Heterozygous variants of CLPB are a cause of severe congenital neutropenia.

Severe congenital neutropenia is an inborn disorder of granulopoiesis. Approximately one third of cases do not have a known genetic cause. Exome sequencing of 104 persons with congenital neutropenia identified heterozygous missense variants of CLPB (caseinolytic peptidase B) in 5 severe congenital neutropenia cases, with 5 more cases identified through additional sequencing efforts or clinical sequencing. CLPB encodes an adenosine triphosphatase that is implicated in protein folding and mitochondrial function. Prior studies showed that biallelic mutations of CLPB are associated with a syndrome of 3-methylglutaconic aciduria, cataracts, neurologic disease, and variable neutropenia. However, 3-methylglutaconic aciduria was not observed and, other than neutropenia, these clinical features were uncommon in our series. Moreover, the CLPB variants are distinct, consisting of heterozygous variants that cluster near the adenosine triphosphate-binding pocket. Both genetic loss of CLPB and expression of CLPB variants result in impaired granulocytic differentiation of human hematopoietic progenitor cells and increased apoptosis. These CLPB variants associate with wild-type CLPB and inhibit its adenosine triphosphatase and disaggregase activity in a dominant-negative fashion. Finally, expression of CLPB variants is associated with impaired mitochondrial function but does not render cells more sensitive to endoplasmic reticulum stress. Together, these data show that heterozygous CLPB variants are a new and relatively common cause of congenital neutropenia and should be considered in the evaluation of patients with congenital neutropenia.

Neutropenia in glycogen storage disease Ib: outcomes for patients treated with granulocyte colony-stimulating factor.

PURPOSE OF REVIEW: Glycogen storage disease Ib (GSD Ib) is characterized by hepatomegaly, hypoglycemia, neutropenia, enterocolitis and recurrent bacterial infections. It is attributable to mutations in G6PT1, the gene for the glucose-6-phosphate transporter responsible for transport of glucose into the endoplasmic reticulum. Neutropenia in GSD Ib is now frequently treated with granulocyte colony-stimulating factor (G-CSF). We formed a cooperative group to review outcomes of the long-term treatment of GSD Ib patients treated with G-CSF. RECENT FINDINGS: The study enrolled 103 patients (48 men and 55 women), including 47 currently adult patients. All of these patients were treated with G-CSF, starting at a median age of 3.8 years (range 0.04-33.9 years) with a median dose of 3.0 mcg/kg/day (range 0.01-93.1 mcg/kg/day) for a median of 10.3 years (range 0.01-29.3 years). Neutrophils increased in response to G-CSF in all patients (median values before G-CSF 0.2 × 10/l, on G-CSF 1.20 x 10/l). Treatment increased spleen size (before G-CSF, 47%, on treatment on G-CSF 76%), and splenomegaly was the dose-limiting adverse effect of treatment (pain and early satiety). Clinical observations and records attest to reduce frequency of infectious events and the severity of inflammatory bowel symptoms, but fever and recurrent infections remain a significant problem. In the cohort of patients followed carefully through the Severe Chronic Neutropenia International Registry, four patients have developed myelodysplasia or acute myeloid leukemia and we are aware of four other cases, (altogether seven on G-CSF, one never treated with G-CSF). Liver transplantation in five patients did not correct neutropenia. Four patients had hematopoietic stem cell transplantation; two adults and two children were transplanted; one adult and one child survived. SUMMARY: GSD Ib is a complex disorder of glucose metabolism causing severe chronic neutropenia. G-CSF is effective to raise blood neutrophil counts and reduce fevers and infections in most patients. In conjunction with other therapies (salicylates, mesalamine sulfasalazine and prednisone), G-CSF ameliorates inflammatory bowel symptoms, but doses must be limited because it increases spleen size associated with abdominal pain.

The diversity of mutations and clinical outcomes for ELANE-associated neutropenia.

PURPOSE OF REVIEW: Mutations in the gene for neutrophil elastase, ELANE, cause cyclic neutropenia (CyN) and severe congenital neutropenia (SCN). This study summarized data from the Severe Chronic Neutropenia International Registry (SCNIR) on genotype-phenotype relationships of ELANE mutations to important clinical outcomes. We also summarize findings for ELANE mutations not observed in SCNIR patients. RECENT FINDINGS: There were 307 SCNIR patients with 104 distinctive ELANE mutations who were followed longitudinally for up to 27 years. The ELANE mutations were diverse; there were 65 single amino acid substitutions; 61 of these mutations (94%) were 'probably' or 'possibly damaging' by PolyPhen-2 analysis, and one of the 'benign' mutations was associated with two cases of acute myeloid leukemia (AML). All frame-shift mutations (19/19) were associated with the SCN. The pattern of mutations in the SCN versus CyN was significantly different (P < 10), but some mutations were observed in both groups (overlapping mutations). The cumulative incidence of severe adverse events, that is, myelodysplasia, AML, stem cell transplantation, or deaths was significantly greater for patients with SCN versus those with CyN or overlapping mutations. Specific mutations (i.e. G214R or C151Y) had a high risk for evolution to AML. SUMMARY: Sequencing is useful for predicting outcomes of ELANE-associated neutropenia.

TCIRG1-associated congenital neutropenia.

Severe congenital neutropenia (SCN) is a rare hematopoietic disorder, with estimated incidence of 1 in 200,000 individuals of European descent, many cases of which are inherited in an autosomal dominant pattern. Despite the fact that several causal genes have been identified, the genetic basis for >30% of cases remains unknown. We report a five-generation family segregating a novel single nucleotide variant (SNV) in TCIRG1. There is perfect cosegregation of the SNV with congenital neutropenia in this family; all 11 affected, but none of the unaffected, individuals carry this novel SNV. Western blot analysis show reduced levels of TCIRG1 protein in affected individuals, compared to healthy controls. Two unrelated patients with SCN, identified by independent investigators, are heterozygous for different, rare, highly conserved, coding variants in TCIRG1.

The CXCR4 antagonist plerixafor is a potential therapy for myelokathexis, WHIM syndrome.

Mutations in CXCR4 cause severe leukopenia in myelokathexis or WHIM syndrome. Plerixafor inhibits binding of CXCR4 to its ligand CXCL12. We investigated the effects of plerixafor (0.04 to 0.24 mg/kg) administered at 2-4 day intervals in 6 patients. Outcome measures were the patients' complete blood cell counts, CD34(+) cell counts and lymphocyte subtypes compared with 5 normal subjects similarly treated with plerixafor. All patients showed prompt leukocytosis with maximum blood neutrophils and lymphocytes at 6-12 hours. Blood neutrophils peaked at 6-12 hours, increasing from a mean baseline of 0.4 ± 0.1 × 109/L, to mean peak of 4.5 ± 0.78 × 109/L. Lymphocytes also increased; the greatest increase was in B cells (CD19(+) cells), a > 40-fold increase over baseline at the 0.08 mg/kg dose. None of the patients experienced any significant adverse effects. Plerixafor is a promising therapy for this condition.

A glycoprotein hormone expressed in corticotrophs exhibits unique binding properties on thyroid-stimulating hormone receptor.

Corticotroph-derived glycoprotein hormone (CGH), also referred to as thyrostimulin, is a noncovalent heterodimer of glycoprotein hormone alpha 2 (GPHA2) and glycoprotein hormone beta 5 (GPHB5). Here, we demonstrate that both subunits of CGH are expressed in the corticotroph cells of the human anterior pituitary, as well as in skin, retina, and testis. CGH activates the TSH receptor (TSHR); (125)I-CGH binding to cells expressing TSHR is saturable, specific, and of high affinity. In competition studies, unlabeled CGH is a potent competitor for (125)I-TSH binding, whereas unlabeled TSH does not compete for (125)I-CGH binding. Binding and competition analyses are consistent with the presence of two binding sites on the TSHR transfected baby hamster kidney cells, one that can interact with either TSH or CGH, and another that binds CGH alone. Transgenic overexpression of GPHB5 in mice produces elevations in serum T(4) levels, reductions in body weight, and proptosis. However, neither transgenic overexpression of GPHA2 nor deletion of GPHB5 produces an overt phenotype in mice. In vivo administration of CGH to mice produces a dose-dependent hyperthyroid phenotype including elevation of T(4) and hypertrophy of cells within the inner adrenal cortex. However, the distinctive expression patterns and binding characteristics of CGH suggest that it has endogenous biological roles that are discrete from those of TSH.

Elastase inhibitors as potential therapies for ELANE-associated neutropenia.

Mutations in ELANE, the gene for neutrophil elastase (NE), a protease expressed early in neutrophil development, are the most frequent cause of cyclic (CyN) and severe congenital neutropenia (SCN). We hypothesized that inhibitors of NE, acting either by directly inhibiting enzymatic activity or as chaperones for the mutant protein, might be effective as therapy for CyN and SCN. We investigated ß-lactam-based inhibitors of human NE (Merck Research Laboratories, Kenilworth, NJ, USA), focusing on 1 inhibitor called MK0339, a potent, orally absorbed agent that had been tested in clinical trials and shown to have a favorable safety profile. Because fresh, primary bone marrow cells are rarely available in sufficient quantities for research studies, we used 3 cellular models: patient-derived, induced pluripotent stem cells (iPSCs); HL60 cells transiently expressing mutant NE; and HL60 cells with regulated expression of the mutant enzyme. In all 3 models, the cells expressing the mutant enzyme had reduced survival as measured with annexin V and FACS. Coincubation with the inhibitors, particularly MK0339, promoted cell survival and increased formation of mature neutrophils. These studies suggest that cell-permeable inhibitors of neutrophil elastase show promise as novel therapies for ELANE-associated neutropenia.

Use of granulocyte colony-stimulating factor during pregnancy in women with chronic neutropenia.

OBJECTIVE: To report outcomes associated with the administration of granulocyte colony-stimulating factor (G-CSF) to women with chronic neutropenia during pregnancy. METHODS: We conducted an observational study of women of childbearing potential with congenital, cyclic, idiopathic, or autoimmune neutropenia enrolled in the Severe Chronic Neutropenia International Registry to determine outcomes of pregnancies, without and with chronic G-CSF therapy, 1999-2014. Treatment decisions were made by the patients' personal physicians. A research nurse conducted telephone interviews of all enrolled U.S. women of childbearing potential using a standard questionnaire. Comparisons used Fisher's exact test analysis and Student's t test. RESULTS: One hundred seven women reported 224 pregnancies, 124 without G-CSF therapy and 100 on chronic G-CSF therapy (median dose 1.0 micrograms/kg per day, range 0.02-8.6 micrograms/kg per day). There were no significant differences in adverse events between the groups considering all pregnancies or individual mothers, for example, spontaneous terminations (all pregnancies: no G-CSF in 27/124, G-CSF in 13/100; P=.11, Fisher's exact test), preterm labors (all pregnancies, no G-CSF in 9/124, G-CSF in 2/100, P=.12). A study with at least 300 per group would be needed to detect a difference in these events with 80% statistical power (α=0.05). Four newborns of mothers with idiopathic or autoimmune neutropenia not on G-CSF (4/101) had life-threatening infections, whereas there were no similar events (0/90) in the treated group, but this difference was also not statistically significant (P=.124). Adverse events in the neonates were similar for the two groups. CONCLUSION: This observational study showed no significant adverse effects of administration of G-CSF to women with severe chronic neutropenia during pregnancy. LEVEL OF EVIDENCE: III.