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Severe congenital neutropenia (SCN) is characterized by early onset of severe bacterial infections due to a paucity of mature neutrophils. There is also an increased risk of leukemia. The genetic causes of SCN are unknown in many patients.
Genome-wide genotyping and linkage analysis were performed on two consanguineous pedigrees with a total of five children affected with SCN. Candidate genes from the linkage interval were sequenced. Functional assays and reconstitution experiments were carried out.
All index patients had susceptibility to bacterial infections and myeloid maturation arrest in the bone marrow; some had structural heart defects and venous angiectasia on the trunk and extremities. Linkage analysis of the two index families yielded a combined multipoint LOD score of 5.74 on a linkage interval on chromosome 17q21. Sequencing of the candidate gene glucose-6-phosphatase catalytic subunit 3 (G6PC3) revealed a homozygous missense mutation in exon 6 in all affected children in the two families, abrogating enzymatic activity of Glucose-6-phosphatase. Neutrophils and fibroblasts of patients had increased susceptibility to apoptosis. Myeloid cells showed evidence of increased endoplasmic reticulum stress and increased activity of GSK3β. We identified seven additional, unrelated SCN patients with syndromic features and distinct biallelic mutations in G6PC3.
Defective function of G6PC3 defines a novel SCN syndrome associated with cardiac and urogenital malformations.
Syndromes associated with congenital neutropenia are a heterogeneous group of disorders 1, 2. Severe congenital neutropenia was described more than 50 years ago by Kostmann 3, 4. In these syndromes, the paucity of neutrophils in peripheral blood causes life-threatening bacterial infections early in life. Most patients respond to recombinant human granulocyte-stimulating factor (rh-G-CSF), which increases peripheral neutrophil counts and decreases the frequency and severity of infections5. Nonetheless, patients may remain at risk for both infectious complications and the development of clonal disorders of hematopoiesis such as myelodysplastic syndrome or acute myeloid leukemia6.
Considerable progress has been made in identifying the molecular defects that cause congenital neutropenia7, 8. Many patients with severe congenital or cyclic neutropenia have a heterozygous mutation in the neutrophil elastase (ELA2) gene 9–11. We recently identified homozygous mutations in HAX1 in a subgroup of patients with autosomal recessive severe congenital neutropenia12. In addition, mutations in WAS 13, 14 and GFI115 have been associated with a phenotype resembling Kostmann’s syndrome. In many patients with congenital neutropenia, however, the underlying molecular cause remains unknown. Despite recent insights into the role of apoptosis 12, 16, 17 the mechanisms of neutropenia and the risk of leukemia in severe congenital neutropenia are incompletely understood.
Here we report a syndrome, which to our knowledge has not been previously recognized, associating severe congenital neutropenia with extra-hematopoietic features, which is caused by bi-allelic mutations in the gene encoding the glucose-6-phosphate catalytic subunit 3 (G6PC3).
Blood and bone marrow samples from patients and healthy individuals were taken upon informed consent. The study was approved by the institutional review board at Hannover Medical School.
We genotyped microsatellite markers in a whole genome scan for family SCN-I. Equipment and protocols for genotyping were as described previously12. The genetic linkage analysis was done using a combination of quantitative and qualitative syllogisms. The quantitative decisions were made using LOD scores and optimal recombination fractions computed with the software Superlink18, 19. For LOD score computations, we modeled neutropenia as a fully penetrant autosomal recessive disease with no phenocopies and disease allele frequency 0.001. The Marshfield map20 was used to select usefully positioned markers for fine mapping. For details, see Supplementary Information.
Exons and flanking intron-exon boundaries from candidate genes were PCR-amplified and analyzed using an ABI Prism 3130 DNA Sequencer and the DNA Sequencing Analysis software version 3.4 (Applied Biosystems, Foster City, CA, USA) and Sequencer version 3.4.1 (Gene Codes Corporation, Ann Arbor, USA). For primer sequences and details on the restriction length polymorphism analysis to analyze the frequency of the R253H mutation in healthy controls, refer to Supplementary Methods.
Promyelocytes were sorted by FACS as described previously with minor modifcations17.
Gene expression analysis of G6PC3 and HSPA5/Bip/Grp78 was performed using a Lightcycles 2.0 (Roche). See Supplement for details.
The complete open reading frames of wild type and mutant G6PC3 were PCR amplified and cloned in pYES-cup1 (modified from pYES-NT (Invitrogen) as described in21 and expressed in Saccharomyces cerevisiae. The 100,000 g microsomal fraction was assayed for glucose-6–phosphate (G6P) hydrolysis to glucose by addition of [14C]G6P (MP Biomedicals). Released [14C]glucose was separated from G6P by anion exchange and measured in the eluate by liquid scintillation.
Whole cell lysates from primary granulocytes were separated by SDS-PAGE, blotted and stained with antibodies against phospho-Mcl-1 (Ser159/Thr163), total GSK3βphospho-GSK3β (Ser9) (all from Cell Signaling/New England Biolabs, Frankfurt am Main, Germany), Bip/Grp78 (BD Biosciences, Heidelberg, Germany) and GAPDH (Santa Cruz Biotechnologies, Heidelberg, Germany). Please refer to the supplement for further information.
Bone marrow samples from patients and healthy controls were subjected to hypotonic lysis. Fixation and electron microscopy were performed as described previously22.
The human G6PC3 cDNA was cloned into a bicistronic retroviral MMP vector23 containing murine cd24 as a marker gene. RD114-pseudotyped retroviral particles were generated by tripartite transfection of MMP-based vectors together with the envelope plasmid and the packaging plasmid mPD.old.gag/pol into the HEK 293T cell line. Transduction of CD34+ cells and myeloid differentiation was performed as described previously12.
Apoptosis in peripheral blood neutrophils or in vitro differentiated myeloid cells was induced using TNF-α (50 ng/ml), thapsigargin (10μM) or tunicamycin (5 μg/ml; all from Sigma) and assessed by Annexin-V (Invitrogen)/propidium iodide (Sigma) staining. In fibroblasts, apoptosis was induced using 5mM dithiothreitol (Roche). Caspase 3/7 activation was assessed as described previously12. For details, refer to Supplementary information.
Table 1 lists the main features of the five patients we studied. The siblings P1 and P2, born to consanguineous parents of Aramean descent, presented with neonatal sepsis. their extended pedigree is denoted SCN-I (Suppl. Fig. 1). Further workup in their first year of life revealed severe neutropenia, apparently congenital, with a paucity of mature neutrophils in peripheral blood and bone marrow. Phenotypically, bone marrow smears showed a pathognomonic maturation arrest at the stage of promyelocytes/myelocytes (Fig. 1a, b and Suppl. Table 1). Erythrocyte counts were normal. Platelet counts in P1 ranged from 73,000–425,000, while P2 had normal platelet counts. Both patients had unusually prominent subcutaneous veins and/or venous angiectasia (Fig 1c); P1 had atrial septal defect (ASD) type II, and P2 had cor triatriatum (Fig. 1d) and hepatosplenomegaly. Genealogical investigations revealed that the SCN-I pedigree could be extended to include two additional sibships each having one child also affected by severe congenital neutropenia and ASD-II (Patients #3 and 4, Suppl. Fig. 1). We also identified a child with severe congenital neutropenia in a second consanguineous pedigree (SCN-II) from the same ethnic background (Patient #5). All patients received recombinant human G-CSF (rh-G-CSF) and responded with an increase in peripheral neutrophil counts.
Mutations in both ELA210 and HAX112 were excluded in all five index patients. Genetic linkage analysis gave statistical evidence that the gene mutated in SCN-I is located on chromosome 17q21 between D17S1299 (36.2Mb, 62.0cM) and D17S1290 (53.7Mb, 82.0cM) (Fig. 2a and Supplementary Results). We carried out a series of fine mapping steps in SCN-I and SCN-II and were able to genotype an additional 13 microsatellite markers between D17S1299 and D17S1290 in SCN-I, and 11 of these in SCN-II. Supplementary Table 2 shows single-marker LOD scores. Assuming that the same gene is mutated in all five affected children, the maximal linkage interval spanned from D17S1789 (39.1Mb, 63.1cM) to D17S791 (42.2Mb, 64.2cM). Using D17S932, D17S950, and D17S806, the peak multipoint LOD score in SCN-I alone was 4.98, and the peak two-pedigree multipoint LOD score was 5.74.
Several candidate genes were identified in the SCN-I linkage interval (Supplementary Table 3). Of these, G6PC3, encoding the glucose-6-phosphatase catalytic subunit-3, and located in the narrowest possible linkage interval, was a plausible candidate, because abnormal glucose metabolism has been implicated in neutropenia. in glycogen storage disease type Ib patients24. DNA sequencing revealed a homozygous missense mutation in exon 6 of the G6PC3 gene (c. G758A, p. R253H) (Fig. 2b). This mutation was found in all four affected children in SCN-I and in the affected child in SCN-II. All parents were heterozygous at this position, confirming autosomal recessive inheritance of a germline missense mutation. With restriction site analysis, the G6PC3R253H allele was not found in 192 healthy central European individuals. An in silico sequence analysis using SIFT25 predicted that the probability of this mutation being benign was 0.01; analysis with Polyphen26 predicted that the R253H mutation is probably damaging to protein function, as expected since R253 is conserved in multiple species including mammals, amphibians, bony fish, and insects.
G6PC3wildtype and G6PC3R253H were expressed in Saccharomyces cerevisiae. Microsomes were isolated from yeast transfected with G6PC3wildtype or G6PC3R253H, and assayed for phosphatase activity. G6PC3wildtype hydrolyzed glucose-6-phosphate and the universal substrate p-Nitrophenylphosphate (pNPP), as demonstrated by radioactive (Fig. 2c) and spectrometric (Suppl. Fig. 3) assays, respectively. In contrast, the level of enzymatic activity of mutant G6PC3R253H did not exceed the phosphatase level in yeast transfected with an empty vector.
Similar to patients with mutations in ELA210 or HAX112, peripheral blood neutrophils had an increased rate of spontaneous apoptosis in all five patients tested. Apoptosis was also markedly accelerated in patients’ neutrophils after induction with either TNF-α (Fig. 3a) or tunicamycin (data not shown), as assessed by Annexin-V staining and a test for caspase-3/7 activation, respectively (see also Suppl. Fig. 4). Since G6PC3 is a ubiquitously expressed gene and since the phenotype of our patients was not restricted to the hematopoietic system, we tested non-hematopoietic cells for susceptibility to apoptosis. Skin fibroblasts from G6PC3-deficient patients displayed an increased susceptibility to apoptosis following DTT-induced stress to the endoplasmic reticulum (Fig. 3b).
To provide further evidence that this novel form of SCN is caused by mutations in G6PC3, we performed reconstitution experiments to correct premature apoptosis in myeloid cells. CD34+ hematopoietic stem cells from two patients were isolated and transduced with retroviral constructs containing either the wildtype G6PC3 cDNA sequence and murine CD24 as a reporter gene (MMP-G6PC3-mCD24) or the reporter gene only (MMP-mCD24). Upon in vitro differentiation in the presence of recombinant human G-CSF and GM-CSF, cells were exposed to tunicamycin to induce apoptosis and analyzed by flow cytometry by gating on mCD24-positive cells. In control-transduced cells from a patient, exposure to tunicamycin induced a high degree of apoptosis (29.99% Annexin-V positive +4.63% AnnexinV/propidium iodide double positive cells), whereas in G6PC3-transduced cells, apoptosis was reduced (17.86% + 1.90%) (Fig 3c; Suppl. Fig. 5). We tested the function of neutrophils in G6PC3-deficient neutrophils. Both phagosomal lysis of E. coli and the oxidative burst were comparable to neutrophils from healthy control individuals (Suppl. Fig. 6).
Endoplasmic reticulum (ER) stress and the unfolded protein response have been linked to the pathophysiology of aberrant organogenesis27, including structural heart defects28, and congenital neutropenia caused by mutations in neutrophil elastase (ELA2)17, 29. We therefore sought evidence for increased endoplasmic reticulum stress in our patients. Transmission electron microscopy of bone marrow cells from all four G6PC3-deficient patients analyzed showed an enlarged rough ER in myeloid progenitor cells as compared with such cells from a healthy individual (Fig. 4a, 4b), consistent with increased ER stress (Suppl. Fig. 7 shows electron microscopy in other patients). BiP mRNA, another marker of increased ER stress, was measured by RT-PCR in bone marrow promyelocytes isolated by flow cytometry. The BiP mRNA level was increased in promyelocytes from both patients tested compared to promyelocytes from healthy individuals (Fig. 4c).
Recently, a signalling circuit linking glucose, GSK3β, and Mcl1 has been established30, 31. GSK3βcontrols glycogen metabolism, Wnt-signalling, and apoptosis32. Mcl1, an anti-apoptotic member of the Bcl2 family, is involved in maintenance of neutrophil viability33. GSK3βphosphorylates Mcl1, thus facilitating its degradation via the proteasome30. We performed Western blot studies to estimate the levels of GSK3β, BiP, and Mcl1 proteins in neutrophils exposed to the ER stress-inducing agent tunicamycin. We observed increased levels of Bip (Fig. 4e), an increase in the enzymatically active dephosphorylated form of GSK3β (Fig. 4d and Suppl. Fig. 8), and increased phosphorylation of Mcl1 in in neutrophils from all patients examined (n= 2) (Fig. 4e). To investigate whether intracellular glucose deprivation causes de-phosphorylation of GSK3β, we inhibited glucose metabolism in neutrophils from two healthy individuals using 2-deoxyglucose (2DG). Treatment with 2DG induced dephosphorylation of GSK3β(Fig. 4f) and increased apoptosis of neutrophils (Fig. 4g), whereas CD3-positive T lymphocytes were resistant to the effects of 2DG (Fig. 4g).
We assessed the frequency and variety of G6PC3 mutations in a cohort of patients with genetically unclassified severe congenital neutropenia. 104 such patients were examined for mutations in G6PC3, and seven of these had distinctive biallelic mutations in G6PC3 (Table 1). The mutations include nonsense mutations (Y47X, Y48X) that would destroy the functional parts of the protein, even if the truncated mRNA is translated rather than being subjected to nonsense mediated decay. The three additional missense mutations were predicted to be deleterious by SIFT22 analysis, with probabilities of being benign of 0.03 for L185P, 0.00 for G262R and 0.00 for G260R, respectively. None of these additional patients with G6PC3 mutations had mutations in ELA2 or HAX1, suggesting that these three genetic defects are distinct variants of severe congenital neutropenia. Of note, none of the G6PC3-deficient patients suffered from hypoglycemia or lactic acidosis (Supplementary Table 4), as seen in patients with glycogen storage disorders.
A comprehensive clinical review of all 12 G6PC3-deficient patients revealed variability of clinical features. Of the 12 patients, eight had various cardiac malformations, and ten had a phenotype of unusually prominent subcutaneous veins and/or venous angiectasia (Fig. 1 and Supplementary Fig. 2). Five patients had urogenital malformations, including cryptorchidism and urachal fistula. Two patients had inner ear hearing loss. While two patients showed growth delay, no consistent dysmorphic features were noted (Table 1).
We describe a novel congenital neutropenia syndrome caused by biallelic mutations in G6PC3. Similar to patients with mutations in HAX112 or ELA216, G6PC3-deficient patients had an evidence of myeloid maturation arrest and increased apoptosis in peripheral neutrophils Of twelve patients, 8 had structural heart defects (e.g., atrial septal defect type II, cor triatriatum, pulmonary stenosis) and 5 had urogenital defects (e.g., cryptorchidism, urachal fistula). In most patients, angiectasia of superficial veins was prominent. The broad spectrum of developmental aberrations may depend on factors other than the mutant G6PC3. Perhaps increased susceptibility to apoptosis also affects cardiac or urogenital development in G6PC3-deficient patients. There is clinical variation in other neutropenia syndromes such as Cohen syndrome and cartilage hair hypoplasia, even though the two syndromes are genetically homogeneous8. All patients in our cohort responded to treatment with rh-G-CSF, and to date no patient has developed a clonal hematopoietic disorder.
Three human genes mediating glucose-6-phosphatase activity have been discovered to date: G6PC1, G6PC2, and G6PC3. G6PC1, the classical glucose-6-phosphatase expressed in liver/kidney/small intestine catalyzes the hydrolysis of glucose-6-phosphate, an essential step in the gluconeogenic and glycogenolytic pathways. Patients without G6PC1 activity suffer from glycogen storage disease type Ia34. G6PC2 is expressed uniquely in pancreas islet cells35, 36 and may be involved in glucose-dependent insulin secretion by controlling free glucose levels37. Statistically significant associations between noncoding polymorphisms in or near G6PC2 and the risk of diabetes have been shown38, 39. In contrast to G6PC1 and G6PC2, G6PC3 is ubiquitously expressed40, 41. Glucose-6-phosphate is transported to the endoplasmic reticulum via a glucose-6-phosphate transporter (G6PT)42. Although the stoichiometry and topological relationships between the catalytic subunits of glucose-6-phosphatase and G6PT remain unclear, their functional link has been recognized42–44. The complex formed between G6PT and G6PC1 (potentially also the complex G6PT/G6PC2) appears to maintain normoglycemia. In contrast, our data show that G6PC3 is needed to maintain neutrophil viability and suggest an important role for glucose in the homeostasis of human neutrophils.
Cheung et al. recently described the phenotype of g6pc3-deficient mice that were generated by gene-targeting45. These mice had neutropenia and neutrophil dysfunction; another group had previously shown that murine g6pc3 deficiency results in lowered plasma cholesterol and elevated glucagon levels46. We could not identify any consistent aberration in neutrophil function nor any metabolic aberrations in G6PC3-deficient patients. The underlying mechanism of increased apoptosis of neutrophils in the absence of G6PC3 involves increased endoplasmic reticulum stress, usually seen in case of deficient protein folding in the endoplasmic reticulum. In an attempt to counteract potentially toxic effects that may ensue, cells initiate a rescue program which leads, if ineffective, ultimately to apoptosis47. Furthermore, we provide evidence that GSK3β, a key enzyme regulating cellular differentiation and apoptosis32, is implicated in this pathway. In the absence of intracellular glucose, GSK3β is activated and thus can phosphorylate the anti-apoptotic molecule Mcl1, thereby mediating its degradation30, 31. Neutrophils from G6PC3-deficient patients contain a relative abundance of non-phosphorylated GSK3β and increased phosphorylation of Mcl1, suggesting that a decrease in antiapoptotic Mcl1 accounts for increased apoptosis in G6PC3-deficient neutrophils. These alterations may at least in part be responsible for the phenotype of G6PC3 deficiency.
While we cannot rule out that additional mechanisms may contribute to the increased level of apoptosis in G6PC3-deficient neutrophils, our data suggest that G6PC3 acts via a pathway involving GSK3β to maintain viability of neutrophils. Evidence of increased endoplasmic reticulum stress has previously been reported in patients with mutations in ELA217, 29, and premature apoptosis of neutrophils is known to cause the phenotype of congenital neutropenia12, 16, 17. Thus, G6PC3 deficiency provides another example of how increased apoptosis of neutrophil granulocytes causes congenital neutropenia.
Five arguments support our claim that G6PC3-deficiency causes neutropenia: 1) distinct biallelic G6PC3 mutations were found in two pedigrees and seven singleton patients with congenital neutropenia; 2) sequence analysis predicted that all four missense mutations are likely to affect the function of G6PC3; 3) heterologous expression of G6PC3wildtype and G6PC3R253H in yeast demonstrated that the R253H mutation abrogates enzymatic activity; 4) g6pc3−/− knockout mice show a similar phenotype characterized by neutropenia and increased myeloid cell apoptosis and 5) the susceptibility to apoptosis in G6PC3-deficient myeloid cells could be reduced upon retroviral transfer of the wildtype G6PC3 gene.
Our findings increase the scope of genetic types of congenital neutropenia. and demonstrate a novel role of glucose and/or glucose metabolism in the homeostasis of neutrophil granulocytes..
Supplementary Figure 1. Full pedigree diagram of SCN-I.
Supplementary Figure 2. Skin phenotype of G6PC3-deficient patients.
Supplementary Figure 3. Defective enzymatic activity of G6PC3R253H using the universal substrate pNPP.
This panel shows activity of the same three constructs as depicted in Fig. 2c using the universal substrate p-Nitrophenylphosphate (pNPP). Presented is the continuous pNP production over time in the presence of saponin (solid lines) and triton-X-100 (dotted lines). Green: wild type, Red: R253H, Blue: empty vector. Data represents the average of three independent microsomal preparations.
Supplementary Figure 4. Increased apoptosis in peripheral neutrophils from P10 and increased Caspase 3/7 activation.
Panel A shows increased spontaneous and TNF-α or Thapsigargin-induced neutrophil apoptosis in patient P10, as compared to a healthy control. Panel B shows increased caspase 3/7 activation (x axis in FACS histogram) upon induction of apoptosis in neutrophils from patients P2 and P3, as compared to a healthy donor with (HD1) or without G-CSF supplementation (HD2), respectively.
Supplementary Figure 5. Decreased tunicamycin-induced apoptosis in G6PC3-transduced myeloid cells from patients P2 and P4 at timepoints 16hr and 24hr post induction. Note that the data for P2 at 16 hours post induction are also shown as FACS plots in Fig. 3c.
Supplementary Figure 6. Assessment of neutrophil function in G6PC3-deficient patients
Panel A shows intact E. coli killing activity of patients (P2, P3) in comparison to healthy donors. Panel B shows normal respiratory burst formation in neutrophils from patients P1 and P3.
Supplementary Figure 7. Electron microscopy in myeloid progenitor cells from patients P2, P3 and P10.
Myeloid cells from Patients P2, P3 and P10 show the same abnormal phenotype of an enlarged endoplasmic reticululum (ER) indicative of enhanced ER stress as P1 (Fig. 4).
Supplementary Figure 8. Western Blot analysis of GSK3β, Mcl1 and Bip expression in neutrophils from an additional G6PC3-deficient patient (P3)
We thank the patients and their families for supporting our study. We also thank all colleagues referring and registering patients at the Severe Chronic Neutropenia International Registry (SCNIR). The RD114 plasmid was a kind gift from François-Loïc Cosset. We thank Edelgard Odenwald for analysis of bone marrow smears and Thomas Jack for his help with echocardiography. The authors gratefully acknowledge the excellent technical assistance from Jessica Pfannstiel, Maren Sievers, Marie Böhm, Marly Dalton, Martina Wackerhahn, Tanja Reinke and Gwendoline Leroy and the excellent collaboration with Dr. Jean Donadieu at the Pediatric Hematology Oncology Service, Hôpital Trousseau, Paris, France.
This research was supported by grants from the Deutsche Forschungsgemeinschaft (DFG KliFo 110–2 (C.K.) and the Junior Research Group “Glycomics” (R.G.-S.). C.K & R.G.-S. are REBIRTH investigators, a DFG cluster of Excellence. Additional support was provided by BMBF Bone Marrow Failure Syndromes (K.W., C.K.), the Intramural Research Program of the U.S. National Institutes of Health, National Library of Medicine (NLM) (A.A.S.), and EU grant MEXT-CT-2006–042316 (B.G.). K.B. is a recipient of an Else Kröner Memorial fellowship.
Author contributionsK.B. designed and performed most of the experiments and identified the first G6PC3 mutation, wrote the initial draft of the manuscript and critically participated in all further revisions of the manuscript. G.A. performed immunoblot analyses and caspase activation assays. A.A. performed phosphatase assays. A.A.S. performed linkage analysis computations, chose microsatellite markers to genotype in the linkage region and wrote parts of the manuscript. U.S. provided laboratory resources for genotyping and tested whether control individuals carry the R253H mutation. J.D. performed sequencing of candidate genes and sequenced patients for ELA2, HAX1, and G6PC3 mutations. M.G. sequenced ELA2, HAX1, G6PC3 and CSFR3 in patient samples. G.B. performed electron microscopy studies. J.L.-G. performed candidate gene sequencing. F.N. helped with cloning of retroviral vectors. A.-K.G. performed E. coli killing assays. M.M., J.G., C.Kr., T.P., I.P., C.B.-C., N.R., K.M. and N.I.-H. obtained clinical samples and provided clinical data. H.B. and R.G.-S. supervised A.A. and were involved in critical discussions. C.Z. cared for patients and collected data in the SCN patient registry. B.G. provided laboratory resources and assisted A.A.S. K.W. provided resources for the SCN registry and significant help to carry out this study. C.Kl. designed and initiated the study, directed the course of investigations, provided laboratory and financial resources, and wrote the manuscript together with K.B.
K.B., A.A.S. and C.Kl. analyzed the data in this study. C.Kl. decided to publish this manuscript and vouches for the data.
K.W. is sponsored by Amgen, receiving royalities on G-CSF patent.