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Gain-of-function variants in SYK cause immune dysregulation and systemic inflammation in humans and mice

Abstract

Spleen tyrosine kinase (SYK) is a critical immune signaling molecule and therapeutic target. We identified damaging monoallelic SYK variants in six patients with immune deficiency, multi-organ inflammatory disease such as colitis, arthritis and dermatitis, and diffuse large B cell lymphomas. The SYK variants increased phosphorylation and enhanced downstream signaling, indicating gain of function. A knock-in (SYK-Ser544Tyr) mouse model of a patient variant (p.Ser550Tyr) recapitulated aspects of the human disease that could be partially treated with a SYK inhibitor or transplantation of bone marrow from wild-type mice. Our studies demonstrate that SYK gain-of-function variants result in a potentially treatable form of inflammatory disease.

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Fig. 1: Clinical phenotype and identification of variants in SYK.
Fig. 2: Functional characterization of SYK variants.
Fig. 3: Phenotype of the SYK-Ser544Tyr mouse model.
Fig. 4: Treatment and bone marrow transplant experiments in SYK-Ser544Tyr mice.

Data availability

The identified SYK variants have been submitted to the ClinVar database (https://www.ncbi.nlm.nih.gov/clinvar/) with the IDs SCV001450452 [c.1649C>A, p.Ser550Tyr], SCV001450453 [c.1649C>T, p.Ser550Phe], SCV001450454 [c.1024C>A, p.Pro342Thr], SCV001450455 [c.1350G>A, p.Met450Ile] and SCV001450456 [c.1057G>A, p.Ala353Thr]. The whole-exome sequencing data will not be made publicly available as they contain information that could compromise research participant privacy/consent. Information on the whole-exome sequencing raw data supporting the findings of this study is available from the corresponding authors upon request. Publicly available datasets/databases used in this study include the COSMIC database (https://cancer.sanger.ac.uk/cosmic), Kinase.com (http://www.kinase.com/human/kinome/) and the RCSB Protein Databank (PDB, https://www.rcsb.org/). Source data are provided with this paper.

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Acknowledgements

We thank the patients, their families and all healthy individuals who actively participated in this study. We thank K. Fiedler for assistance with patient-related materials and H.-B. Shu (Wuhan University, China) for providing plasmids. We acknowledge the contribution to this research through access to the data and findings generated by the 100,000 Genomes Project. The 100,000 Genomes Project is managed by Genomics England Limited (a wholly owned company of the Department of Health). The 100,000 Genomes Project and/or research infrastructure are funded by the National Institute for Health Research, the National Health Service England, the Wellcome Trust, Cancer Research UK and the Medical Research Council. The 100,000 Genomes Project uses data provided by patients and collected by the National Health Service as part of their care and support. We acknowledge the contribution of the Oxford IBD cohort study and the Gastrointestinal Illness Biobank, which are supported by the NIHR Biomedical Research Centre, Oxford. We acknowledge the contribution of the VEOIBD.org Consortium. D.L., Z.Z., X.C. and Liren Wang are funded by the National Key R&D Program of China (2019YFA0110802). Lin Wang and Y.H. are funded by the JiuJiu Charitable Trust. Lin Wang was supported by a Chinese Government Scholarship. C.K. and D.K. are funded by the German Research Foundation CRC1054 Project A05 and the Care-for-Rare Foundation. H.H.U. and D.A. are supported by the NIHR Biomedical Research Centre, Oxford. Q.L. and D.L. were supported by a Crohn’s and Colitis Canada, Canadian Association of Gastroenterology, and Canadian Institutes of Health Research (CIHR) Fellowships. K.B. is supported by a European Research Council Consolidator Grant (iDysChart, ERC grant agreement number 820074). B.G. receives support through the DFG SFB1160/2_B5, under Germany’s Excellence Strategy (CIBSS – EXC-2189 – Project ID 390939984, and RESIST – EXC 2155 – Project ID 39087428); through the E-rare program of the EU, managed by the DFG, grant code GR1617/14-1/iPAD; and through the ‘Netzwerke Seltener Erkrankungen’ of the German Ministry of Education and Research (BMBF), grant code GAIN_ 01GM1910A. W.H.A.K. was supported by operating grants from the CIHR (PJT-156095 and PJT-153168). S.H., K.R.E. and H.R.G. were funded by the Wellcome Trust (207556_Z_17_Z). A.M.M., C.K., S.B.S., H.H.U., I.D. and S.C.N. are funded by the Leona M. and Harry B. Helmsley Charitable Trust. A.M.M., C.K. and S.B.S. are funded by an NIH (RC2DK122532) grant. A.M.M. is funded by a Canada Research Chair (Tier 1) in Pediatric IBD, a CIHR Foundation grant and NIDDK NIH (RC2DK118640). D.L., Z.Z., X.C. and Liren Wang are funded by the National Key R&D Program of China (2019YFA0110802) and National Natural Science Foundation of China (no. 32025023).

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Lin Wang, D.A., Z.Z., X.C., D.M., M.M., M.C., N.W., J.P., Liren Wang, Q.L., T.Z., S.C.-K., J.L., R.C.A., D.J.M., K.P., S.B., A.N.G., M.G.S., C.-Y.L., K.R.E. and H.D. carried out the research under the supervision of S.B.S., C.K., D.K., C.-H.G., I.D., S.C.N., S.A.F., K.B., Y.H., D.L., H.D., S.H., H.H.U. and A.M.M. X.C., D.M., M.M. and M.C. carried out equivalent research. Lin Wang, D.J.M., D.D., Z.H., X.L., Y.W., D.K., X.W., S.L., K.W., A.-I.S., G.K., V.G., W.H.A.K., M.L., I.S., K.R.E., R.R., R.M.L., M.P., S.H., S.E., B.G., K.B., Y.H. and A.M.M. provided resources, clinical care and/or clinical samples. Lin Wang, D.A., Z.Z., X.C., D.M., M.M., N.W., S.A.F., K.B., Y.H., D.L., H.H.U. and A.M.M. wrote the paper with contributions from all authors. All authors approved the final manuscript.

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Correspondence to Ying Huang or Dali Li or Aleixo M. Muise.

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The authors declare no competing interests.

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Peer review information Nature Genetics thanks Ivona Aksentijevich, Geert van Loo and the other, anonymous, reviewer(s) for their contribution to the peer review of this work.

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Extended data

Extended Data Fig. 1 Genetic analysis pipeline and validation of heterozygous variants by sanger sequencing.

Variant identification pipeline and Sanger sequencing validation of WES data for (a) Patient 1 (p.S550Y), (b) Patient 2 (p.S550F) and Patient 3 (p.S550F) and (c) Patient 4 (p.P342T). (d) Chronologic disease course and Sanger sequencing validation for Patient 5 (p.M450I) and (e) Patient 6 (p.A353T).

Extended Data Fig. 2 Patient 4 phenotype summary and SYK variants conservation analyses.

a, Patient 4’s laboratory tests revealed normal white blood cell (WBC) counts. b, Cervical and thoracic spine MRI of Patient 4 taken at 26 years of age showing a signal enhancing intramedullary space-occupying lesion through nearly the entire length of the cervical and thoracic spinal cord. One representative image out of 6 acquired images is shown. All images revealed signs of inflammation. c, Spinal cord biopsy with prominent granulomatous inflammation (clinical data, n = 1). d, Species conservation of SYK amino acids p.P342, p.A353, p.M450, and p.S550. e, Summary of the residue variety in % for each identified variant in SYK across 124 homologues across species following ClustalW-based multiple sequence alignment using the ConSurf server (see Methods). Human wild-type SYK residues are indicated in blue and human SYK variants in red.

Extended Data Fig. 3 SYK hyperphosphorylation in human epithelial cells.

a, SYK hyper-phosphorylation in intestinal tissue from Patient 1 compared to healthy controls. Double immunostaining of normal and p.S550Y SYK variant colon biopsy sections for pSYK (red), β-catenin (green) and merged dual labeling (yellow). The normal case demonstrated a distinctive glandular apical expression of pSYK. The fine apical signal almost reaches to the microvilli. Scattered infiltrated cells show pSYK staining in lamina propria. Unstained patches of glandular epithelium represent goblet cells. Immunostaining of β-catenin as a structural membrane marker indicates an organized glandular architecture in the normal colon section. Staining for pSYK was evident at the glandular epithelial base (membrane and cytoplasm) in colon sections of the patient. The β-catenin labeling in patient 1 intestinal biopsies presented a disorganized glandular architecture compared to the normal control. One representative image is shown out of 3 total images acquired. b, Double immunostaining of pSYK and the myeloid marker CD68 in rectal and duodenal biopsy sections of Patient 1 illustrating strong pSYK expression in intestinal epithelial cells with moderate overlap with CD68+ myeloid cells. One representative image is shown out of 6 total images acquired from rectal biopsies and 4 images acquired from duodenal biopsies.

Extended Data Fig. 4 Comparative assays of human SYK variants and reduction of SYK phosphorylation by R406 SYK inhibitor treatment.

a, Flow cytometry comparison of percent phosphorylated SYK (Y525/526) normalised to ectopic SYK expression in HEK293 cells. b, Non-normalised data presentation of percent phosphorylated SYK (Y525/526). c, Percent ectopic SYK expression in HEK293 cells following transfection. d, Relative mean fluorescence intensity (rMFI) of phosphorylated SYK(Y525/526) normalised to ectopic SYK expression. e, Non-normalised rMFI of phosphorylated SYK(Y525/526) and (f) rMFI of ectopic SYK (a-f: the dotted line indicates WT median; a-c: quartiles and median; d-f: median and interquartile range and minimum to maximum range; n of independent experiments/n of cell culture replicates: eV(8/30), WT(8/24), p.S550Y(8/28), p.S550F(6/21), p.M450I(8/28), p.A353T(8/30), p.P342T(8/30), p.Y323F(3/12); Mann-Whitney test). g, Western blot analysis of SYK phosphorylation (Y525/526), total SYK expression, JNK phosphorylation (T183/Y185), total JNK expression and GAPDH expression in HEK293 cells after transfection with control, p.Y525F/Y526F mutated SYK, wild-type SYK and the range of different identified variants in SYK (p.S550Y, p.S550F, p.A353T, p.P342T and p.M450I). h, Quantification of pJNK normalised to total JNK according to (g) (quartiles and median; n of independent experiments: WT(8), p.Y525/526 F(8) p.S550Y(8), p.S550F(8), p.M450I(3), p.A353T(3), p.P342T(6); Mann-Whitney test). i, Analysis of AP-1 (n = 4) or NF-κB (n = 3) activity by luciferase reporter assay in HEK293 cells following co-transfection with reporter plasmids and empty vector (Control), WT SYK or p.S550Y SYK and 24 hours stimulation with TNF (20 ng/ml) or PMA (20 ng/ml) (Min-max and median; unpaired t-test). j, Western blot analysis of SYK phosphorylation (Y525/526), total SYK expression, JNK phosphorylation (T183/Y185), total JNK expression, ERK phosphorylation (T202/Y204), total ERK expression and GAPDH expression in HEK293 cells after transfection with wild-type SYK, p.S550Y SYK and p.S550F SYK in presence or absence of R406 SYK inhibitor (2 μM). k, Quantification of pSYK (Y525/526) (n = 7), pJNK (T183/Y185) (n = 6), pERK (T202/Y204) (n = 6) western blot signals normalised to the respective total protein expression according to (j) (quartiles and median; Mann-Whitney test). Representative western blot panels have been assembeled from individual gels. Raw data are presented in the respective source data. Source data

Extended Data Fig. 5 SYK response to stimulation in intestinal epithelial cells.

a, Western blot analysis of SYK and pSYK (Y525/526) in SW480 cells transfected with plasmid expressing p.S550Y SYK or WT SYK. b, Time-course of SYK phosphorylation upon zymosan (200 µg/mL) stimulation by western blot analysis of SYK and pSYK (Y525/526) in SW480 cells for the indicated time (NT = untreated cells). c, Western blot analysis of SYK, pSYK (Y525/526) and pSYK (323) in SW480 cells transfected with plasmid expressing p.S550Y SYK or WT SYK left either untreated (–) or stimulated with 20 μg/mL or 100 μg/mL zymosan. d, Western blot analysis of SYK and pSYK (Y525/526) SW480 cells transfected with plasmid expressing p.S550Y SYK or WT SYK left either untreated (NT) or stimulated with curdlan (100 µg/mL) for the indicated time. e,f, Western blot analysis of SYK and pSYK (Y525/526) in SW480 (e) or in SW480 cells transfected with plasmid expressing p.S550Y SYK (f), upon stimulation with zymosan (200 µg/mL) for 1 hour, in the presence or absence of SYK inhibitor (5 µM, Calbiochem, #574711) added 1 hour prior to zymosan stimulation. g, IL-8, CXCL1, GM-CSF and CCL2 secretion by 20 hours zymosan-stimulated SW480 cells expressing WT SYK or p.S550Y SYK. SYK inhibitor (5 µM, Calbiochem, #574711) was added 1 hour prior to zymosan stimulation and analysis performed by ELISA. Median and interquartile range and minimum to maximum range; n of independent experiments/n of cell culture replicates: IL-8: (3/3-10); CXCL1, GM-CSF, CCL2: (2-3/2-7); Mann-Whitney test. h, IL-8, CXCL1, GM-CSF and CCL2 secretion by SW480 cells expressing WT SYK or p.S550Y SYK cells incubated with/without R406 (5 µM) for 60 mins prior to the addition of curdlan and analyzed by ELISA 20 hours following stimulation. Median and interquartile range and minimum to maximum range; n of independent experiments/n of cell culture replicates: 3/3-6; Mann-Whitney test. Representative western blot panels have been assembeled from individual gels. Raw data are presented in the respective source data. Source data

Extended Data Fig. 6 Phenotypic and functional analysis of Patient 1T cells.

a, Dot plot presentation of CD4+, CD8+ and double negative (DN) T cell frequencies among CD3+αβTCR+ cells as determined by surface staining and flow cytometry of PBMC from Patient 1 at 2 years of age and a 3 year-old healthy donor (HD). b, Kinetics of CD4/CD8 ratio based on clinical laboratory measurements. (c) CD4/CD8 ratio as estimated from patient intestinal biopsy sections stained for CD3 and CD8 by immunohistochemistry. d, Intraepithelial CD8+ T cell counts in patient intestinal tissue sections manually counted based on CD4 and CD8 immunofluorescence staining. e, Dot plot presentation of CD3+αβTCR+CD8+CD25 naïve and memory T cell frequencies as assessed by surface staining of CD45RA and CCR7. f, Dot plot presentation of TNF and IFN-γ producing CD3+αβTCR+CD8+CD25 memory T cell frequencies as assessed by intracellular cytokine staining. g, CD3+αβTCR+CD4+CD25 naïve and memory T cell frequencies as assessed by surface staining of CD45RA and CCR7 and flow cytometry analysis. h, Dot plot presentation of CD3+αβTCR+CD4+CD25 memory CCR6+RORγt+ T cell frequencies as assessed by combined surface and intracellular staining. i, Presentation of IL-17A, IL-22 and IFN-γ producing CD3+CD4+CD25 memory T cell frequencies. j, Summary of IL-17A-, IL-22- and IFN-γ-producing CD3+CD4+CD25 memory T cell frequencies as assessed by intracellular cytokine staining following 5 hrs PMA (0.2 μM) and Ionomycin (1 μg/mL) stimulation. Grey areas indicate healthy donor control ranges.

Extended Data Fig. 7 Phenotypic and functional analysis of Patient 1T cells.

a, Summary of CD3+CD8+ T cell frequencies of adult healthy donors (HD) (HD adult: n = 26) and 9 months to 14 years old HD (HD 9 months to 14 years: n = 17) and of Patient 1 at 1.5 years and 2 years of age (n = 2, each 3 technical replicates) expressed as percent of live CD3+ T cells. b, Summary of CD3+CD8+CD25 memory T cell frequencies as assessed by surface staining of CD45RA and CCR7. c,d, Summary of IFN-γ- (c) and TNF-producing (d) CD3+CD8+CD25 memory T cell frequencies as assessed by intracellular cytokine staining following 5 hours PMA (0.2 μM) and Ionomycin (1 μg/mL) stimulation. e, Summary of CD3+CD4+CD25 memory T cell frequencies as assessed by surface staining of CD45RA and CCR7. f, Summary of CD3+CD4+CD25CCR6+ memory T cell frequencies as assessed by surface staining. g, Summary of CD3+CD4+CD25RORγt+ memory T cell frequencies as assessed by combined surface and intracellular staining. h,i,j, Summary of TNF- (h), IL-10- (i) and IL-13-producing (j) CD3+CD4+CD25 memory T cell frequencies as assessed by intracellular cytokine staining following 5 hours PMA (0.2 μM) and Ionomycin (1 μg/mL) stimulation. k, CCR6+CCR4+CXCR3 (Th17-enriched) and CCR6+CXCR3+ CCR4 (Th1/Th17-enriched) CD3+CD4+CD25 memory T cell frequencies as assessed by surface staining and flow cytometry.

Extended Data Fig. 8 Phenotypic and functional characterization of SYKS544 mice.

a, Immunoblot analysis of SYK and pSYK protein levels in ankle of 3 months old mice (wt: n = 3; SYKS544Y: n = 3). b, Hematoxylin and eosin stain of tail tissue sections from 3 months old wild-type and SYKS544 mice showing bone erosion and immune cell infiltration (wt: n = 3; SYKS544Y: n = 3). c, Hyper-phosphorylation of SYK in intestinal tissue from SYKS544 mice compared to wild-type mice (wt: n = 3; SYKS544Y: n = 3). d, Western blot analysis of wild-type and SYKS544 bone marrow derived dendritic cells SYK phosphorylation (Y519/520), total SYK expression, JNK1 (T183/Y185) phosphorylation, total JNK1 expression, ERK1 phosphorylation (T203/Y205) and total ERK1 expression treated or not treated with R406 SYK inhibitor (2 μM, R406 was added 30 minutes prior to LPS (200 ng/mL) stimulation and samples collected after 24 hours) (wt: n = 3; SYKS544Y: n = 3). e, Analysis of IgG and IgM in serum from wild-type and SYKS544Y mice by ELISA at indicated age (n = 3; Unpaired t-test). f, RT–qPCR analysis of Il17f (wt: n = 3; SYKS544Y: n = 6), Csf2 (wt: n = 3; SYKS544Y: n = 9), Ifng (wt: n = 3; SYKS544Y: n = 3), Il17a (wt: n = 6; SYKS544Y: n = 6) and Il4 (wt: n = 3; SYKS544Y: n = 3) expression in blood at the age of 3 months. Unpaired t-test. g, Serum cytokine concentrations in wild-type and SYKS544Y mice at 3 months of age measured by ELISA (n = 4; unpaired t-test). Violin plots indicate quartiles and median. Representative western blot panels have been assembeled from individual gels. Raw data are presented in the respective source data. Source data

Extended Data Fig. 9 Phenotype of immune cells in circulation and tissue of a CRISPR-Cas9-knock-in SYKS544Y mouse model.

a, Frequencies of live CD45+ cells in blood (top) and ankles (bottom) of wild-type and SYKS544Y mice (blood: n = 4, ankle: n = 3; unpaired t-test) at the age of 3 months. b-f, Frequencies and normalised counts of B220+ B cells, CD3+ T cells, CD4+ T cells, Foxp3+CD25+ regulatory T cells (Treg), CD11b+F4/80 mononuclear phagocytes, CD11b+F4/80int mononuclear phagocytes and CD11b+F4/80+ macrophages (Mɸ) in blood (top) and ankles (bottom) of wild-type and SYKS544Y mice at the age of 3 months (blood: n = 4, ankle: n = 3; Treg-blood: n = 3, Treg-ankle (wt): n = 2, Treg-ankle (SYKS544Y): n = 4; unpaired t-test). Normalised counts were calculated as counts/1*105 live CD45+ cells relative to the average change in live CD45+ cells comparing wild-type and SYKS544Y mice. Violin plots indicate quartiles and median.

Extended Data Fig. 10 Full blood counts of wild-type and CRISPR-Cas9-knock-in SYKS544Y mice.

Full blood counts of wild-type (n = 5) and SYKS544Y (n = 6) mice; Mann-Whitney test. Box plots and whiskers indicate median and interquartile range and minimum to maximum range. MCV: mean corpuscular volume; MCH: mean corpuscular haemoglobin; MCHC: mean corpuscular haemoglobin concentration; MPV: mean platelet volume.

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Raw western blot images for Fig. 2e,f.

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Raw western blot images for Extended Data Fig. 5 a–f.

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Raw western blot images for Extended Data Fig. 8d.

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Wang, L., Aschenbrenner, D., Zeng, Z. et al. Gain-of-function variants in SYK cause immune dysregulation and systemic inflammation in humans and mice. Nat Genet 53, 500–510 (2021). https://doi.org/10.1038/s41588-021-00803-4

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