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Severe combined immunodeficiency (SCID) consists of a group of disorders defined by abnormal B and T cell development that typically results in death within the first year of life if undiagnosed or untreated. Reticular dysgenesis (RD) is a rare but especially severe form of SCID that is caused by adenylate kinase 2 deficiency and is characterized not only by lymphopenia but also by profound neutropenia. RD predisposes patients to viral and fungal infections typical of SCID as well as serious bacterial infections atypical in the neonatal period in other SCID types. RD is also associated with sensorineural hearing loss not typically seen in other forms of SCID. Without rapid diagnosis and curative hematopoietic stem cell transplantation, RD is fatal within days to months due to overwhelming bacterial infection. The inclusion of the T cell receptor excision circle assay nationally in 2017 on the newborn screen has facilitated diagnosis of SCID in the neonatal period. This case reports on a male infant with RD who presented after preterm birth with severe cytopenias and a gastrointestinal anomaly and ultimately developed severe bacterial sepsis. Postmortem bone marrow evaluation and panel-based gene sequencing identifying 2 novel variants in the adenylate kinase 2 gene provided confirmation for a diagnosis of RD. This case emphasizes the importance of thorough diagnostic evaluation, including the newborn screen, in neonates and infants with persistent and unexplained cytopenias. Prompt hematology and/or immunology referral is advised for disease management and to facilitate hematopoietic stem cell transplantation to optimize long-term survival.

Reticular Dysgenesis is a particularly grave form of severe combined immunodeficiency (SCID) because it affects the adaptive and innate immune system. Patients suffer from congenital neutropenia, defective lymphocyte development and sensorineural hearing loss. The disease is caused by biallelic loss of function mutations in the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 mediates the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) as substrate for adenosine triphosphate (ATP) synthesis. Accordingly, declining OXPHOS metabolism has been postulated as the driver of disease pathology. The mechanistic basis for the failure of myelopoiesis and lymphopoiesis in Reticular Dysgenesis, however, remains incompletely understood. We have used single cell RNA-sequencing of bone marrow cells from two Reticular Dysgenesis patients to gain insight into the disease mechanism. Gene set enrichment analysis of differentially expressed genes in different subsets of myeloid and lymphoid progenitor cells implicated processes involved in RNA catabolism and ribonucleoprotein synthesis in the pathogenesis of Reticular Dysgenesis. To investigate these findings and precisely mimic the failure of human myelopoiesis in culture, we developed a cell-traceable disease model for Reticular Dysgenesis based on CRISPR-mediated disruption of the AK2 gene in primary human hematopoietic stem cells. In this model, we have shown that AK2-deficienct myeloid progenitor cells not only have compromised mitochondrial energy metabolism and increased AMP levels, but also NAD+ and aspartate depletion, metabolites that rely on TCA-cycle activity for regeneration and synthesis. Furthermore, AK2-deficient cells exhibited strikingly increased levels in the purine nucleotide precursor inosine monophosphate (IMP), decreased cellular RNA content and ribosome subunit expression, reduced protein synthesis and a profoundly hypo-proliferative phenotype. Although NAD+ and aspartate are critical substrates of purine synthesis, AK2-deficient cells did not exhibit purine auxotrophy. Instead, our studies revealed that the rise in IMP levels stemmed from increased AMP deamination. Pharmacologic inhibition of AMP deaminase normalized IMP levels in AK2-deficient cells, but further aggravated the Reticular Dysgenesis phenotype, suggesting AMP catabolism to IMP represents a metabolic adaptation to mitigate AMP-mediated toxicity. This study is the first to identify globally curtailed mitochondrial metabolism resulting in NAD+ and aspartate deficiency and disordered purine metabolism as the two primary cellular consequences of AK2-deficiency. Our data suggests that AMP accumulation and its detrimental effects on ribonucleotide synthesis capacity are the dominant driver of cellular pathology causing failure of myelopoiesis in Reticular Dysgenesis.

We report a rare case of agranulocytosis and lymphopenia complicated with hemophagocytic lymphohistiocytosis. Diagnosis of reticular dysgenesis was made by detection of a pathogenic stop gain variant in the AK2 gene on targeted next generation sequencing and confirmed by Sanger sequencing. Parents were found to be carriers for this variant. Bone marrow aspirate and biopsy was also performed with a clinical diagnosis of severe combined immunodeficiency with HLH. However, no hemophagocytosis was noted in the bone marrow aspirate or trephine biopsy. Instead, it showed aggregates of large histiocyte-like cells, scattered erythroid precursors and megakaryocytes. These cells were confused to be some form of storage cells, but did not resemble storage cells seen in Gaucher's disease or Niemann Pick disease. Myeloid precursors were very few in number. Reticular dysgenesis was not suspected during admission due to a lack of awareness of this entity. Testing for sensorineural deafness in neonates with severe agranulocytosis and lymphopenia would facilitate an early diagnosis of reticular dysgenesis. To the best of our knowledge, hemophagocytic lymphohistiocytosis has not been previously reported in association with reticular dysgenesis.

Mutations in the gene AK2 are responsible for reticular dysgenesis (RD), a rare and severe form of primary immunodeficiency in children. RD patients have a severely shortened life expectancy and without treatment die, generally from sepsis soon after birth. The only available therapeutic option for RD is hematopoietic stem cell transplantation (HSCT). To gain insight into the pathophysiology of RD, we previously created zebrafish models for Ak2 deficiencies. One of the clinical features of RD is hearing loss, but its pathophysiology and causes have not been determined. In adult mammals, sensory hair cells of the inner ear do not regenerate; however, their regeneration has been observed in several non-mammalian vertebrates, including zebrafish. Therefore, we used our RD zebrafish models to determine whether Ak2 deficiency affects sensory organ development and/or hair cell regeneration. Our studies indicated that Ak2 is required for the correct development, survival and regeneration of sensory hair cells. Interestingly, Ak2 deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in the hair cells. Finally, we show that glutathione treatment can partially rescue hair cell development in the sensory organs in our RD models, pointing to the potential use of antioxidants as a therapeutic treatment supplementing HSCT to prevent or ameliorate sensorineural hearing deficits in RD patients., Summary: A zebrafish model of reticular dysgenesis reveals hair cell developmental deficits that can be partially rescued by antioxidants, pointing to their potential use as a therapeutic treatment for reticular dysgenesis patients.

Abstract: Primary immune deficiencies (PIDs) are characterized by quantitative and/or functional abnormalities of the immune system elements. Bone and joint abnormalities are not rare in patients with immunodeficiencies. Joint manifestations, of which arthritis is the most common, occur mainly in humoral PIDs (X-linked agammaglobulinemia, common variable immunodeficiency, and IgA deficiency) and occasionally in defects of the phagocyte system (chronic granulomatous disease, glicogen storage diseases). Monoarthritis or oligoarthritis is the usual pattern, caused by Mycoplasma, Staphylococcus, Streptococcus, Pneumococcus or Haemophilus species. These bacteria can provoke not only synovial infections, but also aseptic arthritogenic inflammatory responses. Infectious arthritis or osteomyelitis must be diagnosed and treated with antimicrobial therapy at the earliest. Bone lesions are far less common and usually present as infectious complications in humoral PID. Larger bone manifestations occur in hyper-IgE syndrome and spondyloepiphyseal dysplasia. Short stature is the most common in reticular dysgenesis, in autoimmune polyendocrinopathy candidiasis ectodermal dysplasia and in DNA repair disorders. Knowledge of PID syndromes both enhances the diagnostic capabilities of physicians and provides understanding the pathophysiology of bone and joint abnormalities associated with immune dysfunction. In children and occasionally in adults, a combination of bone and/or joint manifestations and hypogammaglobulinemia may be the first sign of PID. When lymphoproliferative disease or infection can not be proved, investigations for PID should be accomplished. Orv Hetil. 2018; 159(23): 918–928.

Reticular Dysgenesis (RD) is a particularly grave form of severe combined immunodeficiency (SCID), characterized by maturation arrest of both myeloid and lymphoid lineages paired with sensorineural hearing loss. RD is caused by biallelic mutations in the mitochondrial enzyme adenylate kinase 2 (AK2). AK2 catalyzes the phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP) in the mitochondrial intermembrane space. Using a CRISPR/Cas9 AK2 biallelic knock out model in human hematopoietic stem and progenitor cells (HSPCs), we have shown that AK2 -/- HSPCs mimic the neutrophil maturation defect in RD patients. Mitochondrial respiration is compromised in AK2 -/- HSPCs, which leads to a decreased NAD +/NADH ratio resulting in reductive stress. Metabolomics analysis by LC-MS/MS showed a significant accumulation of AMP, along with increased AMP/ADP and AMP/ATP ratios in AK2 -/- HSPCs, suggesting that purine metabolism is compromised by AK2 deficiency. Purine metabolism defects, such as deficiencies in adenosine deaminase (ADA) and purine nucleotide phosphorylase (PNP), have long been recognized as a cause of SCID. Furthermore, pharmacological interference with purine metabolism is a highly effective antiproliferative strategy in cancer therapy. In this study, we sought to investigate whether impaired purine metabolism contributes to the myelopoietic defect caused by AK2 deficiency. Results We explored how purine metabolism affects myelopoiesis by differentiating HSPCs in media containing no nucleosides (nucleoside-), mixed nucleosides (nucleoside+) or adenosine only (adenosine+). We observed no difference in proliferation or neutrophil maturation between nucleosides- and nucleoside+ media for both control and AK2 -/- HSPCs, suggesting that AK2 -/- HSPCs do not rely on exogenous nucleosides. Interestingly, control HSPCs cultured in adenosine+ media showed severe proliferation and neutrophil maturation defects that mimic AK2 deficiency, suggesting that purine imbalance is detrimental to myelopoiesis. Previous metabolomics analysis showed a significant accumulation of inosine monophosphate (IMP) in AK2 -/- HSPCs. Since IMP can be produced through AMP deamination by AMPD, we asked whether the IMP accumulation in AK2 -/- HSPCs is caused by converting excess AMP to IMP. An LC-MS/MS analysis showed that AMPD inhibitor (AMPDi) treatment significantly lowered IMP levels and increased AMP levels in AK2 -/- HSPCs, indicating that AMP deamination is a major source of IMP accumulation in AK2 -/- HSPCs. Furthermore, AMPDi treatment did not improve, but rather slightly aggravated neutrophil differentiation in AK2 -/- HSPCs, suggesting that the AK2 -/- neutrophil maturation defect is not caused by IMP accumulation. This raises the possibility that AK2 -/- HSPCs employ AMP deamination as a mechanism to curtail the toxicity of excess AMP. Since purine is a building block of RNA, and ribosomal RNA (rRNA) constitutes >85% of cellular RNA content, we asked whether rRNA synthesis is compromised by AK2 deficiency. Pyronin Y staining showed a significant decrease in rRNA content in AK2 -/- HSPCs. Nascent peptide synthesis rate was also decreased in AK2 -/- HSPCs, as quantified by OP-puromycin uptake. These findings are corroborated by RNA-seq analysis of AK2 -/- and control HSPCs, which showed that ribosomal subunits, ribosomal biogenesis and ribonucleoprotein complex assembly are among the top down-regulated pathways. The data suggest that defective purine metabolism in AK2 -/- HSPCs impairs ribosomal biogenesis and protein synthesis. Conclusion Our studies showed that purine imbalance in HSPCs impairs myeloid proliferation and neutrophil maturation. AK2 depletion in HSPCs leads to AMP accumulation and defective ribosomal biogenesis. AK2 -/- HSPCs convert excess AMP to IMP, possibly as a means to mitigate AMP toxicity. However, AMP deamination activities alone are not sufficient to lower AMP levels to those of control HSPCs. We are currently testing whether boosting 5'-nucleotidase activities (cNIA, cN1B and cNII) in AK2 -/- HSPCs can decrease AMP levels and rescue the neutrophil maturation defect. As purine metabolic defects are associated with diverse immune and non-immune abnormalities, further understanding of how purine metabolism governs differentiation of human HSPCs will enable us to develop novel therapeutic strategies for RD and other purine disorders. Disclosures Porteus: CRISPR Therapeutics: Current equity holder in publicly-traded company; Allogene Therapeutics: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Versant Ventures: Consultancy; Ziopharm: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees; Graphite Bio: Current equity holder in publicly-traded company, Membership on an entity's Board of Directors or advisory committees. Morrison: Garuda Therapeutics: Other: founder and SAB member ; Kojin Therapeutics: Other: SAB member ; Frequency Therapeutics: Other: SAB member ; Ona Terapeutics: Other: SAB member ; Protein Fluidics: Other: SAB member .

Reticular dysgenesis (RD) is a rare congenital disorder defined clinically by the combination of severe combined immunodeficiency (SCID), agranulocytosis, and sensorineural deafness. Mutations in the gene encoding adenylate kinase 2 were identified to cause the disorder. Hematopoietic stem cell transplantation (HSCT) is the only option to cure this otherwise fatal disease. Retrospective data on clinical presentation, genetics, and outcome of HSCT were collected from centers in Europe, Asia, and North America for a total of 32 patients born between 1982 and 2011. Age at presentation was

Severe combined immunodeficiency (SCID) represents one of the most severe forms of primary immunodeficiency (PID) disorders characterized by impaired cellular and humoral immune responses. Here, we report the clinical, immunological, and molecular findings in 57 patients diagnosed with SCID from India. Majority of our patients (89%) presented within 6 months of age. The most common clinical manifestations observed were recurrent pneumonia (66%), failure to thrive (60%), chronic diarrhea (35%), gastrointestinal infection (21%), and oral candidiasis (21%). Hematopoietic Stem Cell Transplantation (HSCT) is the only curative therapy available for treating these patients. Four patients underwent HSCT in our cohort but had a poor survival outcome. Lymphopenia (absolute lymphocyte counts/μL

Reticular dysgenesis is a form of severe combined immunodeficiency (SCID) caused by biallelic pathogenic variants in AK2. Here we present the case of a boy diagnosed with SCID following a positive newborn screen (NBS). Genetic testing revealed a homozygous variant: AK2 c.330 + 5G > A. In silico analyses predicted weakened native donor splice site. However, this variant was initially classified as a variant of uncertain significance (VUS) given lack of direct evidence. To determine the impact on splicing, we analyzed RNA from the proband and his parents, using massively parallel RNA-seq of cloned RT-PCR products. Analysis showed that c.330 + 5G > A results in exon 3 skipping, which encodes a critical region of the AK2 protein. With these results, the variant was upgraded to pathogenic, and the patient was given a diagnosis of reticular dysgenesis. Interpretation of VUS at noncanonical splice site nucleotides presents a challenge. RNA sequencing provides an ideal platform to perform qualitative and quantitative assessment of intronic VUS, which can lead to reclassification if a significant impact on mRNA is observed. Genetic disorders of hematopoiesis and immunity represent fruitful areas to apply RNA-based analysis for variant interpretation given the high expression of RNA in blood.

Reticular dysgenesis is an autosomal recessive form of severe combined immunodeficiency (SCID) that usually manifests in newborns. It is a unique example of an immune deficiency that is linked to dysfunctional mitochondrial energy metabolism and caused by adenylate kinase 2 (AK2) deficiency. It is characterized by an early differentiation arrest in the myeloid lineage, impaired lymphoid maturation, and sensorineural hearing loss. In this study, a novel AK2 homozygous mutation, c.622 T > C [p.Ser208Pro], was identified in an Old Order Amish patient through whole exome sequencing. Functional studies showed that the patient’s cells have no detectable AK2 protein, as well as low oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and proton production rate (PPR). An increased production of reactive oxygen species, mitochondrial membrane permeability, and mitochondrial mass, and decreased ATP production, were also observed. The results confirm the pathogenicity of the AK2 mutation and demonstrate that reticular dysgenesis should be considered in Amish individuals presenting with immune deficiency. We also describe other pathophysiological aspects of AK2 deficiency not previously reported.

By establishing biochemical, molecular and animal models, this thesis generated data for the understanding of AK2-deficiency. One aim of this thesis was the expression and purification of recombinant adenylate kinase 2 (AK2) protein and the establishment of an enzymatic assay to determine the enzymatic activities of different published disease-causing AK2 missense mutations. An enzymatic adenylate kinase assay was established and validated which now enables the functional testing of adenylate kinase missense mutations. The determination of the enzymatic activity of five previously published AK2 mutations p.[Gly47Arg], p.[Arg103Trp], p.[Arg175Gln], p.[Arg175Pro], and p.[Arg186Cys] showed for the first time that the clinical phenotype of Reticular Dysgenesis (RD) is caused by the absence of the enzymatic kinase activity of AK2. The hypothesised correlation between residual enzymatic activity and normal numbers of B cells, monocytes, or NK cells was disproven. The method established in this thesis allows the future analysis of the enzymatic activity of any unknown adenylate kinase mutation or variation, even of patients with atypical clinical phenotypes and will be helpful to decide if a novel AK2 mutation may be the cause of the disease or not. The pathophysiology of RD, especially why an adenylate kinase 2 deficiency leads to the observed cell-type specific effects, is unknown. The second aim of this thesis was to investigate the expression and regulation of AK1 in haematopoietic cell lines compared to non-haematopoietic cell lines. The expression of AK1 and AK2 in haematopoiesis was characterised in order to support or disprove the previously published hypothesis, that leukocytes might be susceptible to genetic defects of mitochondrial AK2, as they do not express the cytosolic adenylate kinase 1 (AK1) in sufficient amounts to compensate for functionally impaired AK2 (Pannicke et al., 2009). Protein and RNA expression analyses of AK1 and AK2 in human and murine cell lines and in murine primary cells confirmed that AK2 is ubiquitously expressed in almost all haematopoietic and non-haematopoietic cells. AK1 is only expressed in cells which are not affected by an adenylate kinase 2 deficiency. This supports the previously published hypothesis that AK1 is able to compensate for an AK2 deficiency and shows that this hypothesis, established in the human system, is still concordant with the murine system. In addition it was shown that there are some cell types, like low-energy requiring hematopoietic stem and progenitor cells and mature murine NK cells, which need neither AK2 nor AK1 protein expression to survive. However, proliferation and maturation of cells seems to depend on AK2 or AK1 protein expression. Further investigations on the regulation of the AK1 expression revealed that human AK1 protein expression does not directly correlate with total AK1 mRNA amount because several splice variants with alternative 5’UTR exons upstream of exon 2 were identified which do not enable protein translation. It was shown that AK1 protein expression correlates with an AK1 mRNA with exon 1 upstream of exon 2. This AK1 mRNA with exon 1 is regulated mainly epigenetically by CpG methylation of the Ak1 promoter region upstream of exon 1. In addition, AK1 mRNA expression depends on the availability of non-ubiquitous transcription factors. It was also shown that AK1 mRNA can be stably introduced in cells which do not express AK1 protein endogenously and this results in robust protein expression. These new findings are important for AK1 protein regulation in the future and for testing whether AK1 protein is able to compensate for a functional AK2 deficiency. This will lead to a better understanding of the pathophysiology of RD with the aim to eventually improve patients’ treatment. As a third aim, an Ak2 knockout mouse model was established and characterised to provide a model system for further studies on the molecular biology and pathophysiology of RD. A homozygous knockout of Ak2 in mice leads to early embryonic lethality shortly after nidation around embryonic day 6.5 due to a non-haematopoietic defect. A haematopoiesis-specific AK2-deficient mouse model shows also a much more severe phenotype than human AK2-deficiency with embryonic lethality between E13 and E15 due to severe anaemia. Flow cytometry analyses and colony assays revealed an early onset of erythroid development failure in conditional Ak2 knockout embryos. Megakaryocyte-erythrocyte progenitor (MEP) numbers were significantly decreased and a developmental block of early erythroid progenitors from erythropoietin independent progenitors to erythropoietin dependent progenitors leads to an impaired definitive erythropoiesis and results in severe anaemia. It was shown that a main difference between human and murine erythrocytes is the adenylate kinase protein expression. Human erythrocytes express AK1 protein but no AK2 protein and are therefore expected to be AK2-independent, whereas murine erythroid progenitors only express AK2 protein and may be sensitive to an AK2 deficiency. The number of stem cell and progenitor cells (except for MEP cells) is independent of the Ak2 genotype, but the viability of the progenitor cells is slightly reduced. The numbers of CD11b+Gr-1- monocytes and CD11b+Gr-1+ granulocytes, are not altered by murine AK2-deficiency. Nevertheless, murine AK2-deficient CFU-M, CFU-G, and CFU-GM colonies exhibited an abnormal morphology and a negative effect of an AK2-deficiency on the further development and possibly on the function of monocytes, macrophages, and granulocytes may be predicted. A complete characterisation of the B-, T- and NK-cell development and function was not possible due to the early embryonic death of AK2-deficient mice and no pre-B-cell colonies nor single B220+CD19+ pre-B-cells were detectable in Ak2 knockout samples after IL-7 stimulation of foetal liver cells. As matters stand, the AK2-deficient mouse model and the haematopoiesis-specific AK2-deficient mouse model are not suitable to model human RD in mice, but the models offer the potential to discover and understand the cellular and molecular pathomechanism of AK2-deficieny by analysing common features and differences of affected and unaffected cell populations in the human and the murine system.

Reticular Dysgenesis is a rare immunodeficiency which is clinically characterized by the combination of Severe Combined Immunodeficiency (SCID) with agranulocytosis and sensorineural deafness. Mutations in the gene encoding adenylate kinase 2 (AK2) were identified to cause this phenotype. In this review, we will demonstrate important clinical differences between reticular dysgenesis and other SCID entities and summarize recent concepts in the understanding of the pathophysiology of the disease and the management strategies for this difficult condition.

Rissone et al. demonstrate that adenylate kinase AK2, an enzyme mutated in reticular dysgenesis (RD) in humans, prevents oxidative stress during hematopoiesis. Using a zebrafish model, as well as induced pluripotent stem cells derived from an RD patient, they find that AK2 deficiency affects hematopoietic stem and progenitor development with increased oxidative stress. Antioxidant treatment rescues the hematopoietic phenotypes., Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.

Rare cases of extrapulmonary involvement in Legionella spp. infections have been described, mostly in immunocompromised adults. We report a case of a 2-month old male with reticular dysgenesis variant of severe combined immune deficiency with multiple liver lesions. Core-needle biopsies of one liver lesion demonstrated Gram-negative bacilli and a broad-spectrum polymerase chain reaction assay detected Legionella pneumophila.

The Primary Immune Deficiency Treatment Consortium (PIDTC) is enrolling children with severe combined immunodeficiency (SCID) to a prospective natural history study. We analyzed patients treated with allogeneic hematopoietic cell transplantation (HCT) from 2010 to 2014, including 68 patients with typical SCID and 32 with leaky SCID, Omenn syndrome, or reticular dysgenesis. Most (59%) patients were diagnosed by newborn screening or family history. The 2-year overall survival was 90%, but was 95% for those who were infection-free at HCT vs 81% for those with active infection (P = .009). Other factors, including the diagnosis of typical vs leaky SCID/Omenn syndrome, diagnosis via family history or newborn screening, use of preparative chemotherapy, or the type of donor used, did not impact survival. Although 1-year post-HCT median CD4 counts and freedom from IV immunoglobulin were improved after the use of preparative chemotherapy, other immunologic reconstitution parameters were not affected, and the potential for late sequelae in extremely young infants requires additional evaluation. After a T-cell-replete graft, landmark analysis at day +100 post-HCT revealed that CD3 < 300 cells/μL, CD8 < 50 cells/μL, CD45RA < 10%, or a restricted Vβ T-cell receptor repertoire (

Severe combined immunodeficiencies (SCID) are a heterogeneous group of inherited disorders characterized by a profound reduction or alteration of T lymphocyte function. They arise from a variety of molecular defects which affect T lymphocytes development and function. The number of infections prior hematopietic stem cells tansplantaton (HSCT), genotype, and the type of donor are described as prognostic factors for stem cell transplants. In this retrospective study, we included 30 pediatric patients suffering from SCID who underwent to CD34+-selected grafts between January 2008 to December 2017 in our center. Diagnosis of reticular dysgenesis, ADA deficiency, or leaky SCIDs and intra thymic deficiency were excluded. A mechanistic mathematical model of all available data was performed and provided a dynamic appreciation of immune reconstitution, while removing bias. T-cell populations were maintained through proliferation and loss model and thymic output have been integrated to the production function. This joint modeling approach aimed to predict rate and extent of T cell immune reconstitution over time (mainly CD3+ T cells, CD3+CD4+ helper T cells, and the CD3+CD4+ CD45RA+ cells). With a median follow-up time of 97.28 months [range 0.85; 131.54], there were 345 points of T cell phenotyping concentrations in total with a median of 12 samples per patient (range, 0 -35 samples) taken post-transplantation. In this data-set, 13 % of the patients (n= 4) died from infections. Time to reach half of the maximal T cell concentrations was estimated to 3.4 months, 95%CI [2.5 - 4.6]. In covariate analysis, genetic diagnosis (p= 0.0047) and conditioning regimen (p= 0.01) were found to be significant pre transplant covariates which impacted the trajectory of T cell concentration with time. This modeling approach appeared to be the best method to learn about the dynamic T cell reconstitution after transplant in patients suffering from SCID. In the context of new cell therapy approach for T cell depletion and in vitro thymic maturation, this mechanistic joint model can be used for the design and analysis of incoming clinical trials. Figure Disclosures Cavazzana: Smartimmune: Other: Founder of Smartimmune.

To the Editor: Adenosine deaminase (ADA) deficiency is a rare autosomal recessive form of severe combined immunodeficiency disorder (SCID) caused by mutations in the ADA gene [1]. ADA-SCID is associated with both Bearly^ and Blate onset^ of presentation due to wide variability in the genetic mutations. No data is available about its incidence in India, but it is expected to be high due to high consanguinity. We are presenting a case of ADA-SCID from India. Informed consent was obtained from the participants included in the study. A 6-mo boy, born of 2 degree consanguineous marriage presented with respiratory infections, failure to thrive, developmental delay and dysmorphic features like hairy forehead, frontal bossing, fused eyebrows and clinodactyly. Chest radiograph showed an absence of thymic shadow, the anterior end of ribs and costochondral junctions appeared normal. Complete blood count (CBC) revealed a low lymphocyte percentage (6 %) and absolute lymphocyte count (ALC – 771/ cumm) with normal blood indices (Hb 9.3 g/dl, platelet count 266 x10/L). Serum immunoglobulin and uric acid levels were normal. Patient was immunized as per schedule including BCG at birth. The infectious work up was negative. RBCADA levels were low (0.7 U/g Hb) [2]. The lymphocyte subset analysis (LSS) by flowcytometry showed marked reduction in absolute counts of T (655/cumm), B (23/cumm), NK (85/cumm) cells. Sanger sequencing revealed a novel homozygous missense mutation (c.42 T > C; p.Leu14Pro) in exon 2 ofADA gene and was predicted deleterious using SIFT, Polyphen and Panther software. Parents were carrier for the same mutation. This change was not detected in a statistically significant number of control samples. As reported, depletion of T, B and NK cells with severe infections and developmental delay was noted in our patient at an early age [3]. A low ALC and TBNK phenotype is associated with ADA deficiency, Purine Nucleoside Phosphorylase (PNP) deficiency and Reticular Dysgenesis. The present case showed low ADA levels with normal uric acid levels and normal ANC on CBC; hence, was evaluated for ADA gene studies. Developmental delay and neurological abnormalities are well described in ADA–SCID, however, the dysmorphic features with clinodactyly seen in this child is rare and not been reported in the literature. It is difficult to say if any other additional genetic abnormalities were responsible for the same, however we could not evaluate it by cytogenetic studies. Though, live vaccines are contraindicated in ADA–SCID, no immunization complication was observed in our patient. Murine ADA gene studies revealed that H15D mutation alters an ability of zinc to activate a water molecule postulated to play a role in the catalytic mechanism [4]. The L14P in our patient is located just before H15D mutation and may result in similar effects. Apart from hematopoietic stem cell transplantation (HSCT) or gene therapy or enzyme replacement therapy, supportive measures like immunoglobulin supplements, prophylactic antifungals, and antibiotic must be initiated immediately after diagnosis to reduce the morbidity [5]. Inspite of adequate supportive therapy our patient expired due to respiratory complications. Typical cl inical manifesta t ions, lymphopenia, immunophenotypic studies and RBC ADA levels provide a clue towards the underlying ADA deficiency that can be * Manisha Madkaikar madkaikarm@icmr.org.in

Hematopoietic stem cell (HSC) differentiation is accompanied by a metabolic shift from glycolysis to oxidative phosphorylation (OXPHOS) to meet the increasing energy demand during proliferation and differentiation. However, the role of mitochondrial metabolism in HSC differentiation goes beyond ATP production. Metabolites generated during mitochondrial metabolism may impact in HSC fate decisions through stable epigenetic modifications. Despite some progress in understanding mitochondrial communication during HSC development, their role in human hematopoiesis remains largely elusive, where the lack of appropriate model systems poses a major obstacle. Reticular Dysgenesis (RD), a rare and particularly severe form of severe combined immunodeficiency (SCID), offers an attractive model for studying the role of mitochondrial metabolism in hematopoiesis. RD is an autosomal recessive disease caused by biallelic mutations of the mitochondrial enzyme Adenylate Kinase 2 (AK2). AK2 catalyzes the reversible phosphorylation of adenosine monophosphate (AMP) to adenosine diphosphate (ADP), which serves as the substrate for the ATP synthase. In addition to defective lymphocyte development typical of classic SCID, RD patients also suffer from impaired myeloid development, suggestive of a global defect in hematopoiesis. In a human induced pluripotent stem cell (iPSC) model for RD, hematopoietic stem and progenitor cells (HSPCs) recapitulate a profound maturation arrest of the myeloid lineage, increased oxidative stress and an energy-depleted metabolite and transcriptional profile. We hypothesize that AK2 defects drive hematopoietic cell fate decisions through changes in metabolites that regulate the activities of DNA/histone modifying enzymes and result in stable epigenetic modifications. Methods: Since iPSCs are not suitable to model the epigenetic characteristics of definitive hematopoiesis, we developed a novel model system in which we deleted AK2 in primary human HSCs using CRISPR/Cas9 gene editing technique. We found a highly effective single-guide RNA (sgRNA) targeting the catalytic LID domain of the AK2 gene to introduce directed DNA double stranded breaks (DSBs), and use a homologous recombination (HR)-mediated dual reporter system to track and isolate cells with biallelic AK2 disruption. Results: Our single-color GFP reporter system consistently produces a >60% GFP+ population of AK2-targeted CD34+ umbilical cord blood (UCB) cells. With dual GFP/BFP reporters, we were able to achieve 6% GFP/BFP double positive cells with confirmed biallelic AK2 knock-out. Since HR events on one allele are biologically linked to CRISPR/Cas9 mediated DSBs on the other, we assessed insertion and/or deletion (INDEL) frequency and protein expression in a single reporter (GFP+) population of AK2-targeted UCBs. We detected an INDEL frequency of over 90% on the non-HR alleles along with nearly absent AK2 protein expression by Western Blot. These results indicated that the highly efficient single-color reporter system with >60% targeting efficiency is sufficient to achieve an AK2 biallelic knock-out population in primary HSCs. in vitro myeloid differentiation of these AK2-targeted HSCs recapitulates the RD phenotype with impaired neutrophil but preserved monocyte development. Conclusion: This novel disease model for RD will now allow us to examine the cellular and molecular impact of perturbations in metabolism on human HSC development. We will investigate the effect on differentiation potential, metabolite profile, transcriptome and epigenome in vitro as well as in a xenograft mouse model. Elucidating how metabolism governs differentiation and self-renewal of human HSCs will not only advance our basic understanding of many blood and immune diseases, but has important translational implications for improving the use of HSCs in hematopoietic stem cell transplantation, gene and cell therapy. Disclosures Porteus: CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Reticular dysgenesis (RD) is a rare form of severe combined immunodeficiency (SCID). The underlying genetic defect for most cases of RD was recently identified in the gene encoding adenylate kinase 2 (AK2). However, rare patients with RD and no mutations in AK2 exist, suggesting that mutations in other genes may also cause RD. Although rare, RD has a devastating presentation involving severe neutropenia and T cell lymphopenia, in addition to life non-threatening, but still disabling sensori-neural deafness. An identical phenotype is observed in mice deficient for growth factor independence-1 (Gfi-1) or transgenic for Gfi-1b, related nucleoproteins with opposing, antagonizing roles in development. We hypothesize that a genetically based, altered functional balance between these two factors may be an alternative cause of RD.

Thymocytes and thymic epithelial cell (TEC) cross-talk is crucial to preserve thymic architecture and function, including maturation of TECs and dendritic cells, and induction of mechanisms of central tolerance. We have analyzed thymic maturation and organization in 9 infants with various genetic defects leading to complete or partial block in T-cell development. Profound abnormalities of TEC differentiation (with lack of AIRE expression) and severe reduction of thymic dendritic cells were identified in patients with T-negative severe combined immunodeficiency, reticular dysgenesis, and Omenn syndrome. The latter also showed virtual absence of thymic Foxp3+ T cells. In contrast, an IL2RG-R222C hypomorphic mutation permissive for T-cell development allowed for TEC maturation, AIRE expression, and Foxp3+ T cells. Our data provide evidence that severe defects of thymopoiesis impinge on TEC homeostasis and may affect deletional and nondeletional mechanisms of central tolerance, thus favoring immune dysreactive manifestations, as in Omenn syndrome.

Human adenylate kinase 2 deficiency causes the most profound human haematopoietic defect associated with sensorineural deafness. Reticular dysgenesis (RD) is an autosomal recessive form of human severe combined immunodeficiency (SCID) characterized by an early differentiation block in the myeloid lineage and a profound impairment in lymphoid maturation associated with bilateral sensorineural deafness. The lack of polymorphonuclear neutrophils in affected newborns is responsible for the occurrence of severe infections earlier than usually observed in the other forms of SCID. Furthermore, RD associated neutropenia is characterized by the lack of responsiveness to G-CSF. We have identified bi-allelic mutations in the adenylate kinase 2 (AK2) gene is seven patients affected with RD. These mutations resulted in the absence or a strong decrease in protein expression. Restoration of AK2 expression in the bone marrow cells of RD patients overcomes the neutrophil differentiation arrest, underlying its specific requirement in the development of a restricted set of haematopoietic lineages. Lastly, we established that AK2 is specifically expressed in the stria vascularis region of the inner ear. The function of this gene in the differentiation of a given set of cell lineages is rapidly in progress, showing that studies of primary immunodeficiencies continue to provide key information on human lympho-haematopoietic development. Moreover the AK2 enzyme seems a key molecule in different biological systems such as the lympho-haematopoiesis and the brain development.

Hematopoietic stem cell transplantation is the treatment of choice for severe primary T-cell immunodeficiencies. When an HLA-identical sibling as the donor is not available, an alternative donor stem cell source is needed. In primary T-cell immunodeficiencies, T-cell–depleted HLA-haploidentical bone marrow transplantation has been particularly successful in reconstituting the immune system in many but not all of the severe T-cell immune deficiency disorders. This study reports the use of umbilical cord blood (UCB) stem cell transplantation in 16 patients with severe T cell immune deficiency disorders. UCB was evaluated as a stem cell source for immune reconstitution in children with severe primary T-cell immunodeficiency disorders, such as severe combined immunodeficiency syndrome (SCID), reticular dysgenesis, thymic dysplasia, combined immunodeficiency disease (CID), and Wiskott-Aldrich syndrome (WAS) when an HLA-matched sibling donor was unavailable. From 1996 through 2004, 16 children diagnosed with se...

Background The approach to the diagnosis of severe combined immunodeficiency disease (SCID) and related disorders varies among institutions and countries. Objectives The Primary Immune Deficiency Treatment Consortium attempted to develop a uniform set of criteria for diagnosing SCID and related disorders and has evaluated the results as part of a retrospective study of SCID in North America. Methods Clinical records from 2000 through 2009 at 27 centers in North America were collected on 332 children treated with hematopoietic stem cell transplantation (HCT), enzyme replacement therapy, or gene therapy for SCID and related disorders. Eligibility for inclusion in the study and classification into disease groups were established by using set criteria and applied by an expert review group. Results Two hundred eighty-five (86%) of the patients were determined to be eligible, and 47 (14%) were not eligible. Of the 285 eligible patients, 84% were classified as having typical SCID; 13% were classified as having leaky SCID, Omenn syndrome, or reticular dysgenesis; and 3% had a history of enzyme replacement or gene therapy. Detection of a genotype predicting an SCID phenotype was accepted for eligibility. Reasons for noneligibility were failure to demonstrate either impaired lymphocyte proliferation or maternal T-cell engraftment. Overall (n = 332) rates of testing were as follows: proliferation to PHA, 77%; maternal engraftment, 35%; and genotype, 79% (mutation identified in 62%). Conclusion Lack of complete laboratory evaluation of patients before HCT presents a significant barrier to definitive diagnosis of SCID and related disorders and prevented inclusion of subjects in our observational HCT study. This lesson is critical for patient care, as well as the design of future prospective treatment studies for such children because a well-defined and consistent study population is important for precision in outcomes analysis. © 2013 American Academy of Allergy, Asthma & Immunology.

SUMMARY We report a patient with reticular dysgenesis (RD) who received an HLA-identical marrow graft and remained free of infection in spite of incomplete haematological recovery. Mixed chimerism was achieved and resulted from the presence of autologous B cells and monocytes and grafting of donor T cells. Granulocyte recovery was impaired. The B cells were CD5+ (B1 cells) and appeared to be functional, since serum immunoglobulin levels became normal after the graft. The findings described here suggest that in some cases the defect selectively affects different cell types, including the more abundant leucocyte populations, granulocytes and T lymphocytes. However, B cells and monocytes appear to be relatively spared in this case of RD. Furthermore, the present case may provide insight into the mechanism involved in the expansion of distinct B cell subpopulations (B1 and B2 cells).

Reticular dysgenesis (RD) is a rare congenital immunodeficiency classified within the severe combined immunodeficiencies (SCIDs) and characterized by impairment of both lymphoid and myeloid cell development. Neutropenia unresponsive to recombinant human granulocyte colony-stimulating factor (rGCSF) is the hallmark of RD and the clinical course is rapidly fatal due to overwhelming infections. The authors report a female newborn at 32 weeks of gestation presenting with severe leukopenia at birth. The bone marrow showed a maturation arrest in the myeloid and lymphoid lineage. She had no response to granulocyte colony stimulating factor (rGCSF) treatment and died with sepsis at age of 2 months.

Human severe combined immunodeficiencies (SCID) are phenotypically and genotypically heterogeneous diseases. Reticular dysgenesis is the most severe form of inborn SCID. It is characterized by absence of granulocytes and almost complete deficiency of lymphocytes in peripheral blood, hypoplasia of the thymus and secondary lymphoid organs, and lack of innate and adaptive humoral and cellular immune functions, leading to fatal septicemia within days after birth. In bone marrow of individuals with reticular dysgenesis, myeloid differentiation is blocked at the promyelocytic stage, whereas erythro- and megakaryocytic maturation is generally normal. These features exclude a defect in hematopoietic stem cells but point to a unique aberration of the myelo-lymphoid lineages. The dramatic clinical course of reticular dysgenesis and its unique hematological phenotype have spurred interest in the unknown genetic basis of this syndrome. Here we show that the gene encoding the mitochondrial energy metabolism enzyme adenylate kinase 2 (AK2) is mutated in individuals with reticular dysgenesis. Knockdown of zebrafish ak2 also leads to aberrant leukocyte development, stressing the evolutionarily conserved role of AK2. Our results provide in vivo evidence for AK2 selectivity in leukocyte differentiation. These observations suggest that reticular dysgenesis is the first example of a human immunodeficiency syndrome that is causally linked to energy metabolism and that can therefore be classified as a mitochondriopathy.

Reticular dysgenesis is a rare congenital disorder characterized by severe combined immunodeficiency and profound neutropenia, curable to date, only by bone marrow transplantation. This report describes the association of bilateral sensorineural deafness with this disease.

Adenylate kinases (AKs) are phosphotransferases that regulate the cellular adenine nucleotide composition and play a critical role in the energy homeostasis of all tissues. The AK2 isoenzyme is expressed in the mitochondrial intermembrane space and is mutated in reticular dysgenesis (RD), a rare form of severe combined immunodeficiency (SCID) in humans. RD is characterized by a maturation arrest in the myeloid and lymphoid lineages, leading to early onset, recurrent, and overwhelming infections. To gain insight into the pathophysiology of RD, we studied the effects of AK2 deficiency using the zebrafish model and induced pluripotent stem cells (iPSCs) derived from fibroblasts of an RD patient. In zebrafish, Ak2 deficiency affected hematopoietic stem and progenitor cell (HSPC) development with increased oxidative stress and apoptosis. AK2-deficient iPSCs recapitulated the characteristic myeloid maturation arrest at the promyelocyte stage and demonstrated an increased AMP/ADP ratio, indicative of an energy-depleted adenine nucleotide profile. Antioxidant treatment rescued the hematopoietic phenotypes in vivo in ak2 mutant zebrafish and restored differentiation of AK2-deficient iPSCs into mature granulocytes. Our results link hematopoietic cell fate in AK2 deficiency to cellular energy depletion and increased oxidative stress. This points to the potential use of antioxidants as a supportive therapeutic modality for patients with RD.