Adaptive NK cells can persist in patients with GATA2 mutation depleted of stem and progenitor cells.

Heterozygous GATA2 mutation is associated with immunodeficiency, lymphedema, and myelodysplastic syndrome. Disease presentation is variable, often coinciding with loss of circulating dendritic cells, monocytes, B cells, and natural killer (NK) cells. Nonetheless, in a proportion of patients carrying GATA2 mutation, NK cells persist. We found that peripheral blood NK cells in symptomatic patients uniformly lacked expression of the transcription factor promyelocytic leukemia zinc finger (PLZF), as well as expression of intracellular signaling proteins FcεRγ, spleen tyrosine kinase (SYK), and EWS/FLI1-Activated Transcript 2 (EAT-2) in a variegated manner. Moreover, consistent with an adaptive identity, NK cells from patients with GATA2 mutation displayed altered expression of cytotoxic granule constituents and produced interferon-γ upon Fc-receptor engagement but not following combined interleukin-12 (IL-12) and IL-18 stimulation. Canonical, PLZF-expressing NK cells were retained in asymptomatic carriers of GATA2 mutation. Developmentally, GATA-binding protein-2 (GATA-2) was expressed in hematopoietic stem cells, but not in NK-cell progenitors, CD3-CD56bright, canonical, or adaptive CD3-CD56dim NK cells. Peripheral blood NK cells from individuals with GATA2 mutation proliferated normally in vitro, whereas lineage-negative progenitors displayed impaired NK-cell differentiation. In summary, adaptive NK cells can persist in patients with GATA2 mutation, even after NK-cell progenitors expire. Moreover, our data suggest that adaptive NK cells are more long-lived than canonical, immunoregulatory NK cells.

The evolution of cellular deficiency in GATA2 mutation.

Constitutive heterozygous GATA2 mutation is associated with deafness, lymphedema, mononuclear cytopenias, infection, myelodysplasia (MDS), and acute myeloid leukemia. In this study, we describe a cross-sectional analysis of 24 patients and 6 relatives with 14 different frameshift or substitution mutations of GATA2. A pattern of dendritic cell, monocyte, B, and natural killer (NK) lymphoid deficiency (DCML deficiency) with elevated Fms-like tyrosine kinase 3 ligand (Flt3L) was observed in all 20 patients phenotyped, including patients with Emberger syndrome, monocytopenia with Mycobacterium avium complex (MonoMAC), and MDS. Four unaffected relatives had a normal phenotype indicating that cellular deficiency may evolve over time or is incompletely penetrant, while 2 developed subclinical cytopenias or elevated Flt3L. Patients with GATA2 mutation maintained higher hemoglobin, neutrophils, and platelets and were younger than controls with acquired MDS and wild-type GATA2. Frameshift mutations were associated with earlier age of clinical presentation than substitution mutations. Elevated Flt3L, loss of bone marrow progenitors, and clonal myelopoiesis were early signs of disease evolution. Clinical progression was associated with increasingly elevated Flt3L, depletion of transitional B cells, CD56(bright) NK cells, naïve T cells, and accumulation of terminally differentiated NK and CD8(+) memory T cells. These studies provide a framework for clinical and laboratory monitoring of patients with GATA2 mutation and may inform therapeutic decision-making.

GATA2 deficiency phenotype associated with tandem duplication of GATA2 and overexpression of GATA2-AS1.

A 3-year-old girl of nonconsanguineous healthy parents presented with cervical and mediastinal lymphadenopathy due to Mycobacterium fortuitum infection. Routine blood analysis showed normal hemoglobin, neutrophils, and platelets but profound mononuclear cell deficiency (monocytes < 0.1 × 109/L; B cells 78/µL; NK cells 48/µL). A 548 902-bp region containing GATA2 was sequenced by targeted capture and deep sequencing. This revealed a de novo 187-kb duplication of the entire GATA2 locus, containing a maternally inherited copy number variation deletion of 25 kb (GRCh37: esv2725896 and nsv513733). Many GATA2-associated phenotypes have been attributed to amino acid substitution, frameshift/deletion, loss of intronic enhancer function, or aberrant splicing. Gene deletion has been described, but other structural variation has not been reported in the germline configuration. In this case, duplication of the GATA2 locus was paradoxically associated with skewed diminished expression of GATA2 messenger RNA and loss of GATA2 protein. Chimeric RNA fusion transcripts were not detected. A possible mechanism involves increased transcription of the anti-sense long noncoding RNA GATA2-AS1 (RP11-472.220), which was increased several fold. This case further highlights that evaluation of the allele count is essential in any case of suspected GATA2-related syndrome.

The SLIT-ROBO pathway: a regulator of cell function with implications for the reproductive system.

The secreted SLIT glycoproteins and their Roundabout (ROBO) receptors were originally identified as important axon guidance molecules. They function as a repulsive cue with an evolutionarily conserved role in preventing axons from migrating to inappropriate locations during the assembly of the nervous system. In addition the SLIT-ROBO interaction is involved in the regulation of cell migration, cell death and angiogenesis and, as such, has a pivotal role during the development of other tissues such as the lung, kidney, liver and breast. The cellular functions that the SLIT/ROBO pathway controls during tissue morphogenesis are processes that are dysregulated during cancer development. Therefore inactivation of certain SLITs and ROBOs is associated with advanced tumour formation and progression in disparate tissues. Recent research has indicated that the SLIT/ROBO pathway could also have important functions in the reproductive system. The fetal ovary expresses most members of the SLIT and ROBO families. The SLITs and ROBOs also appear to be regulated by steroid hormones and regulate physiological cell functions in adult reproductive tissues such as the ovary and endometrium. Furthermore several SLITs and ROBOs are aberrantly expressed during the development of ovarian, endometrial, cervical and prostate cancer. This review will examine the roles this pathway could have in the development, physiology and pathology of the reproductive system and highlight areas for future research that could further dissect the influence of the SLIT/ROBO pathway in reproduction.

Involvement of the SLIT/ROBO pathway in follicle development in the fetal ovary.

In humans and domestic mammals, pivotal processes in ovary development, including primordial follicle assembly, occur prenatally. These events are essential for determining fertility in adult life; however, they remain poorly understood at the mechanistic level. In mammals, the SLITs (SLIT1, SLIT2 and SLIT3) and their ROBO (ROBO1, ROBO2, ROBO3/RIG-1 and ROBO4/MAGIC ROBO) receptors regulate neural, leukocyte, vascular smooth muscle cell and endothelial cell migration. In addition, the SLIT/ROBO pathway has functional roles in embryonic development and in the adult ovary by inhibiting cell migration and promoting apoptosis. We therefore characterised follicle formation and investigated the expression and localisation of the ROBO/SLIT pathway in the ovine fetal ovary. Using RT-PCR, we identified SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 in sheep ovaries harvested across gestation. The real-time quantitative PCR results implied that ROBO2 expression and ROBO4 expression were elevated during the early stages of follicle formation and stayed abundant during primordial follicle maturation (P<0.05). Immunohistochemistry examination demonstrated that ROBO1 was localised to the pre-granulosa cells, while ROBO2, ROBO4 and SLIT2 were expressed in the oocytes of the developing primordial follicle. This indicates that in the fetal ovary, SLIT-ROBO signalling may require an autocrine and paracrine interaction. Furthermore, at the time of increased SLIT-ROBO expression, there was a significant reduction in the number of proliferating oocytes in the developing ovary (P<0.0001). Overall, these results suggest, for the first time, that the SLIT-ROBO pathway is expressed at the time of follicle formation during fetal ovary development.

Novel regulated expression of the SLIT/ROBO pathway in the ovary: possible role during luteolysis in women.

The human corpus luteum (CL) undergoes luteolysis, associated with marked tissue and vascular remodeling, unless conception occurs and the gland is rescued by human chorionic gonadotropin (hCG). In Drosophila the Slit gene product, a secreted glycoprotein, acts as a ligand for the roundabout (robo) transmembrane receptor. Together they influence the guidance and migration of neuronal and nonneuronal cells. In vertebrates three Slit (Slit1, Slit2, Slit3) and four Robo (Robo1, Robo2, Robo3/Rig-1, Robo4/Magic Robo) genes have been identified. ROBO1, SLIT2, and SLIT3 are also inactivated in human cancers and may regulate apoptosis and metastasis. Because processes such as apoptosis and tissue remodeling occur during the regression of the CL, the aim of this study was to investigate the expression, regulation, and effects of the SLIT and ROBO genes in human luteal cells. Immunohistochemistry and RT-PCR revealed that SLIT2, SLIT3, ROBO1, and ROBO2 are expressed in luteal steroidogenic cells and fibroblast-like cells of the human CL. Furthermore, using real-time quantitative PCR, expression of SLIT2, SLIT3, and ROBO2 was maximal in the late-luteal phase and significantly reduced after luteal rescue in vivo with exogenous hCG (P<0.05). Additionally, hCG significantly inhibited SLIT2, SLIT3, and ROBO2 expression in cultured luteinized granulosa cells (P<0.05). Blocking SLIT-ROBO activity increased migration and significantly decreased levels of apoptosis in primary cultures of luteal cells (P<0.05). Overall, these results suggest the SLIT/ROBO pathway could play an important role in luteolysis in women.

Glucocorticoid regulation of SLIT/ROBO tumour suppressor genes in the ovarian surface epithelium and ovarian cancer cells.

The three SLIT ligands and their four ROBO receptors have fundamental roles in mammalian development by promoting apoptosis and repulsing aberrant cell migration. SLITs and ROBOs have emerged as candidate tumour suppressor genes whose expression is inhibited in a variety of epithelial tumours. We demonstrated that their expression could be negatively regulated by cortisol in normal ovarian luteal cells. We hypothesised that after ovulation the locally produced cortisol would inhibit SLIT/ROBO expression in the ovarian surface epithelium (OSE) to facilitate its repair and that this regulatory pathway was still present, and could be manipulated, in ovarian epithelial cancer cells. Here we examined the expression and regulation of the SLIT/ROBO pathway in OSE, ovarian cancer epithelial cells and ovarian tumour cell lines. Basal SLIT2, SLIT3, ROBO1, ROBO2 and ROBO4 expression was lower in primary cultures of ovarian cancer epithelial cells when compared to normal OSE (P<0.05) and in poorly differentiated SKOV-3 cells compared to the more differentiated PEO-14 cells (P<0.05). Cortisol reduced the expression of certain SLITs and ROBOs in normal OSE and PEO-14 cells (P<0.05). Furthermore blocking SLIT/ROBO activity reduced apoptosis in both PEO-14 and SKOV-3 tumour cells (P<0.05). Interestingly SLIT/ROBO expression could be increased by reducing the expression of the glucocorticoid receptor using siRNA (P<0.05). Overall our findings indicate that in the post-ovulatory phase one role of cortisol may be to temporarily inhibit SLIT/ROBO expression to facilitate regeneration of the OSE. Therefore this pathway may be a target to develop strategies to manipulate the SLIT/ROBO system in ovarian cancer.

Differential expression and functional characterization of luteinizing hormone receptor splice variants in human luteal cells: implications for luteolysis.

The human LH receptor (LHR) plays a key role in luteal function and the establishment of pregnancy through its interaction with the gonadotropins LH and human chorionic gonadotropin. We previously identified four splice variants of the LHR in human luteinized granulosa cells (LGCs) and corpora lutea (CL). Real-time quantitative PCR revealed that expression of the full-length LHR (LHRa) and the most truncated form (LHRd) changed significantly in CL harvested at different stages of the ovarian cycle (P < 0.01, ANOVA). LHRa expression was reduced in the late luteal CL (P < 0.05). Conversely, an increase in LHRd expression was observed in the late luteal CL (P < 0.01). Chronic manipulation of human chorionic gonadotropin in LGC primary cultures supported the in vivo findings. LHRd encodes a protein lacking the transmembrane and carboxyl terminal domains. COS-7 cells expressing LHRd were unable to produce cAMP in response to LH stimulation. COS-7 cells coexpressing LHRd and LHRa also failed to generate cAMP in response to LH, suggesting that this truncated form has a negative effect on the signaling of LHRa. Immunofluorescence staining of LGC and COS-7 cells implied that there is a reduction in cell surface expression of LHRa when LHRd is present. Overall, these results imply expression of LHR splice variants is regulated in the human CL. Furthermore, during functional luteolysis a truncated variant could modulate the cell surface expression and activity of full-length LHR.

Frequent epigenetic inactivation of the SLIT2 gene in chronic and acute lymphocytic leukemia.

Recently a mouse model of T/natural killer acute lymphoblastic leukemia was used to assess global promoter methylation across the mouse genome using the restriction landmark genomic scanning technique. One of the methylated mouse genes identified in this way was Slit2. There are three mammalian SLIT genes (SLIT1, SLIT2, SLIT3), that belong to a highly conserved family of axon guidance molecules. We have previously demonstrated that SLIT2 is frequently inactivated in lung, breast, colorectal and glioma tumors by hypermethylation of a CpG island in its promoter region, whilst inactivating somatic mutations are rare. Furthermore, we demonstrated that SLIT2 acts as a tumor suppressor gene in breast and colorectal cancer cells. In this report we determined the methylation status of the SLIT2 gene in leukemias (CLL and ALL). SLIT2 was methylated in all ten leukemia cell lines analyzed (eight completely and two partially methylated). SLIT2 expression was restored after treating ALL lines with 5-aza-2dC. In primary ALL and CLL samples, SLIT2 was also frequently methylated, 58% (30/52) B-ALL; 83% (10/12) T-ALL and in 80% (24/30) CLL. Whilst DNA from peripheral blood and bone marrow from healthy control samples showed no SLIT2 methylation. Methylation results in leukemia cell lines and ALL and CLL primary samples were confirmed by direct sequencing of bisulfite modified DNA. Our results demonstrate that methylation of the SLIT2 5' CpG island is conserved between mice and humans, and therefore is likely to be of functional importance.