The analysis of individuals with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling the differentiation, maintenance and decay of neutrophils. We identify 9 distinct homozygous mutations in the JAGN1 gene encoding Jagunal homolog 1 in 14 individuals with SCN. JAGN1-mutant granulocytes are characterized by ultrastructural defects, a paucity of granules, aberrant N-glycosylation of multiple proteins and increased incidence of apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte colony-stimulating factor receptor–mediated signaling. JAGN1 emerges as a factor that is necessary in the differentiation and survival of neutrophils.
This is a preview of subscription content, access via your institution
Subscribe to this journal
Receive 12 print issues and online access
$189.00 per year
only $15.75 per issue
Rent or buy this article
Get just this article for as long as you need it
Prices may be subject to local taxes which are calculated during checkout
Kostmann, R. Infantile genetic agranulocytosis; agranulocytosis infantilis hereditaria. Acta Paediatr. Suppl. 45 (suppl. 105), 1–78 (1956).
Klein, C. Genetic defects in severe congenital neutropenia: emerging insights into life and death of human neutrophil granulocytes. Annu. Rev. Immunol. 29, 399–413 (2011).
Grenda, D.S. et al. Mutations of the ELA2 gene found in patients with severe congenital neutropenia induce the unfolded protein response and cellular apoptosis. Blood 110, 4179–4187 (2007).
Köllner, I. et al. Mutations in neutrophil elastase causing congenital neutropenia lead to cytoplasmic protein accumulation and induction of the unfolded protein response. Blood 108, 493–500 (2006).
Boztug, K. et al. A syndrome with congenital neutropenia and mutations in G6PC3. N. Engl. J. Med. 360, 32–43 (2009).
Klein, C. et al. HAX1 deficiency causes autosomal recessive severe congenital neutropenia (Kostmann disease). Nat. Genet. 39, 86–92 (2007).
Dell'Angelica, E.C., Shotelersuk, V., Aguilar, R.C., Gahl, W.A. & Bonifacino, J.S. Altered trafficking of lysosomal proteins in Hermansky-Pudlak syndrome due to mutations in the β3A subunit of the AP-3 adaptor. Mol. Cell 3, 11–21 (1999).
Bohn, G. et al. A novel human primary immunodeficiency syndrome caused by deficiency of the endosomal adaptor protein p14. Nat. Med. 13, 38–45 (2007).
Kolehmainen, J. et al. Cohen syndrome is caused by mutations in a novel gene, COH1, encoding a transmembrane protein with a presumed role in vesicle-mediated sorting and intracellular protein transport. Am. J. Hum. Genet. 72, 1359–1369 (2003).
Stepensky, P. et al. The Thr224Asn mutation in the VPS45 gene is associated with congenital neutropenia and primary myelofibrosis of infancy. Blood 121, 5078–5087 (2013).
Vilboux, T. et al. A congenital neutrophil defect syndrome associated with mutations in VPS45. N. Engl. J. Med. 369, 54–65 (2013).
Person, R.E. et al. Mutations in proto-oncogene GFI1 cause human neutropenia and target ELA2. Nat. Genet. 34, 308–312 (2003).
Devriendt, K. et al. Constitutively activating mutation in WASP causes X-linked severe congenital neutropenia. Nat. Genet. 27, 313–317 (2001).
Glocker, E.-O. et al. A homozygous CARD9 mutation in a family with susceptibility to fungal infections. N. Engl. J. Med. 361, 1727–1735 (2009).
Jun, H.S. et al. Lack of glucose recycling between endoplasmic reticulum and cytoplasm underlies cellular dysfunction in glucose-6-phosphatase-β-deficient neutrophils in a congenital neutropenia syndrome. Blood 116, 2783–2792 (2010).
Lee, S. & Cooley, L. Jagunal is required for reorganizing the endoplasmic reticulum during Drosophila oogenesis. J. Cell Biol. 176, 941–952 (2007).
Dale, D.C. et al. Mutations in the gene encoding neutrophil elastase in congenital and cyclic neutropenia. Blood 96, 2317–2322 (2000).
Horwitz, M.S. et al. Neutrophil elastase in cyclic and severe congenital neutropenia. Blood 109, 1817–1824 (2007).
Boztug, K. et al. Extended spectrum of human glucose-6-phosphatase catalytic subunit 3 deficiency: novel genotypes and phenotypic variability in severe congenital neutropenia. J. Pediatr. 160, 679–683 (2012).
Babu, P. et al. Structural characterisation of neutrophil glycans by ultra sensitive mass spectrometric glycomics methodology. Glycoconj. J. 26, 975–986 (2009).
Hayee, B. et al. G6PC3 mutations are associated with a major defect of glycosylation: a novel mechanism for neutrophil dysfunction. Glycobiology 21, 914–924 (2011).
Horwitz, M.S., Corey, S.J., Grimes, H.L. & Tidwell, T. ELANE mutations in cyclic and severe congenital neutropenia: genetics and pathophysiology. Hematol. Oncol. Clin. North Am. 27, 19–41 (2013).
Tidwell, T. et al. Neutropenia-associated ELANE mutations disrupting translation initiation produce novel neutrophil elastase isoforms. Blood 123, 562–569 (2014).
Rudashevskaya, E.L. et al. A method to resolve the composition of heterogeneous affinity-purified protein complexes assembled around a common protein by chemical cross-linking, gel electrophoresis and mass spectrometry. Nat. Protoc. 8, 75–97 (2013).
Duden, R. ER-to-Golgi transport: COP I and COP II function. Mol. Membr. Biol. 20, 197–207 (2003).
Dale, D.C. et al. A randomized controlled phase III trial of recombinant human granulocyte colony-stimulating factor (filgrastim) for treatment of severe chronic neutropenia. Blood 81, 2496–2502 (1993).
Li, J. & Sartorelli, A.C. Evidence for the glycosylation of the granulocyte colony-stimulating factor receptor. Biochem. Biophys. Res. Commun. 205, 238–244 (1994).
Welte, K., Zeidler, C. & Dale, D.C. Severe congenital neutropenia. Semin. Hematol. 43, 189–195 (2006).
de Koning, J.P. et al. The membrane-distal cytoplasmic region of human granulocyte colony-stimulating factor receptor is required for STAT3 but not STAT1 homodimer formation. Blood 87, 1335–1342 (1996).
Jung, J. et al. Identification of a homozygous deletion in the AP3B1 gene causing Hermansky-Pudlak syndrome, type 2. Blood 108, 362–369 (2006).
Seifert, W. et al. Cohen syndrome–associated protein, COH1, is a novel, giant Golgi matrix protein required for Golgi integrity. J. Biol. Chem. 286, 37665–37675 (2011).
Bryant, N.J. & James, D.E. The Sec1p/Munc18 (SM) protein, Vps45p, cycles on and off membranes during vesicle transport. J. Cell Biol. 161, 691–696 (2003).
Carpp, L.N., Ciufo, L.F., Shanks, S.G., Boyd, A. & Bryant, N.J. The Sec1p/Munc18 protein Vps45p binds its cognate SNARE proteins via two distinct modes. J. Cell Biol. 173, 927–936 (2006).
Wirnsberger, G. et al. Jagunal homolog 1 is a critical regulator of neutrophil function in fungal host defense. Nat. Genet. 10.1038/ng.3070 (17 August 2014).
Pfeifer, D. et al. The hyper-IgE syndrome is not caused by a microdeletion syndrome. Immunogenetics 59, 913–926 (2007).
Fishelson, M. & Geiger, D. Exact genetic linkage computations for general pedigrees. Bioinformatics 18 Suppl 1, S189–S198 (2002).
Hamada, T. et al. Lipoid proteinosis maps to 1q21 and is caused by mutations in the extracellular matrix protein 1 gene (ECM1). Hum. Mol. Genet. 11, 833–840 (2002).
International HapMap Consortium. A second generation human haplotype map of over 3.1 million SNPs. Nature 449, 851–861 (2007).
Li, H. & Durbin, R. Fast and accurate long-read alignment with Burrows-Wheeler transform. Bioinformatics 26, 589–595 (2010).
DePristo, M.A. et al. A framework for variation discovery and genotyping using next-generation DNA sequencing data. Nat. Genet. 43, 491–498 (2011).
Salzer, E. et al. Combined immunodeficiency with life-threatening EBV-associated lymphoproliferative disorder in patients lacking functional CD27. Haematologica 98, 473–478 (2013).
Wang, K., Li, M. & Hakonarson, H. ANNOVAR: functional annotation of genetic variants from high-throughput sequencing data. Nucleic Acids Res. 38, e164 (2010).
Ng, P.C. & Henikoff, S. Accounting for human polymorphisms predicted to affect protein function. Genome Res. 12, 436–446 (2002).
Adzhubei, I.A. et al. A method and server for predicting damaging missense mutations. Nat. Methods 7, 248–249 (2010).
Ashkenazy, H., Erez, E., Martz, E., Pupko, T. & Ben-Tal, N. ConSurf 2010: calculating evolutionary conservation in sequence and structure of proteins and nucleic acids. Nucleic Acids Res. 38, W529–W533 (2010).
Altschul, S.F. et al. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 25, 3389–3402 (1997).
Larkin, M.A. et al. Clustal W and Clustal X version 2.0. Bioinformatics 23, 2947–2948 (2007).
Girish, V. & Vijayalakshmi, A. Affordable image analysis using NIH Image/ImageJ. Indian J. Cancer 41, 47 (2004).
Jang-Lee, J. et al. Glycomic profiling of cells and tissues by mass spectrometry: fingerprinting and sequencing methodologies. Methods Enzymol. 415, 59–86 (2006).
Pichlmair, A. et al. Viral immune modulators perturb the human molecular network by common and unique strategies. Nature 487, 486–490 (2012).
Colinge, J., Masselot, A., Giron, M., Dessingy, T. & Magnin, J. OLAV: towards high-throughput tandem mass spectrometry data identification. Proteomics 3, 1454–1463 (2003).
The authors thank the patients and their families for their participation in this study. The help of all contributing medical, technical and administrative staff is greatly appreciated. The authors thank J. Colinge for bioinformatics analysis of the mass spectrometry data. The authors thank G. Leverger, H. Ducou Lepointe, J. Levin, the association IRIS (Immunodéficience Primitive, Recherche, Information, Soutien) and V. Grosjean for their support. This study was supported by CeMM intramural funds and the FWF (Austrian Science Fund) START Programme (to K.B.) and by grants from the European Research Council (Advanced Grants to C.K. and J.P.; Starting Grant to K.B.), the European Program on Rare Diseases (E-RARE Neutro-Net) and the German Research Foundation (SFB914 and Gottfried-Wilhelm-Leibniz Program). The work at Imperial College was supported by the Biotechnology and Biological Sciences Research Council (BBSRC; grants BB/F0083091 and BB/K016164/1). The French SCN registry is supported by grants from Amgen, Chugai, Fondation Maladies Rares, the Institut de Veille Sanitaire, INSERM, the Association Sportive de Saint Quentin Fallavier, CEREDIH (Reference Center for Hereditary Immunodeficiencies) and the Société d'Hémato-Immunologie Pédiatrique. This research is supported in part by the Intramural Research Program of the US National Institutes of Health, the National Library of Medicine, the DZIF (German Center for Infection Research) and the Care-for-Rare Foundation.
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Maturation arrest in bone marrow smears from a JAGN1-deficient patient (P4).
JAGN1-deficient patients in this study showed (sometimes intermittently, as in this figure) signs of maturation arrest at the promyelocyte/myelocyte stage, a hallmark feature of classical severe congenital neutropenia.
Supplementary Figure 2 Segregation analysis of the JAGN1 mutation identified in the index family (pedigree A).
The mutation shows perfect segregation with the disease, with all affected individuals (P1–P5) homozygous for the mutation (c.3G>A; p.Met1Ile). All parents are heterozygous, and the non-affected sibling II-1 is a heterozygous carrier of the mutation. No DNA material was available for the study of the non-affected sibling II-5.
Supplementary Figure 3 Segregation analysis of families B through I.
The figure illustrates perfect segregation with the disease in pedigrees B–I in this study, consistent with autosomal recessive inheritance. The initial L next to P14 indicates that this patient developed leukemia (see also Supplementary Table 1 for clinical details on the individual patients).
Supplementary Figure 4 Immunoblot analysis of JAGN1 protein expression in fibroblasts.
Fibroblasts were obtained from patients P12, P13 and P14 as well as from the heterozygous father of P13 (P13F) and one unrelated, healthy donor. GAPDH was used as a loading control. Immunoblot analysis for JAGN1 expression confirmed the findings seen in EBV-immortalized B cell lines that JAGN1 protein expression is decreased in P12 and absent in P13. Furthermore, for patient P14, for whom no EBV-immortalized B cell lines were available, the results show that the mutation leads to markedly decreased protein expression for JAGN1.
Supplementary Figure 5 Transmission electron microscopy analysis of healthy donor neutrophils with or without rhG-CSF treatment.
The figure illustrates that there are no obvious ultrastructural differences between peripheral blood neutrophils in healthy donors with or without injections with recombinant G-CSF.
Supplementary Figure 6 MALDI-TOF mass spectrometry of permethylated N-glycans from healthy human neutrophil samples.
N-glycomic profiles of permethylated N-glycans from human neutrophil samples from the healthy mother (a) and father (b) of pedigree C. Profiles of permethylated N-glycans are from the 50% MeCN fraction (Online Methods). Putative structures are based on composition, tandem mass spectrometry and biosynthetic knowledge. Healthy human neutrophil N-glycans included high mannose and complex bi-, tri- and tetra-antennary structures, of which the latter were core fucosylated and non-bisected and mainly terminated with sialylated or fucosylated epitopes. Immature truncated N-glycan structures were found to be relatively minor and were restricted to two core-fucosylated bi- and triantennary structures (m/z = 1,835.9 and 2,040.0; top). Most of the N-glycans are multibranched and are rich in the LeX epitope (Galβ1-4(Fucα1-3)GlcNAc). Of note is the presence of up to three LeX moieties on tri- and tetra-antennary N-glycans (for the mother sample, m/z = 3,402.6, 3,851.7, 4,025.7, 4,212.8, 4,300.8, 4,474.9, 4,749.9 and 4,924.0; bottom). These structures were found to be higher in relative abundance than corresponding glycans with absent or single fucose residues attached on their antennae (for the mother sample; m/z = 3,415.6, 3,864.7, 4,038.8, 4,313.8, 4,487.9, 4,762.9 and 4,937.8).
Supplementary Figure 7 G-CSF treatment does not alter the glycomic profile of healthy human neutrophils.
MALDI-TOF mass spectrometry of permethylated N-glycans from (a) an unrelated healthy donor and (b) an unrelated healthy donor treated additionally with G-CSF. Other conditions are identical to those in Supplementary Figure 6. G-CSF treatment does not alter the composition or the relative abundance of the mature N-glycans found in the lower panels of the spectra.
Supplementary Figure 8 JAGN1-mutated human neutrophils exhibit aberrant glycosylation.
MALDI-TOF mass spectrometry of permethylated N-glycans from JAGN1-mutated neutrophils from (a) patient P8 and (b) patient P7. Other conditions are identical to those in Supplementary Figure 6. The N-glycans of the JAGN1-mutated human neutrophils are characterized by decreased abundance or almost a complete absence of the tri- and tetra-antennary core-fucosylated structures containing double and triple antenna-fucosylated residues.
Supplementary Figure 9 JAGN1-mutated human neutrophils exhibit severely aberrant glycosylation.
MALDI-TOF mass spectrometry of permethylated N-glycans from (a) JAGN1-mutated peripheral blood neutrophils from patient P12 and (b) bone marrow–derived neutrophil granulocytes from patient P3. Red peaks depict the glycans whose abundance is markedly altered in patients. Other conditions are identical to those in Supplementary Figure 6. N-glycans from JAGN1-mutated human neutrophil samples from these patients exhibited an abnormal increase in the agalactosylated structures found at m/z = 1,835.9 and 2,081.0. Their low-mass N-glycome was characterized by abnormally high abundance of truncated bi- and triantennary agalactosylated structures (m/z = 1,835.9 and m/z = 2,081.0, respectively). Concomitant with this increase in immature glycans was a substantial reduction in all high-molecular-weight tri- and tetra-antennary glycans, including the complete absence of components above an m/z of 3,800. Consequently, these patients’ neutrophils have a dramatic reduction in functional glycan epitopes, including LeX and sialylated sequences.
Supplementary Figure 10 MALDI-TOF mass spectrometry of permethylated O-glycans from healthy and JAGN1-mutated human peripheral blood and bone marrow neutrophil samples.
MALDI-TOF mass spectrometry of permethylated Ο-glycans from (a) the healthy mother from pedigree C, (b) the healthy father from pedigree C, (c) an unrelated healthy donor, (d) an unrelated healthy donor additionally stimulated with G-CSF, (e) patient P8, (f) patient P7, (g) patient P12 and (h) patient P3. Peripheral blood neutrophil granulocytes were available for a–g; for patient P3, neutrophil granulocytes from bone marrow were assessed. O-glycomic profiles of permethylated O-glycans are from the 35% MeCN fraction (Online Methods). Putative structures are based on composition, tandem mass spectrometry and biosynthetic knowledge.
Supplementary Figure 11 Investigation of the effects of JAGN1 knockdown on the protein expression levels, glycosylation and localization of neutrophil elastase (ELANE).
To study the effect of JAGN1 silencing on the expression of neutrophil elastase, wild-type HA-tagged ELANE was expressed in JAGN1-silenced HeLa cells. In protein blotting, neutrophil elastase and JAGN1 were detected, and actin was used as a loading control. siRNA-mediated knockdown of JAGN1 did not influence the protein expression levels or the molecular weight of neutrophil elastase (a). To study further the possible effect of JAGN1 on the glycosylation of neutrophil elastase, HA-tagged ELANE was expressed in HeLa cells in combination with siRNA-mediated JAGN1 knockdown. Treatment of protein lysates with either the EndoH or PNGase enzymes revealed no difference in the glycosylation pattern between control and siRNA-treated samples (b). Immunofluorescence studies were performed in HeLa cells to assess the potential effects of JAGN1 knockdown on neutrophil elastase (green) subcellular localization. Nuclei were visualized by DAPI staining (blue). Scale bar, 10 μm. No difference in subcellular localization between control and JAGN1 knockdown cells was detected (c).
Supplementary Figure 12 Assessment of apoptosis in neutrophil granulocytes in patient P13.
The figure illustrates increased numbers of apoptotic cells following apoptosis induction using staurosporine, similar to the data for patient P12 displayed in Figure 2d.
Supplementary Figure 13 Enhanced loss of mitochondrial membrane potential in patient P13.
The figure illustrates the accelerated loss of mitochondrial membrane potential in patient P13, similar to the data for patient P7 displayed in Figure 2e.
Supplementary Figure 14 Immunofluorescence-based localization studies using GFP-tagged JAGN1 constructs.
HeLa cells (a,b) and fibroblasts (c,d) transiently transfected with constructs expressing GFP or fusion proteins (N-terminally fused EGFP-JAGN1 or C-terminally fused JAGN1-EGFP) were counterstained using an antibody either against the ER marker protein calnexin or the Golgi apparatus marker protein golgin-97. Live-cell staining was performed with MitoTracker for mitochondria staining. In all cases, the characteristic reticulated staining pattern of the ER is observed. The far right panel depicts the merged images for GFP (green) and the specific cell organelle (red), where colocalization of the signals is demonstrated in yellow. The overlay shows that colocalization of calnexin and JAGN1 is extensive but incomplete. There is no colocalization of JAGN1 and the Golgi apparatus or mitochondria.
Supplementary Figure 15 Investigation of the effects of JAGN1 knockdown on ER stress and the glycosylation, localization and functionality of G-CSF-R (CSF3R).
(a) The induction of ER stress was studied in HeLa cells by transfecting the cells first with JAGN1-specific oligonucleotides (numbered 1 and 2) and then treating them with thapsigargin. In protein blotting, BIP levels were detected as an indicator of ER stress, and knockdown of JAGN1 is shown. Actin was used as a loading control. No differences in induced BIP levels were observed between control siRNA and JAGN1-silenced samples. (b) Glycosylation of G-CSF-R was studied in more detail in JAGN1-silenced HeLa cells. Cells were first transfected with either control siRNA or JAGN1-specific siRNAs and were then transfected with construct encoding HA-tagged G-CSF-R. The samples were then either left untreated or treated with EndoH or PNGase enzymes. Protein blotting analysis shows HA-tagged G-CSF-R and actin as a loading control. No differences in glycosylation between control siRNA and JAGN1-silenced samples were detected. (c) The localization of G-CSF-R was further studied by immunofluorescence staining in JAGN1-silenced HeLa cells. The cells were first transfected with siRNAs as explained above and were then transiently transfected with a construct encoding GFP-tagged G-CSF-R (in the pMMP vector). G-CSF-R fused to GFP is detected on a green channel, and anti-G-CSF-R is detected on a red channel. DAPI was used to stain nuclei (blue). No differences in the localization of G-CSF-R between control siRNA and JAGN1-silenced samples were detected. Scale bar, 10 μm. (d) To study the effect of JAGN1 knockdown on G-CSF-R–dependent signaling, G-CSF-R–GFP was expressed in JAGN1-silenced HeLa cells. In contrast to cells treated with control siRNA, JAGN1 knockdown HeLa cells showed reduced phosphorylation of STAT3 upon exposure to rhG-CSF.
Supplementary Text and Figures
Supplementary Figures 1–15 and Supplementary Tables 1–5. (PDF 3898 kb)
Supplementary Data Set 1
Identified proteins of all pulldowns using C- or N-terminally Strep-HA–tagged JAGN1 (msrun_ac P4361, P4362, P4367, P4368) and pulldowns using N-terminally Strep-HA–tagged GFP (P3208, P3209) (XLSX 1192 kb)
Rights and permissions
About this article
Cite this article
Boztug, K., Järvinen, P., Salzer, E. et al. JAGN1 deficiency causes aberrant myeloid cell homeostasis and congenital neutropenia. Nat Genet 46, 1021–1027 (2014). https://doi.org/10.1038/ng.3069
This article is cited by
Associating physiological functions with genomic variability in hibernating bats
Evolutionary Ecology (2021)
Novel Frameshift Autosomal Recessive Loss-of-Function Mutation in SMARCD2 Encoding a Chromatin Remodeling Factor Mediates Granulopoiesis
Journal of Clinical Immunology (2021)
Neutrophils as emerging therapeutic targets
Nature Reviews Drug Discovery (2020)
Serpin B1 defect and increased apoptosis of neutrophils in Cohen syndrome neutropenia
Journal of Molecular Medicine (2019)
Novel Mutation in CECR1 Leads to Deficiency of ADA2 with Associated Neutropenia
Journal of Clinical Immunology (2018)