Autosomal recessive severe congenital neutropenia (SCN) constitutes a primary immunodeficiency syndrome associated with increased apoptosis in myeloid cells, yet the underlying genetic defect remains unknown. Using a positional cloning approach and candidate gene evaluation, we identified a recurrent homozygous germline mutation in HAX1 in three pedigrees. After further molecular screening of individuals with SCN, we identified 19 additional affected individuals with homozygous HAX1 mutations, including three belonging to the original pedigree described by Kostmann. HAX1 encodes the mitochondrial protein HAX1, which has been assigned functions in signal transduction and cytoskeletal control. Here, we show that HAX1 is critical for maintaining the inner mitochondrial membrane potential and protecting against apoptosis in myeloid cells. Our findings suggest that HAX1 is a major regulator of myeloid homeostasis and underline the significance of genetic control of apoptosis in neutrophil development.

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We describe a novel clinical phenotype associating T-and B-cell lymphopenia, intermittent neutropenia, and atrial septal defects in 3 members of a consanguineous kindred. Their clinical histories included recurrent bacterial infections, viral infections, mucocutaneous candidiasis, cutaneous warts, and skin abscesses. Homozygosity mapping and candidate gene sequencing revealed a homozygous premature termination mutation in the gene STK4 (serine threonine kinase 4, formerly having the symbol MST1). STK4 is the human ortholog of Drosophila Hippo, the central constituent of a highly conserved pathway controlling cell growth and apopto- IntroductionMonogenic disorders of the human immune system have provided important insights into the function of host defense mechanisms. 1 Despite remarkable progress in the field, many disorders remain poorly understood. 2,3 Identifying genetic mutations in patients with immunodeficiency syndromes may reveal novel insights into basic mechanisms of the human immune system.Here, we describe the first human patients with a biallelic mutation of serine threonine kinase 4 (STK4; MIM: 604965). STK4 (previously sometimes named MST1) was originally identified as a ubiquitously expressed kinase with structural homology to yeast Ste20. 4,5 STK4 and STK3 (MST2; MIM: 605030) are the mammalian homologs of the Drosphila Hpo protein, the central constituent of the highly conserved HIPPO pathway controlling cell growth, apoptosis, and tumorigenesis. 6 Mice lacking either Stk3 or Stk4 are viable, but those lacking both proteins are not. This indicates that each protein can substitute for the other in the most essential functions. 7 When both Stk3 and Stk4 are conditionally deleted, however, their respective role as growth control regulators becomes manifest, exemplified by liver-specific double-knockout mice that develop massive hepatomegaly and hepatocellular carcinoma. 8,9 STK4 has both proapoptotic and antiapoptotic functions. Earlier papers focused on the proapoptotic functions, and STK4 was described with the adjective "proapoptotic" in the title of a paper as recently as 2007. 10 The strongest evidence that STK4 delivers proapoptotic signals is that STK4 is cleaved by caspases 11,12 ; caspase activity is unambiguously proapoptotic. In resting conditions, STK4 is a cytoplasmic protein. In response to apoptotic stimuli, the 63-kDa full-length protein is cleaved by caspases and a 36-kDa N-terminal fragment translocates to the nucleus and phosphorylates histones, 13,14 suggesting that STK4 plays a proapoptotic role. STK4 is also in a proapoptotic regulatory loop with JNK. [15][16][17] Finally, the interaction between RASSF1A and STK4 was shown to promote Fas-mediated apoptosis. 18 There was also some evidence, before the generation of Stk4-deficient mice, that STK4 has antiapoptotic functions. For example, a study in Caenorhabditis elegans showed that phosphorylation of FOXO proteins by the STK4 ortholog DAF16 protects against cell death induced by oxidative stress. Furthermore, when DAF16 canno...

Analysis of patients with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling differentiation, maintenance, and decay of neutrophils. We identify 9 distinct homozygous mutations in the gene encoding Jagunal homolog 1 (JAGN1) in 14 SCN patients. JAGN1-mutant granulocytes are characterized by ultrastructural defects, paucity of granules, aberrant N-glycosylation of multiple proteins, and increased apoptosis. JAGN1 participates in the secretory pathway and is required for granulocyte-colony stimulating factor receptor-mediated signaling. JAGN1 emerges as a factor necessary in differentiation and survival of neutrophils.

692 Congenital neutropenia syndromes comprise a heterogeneous group of disorders, whose genetic etiology remains often unknown. We describe a consanguineous pedigree with several affected individuals characterized by predisposition to recurrent and chronic bacterial and viral infections. Affected patients had chronic bronchitis/bronchiectasis, recurrent bacterial and herpes simplex skin infections, and disseminated warts associated with human papillomavirus and molluscum contagiosum virus. One patient developed a lymphoproliferative disorder associated with EBV-infection. Patients had congenital neutropenia with fluctuating absolute neutrophil granulocyte counts (180-4000/μl), yet no evidence of cyclic neutropenia. Immunophenotyping of peripheral blood revealed a paucity of peripheral T- and B-cells. Interestingly all patients showed evidences of autoimmunity. In addition all affected patients had subtle and hemodynamically not relevant cardiac defects such as ASD-II (P1), patent foramen ovale (P2) and patent foramen ovale associated with mitral, tricuspid and pulmonary insufficiency (P3). Genome-wide genotyping and linkage analysis of the index family yielded a LOD score of 4.3 on a linkage interval on chromosome 20. Candidate gene sequencing revealed a homozygous nonsense mutation in exon 7 of the gene STK4 (formerly MST1). STK4 is the human ortholog of Drosophila Hippo, the central constituent of a highly conserved pathway controlling cell growth and apoptosis. Isolated STK4-deficient lymphocytes and neutrophils of these patients exhibit enhanced loss of mitochondrial membrane potential and increased susceptibility to apoptosis in response to various proapoptotic stimuli. Lymphopenia and congenital neutropenia may therefore be a consequence of increased loss of peripheral lymphocytes and neutrophils, similar to other well defined monogenetic diseases of the immune system. STK4 deficiency is a novel human primary immunodeficiency syndrome and highlights the role of the HIPPO pathway for the development of the human immune and cardiac systems. Disclosures: No relevant conflicts of interest to declare.

Biallelic mutations in the gene encoding HCLS-associated protein X-1 (HAX1) cause autosomal recessive severe congenital neutropenia. Some of these patients display neurological abnormalities including developmental delay, cognitive impairment and/or epilepsy. Recent genotype-phenotype studies have shown that mutations in HAX1 affecting transcripts A (NM_006118.3) and B (NM_001018837.1) cause the phenotype of SCN with neurological impairment, while mutations affecting isoform A but not B lead to SCN without neurological aberrations. In this study, we identified a consanguineous family with two patients suffering from SCN and neurological disease caused by a novel, homozygous genomic deletion including exons 4–7 of the HAX1 gene. Quantitative MRI analyses revealed general alterations in cerebral proton density in both of the patients, as well as in an additional unrelated patient with another HAX1 mutation (Arg86X) known to be associated with neurological manifestations. This study provides first in vivo evidence of general neurodegeneration associated with HAX1 deficiency in SCN patients.

Analysis of patients with severe congenital neutropenia (SCN) may shed light on the delicate balance of factors controlling differentiation, maintenance, and decay of neutrophil granulocytes. Mutations in ELANE, GFI1, HAX1, G6PC3, WAS, and VPS45 are known to cause SCN. We here describe a new monogenetic SCN variant with biallelic mutations in the gene encoding Jagunal homolog 1 (JAGN1). We studied an index family from Northern Africa with a total of 5 children suffering from SCN. An Affymetrix SNP array-based genetic linkage analysis was performed and identified a single interval of perfect segregation with highly significant multi-marker LOD scores of at least 4.5spanning approximately 1.5Mbp from 9.52Mb to 11.04Mb on chromosome 3 of NCBI’s human genome build 36.3. This interval contained a total of 30 genes, including JAGN1 which encodes an ER-resident protein. Sanger sequencing revealed a homozygous mutation c.3G>A in exon 1 of the JAGN1 gene; this mutation leads to disruption of the defined start of translation. Systematic analysis of a cohort of 90 SCN patients identified 9 distinct homozygous mutations in the gene encoding Jagunal homolog 1 (JAGN1) in 14 SCN patients, thus accounting for approximately 10% of SCN patients. The clinical phenotype was variable and included failure to thrive, developmental delay and bone skeletal abnormalities. The only consistent finding in all JAGN1-deficient patients was SCN and partial or complete refractoriness to therapy with rh-GCSF. JAGN1 is the human ortholog of a gene originally identified in Drosophila melanogaster. Jagunal-deficient oocytes are characterized by defective ER reorganization and aberrant membrane trafficking during vitellogenesis. We found that JAGN1-mutant human granulocytes showed aberrantly enlarged ER structures and paucity of secretory vesicles. We hypothesized that that ER aberrations may be associated with defective N-glycosylation of multiple proteins in neutrophil granulocytes and found that JAGN1-mutant neutrophil granulocytes exhibited anomalous N-glycomic profiles characterized by a marked reduction in fucosylation of all their multi-antennary glycans. JAGN1-deficient neutrophil granulocytes showed increased apoptosis in response to TNFa and staurosporine, likely accounting for the lack of mature neutrophils in these patients. Additional studies in JAGN1-knockdown cells indicate that JAGN1 participates in the secretory pathway and is required for granulocyte-colony stimulating factor receptor-mediated signaling. Global proteomic analysis of the JAGN1-interactome identified a limited number of interaction partners including members of the Coat Protein I (COPI) complex (COPA, COPB2, and COPG2) which suggest a role for JAGN1 in vesicular trafficking from Golgi to ER. Taken together, JAGN1 emerges as a hitherto unrecognized factor necessary in differentiation and survival of neutrophil granulocytes and a novel gene implicated in SCN. Disclosures: No relevant conflicts of interest to declare.

550 Severe congenital neutropenia is a heterogeneous disorder with the phenotypic hallmark of ‘myeloid maturation arrest‘. Recent work in our group identified that HS-1 associated protein X1 (HAX1), a mitochondrial protein is essential for survival of neutrophils through maintenance of mitochondrial membrane potential (MMP). A large group of patients have mutations in ELANE/ELA2 or HAX1, respectively, causing increased susceptibility to apoptosis. Although phenotypically indistinguishable, it is controversial whether HAX1 and ELA2 mutant cells share a joint pathway. To further address the biological abnormalities, we measured reactive oxygen species (ROS) production and autophagy in primary human neutrophil granulocytes from SCN patients. HAX1 deficient neutrophils, but not SCN neutrophils expressing functional HAX1 showed evidence of enhanced production of ROS, a continuous byproduct of the oxidative phosphorylation. Antioxidant defense system in a cell constitutes proteins that restrain ROS mediated cellular damage. The degradation of catalase, a primary antioxidant defense protein was accelerated in HAX1 deficient neutrophils as compared to healthy controls. This effect was not observed in ELA2 deficient neutrophils or in SCN neutrophils expressing functional HAX1. Oxidative phosphorylation coupled ATP synthesis; a prime function of the mitochondria is hampered with mitochondrial dysfunction, resulting from loss of MMP. We observed that the HAX1-deficient neutrophils but not ELA2-deficient neutrophils have an elevated AMP/ATP ratio as measured by the increased activation of AMP activated protein kinase α (AMPKα). In view of increased ROS production hampering the cellular organelles, and decreased levels of cellular ATP, we hypothesized that autophagy may play a role in HAX1-deficient neutrophils. Autophagy describes a process of organelle digestion within a cell aiming at sparing energy and disposal of damaged organelles. In case these rescue mechanisms are futile, the cell undergoes apoptosis. Expression of beclin-1, an autophagy essential protein was increased in HAX1-deficient neutrophils, accompanied by an increase in the ATG12-5 complex formation as compared to healthy control cells. Furthermore, transmission electron microscopy studies revealed evidence of increased autophagy in HAX1-deficient neutrophils. These observed effects are specific to HAX1 deficiency, as these biochemical aberrations were not observed in ELA2-deficient patients or SCN patients expressing functional HAX1. Moreover the phenotype was observed only in neutrophils and not in HAX1-deficient lymphoid or monocytic cells. Our results unravel a novel role of the antiapoptotic protein HAX1 in maintenance of cellular homeostasis in neutrophil granulocytes by regulating the autophagic machinery through control over ROS production. Disclosures: No relevant conflicts of interest to declare.