift p.his786glnfs frameshift c g>a splice acc p.arg98thrfs frameshift p.gly92ter* stop gained p.met76valfs frameshift p.val55ter frameshift p.gln545ter stop gained p.thr201ilefs frameshift c.570-1g>a splice acc c.570-1g>t splice acc splice c g>a donor 1 p.asp371ilefs frameshift p.glu367ter stop gained p.ile354phefs frameshift c.327-1c>g splice acc p.arg48ter stop gained p.glu27argfs frameshift p.phe3_serfs frameshift p.pro8hisfs frameshift Allele count SUM of putative loss-of-function population alleles Total population alleles loss-of-function carrier frequency E E E E E E- 04 Expected rate of nullizygous per million (1 in X non-consanguineous conceptions) < <
12 Supplementary Table 5b. RASGRP1 conservation evaluation. CADD Score, RVIS Score and expected versus observed number of nonsense variants in RASGRP1 are summarized below. Performed Test Score Chr15: : G>A variant CADD (v1.3) score 35 CCDS Size (including canonical splice) ENSG Number of unique protein-coding variants (ExAC) 594 RVIS Score Version 2 of RASGRP (11.77% most intolerant) CCDS ExAC Expected number of nonsense variants 28.3 CCDS Observed nonsense variants (ExAC) 7 Binomial Exact test p-value E-06
13 Supplementary Table 6. Comparison of human RASGRP1 deficiency with findings from Rasgrp1 -/- knockout mice 2, 3, 4, 5. Human RASGRP1 Rasgrp1-/- mouse model Deficiency Peripheral T-cells CD3+ CD4+ 8 CD3+CD8+ 8 TCRαβ n.d. TCRγδ 8 Proliferation 8 ERK signaling 8 inkt cells 18 CD19+ NK cells CD16+CD56+ normal normal Cytotoxicity n.d. Peripheral B-cells CD19+ normal 8 CD19+IgD+ normal n.d. CD19+IgM+ 8 CD19+CD21 low n.d. CD19+ CD38+ n.d. Autoimmunity Splenomegaly no yes, older mice 24 Autoantibodies no Yes, ANCA 24 Immunodeficiency Viral infections n.d. Bacterial Infectios n.d.
14 Supplementary Table 7. Absence of all autoantibodies tested in index patient P1. Nuclear Antibodies Anti-nuclear antibody (ANA) Rheuma Serology Rheumatoid-factor IgG Rheumatoid -factor IgA Rheumatoid -factor IgM Rheumatoid factor latex test Anti-streptolysine O Collagen type I Collagen type II Collagen type III Collagen type IV Thyreoid gland Antibodies Thyreoglobulin antibody Vessel & kidney Antibodies Neutrophil cytoplasmic antibody (ANCA) Proteinase-3 antibody Myeloperoxidase Phospholipid antibodies Cardiolipin IgG Cardiolipin IgA Cardiolipin IgM B2-glycoprotein IgG B2-glycoprotein IgA b2-glycoprotein IgM Intestinal Antibodies Gliadin IgG Gliadin IgA Tissue transglutaminase IgG Tissue Transglutaminase IgA
15 Supplementary Table 8a. FCA, FCB and SAINT Score of identified RASGRP1 interactors in HEK293 cells. BAIT PREY FC_A FC_B SAINT RASGRP1 RASGRP RASGRP1 DYNLL RASGRP1 DYNLL RASGRP1 TUBA1A RASGRP1 TUBA1C RASGRP1 TUBB4B RASGRP1 TUBB RASGRP1 TUBB RASGRP1 TUBA RASGRP1 TUBB2B RASGRP1 TUBA1B RASGRP1 SLC25A RASGRP1 EMD RASGRP1 HSPA1B RASGRP1 RPS RASGRP1 SLC25A RASGRP1 ATP5A RASGRP1 CAD RASGRP1 IRS RASGRP1 PPP6R RASGRP1 SLC25A RASGRP1 LGALS3BP RASGRP1 RHOT RASGRP1 HSPA1L RASGRP1 HNRNPH RASGRP1 HNRNPF RASGRP1 RPS RASGRP1 ATP1A RASGRP1 HSPD RASGRP1 TUFM RASGRP1 ATP2A RASGRP1 DCAF RASGRP1 PPP6C RASGRP1 RCN RASGRP1 SLC25A RASGRP1 SLC25A RASGRP1 RMDN RASGRP1 ACSL RASGRP1 HLA-B RASGRP1 HSP90AA RASGRP1 HSPA RASGRP1 HSPA
16 RASGRP1 CHUK RASGRP1 RPL RASGRP1 EEF1A RASGRP1 RPLP RASGRP1 FASN RASGRP1 ANKRD RASGRP1 BAG RASGRP1 SLC25A RASGRP1 ACTA RASGRP1 HMGB1P RASGRP1 HSPA RASGRP1 MYL RASGRP1 KRT RASGRP1 TBC1D RASGRP1 HNRNPU RASGRP1 HSP90AB RASGRP1 GFP RASGRP1 FBXO RASGRP1 UBB RASGRP1 ACTB Supplementary Table 8b. FCA, FCB and SAINT Score of identified RASGRP1 interactors in Jurkat T cells. BAIT PREY FC_A FC_B SAINT RASGRP1 RASGRP RASGRP1 NA RASGRP1 TUBB4B RASGRP1 DYNLL RASGRP1 TUBB RASGRP1 EIF3I RASGRP1 EMD RASGRP1 TUBA1B RASGRP1 EEF RASGRP1 TUBB RASGRP1 HSPA RASGRP1 PDIA RASGRP1 TKT RASGRP1 RPS RASGRP1 EEF1A1P RASGRP1 NME RASGRP1 ATP5A RASGRP1 SERBP RASGRP1 VCP RASGRP1 HNRNPM
17 RASGRP1 MSN RASGRP1 CCT RASGRP1 PGK RASGRP1 HSPA RASGRP1 SSBP RASGRP1 HSPD RASGRP1 RBBP RASGRP1 HMGB RASGRP1 CCT RASGRP1 TRMT RASGRP1 CCT RASGRP1 NME RASGRP1 CCT RASGRP1 SUB RASGRP1 PKM RASGRP1 MDH RASGRP1 HINT RASGRP1 PAFAH1B RASGRP1 PGAM RASGRP1 DUT RASGRP1 SFPQ RASGRP1 PCNP RASGRP1 SOD RASGRP1 CCT RASGRP1 EZR RASGRP1 PRKCSH RASGRP1 YWHAG RASGRP1 RPL RASGRP1 LAP RASGRP1 ADA RASGRP1 CCT RASGRP1 CCT6A RASGRP1 RBBP RASGRP1 PPM1G RASGRP1 PSMA RASGRP1 RPL RASGRP1 ATP5H RASGRP1 PRDX RASGRP1 NPM RASGRP1 NUDC RASGRP1 SF3A RASGRP1 STMN RASGRP1 PPIA
18 RASGRP1 PAICS RASGRP1 HMGB RASGRP1 CNPY RASGRP1 MTHFD RASGRP1 PSME RASGRP1 LUC7L RASGRP1 P4HB RASGRP1 PA2G RASGRP1 ENO RASGRP1 PRSS RASGRP1 TPM RASGRP1 SHMT RASGRP1 NAP1L RASGRP1 SRRM RASGRP1 PFDN RASGRP1 RANBP RASGRP1 TPI RASGRP1 CAPZB RASGRP1 GDI RASGRP1 AIP RASGRP1 IDH RASGRP1 TALDO RASGRP1 RPS RASGRP1 PFDN RASGRP1 YWHAB RASGRP1 HSPE RASGRP1 RNASEH2C RASGRP1 LCP RASGRP1 THYN RASGRP1 MYL RASGRP1 PARK RASGRP1 RPS RASGRP1 ATP5B RASGRP1 NONO RASGRP1 PSMA RASGRP1 MANF RASGRP1 COX4I RASGRP1 DPYSL RASGRP1 PSME RASGRP1 SARNP RASGRP1 SEP RASGRP1 ANXA RASGRP1 EEF1D
19 RASGRP1 DSTN RASGRP1 EIF4A RASGRP1 CALR RASGRP1 SET RASGRP1 NEDD RASGRP1 LASP RASGRP1 TBCA RASGRP1 EIF3A RASGRP1 PHGDH RASGRP1 PAK RASGRP1 KHDRBS RASGRP1 SF3B RASGRP1 YWHAH RASGRP1 ATIC RASGRP1 MCM RASGRP1 ATP5I RASGRP1 PDIA RASGRP1 G3BP RASGRP1 PCNA RASGRP1 HNRNPK RASGRP1 PRDX RASGRP1 ILF RASGRP1 RPS RASGRP1 TCEB RASGRP1 CCDC RASGRP1 FLNA RASGRP1 ACAT RASGRP1 COX5B RASGRP1 PSMC RASGRP1 LRRC RASGRP1 TLN RASGRP1 MDH RASGRP1 KHSRP RASGRP1 MYH RASGRP1 TFG RASGRP1 NSFL1C RASGRP1 GPI RASGRP1 SMAP RASGRP1 HN RASGRP1 FAM136A RASGRP1 CDV RASGRP1 PSMB RASGRP1 CACYBP
20 RASGRP1 YWHAQ RASGRP1 EIF5A RASGRP1 ACP RASGRP1 COX5A RASGRP1 NAP1L RASGRP1 PPA RASGRP1 EIF5A RASGRP1 LSM RASGRP1 GLO RASGRP1 FARSA RASGRP1 MCM RASGRP1 STMN RASGRP1 PFDN RASGRP1 PSMB RASGRP1 HIST1H1A RASGRP1 RPS RASGRP1 GSTP RASGRP1 MZB RASGRP1 RPS3A RASGRP1 TCOF RASGRP1 WDR RASGRP1 DBNL RASGRP1 GART RASGRP1 PSAP RASGRP1 HINT RASGRP1 PGD RASGRP1 RUVBL RASGRP1 EIF4H RASGRP1 TCP RASGRP1 TCEB RASGRP1 HCLS RASGRP1 YWHAE RASGRP1 MAT2A RASGRP1 H2BFS RASGRP1 PHB RASGRP1 PSMA RASGRP1 FEN RASGRP1 SRP RASGRP1 HNRNPUL RASGRP1 SNRPG RASGRP1 EIF RASGRP1 EEF1G RASGRP1 ENO
21 RASGRP1 ANXA RASGRP1 ALDOA RASGRP1 RPS RASGRP1 PSMA RASGRP1 SUMF RASGRP1 TUBB4A RASGRP1 STIP RASGRP1 GDI RASGRP1 ACAT RASGRP1 EIF3C RASGRP1 BTF3L RASGRP1 CCDC RASGRP1 RANBP RASGRP1 PFKP RASGRP1 EDF RASGRP1 SRSF RASGRP1 HSPH RASGRP1 ARPP RASGRP1 C9orf RASGRP1 MAP RASGRP1 CHORDC RASGRP1 CRIP RASGRP1 CANX RASGRP1 ACTR RASGRP1 VBP RASGRP1 USP RASGRP1 RPSA RASGRP1 RPS RASGRP1 DDX RASGRP1 YWHAZ RASGRP1 RPS RASGRP1 SNRPGP RASGRP1 LDHB RASGRP1 ANP32E RASGRP1 ANP32B RASGRP1 APRT RASGRP1 ANP32A RASGRP1 GANAB RASGRP1 AK RASGRP1 PSMC RASGRP1 COA RASGRP1 REXO RASGRP1 CBFB
22 RASGRP1 EIF4B RASGRP1 MTHFD RASGRP1 ADSS RASGRP1 IFI RASGRP1 RTCB RASGRP1 ST13P RASGRP1 MAT2B RASGRP1 HN1L RASGRP1 TPD RASGRP1 SF RASGRP1 SRSF RASGRP1 SEP RASGRP1 PSMA RASGRP1 TIMM RASGRP1 DPP RASGRP1 ANXA RASGRP1 MCM RASGRP1 EIF1AX RASGRP1 EIF1AY RASGRP1 PFAS RASGRP1 CIAPIN RASGRP1 PPP2CA RASGRP1 PPP2CB RASGRP1 CHTF RASGRP1 ST RASGRP1 CAP RASGRP1 POLDIP RASGRP1 HIST1H1E RASGRP1 THRAP RASGRP1 HCFC RASGRP1 PRPF RASGRP1 DNASE RASGRP1 LRRC RASGRP1 G3BP RASGRP1 MIF RASGRP1 RAN RASGRP1 HAGH RASGRP1 RPS RASGRP1 RPL RASGRP1 TAGLN RASGRP1 CFL RASGRP1 HSPA RASGRP1 ZC3H
23 RASGRP1 LARP RASGRP1 PCBP RASGRP1 RPS17L RASGRP1 TIMM RASGRP1 PDLIM RASGRP1 FLNB RASGRP1 SMS RASGRP1 RAP1B RASGRP1 RAP1A RASGRP1 DCTN RASGRP1 CDC RASGRP1 PBK RASGRP1 RBM RASGRP1 PSMD RASGRP1 IMPDH RASGRP1 RPL RASGRP1 PSMC RASGRP1 CLTA RASGRP1 PSMD RASGRP1 EIF3G RASGRP1 THOC RASGRP1 GTF2F RASGRP1 COPS RASGRP1 RNASEH2A RASGRP1 RPS26P RASGRP1 RCSD RASGRP1 ARHGDIA RASGRP1 LSM RASGRP1 TXNDC RASGRP1 PPP1R RASGRP1 EPB RASGRP1 MRPS RASGRP1 BTF RASGRP1 RPS RASGRP1 RBM8A RASGRP1 RPS RASGRP1 PPIH RASGRP1 PPIF RASGRP1 PNPT RASGRP1 SUCLG RASGRP1 NDUFS RASGRP1 DHX RASGRP1 APEH
24 RASGRP1 HIST1H1B RASGRP1 RBM RASGRP1 RBM RASGRP1 RCC RASGRP1 PSMB RASGRP1 PPP1R2P RASGRP1 SLC9A3R RASGRP1 WBP RASGRP1 LDHA RASGRP1 XRCC RASGRP1 FAU RASGRP1 DDX39B RASGRP1 DDX39A RASGRP1 XRCC RASGRP1 ATP5O RASGRP1 MFAP RASGRP1 CNDP RASGRP1 SNAP RASGRP1 CSNK2B RASGRP1 PSMD RASGRP1 DDB RASGRP1 ENSA RASGRP1 CPOX RASGRP1 EIF RASGRP1 AK RASGRP1 EWSR RASGRP1 NUDT RASGRP1 TOR1AIP RASGRP1 FKBP RASGRP1 FUBP RASGRP1 COX RASGRP1 CNBP RASGRP1 HYPK RASGRP1 ETFB RASGRP1 CDK RASGRP1 SLIRP RASGRP1 CSTF RASGRP1 TARS RASGRP1 CAPRIN RASGRP1 MAPRE RASGRP1 MARCKSL RASGRP1 ANXA RASGRP1 HSPA4L
25 RASGRP1 ACTN RASGRP1 RPA RASGRP1 PSAT RASGRP1 MGMT RASGRP1 EIF4G RASGRP1 CTPS RASGRP1 TXNL RASGRP1 NPM RASGRP1 SNRPE RASGRP1 RPL RASGRP1 ENO RASGRP1 EEF1B RASGRP1 NELFE RASGRP1 GTF2F RASGRP1 DDX RASGRP1 SRSF RASGRP1 FDX RASGRP1 HMGN RASGRP1 FKBP RASGRP1 LSM14A RASGRP1 TBCB RASGRP1 PSMG RASGRP1 TIMM RASGRP1 PDAP RASGRP1 DNAJC RASGRP1 DCTN RASGRP1 PHAX RASGRP1 PSMD RASGRP1 EIF2S RASGRP1 DYNLRB RASGRP1 NOLC RASGRP1 EIF1AD RASGRP1 PNP RASGRP1 HDAC RASGRP1 COTL RASGRP1 POLR2C RASGRP1 FIP1L RASGRP1 NTPCR RASGRP1 VAT RASGRP1 DYNLRB RASGRP1 SNX RASGRP1 NASP RASGRP1 PPP5C
26 RASGRP1 NUDCD RASGRP1 APIP RASGRP1 CIRBP RASGRP1 ATP6V1F RASGRP1 RAB RASGRP1 CLIC RASGRP1 CDK RASGRP1 SF3B RASGRP1 PRDX RASGRP1 MRPS RASGRP1 BID RASGRP1 PSME RASGRP1 TUFM RASGRP1 MLEC RASGRP1 PPP4R RASGRP1 UQCRH RASGRP1 TOMM RASGRP1 RHOA RASGRP1 PPP1R RASGRP1 HDAC RASGRP1 ZNF RASGRP1 ACOT RASGRP1 ARCN RASGRP1 PPM1A RASGRP1 PARP RASGRP1 PHF5A RASGRP1 UBE2S RASGRP1 BAG RASGRP1 UBA RASGRP1 HP1BP RASGRP1 ADI RASGRP1 SEP RASGRP1 BCL7A RASGRP1 PPIE RASGRP1 PPID RASGRP1 AIMP RASGRP1 ELF RASGRP1 SOD RASGRP1 APEX RASGRP1 ALDOC RASGRP1 ZC3HAV RASGRP1 BPNT RASGRP1 MEA
27 RASGRP1 CFDP RASGRP1 AHCY RASGRP1 NMT RASGRP1 LYRM RASGRP1 PITRM RASGRP1 CSNK2A RASGRP1 CSNK2A RASGRP1 FDPS RASGRP1 CD2AP RASGRP1 NIT RASGRP1 PCBP RASGRP1 NACA RASGRP1 RAB5C RASGRP1 HADH RASGRP1 CD2BP RASGRP1 RPL35A RASGRP1 SNRPB RASGRP1 TAL RASGRP1 SNRPA RASGRP1 MRPS RASGRP1 SNRNP RASGRP1 POMP RASGRP1 GLUD RASGRP1 BCL7B RASGRP1 EPB41L RASGRP1 PRPS RASGRP1 CMPK RASGRP1 GCLC RASGRP1 ESD RASGRP1 SNRPN RASGRP1 CAT RASGRP1 DDX RASGRP1 CORO1A RASGRP1 TUBB2A RASGRP1 RPS4X RASGRP1 ATP6V1B RASGRP1 SMARCC RASGRP1 C21orf RASGRP1 B2M RASGRP1 MZT2A RASGRP1 DBN RASGRP1 ZNF RASGRP1 DRAP
28 RASGRP1 OSGEP RASGRP1 DIAPH RASGRP1 FKBP RASGRP1 GRAP RASGRP1 PDRG RASGRP1 EPDR RASGRP1 MYH RASGRP1 ACO RASGRP1 MRPL RASGRP1 NDE RASGRP1 SNW RASGRP1 USP RASGRP1 CYB5B RASGRP1 ACTR1B RASGRP1 ACTR1A RASGRP1 DHRS RASGRP1 DPY RASGRP1 ATP6V1B RASGRP1 H1FX RASGRP1 UQCRC RASGRP1 XPNPEP RASGRP1 FTSJ RASGRP1 SDF2L RASGRP1 TCF RASGRP1 TPT RASGRP1 MLLT RASGRP1 C1orf RASGRP1 LARS RASGRP1 MCM RASGRP1 PROSC RASGRP1 TPM RASGRP1 RPLP RASGRP1 ADSL RASGRP1 WDR RASGRP1 RAB RASGRP1 RAB1C RASGRP1 UNG RASGRP1 BOLA2B RASGRP1 OGFR RASGRP1 RAB1B RASGRP1 BDH RASGRP1 UBAP2L RASGRP1 WIBG
29 RASGRP1 MYEOV RASGRP1 NT5C RASGRP1 ADD RASGRP1 DNAJA RASGRP1 API RASGRP1 TMEM RASGRP1 LSM14B RASGRP1 ARMC RASGRP1 SAE RASGRP1 SLC3A RASGRP1 ZPR RASGRP1 POLE RASGRP1 ATP6V1G RASGRP1 TP53RK RASGRP1 DUS3L RASGRP1 SRI RASGRP1 RRM RASGRP1 EFHD RASGRP1 GNS RASGRP1 MAGOHB RASGRP1 PIN RASGRP1 PRRC2A RASGRP1 LAMTOR RASGRP1 CPNE RASGRP1 ZRANB RASGRP1 GOT RASGRP1 MAGOH RASGRP1 UPF RASGRP1 GLUL RASGRP1 COPA RASGRP1 MZT2B RASGRP1 SSNA RASGRP1 NPC RASGRP1 HMBS RASGRP1 RAB7A RASGRP1 ARPC RASGRP1 C1orf RASGRP1 DNAJB RASGRP1 HNRNPF RASGRP1 CLPP RASGRP1 IGBP RASGRP1 HADHA RASGRP1 MAP7D
30 RASGRP1 NDEL RASGRP1 SNRPC RASGRP1 RCC RASGRP1 RUVBL RASGRP1 RPL RASGRP1 PDCD6IP RASGRP1 ACTA RASGRP1 ACTC RASGRP1 HSP90B RASGRP1 NIPSNAP RASGRP1 RPS RASGRP1 RPS RASGRP1 SNRPD RASGRP1 GAPDH RASGRP1 PRDX RASGRP1 RPS RASGRP1 HIST1H1D RASGRP1 HIST1H1C RASGRP1 CAPZA RASGRP1 HNRNPH RASGRP1 TRAP RASGRP1 RBMX RASGRP1 RPL RASGRP1 ILF RASGRP1 RPS RASGRP1 RPS RASGRP1 RPS RASGRP1 TUBB RASGRP1 HNRNPR RASGRP1 HSPA RASGRP1 NCL RASGRP1 SPTBN RASGRP1 ALYREF RASGRP1 RPL RASGRP1 EEF1A RASGRP1 RPL10L RASGRP1 RPL RASGRP1 HNRNPD RASGRP1 DDX RASGRP1 RPL RASGRP1 RPL23A RASGRP1 SNAP RASGRP1 PFN
31 RASGRP1 RPL RASGRP1 RPL RASGRP1 ACACA RASGRP1 DDX RASGRP1 PRDX RASGRP1 ARHGDIB RASGRP1 RPS RASGRP1 RPL36A RASGRP1 ACTG RASGRP1 RPL36AL RASGRP1 YBX RASGRP1 RPL RASGRP1 VIM RASGRP1 RPS RASGRP1 RPL7A RASGRP1 GRPEL RASGRP1 SPTAN RASGRP1 HNRNPA RASGRP1 EEF1A RASGRP1 ACTB RASGRP1 HSP90AB RASGRP1 HNRNPA2B RASGRP1 RPL RASGRP1 RPL RASGRP1 RPL RASGRP1 RPL RASGRP1 RPL RASGRP1 HSP90AA RASGRP1 TUBA1C RASGRP1 YBX RASGRP1 HNRNPU RASGRP1 RPL RASGRP1 LMNB RASGRP1 HIST3H2A RASGRP1 HSPA1B RASGRP1 HSPA1A RASGRP1 HIST1H2AC RASGRP1 HIST1H2AB RASGRP1 HIST2H2AC RASGRP1 HIST2H2AA RASGRP1 H2AFJ RASGRP1 HIST1H2AM RASGRP1 HIST1H2AH
32 RASGRP1 HIST1H2AJ RASGRP1 HIST1H2AD RASGRP1 HIST1H2BB RASGRP1 HIST1H2BO RASGRP1 HIST1H2BJ RASGRP1 HIST2H2BE RASGRP1 HIST2H2BF RASGRP1 HIST1H2BD RASGRP1 HIST1H2BF RASGRP1 HIST1H2BL RASGRP1 HIST1H2BM RASGRP1 HIST1H2BN RASGRP1 HIST1H2BH RASGRP1 HIST1H2BK RASGRP1 ATXN2L RASGRP1 PCCB RASGRP1 KRT RASGRP1 KRT RASGRP1 KRT RASGRP1 KRT RASGRP1 KRT RASGRP1 SNRPF RASGRP1 SH RASGRP1 Trypsin RASGRP1 GFP 0 0 0
33 Supplementary References 1. Espinos, C. et al. Mutations in the urocanase gene UROC1 are associated with urocanic aciduria. J Med Genet 46, (2009). 2. Chen, Y. et al. Differential requirement of RasGRP1 for gammadelta T cell development and activation. J Immunol 189, (2012). 3. Coughlin, J.J., Stang, S.L., Dower, N.A. & Stone, J.C. RasGRP1 and RasGRP3 regulate B cell proliferation by facilitating B cell receptor-ras signaling. J Immunol 175, (2005). 4. Dower, N.A. et al. RasGRP is essential for mouse thymocyte differentiation and TCR signaling. Nat Immunol 1, (2000). 5. Shen, S. et al. Critical roles of RasGRP1 for invariant NKT cell development. J Immunol 187, (2011).
NES: normalized enrichment score (analyzed using KEGG pathway gene sets in the GSEA software); FDR:
Supplementary table1. List of gene sets with simultaneously altered the enrichment upon SAP18 overexpression and knockdown. Gene sets enriched by SAP18 and reduced by shsap18 SAP18 against LacZ shsap18
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encouraged to use the Version of Record that, when published, will replace this version. The most /BCJ
Biochemical Journal: this is an Accepted Manuscript, not the final Version of Record. You are encouraged to use the Version of Record that, when published, will replace this version. The most up-to-date
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