The main objective of WG-1 is to identify novel mutations in patients/families with Congenital Neutropenias and investigate the implicated pathogenetic mechanisms using advanced technologies.

Task 1: Investigation for novel mutations in patients with Congenital Neutropenias.
Severe Congenital Neutropenia is a multigene BM failure syndrome commonly associated with mutations in ELANE and less frequently in HAX1, G6PC3, JAGN1 [2,8]. Disease-causing gene mutations are unknown in more that 20% of these patients. Mechanisms of CNP downstream of known mutations are also not fully understood. To identify novel or known rare gene mutations associated with severe CNP, partners of this WG will perform whole exome sequencing (WES) of genomic DNA of Severe Congenital Neutropenia patients with unknown mutations and healthy family members. Candidate genes will be studied in vitro for their role in HSC granulocytic differentiation. To overcome common limitations in studies of rare CNP syndromes, iPSC lines will be generated from each patient. Candidate genes will be corrected in patient-derived iPSCs using CRISPR-Cas9 technology and improvements/full restoration of in vitro granulocytic differentiation of gene corrected iPSC lines will be evaluated. Candidate gene mutations will be also introduced in iPSCs of healthy individuals and "maturation arrest of granulopoiesis" will be evaluated in vitro. Fresh BM and blood cells will be also investigated. CRISPR/Cas9 will be used to generate zebrafish deficient for candidate genes or transgenic fish carrying patient-specific gene mutations.

Task 2: Investigation of cellular mechanisms associated with Congenital Neutropenias.
A prototype disease associated with Congenital Neutropenia is Shwachman-Diamond (SDS), caused by autosomal recessive mutations in SBDS and less frequently in DNAJC21 [43]. To better understand the role of SBDS and DNAJC21 in ribosome assembly and to provide templates for the development of novel therapeutics, partners of this WG will determine the structures of informative native 60S ribosomal subunit assembly intermediates using single particle cryo-electron microscopy. To answer the question what is the level of residual global protein translation in SDS patient cells and whether this parameter might have utility as a predictive biomarker, partners of this WG will measure translation rates in HSC and progenitor cells from a series of clinically well-characterised patients with SDS through the EuNet-INNOCHRON network. They will also develop a device that will enable monitoring protein synthesis in patient cells. Finally, they will develop robust, viable animal models that can be exploited to better understand disease pathogenesis and to develop new therapeutics.

Milestones:

• M1.1. WG Meetings (1st quarter of each year) which will be based on the progress made in WG-1 followed by the respective reports (months 12, 24, 36, 48).
• M1.2. Calls for STSMs (2nd and 4th quarter of each year) for training of young scientists on the technologies developed in WG-1 followed by the relative technical and scientific reports (months 12, 24, 36, 48).
• M1.3. Standard Operating Procedures (SOPs) on the technologies developed by WG-1 (months 12, 24, 36, 48).

Deliverables:

• D1.1. In silico pipeline for the filtering and identification of neutropenia-causing genes from WES data (months 12-24).
• D1.2. Identification of new gene mutations in patients with Congenital Neutropenias (months 12-48).
• D1.3. Generation of iPSC lines of Severe Congenital Neutropenia patients (months 12- 24).
• D1.4. Methods of haematopoietic differentiation of iPSCs and ΒΜ HSCs (months 12-24).
• D1.5. Methods of CRISPR/Cas9-based gene editing in iPSCs and HSCs (months 12-24).
• D1.6. Zebrafish models of Congenital Neutropenia and viable mouse models for SDS (months 12-36).
• D1.7. Structures of native ribosome assembly intermediates (months 12-36).
• D1.8. Methods to measure translation rates in human ribosomopathies (months 12-36).
• D1.9. Publications (papers/conference abstracts), interim and final progress reports (months 24-48).  

The main objective of WG-2 is to investigate the pathogenetic mechanisms, develop innovative diagnostic approaches and establish clinical-laboratory diagnostic algorithms for Acquired CNPs.

Task 1: Investigation of antibody-mediated CNP.
Anti-neutrophil antibodies are a common cause of CNPs (antibody-mediated CNP); however deep insight is currently lacking on the mode of diagnosis of different antibody types. Knowledge of the different kind of anti-neutrophil antibodies, their diagnostic value, and high quality detection will improve diagnosis and treatment; will result in less inconvenience for the patients and lower costs for the health system. For secondary antibody-mediated CNPs, novel diagnostic tools like NGS contribute to identification of rare diseases such as autoimmune lymphoproliferative syndrome (ALPS) and PID, hence optimizing the diagnostic itinerary and treatment. The partners of this WG will standardize and disseminate algorithms and technologies for the detection of anti-neutrophil antibodies. Since commercial tests are not available for all antineutrophil antibodies, workshops, trainings and exchange of techniques will be organized to improve the knowledge base. Finally, large scale investigation by means of NGS or WES will be performed for the identification of CNPs secondary to rare diseases such as ALPS or PID.

Task 2: Investigation of non-antibody-mediated CNPs.
Immune mechanisms and molecular defects have been implicated in the pathophysiology of non-antibody mediated idiopathic CNPs. NGS analysis in combination with individual clinical-laboratory characteristics of a large number of patients will allow the distinction between different types of acquired CNPs including ICUS and idiopathic CNP. The partners of this WG will investigate: (a) The immune profile of different CNPs at the molecular level looking whether certain immune features (e.g. B-cell and T-cell receptor gene repertoires, NK populations) associate with particular phenotypes and patient outcomes. Characterization of immune repertoires using NGS on cell subpopulations and comparison to public databases or to databases from other diseases available to the partners, will contribute to identifying disease-biased clonotypes. Results, analysed with advanced bioinformatics and immunological approaches, will be used for recognizing cognate antigens and designing tailored immunotherapeutic approaches; (b) the mutational status of HSCs of adult patients with unexplained CNP in correlation with BM morphologic, flowcytometric, cytogenetic data and outcome; (c) the immune suppressive function of the BM MSC and the BM and peripheral blood myeloid derived suppressor cells (MDSC) and monocyte subsets.

Milestones:

• M2.1. WG Meetings (1st quarter of each year) which will be based on the progress made in WG-2 followed by the respective reports (months 12, 24, 36, 48).
• M2.2. Calls for STSMs (2nd and 4th quarter of each year) for training of young scientists on the technologies developed in WG-2 followed by the relative technical and scientific reports (months 12, 24, 36, 48).

Deliverables:

• D2.1. SOPs for the anti-neutrophil antibody screening (auto- and allo-antibodies) (months 2-12).
• D2.2. SOPs for T-cell and B-cell receptor repertoire analysis by NGS (months 2-12).
• D2.3. Standardization of the panel of genes for mutational analysis using NGS or WES (months 12-24).
• D2.4. Generation and dissemination of algorithm for the diagnosis of acquired CNPs (montns 24-36).
• D2.5. Publications (abstracts and/or papers) of data on anti-neutrophil antibody screening, B- and T-cell clonotypes, NKcell populations, BM mutational analysis, BM MSC reserves/function, MDSC and monocyte subsets based on large number of acquired CNP patients (months 12-48). 

The main objective of WG-3 is to investigate the process of clonal evolution and the cellular/molecular mechanisms associated with leukaemogenesis in patients with Congenital and Acquired CNPs.

Task1: Investigation of clonal evolution.
Leukaemic evolution is a multistep process of sequential hits. Predictive biomarkers in CNP are not available, although distinct cytogenetic changes are strongly associated with MDS/AML development. Congenital Neutropenia is an interesting model considering that the very first mutation of the process is acting in both haemopoietic cells and BM microenvironment since embryogenesis. Large-scale application of NGS in these cases as well as in Acquired Neutropenias will significantly improve the diagnosis and risk stratification. Longitudinal NGS studies will provide new insights into driver mutations and stepwise transformation. Moreover, clonality underlying benign conditions will assist in classifying CNP among the so-called clonal haemopoiesis of indeterminate potential (CHIP) conditions [23] and assessing propensities for MDS/AML progression.

Task 2: Investigation of cellular stress mechanisms.
The disease course of pre-leukaemia syndromes such as Severe Congenital Neutropenia strongly indicates that cancer-initiating events precede the occurrence of “classical” driver mutations, which cannot be inferred from clonal architectures. Cellular stresses are likely involved in these early events, but their exact nature is unclear. Novel tools, based on iPSC and genome editing technologies need to be developed to elucidate these stress mechanisms and to interrogate the sequential events leading to MDS/AML. Key findings may be exploited to develop strategies to redirect premalignant progenitors back to normal, ultimately to therapeutically manipulate this process.

Task 3: Molecular mechanisms of leukaemogenesis. Partners of the WG-3 will establish ultra-deep sequencing of the critical region of CSF3R and RUNX1. They will identify, through longitudinal retrospective analysis, HSC clones with acquired CSF3R and/or RUNX1 mutations using multiple BM samples of Congenital Neutropenia and Congenital Neutropenia/AML patients. Correlation of the NGS results with clinical data e.g. inherited mutations, age, G-CSF dose, neutrophil counts will be performed and animal models (mice and zebrafish) will be used to study molecular mechanisms of leukaemogenesis triggered by CSF3R and/or RUNX1 mutations. To inform the mechanisms of progression to MDS/AML in SDS, partners of this WG will determine the landscape of genome-wide somatic mutations in single-cell derived HSC and progenitor cell colonies to identify mutational signatures, reconstruct somatic cell dynamics and estimate the HSC numbers actively making white blood cells.

Milestones:

• M3.1. WG Meetings (1st quarter of each year) which will be based on the progress made in WG-3 followed by the respective reports (months 12, 24, 36, 48).
• M3.2. Calls for STSMs (2nd and 4th quarter of each year) for training of young scientists on the technologies developed in WG-3 followed by the relative technical and scientific reports (months 12, 24, 36, 48). 

Deliverables:

• D3.1. Identification of driver and stepwise mutations associated with leukaemogenesis in CNP and CHIP patients (months 0-24).
• D3.2. Knowledge of cellular responses in iPSC cell models derived from Congenital Neutropenia patients and healthy subjects, including CRIPR/Cas9-introduced clonal variants with somatic mutations implicated in leukaemic progression (months 12-36).
• D3.3. Knowledge of critical pathways involved in cellular stress in Congenital Neutropenia, based on transcriptome profiles, activation of signalling pathways, etc. (months 18-46).
• D3.4. Time-course and differences in the acquisition of CSF3R and RUNX1 mutations in sub-groups of Congenital Neutropenia patients(months 12-46).
• D3.5. Identify genome-wide mutational signatures, estimate the size of the stem cell population and reconstruct somatic cell dynamics in SDS (Months 12-24).
• D3.6. Publications (papers/meeting abstracts), interim and final progress reports (months 12-48).

The main objective of WG-4 is to identify targets for the development of novel therapies using advanced technologies.

Task 1: Activation of NAMPT/SIRT-mediated emergency granulopoiesis in Congenital Neutropenia.
It has been shown that activation of the NAMPT/NAD+ /SIRTs-mediated signalling as compensatory mechanisms to G-CSF-mediated granulopoiesis results in induction of granulopoiesis in healthy individuals and CNP patients [40]. Partners of WG-4 will evaluate the effects of different NAMPTand SIRT1 activators as well as of NAMPT substrate, nicotinamide on the G-CSF-induced granulopoiesis in vitro using iPSC-based hematopoietic differentiation, primary BM HSCs, and in vivo in zebrafish and xenograft mouse models.

Task 2: Establishment of the iPSCs based screening platform for testing of small molecules that restore granulopoiesis in Congenital Neutropenia.
Partners of WG-4 will establish iPSC lines of Congenital Neutropenia patients with different gene mutations and will study the effects of different small molecule libraries on the granulocytic differentiation. The small molecules identified as capable of inducing differentiation in Congenital Neutropenias will be tested in animal models (mice, zebrafish).

Milestones:

• M4.1. WG Meetings (1st quarter of each year) which will be based on the progress made in WG-4 followed by the respective reports (months 12, 24, 36, 48).
• M4.2. Calls for STSMs (2nd and 4th quarter of each year) for training of young scientists on the technologies developed in WG-4 followed by the relative technical and scientific reports (months 12, 24, 36, 48).

Deliverables:

• D4.1. Results from the effect of different NAMPT- and SIRT1 activators and nicotinamide on the GCSFinduced granulopoiesis of Congenital Neutropenia patients using iPSCs, primary BM HSCs and animal models (months 12-36).
• D4.2. Results from the effect of different small molecules on the granulocytic differentiation using patient-derived iPSCs and animal models (months 12-48).
• D4.3. Publications (papers/meeting abstracts), interim and final progress reports (months 24-48). 

Registries of patients with different types of CNPs are required to gather real-world data on the epidemiology, clinical presentation and natural course of CNPs and to identify markers for improved decision-making and risk-adapted treatment strategies. 

Task 1: Development of a reliable classification for CNP.
The International Classification of Diseases (ICD) versions 9 and 10 offer some possibilities for coding CNP (i.e. codes 284.0, 288.0, 288.2, and 288.5 in ICD9 and D70 and P61.5 in ICD10); however, in practice, large databases, including hospital discharge and national death records, have proved inappropriate for identifying CNP patients. Even the more recent Orpha-codes are not univocal and hardly identify the underlying diseases as different codes correspond to the same disease while many diseases are not coded. WG5 will propose a single classification of all different diseases associated with CNP.

Task 2: Organization of Registries of patients with CNP.
So far, no homogeneous approach exists between European countries for registration of CNP patients. These patients are thus included in Registries of BM Failure Syndromes, in the SCNIR, in Registries of PIDs, in the database of the European Society for ImmunoDeficiencies, or in disease-specific or national databases. This WG will contribute to the standardization of the modus operandi of the national CNP Registries to provide statistics and standard indicators on incidence, prevalence, morbidity, mortality, infections’ rate, quality of life, proportion of patients receiving G-CSF and dose, number of patients undergoing HSC transplantation, comorbidities and rate of MDS/AML. Within the framework of this WP opportunities for expansion of clinical trials on the use of already existing drugs like Pegylated G-CSF [44] will be given.

Task 3: Biobanking for patients with CNP.
The availability of Biobanks is a pre-requisite for progress in understanding CNPs and improving patient care. Patient samples will be collected systematically for functional studies, investigation of genetic factors related to CNP pathogenesis, retrospective analysis of cases with MDS/AML transformation, investigation of known genetic defects in untested patients and search for novel ones. In addition to addressing the ethical and regulatory aspects according to the standards of the European Legal Framework [42], the aim of this WG is to propose guidelines for biological data collection, management, sharing and transfer for a given project. In addition, issues specific to type of samples (serum, blood and/or BM cells, others) and the time points of collection in order to allow pertinent study in close link with clinical data will be defined.

Milestones:

• M5.1. WG Meetings (1st quarter of each year) which will be based on the progress made in WG-5 followed by the respective reports (months 12, 24, 36, 48). 

Deliverables:

• D5.1. Working classification of all different diseases belonging to the CNP family for incorporation in the ICD system (months 24-36).
• D5.2. Guidelines for standardization of the mode of operation of the national Registries in Europe for patients with CNPs (months 2-12).
• D5.3. Guidelines including ethical and regulatory rules for biological data collection, management, sharing and transfer (months 2-12).
• D5.4. Publications (papers/meeting abstracts), interim and final progress reports (months 24-48).