Molecular basis of congenital neutropenia

In this issue of The journal, Beel and Vandenberghe1 describe 2 patients with an activating mutation in Wiskott-Aldrich syndrome (WAS) who developed myelodysplastic syndrome and acute myeloid leukemia, associated with mutations in G-CSF receptor (CSF3R) and monosomy 7. This report is interesting in various aspects. First, it adds a new variant of congenital neutropenia to the growing list of pre-leukemic syndromes. Second, it illuminates the clinical diversity of congenital neutropenia syndromes and their long-term complications. And third, this observation follows up on a potential link between control of the actin cytoskeleton and cytogenetic stability by documenting that a constitutive active WASp mutation predisposes to MDS/AML.2

In 1950, Rolf Kostmann discovered autosomal recessive severe congenital neutropenia (SCN) and thus described the first hereditary human immunodeficiency syndrome.3,4 For more than 50 years, the molecular etiology has remained enigmatic. Marshall Horwitz and David Dale opened the field of molecular genetics in SCN research when they identified heterozygous mutations in the gene encoding neutrophil elastase (ELANE/ELA2) in patients with cyclic neutropenia5 and in patients with severe congenital neutropenia.6 Ten years later, more than a dozen genes have been involved in the molecular pathophysiology of congenital neutropenia. And the number continues to grow. Based on these recent genetic insights, many patients with SCN can now be classified according to the underlying mutation.

From a clinical perspective, congenital neutropenia may be an isolated hematologic finding or may be part of a syndrome associating abnormalities in other organs, such as the skin, the brain, the heart, the pancreas, or the urogenital tract. The clinical presentation may offer important clues to the genetic diagnosis (Table 1). Even in the era of molecular medicine, the importance of the clinical approach and systematic physical examination cannot be underestimated.

Patients with congenital neutropenia typically present with recurrent bacterial infections due to a lack of sufficient numbers of neutrophils. In addition to quantitative abnormalities, many variants of congenital neutropenia also comprise a spectrum of qualitative aberrations, such as defective production of bactericidal proteins or defective activation. A prominent feature in many, but not all, variants of severe congenital neutropenia is a so-called maturation arrest in the bone marrow. Myeloid maturation appears arrested at the stage of promyelocytes. Congenital neutropenia must be differentiated from immune-mediated neutropenia or benign ethnic neutropenia, two conditions rarely associated with severe infections. Benign ethnic neutropenia, seen in patients of African or Arabic descent, has recently been linked to a polymorphism in the gene encoding the Duffy antigen receptor for chemokines (DARC).7 This Duffy Null polymorphism (SNPrs2814778) is also associated with protection against plasmodium vivax malaria, yet both of these mechanisms remain unknown.

In Central Europe and Northern America, approximately 50% of SCN patients have heterozygous mutations in ELANE/ELA2. Patients either inherit a mutation in an autosomaldominant pattern of inheritance or acquire mutations during embryogenesis. If the mutations are transmitted into the hematopoietic system, they may cause cyclic or severe congenital neutropenia. ELANE is exclusively expressed in neutrophil granulocytes and monocytes and functions as a serine protease cleaving a wide array of cellular and extracellular proteins. Cells expressing mutated ELANE are more likely to undergo apoptosis. While the exact molecular mechanism accounting for the phenotype of increased apoptosis is still under investigation, recent studies suggest that mutated ELANE cause increased stress in the endoplasmic reticulum (ER), initiating apoptosis.8,9 Increased ER-stress elicits a cellular response collectively termed the unfolded protein response. Three ER-localized protein sensors are known: IRE1alpha (inositol-requiring 1alpha), PERK (double-stranded RNA-dependent protein kinase (PKR)-like ER kinase), and activating transcription factor 6 (ATF6).10 In cases of ER-stress, these sensors are being activated and trigger a complex series of events destined to maintain the homeostasis of the ER and to promote protein folding, maturation, secretion, and ER-associated protein degradation. In cases in which these rescue mechanisms fail, the unfolded protein response initiates an apoptosis program to protect cells and the organism from dysfunctional or toxic proteins.

Autosomal-recessive SCN can be caused by biallelic mutations in HAX1 (HCLS1 associated protein X-1).11 HAX1 is an antiapoptotic molecule which is predominantly, but not exclusively, localized in the mitochondria. Initially identified as as a binding partner of HS1, a protein kinase involved in BCR-mediated signaling, HAX1 has emerged as a ubiquitously expressed protein promiscuously interacting with multiple partners, apparently including proteins controlling endocytosis, nuclear factors, and even RNA.12 No unifying theory has been proposed that would explain this diversity of networks. With respect to the function of HAX1 in mitochondria, a murine knockout model has provided some mechanistic insights.13 Murine Hax1 interacts with Parl, thus controlling processing of HtrA2/Omi, a factor involved in mitochondrial apoptosis pathways. However, Hax1-deficient mice only partly mirror the phenotype of HAX1-deficiency in human patients. Hax1 mice are not neutropenic and develop a severe lethal neurodegenerative disease. Interestingly a subset of HAX1-deficient patients associate severe congenital neutropenia and neurological symptoms such as cognitive dysfunction or developmental delay and epilepsy, depending on whether the mutation affects a HAX1 isoform expressed in neuronal cells or not.14

Table 1.Organ involvement in congenital neutripenia syndromes.

Reticular dysgenesis (RD) is the most severe variant of severe combined immunodeficiency (SCID), associating an early differentiation arrest in the myeloid lineage associated with severe lymphopenia due to impaired lymphoid development. Furthermore, affected patients suffer from sensorineural hearing loss. Mutations in adenylate kinase 2 (AK2) have recently been identified by two independent groups.15,16 Like HAX1, AK2 is localized in the mitochondrial and is important in mitochondrial energy metabolism and control of apoptosis.

Growth factor independent (GFI)-1 is a zinc finger molecule with transcriptional repressor and splice control function. GFI1 regulates differentiation of hematopoietic and non-hematopoietic cells. Heterozygous mutations in the zinc finger domain have been described in rare patients with SCN.17 The hematologic phenotype of GFI1-mutant patients resembles Gfi1mice characterized by decreased maturation of mature neutrophils and an increased number of immature myeloid cells.18,19 A great variety of target genes is under the control of GFI1, so the exact mechanisms accounting for congenital neutropenia are still not completely clear. Recent studies in a mouse model system indicate that the particular mutation in GFI1 found in SCN patients confers a dominant negative block in granulopoiesis associated with dysregulation of CSF1 and its receptor.20

The most recent asset to the list of the genes causing congenital neutropenia is glucose-6-phosphatase catalytic subunit 3 (G6PC3),21 encoding a paralog of glucose-6-phosphatase. G6PC3 is ubiquitously expressed and localized in the endoplasmic reticulum. Biallelic mutations in G6PC3 abrogate its function, leading to premature apoptosis of neutrophils and associated morphological and biochemical evidence of ER-stress. Originally identified in a religious minority, G6PC3 deficiency has now been recognized in many ethnic groups. In addition to severe congenital neutropenia, affected patients display a wide array of constitutional problems involving structural heart defects (e.g. ASD-II, pulmonary stenosis) and urogenital malformations (e.g. cryptorchidism, urachal fistula). Furthermore, many patients have a characteristic appearance of superficial veins.

It has been known for many years that a subtype of glycogen storage disease, GSD-Ib, is characterized by signs of deficient metabolic alterations due to glcycogen storage and by congenital neutropenia.22 The underlying mutations affect the endosomal transporter for glucose-6-phosphate, glucose-6-phosphate translocase (G6PT), encoded by SLC37A4. The discovery of human G6PC3 deficiency has shed light on the unexplained finding of neutropenia in glycogen storage disease type Ib. Although the stoichiometric and biochemical relationships between glucose-6-phosphate translocase and glucose-6-phosphatase remain to be resolved, both proteins appear to be functionally linked. The G6PC1/G6PT complex (in liver, kidney and intestine) regulates glucose levels, and the ubiquitous G6PC3/G6PT complex appears to be of critical importance in the differentiation and homeostasis of mature neutrophil granulocytes. Nevertheless, both clinical presentation and neutrophil function assays differ between G6PT and G6PC3 deficiency, suggesting that their epistatic relationship is complex.

Classically, mutations in WASP cause Wiskott-Aldrich syndrome characterized by microthrombocytopenia, lymphoid and myeloid immunodeficiency, and eczema or X-linked thrombocytopenia. This disease is caused by a loss of function of WASP, leading to reduced WASP levels and/or activity and diminished actin polymerization. As a consequence, many aspects of hematologic and immune cell function are perturbed, including signal transduction, migration, and phagocytosis.23,24 WASP is subject to autoinhibition by adopting a conformation which prevents its C-terminal VCA domain from interacting with the actin-related protein 2/3 (ARP2/3) complex. Therefore, certain mutations may abolish conformation-dependent autoinhibition. In fact, such gain-of-function mutations have been described in patients with X-linked congenital neutropenia.25–27 In addition to congenital neutropenia, affected patients show reduced numbers and function of lymphocytes. Previous studies have reported that X-linked neutropenia patients may have a susceptibility to myelodysplastic syndromes25 and elegant experimental strategies have confirmed that murine hematopoietic stem cells engineered to express a mutant WASP (I294T) showed enhanced and delocalized actin polymerization throughout the cell, decreased proliferation, and increased apoptosis. Cells became binucleated, suggesting a failure of cytokinesis, and micronuclei were formed, indicative of genomic instability.2 It remains to be shown whether other genetic defects leading to congenital neutropenia may also involve aberrant regulation of the actin cytoskeleton.

There is preliminary evidence that at least two additional genetic defects cause autosomal recessive severe congenital neutropenia, nourishing hope that discovery of novel genes controlling differentiation and function of neutrophil granulocytes will help us understand molecular pathways more comprehensively. Furthermore, these insights may shed light on general principles that determine clonal expansion and onset of myelodysplasia or leukemia.

Footnotes

• Christoph Klein is the Director of the Department of Pediatric Hematology/Oncology at the Hannover Medical School in Germany.