High Insta High Instability Of The CSF3R Gene In Severe Congenital Neutropenia Patients As Judged By Multiple CSF3R Mutations In The Majority Of Patients

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 2264-2264
Author(s):  
Maksim Klimiankou ◽  
Murat Uenalan ◽  
Siarhei Kandabarau ◽  
Lutz Wiehlmann ◽  
Anna-Lena Hagemann ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Conflicts Of Interest ◽  
Cell Clone ◽  
Stop Codon ◽  
Severe Congenital Neutropenia ◽  
Sequencing Data ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia ◽  
Extracellular Region ◽  
Inherited Mutations

Abstract It has been reported by us and others that approx. 30 % of all patients with congenital neutropenia (CN) acquire CSF3R mutations in their life time. More than 80 % of the CN patients who develop myeloid leukemia (CN/AML) harbour CSF3R mutations. This suggests that they are the first hit in leukemogenesis. However, detecting sequence changes e.g. by Sanger sequencing reveals only mutations presented in more than 20 % of the cells due to its technical detection limit. Therefore, we asked whether there is a systematic underestimation of cell clones harbouring CSF3R mutations, which might have been traditionally overlooked. We applied the deep-sequencing technology (SOLID 5500xl) to identify CSF3R mutations in myeloid cells from 158 patients with different types of neutropenia (86 severe congenital neutropenia (CN) patients with known inherited mutations (ELANE, HAX1, G6PC3), 21 cyclic neutropenia (CyN) patients, 28 patients with severe chronic neutropenia with so far unknown inherited mutations, 11 patients with SBDS-associated neutropenia) as well as a group of 12 healthy individuals. All neutropenia patients were treated with G-CSF and notably 21 CN patients developed leukemia or MDS. Deep sequencing data were processed according to our custom NGS pipeline (annotation of sequences and prediction of damaging effects on the coding sequence by Polyphen2, removal of known dbSNP variants, and accepting significant Phred-scores at the variant calling stage). Overall the read numbers ranged between 18 and 128069 (median 716), while only variants with at least two percent of the reads were accepted for further consideration (the statistically significant limit is between one and two percent of all reads). All together, we detected 92 CSF3R mutations in 42 CN patients leading to 49 distinct amino acid exchanges (38 missense and 11 stop-codon mutations). The frequency of the mutant alleles ranged from 2 to 96 %. In contrast, in CyN only five out of 21 patients harbour CSF3R mutations; interestingly, two of them in isoform IV of CSF3R (p.P752T). Most notably, whereas 18 patients displayed only one CSF3R mutation, 24 individuals had more than one CSF3R mutation (2-10 mutations, in total 74 mutations). During follow up of some patients, we could demonstrate that the number of mutations increased over time. The majority of mutations were located in the cytoplasmatic region (aa 651-831) of CSF3R, while 15 patients presented mutations within the extracellular region of CSF3R. Intriguingly, in 16 patients we detected 23 non-sense mutations, where 20 of these are stop-codon mutations affecting glutamine (Q) 768, 770, 776, and 781. This suggests that this part of CSF3R is highly instable. In two patients who did not respond to Filgrastim treatment, we detected a stop codon at aa 546 and 547, respectively, affecting the Fibronectin type-III like part of the CSF3R. Twelve patients who developed leukemia (CN/AML) had more than one CSF3R mutations (two to ten) , whereas eight with CN/AML harbored only one mutation. None of the healthy controls, only three neutropenia patients with unknown inheritance, and only one SBDS patient revealed mutations in CSF3R. Taken together, this data suggests that CSF3R is highly prone to genetic instability in severe congenital neutropenia, because more than one mutation in half of the patients was observed and various CSF3R mutations during the course of life accumulated. Once a cell clone harboring CSF3R mutation obtains a second hit (e.g. RUNX1 mutation), they are prone to undergo leukemic transformation. Disclosures: No relevant conflicts of interest to declare.

Optimization of CSF3R Mutation Detection in Severe Congenital Neutropenia and Cyclic Neutropenia Patients for Routine Diagnostics Using Next Generation Sequencing

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 3685-3685
Author(s):  
Maksim Klimiankou ◽  
Cornelia Zeidler ◽  
Sabine Mellor-Heineke ◽  
Ingeborg Steiert ◽  
Karin Haehnel ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Deep Sequencing ◽  
Mutation Detection ◽  
Low Frequency ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia ◽  
Routine Diagnostics ◽  
Inherited Mutations ◽  
Generation Sequencing

Abstract Severe congenital neutropenia (CN) is a group of bone marrow failure syndromes characterized by absolute neutrophil counts below 0.5x109/L, susceptibility to bacterial infections and frequently associated with maturation arrest at promyelocyte stage in the bone marrow (BM). There is a high incidence of malignant transformation among CN patients with a cumulative rate of MDS/AML 22 % after 15 years of G-CSF treatment. The acquisition of G-CSFR truncating mutations is a risk factor for leukemic transformation in CN patients. Therefore, annual monitoring of CSF3Rmutations by means of next generation sequencing (NGS) is required for identification of CN patients with high risk of MDS/AML development. Since CSF3R mutations usually occur at low frequency without additional clinical features, it is important to carefully select suitable clinical sample type and methods for mutation detection. Next, it remains to be evaluated which CN genetic subgroups should be considered for annual screening of CSF3Rmutations. We performed CSF3R mutational screening in DNA and/or cDNA in 101 patients (ELANE, n = 42; HAX1, n = 16; G6PC3, n = 7; JAGN1, n = 2; WASP, n = 1; digenic ELANE, HAX1, n =1; digenic HAX1 and G6PC3, n =1; inherited mutations in CSF3R, n = 2; genetically unclassified CN, n = 9; cyclic neutropenia (CyN), n = 20) from the European Branch of the Severe Congenital Neutropenia Registry (SCNIR). Using DNA deep sequencing we screened 63 of 81 CN-patients and 20 CyN patients. Using this method, we identified CSF3R mutations in 22.2% (14/63) of CN patients and 10% (2/20) of CyN patients. The frequency of CSF3R mutations in CN patients with known inherited mutations was 20% (11/55): 30 % (3/10) in CN-HAX1 patients, and 22.9 % (8/35) in CN-ELANE patients. Interestingly, 3/8 (37.5 %) patients harbouring CSF3R mutations were observed in genetically unclassified CN. We did not detect any acquired CSF3R mutations in the small groups of CN patients (n=10) harbouring inherited G6PC3, JAGN1, CSF3Ror digenic mutations. In order to increase the sensitivity of mutation detection we performed cDNA deep sequencing of the critical region of G-CSFR. We sequenced 38 CN patients (ELANE, n = 15; HAX1, n = 11; JAGN1, n = 2; G6PC3, n = 2; WASP, n = 1; germline CSF3R, n = 1; genetically unclassified, n = 6). We found 13% (2/15) CN-ELANE, 27% (3/11) CN-HAX1 and 33% (2/6) genetically unclassified CN patients to be positive for acquired mutations in the critical region of G-CSFR. One CN patient with WASP mutation also acquired CSF3R mutation. Based on our sequencing data we would suggest CSF3Rmutation sequencing in all studied groups of patients regardless of mutations in ELANE and HAX1 genes. Intriguingly, 3 out of 5 CN patients with CSF3R mutations detected by cDNA deep sequencing were negative based on results of previous DNA deep sequencing. All of them were found to acquire low frequency CSF3R mutant clones (ELANE pos. patient with 0.3% of p.Q739* clone; genetically unclassified CN patient with 2% of p.Q749*clone; HAX1pos. patient with 0.9% of p.Q749* clone) in cDNA deep sequencing. In 2 patients (one CyN-ELANE and one CN-HAX1) with multiple acquired CSF3R mutations we compared mutant clone enrichment in different cell types (BM MNC; BM PMN; PB MNC and PB PMN) by means of cDNA and DNA deep sequencing. In the CyN-ELANE patient with 2 CSF3R mutant clones, the highest mutant allele frequency (MAF) was detected in the cDNA sample of PB PMN (11% of p.Q749* clone and 0.44% of p.Q739* clone), whereas in the PB MNC cDNA sample clone p.Q749* had only 2.5% MAF and clone P.Q739 was not detectable. Similar to that, in the CN-HAX1 patient the highest MAFs for all 3 CSF3R mutant clones were in PB PMN cDNA and the lowest in PB MNC DNA sample. Frequency of mutated CSF3Rclones in BM PMNs of both patients was comparable to PB PMN samples. Taken together, sequencing of cDNA extracted from peripheral blood or bone marrow PMN samples may provide better results than from MNC in terms of frequency of CSF3R mutation detection in CN and CyN patients. Sequencing of cDNA extracted from BM or PB samples allows enrichment of G-CSFR expressing mutant cells, but due to intrinsic low fidelity of reverse transcriptase the threshold level for positive calls could not be improved significantly (current threshold for candidate calls is 0.2-0.5%). We would suggest CSF3R mutation screening using deep-sequencing of cDNA from peripheral blood PMN in all patient groups (CN and CyN) for routine diagnostics. Disclosures No relevant conflicts of interest to declare.

Novel mechanism of G-CSF refractoriness in patients with severe congenital neutropenia

Blood ◽  
2005 ◽  
Vol 105 (2) ◽  
pp. 584-591 ◽  
Author(s):  
Lawrence J. Druhan ◽  
Jing Ai ◽  
Pam Massullo ◽  
Tamila Kindwall-Keller ◽  
Mark A. Ranalli ◽  
...  
Keyword(s):  
Stop Codon ◽  
Novel Mutation ◽  
Case Reports ◽  
Premature Stop Codon ◽  
Lymphoid Cells ◽  
Common Mechanism ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Extracellular Region ◽  
Single Allele

AbstractSevere congenital neutropenia (SCN) is a rare disease diagnosed at or soon after birth, characterized by a myeloid maturation arrest in the bone marrow, ineffective neutrophil production, and recurrent infections. Most patients respond to treatment with granulocyte colony-stimulating factor (G-CSF), and the majority harbor mutations in the neutrophil elastase gene. In the subset of patients with SCN transforming to acute myeloid leukemia (AML), mutations that truncate the cytoplasmic tail of the G-CSF receptor (G-CSFR) have been detected. Here, we report a novel mutation in the extracellular portion of the G-CSFR within the WSXWS motif in a patient with SCN without AML who was refractory to G-CSF treatment. The mutation affected a single allele and introduced a premature stop codon that deletes the distal extracellular region and the entire transmembrane and cytoplasmic portions of the G-CSFR. Expression of the mutant receptor in either myeloid or lymphoid cells was shown to alter subcellular trafficking of the wild-type (WT) G-CSFR by constitutively heterodimerizing with it. WT/mutant G-CSFR heterodimers appeared to be retained in the endoplasmic reticulum and/or Golgi and accumulate intracellularly. These findings together with 2 previous case reports of extracellular mutations in the G-CSFR in patients with SCN unresponsive to G-CSF suggest a common mechanism underlying G-CSF refractoriness.

Mechanism of the Elevated UPR in CN Patients but Not in CyN Patients Carrying Same ELANE Mutations

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 14-14
Author(s):  
Rainer Nustede ◽  
Inna Kuznetsova ◽  
Karl Welte ◽  
Julia Skokowa
Keyword(s):  
Cell Lines ◽  
Mammalian Cells ◽  
Target Genes ◽  
Conflicts Of Interest ◽  
Myeloid Cells ◽  
Myeloid Cell ◽  
Severe Congenital Neutropenia ◽  
Healthy Individuals ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia

Abstract Abstract 14 Several studies found that in patients with severe congenital neutropenia (CN) harboring mutations in the ELANE gene mutated NE protein induced unfolded protein response (UPR) leading to elevated apoptosis and diminished differentiation of myeloid cells. However, it is unclear, why UPR was not detected in patients with cyclic neutropenia (CyN) carrying the same ELANE mutations, which have been found in CN patients. Several UPR components have been identified in mammalian cells, which include three transducers (IRE1, PERK, and activating transcription factor 6 (ATF-6) as well as one master regulator (BiP/GRP78). BiP is known to be regulated by ATF6. The activation of ATF6 and its target genes (GADD34, CHOP and BiP) in CN patients has not been studied yet. We were able to detect significantly elevated levels of ATF6 and BiP in myeloid cells of CN patients with ELANE mutations, in comparison to CyN patients and to healthy individuals. Therefore, we investigated the mechanism of UPR and activation of ATF6 and ATF6 target genes in CN patients in comparison to CyN patients. We transduced the myeloid cell lines HL60 and NB4 with lentiviral constructs contained either wild type (WT) ELANE cDNA, or mutated (MUT) ELANE cDNA and measured mRNA and protein expression of ATF6 as well as mRNA expression of ATF6 target genes. We compared the effects of three ELANE mutations: C42R, V145-C152del (both mutations presented in CN patients, but not in CyN patients) and S97L (typical for CN and CyN patients) with WT ELANE. We found that in both cell lines only C42R ELANE MUT, but not V145-C152del ELANE MUT or S97L ELANE MUT induced expression of ATF6, GADD34, CHOP and BiP, as compared to control transduced cells. Furthermore, we hypothesize that degradation of mutated NE protein by Secretory Leukocyte Protease Inhibitor (SLPI) might be involved in UPR induction. However, we detected only very low levels of SLPI mRNA in CD33+ myeloid cells and in PMNs of patients with severe congenital neutropenia (CN), as compared to patients with cyclic neutropenia (CyN) and to healthy individuals. The lack of the NE inhibitor, SLPI in CN patients may further contribute to elevated UPR triggered by ELANE MUT and normal levels of SLPI in CyN patients might protect from ELANE MUT-induced UPR. Indeed, inhibition of SLPI using SLPI-specific shRNA led to a significantly elevated expression levels of ATF6, GADD34 and BIP, as compared to ctrl shRNA transduced cells. More importantly, co-transduction of NB4 cells with SLPI shRNA in combination with ELANE S97L MUT (which is common for both CN and CyN patients), but not with WT ELANE led to elevated levels of ATF6, GADD34 and BIP. In summary, different ELANE mutations have different effects on UPR as judged by ATF6 activation and the level of ELANE-triggered UPR is regulated by SLPI. Disclosures: No relevant conflicts of interest to declare.

Stop-Codon Mutation In The Extracellular Part Of G-CSFR Leading To G-CSF Unresponsiveness Due To Defects In Erk1/2 Phopsphorylation In a Patient With Severe Congenital Neutropenia

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3461-3461
Author(s):  
Olga Klimenkova ◽  
Cornelia Zeidler ◽  
Julia Skokowa ◽  
Karl Welte
Keyword(s):  
Bone Marrow ◽  
Intracellular Signaling ◽  
Conflicts Of Interest ◽  
Stop Codon ◽  
Novel Mutation ◽  
Myeloid Cells ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Stop Codon Mutation ◽  
Codon Mutation

Abstract We identified a 10 year old female severe congenital neutropnia patient who did not respond to the treatment with G-CSF (Filgrastim) up to 100 µg/kg/day, s.c. By search of possible reasons of G-CSF unresponsiveness, we detected by deep sequencing of the CSF3R (SOLID 5500xl, 3125 reads) a novel mutation within the extracellular part of CSF3R. The mutation was also confirmed by convential Sanger sequencing. It is a heterozygous stop-codon mutation C1641T (NM_000760), p.W547* (NP_000751) which is localized proximal to the cell membrane in the fifth fibronectin type III domain of the CSF3R. This is a first report describing a stop-codon mutation in the extracellular part of CSF3R in severe congenital neutropenia. FACS analysis revealed severely diminished surface expression levels of G-CSFR on CD33+ bone marrow myeloid progenitor cells of this patient as compared to G-CSFR levels on CD33+ cells of healthy individuals. In line with reduced expression of G-CSFR, we found disturbed G-CSFR-triggered intracellular signaling pathways activated by G-CSFR. MAPK/Erk1/2 activation by phosphorylation is known to be crucial for G-CSF- induced myeloid differentiation and STAT5 signaling is important for proliferation and survival of myeloid cells upon stimulation with G-CSF. We demonstrated abrogated phosphorylation of ERK1/2 and of STAT5 in bone marrow CD33+ myeloid cells of CN patient treated with G-CSF in vitro for 2, 5, 10 and 15 minutes, comparing with healthy donor. Since this patient has no known inherited CN-associated mutations (e.g. ELANE, HAX1, G6PC3), it may represent a new subgroup of CN patients characterized by non-responsiveness to G-CSF due to lack of binding of G-CSF to its receptor. Since the majority of investigations on CSF3R mutations are targeting the cytoplasmic part of the receptor only, we suggest that CN patients who do not respond to G-CSF treatment should be tested also for mutations within the extracellular part of the CSF3R. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Ultra-Sensitive CSF3R Deep Sequencing in Patients With Severe Congenital Neutropenia

2019 ◽  
Vol 10 ◽  
Author(s):  
Maksim Klimiankou ◽  
Murat Uenalan ◽  
Siarhei Kandabarau ◽  
Rainer Nustede ◽  
Ingeborg Steiert ◽  
...  
Keyword(s):  
Deep Sequencing ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia

Deficiency Of JAGN1 Causes Severe Congenital Neutropenia Associated With Defective Secretory Pathway and Aberrant Myeloid Cell Homeostasis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 439-439
Author(s):  
Kaan Boztug ◽  
Päivi M Järvinen ◽  
Elisabeth Salzer ◽  
Tomas Racek ◽  
Sebastian Mönch ◽  
...  
Keyword(s):  
Secretory Pathway ◽  
Conflicts Of Interest ◽  
Snp Array ◽  
Neutrophil Granulocytes ◽  
Homozygous Mutation ◽  
Genetic Linkage Analysis ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Gene Encoding ◽  
Consistent Finding

Abstract 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.

Defective Expression of LEF-1 Transcription Factor mRNA and - Protein in Patients with Severe Congenital Neutropenia (Kostmann’S Syndrome).

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 782-782
Author(s):  
Julia Skokowa ◽  
Gunnar Cario ◽  
Zheng Wang ◽  
Cornelia Zeidler ◽  
Martin Stanulla ◽  
...  
Keyword(s):  
Protein Expression ◽  
Mrna Expression ◽  
Progenitor Cells ◽  
Healthy Controls ◽  
Severe Congenital Neutropenia ◽  
Healthy Individuals ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia ◽  
Myeloid Progenitor ◽  
Myeloid Progenitor Cells

Abstract Severe congenital neutropenia (SCN) is characterized by “maturation arrest” of myeloid progenitor cells at the promyelocytic/myelocytic stage with the absence or only few mature neutrophils in the bone marrow and peripheral blood. Significant progress in the treatment of SCN patients has been achieved in the last 15 years by administration of granulocyte colony-stimulating factor (G-CSF), which significantly increases the number of neutrophils leading to an improvement of the quality of life. To date, the pathophysiology and underlying genetic defect in patients with congenital neutropenia is still under investigation. Wnt signalling pathway orchestrates a number of cellular programs such as proliferation, differentiation and cell fate determination in many tissues. In the present study we investigated the mRNA and protein expression patterns of Wnt signalling peptides, such as the High Mobility Group (HMG) box containing transcription factors such as lymphoid enhancer factor-1 (LEF-1) and T cell factors (TCFs), as well as β-catenin in CD33+ bone marrow myeloid progenitor cells from SCN patients (n = 6) in comparison to those of patients with cyclic neutropenia (n = 4) and G-CSF-treated healthy controls (n = 3). All SCN and cyclic neutropenia patients are under G-CSF therapy. mRNA expression of genes of interest was measured by quantitative real-time PCR. Protein expression was assessed by immunofluorescence staining, visualized and recorded by confocal microscopy. We found that CD33+ cells from patients with SCN exhibited 20 times lower or even absent expression of LEF-1 mRNA and protein, as compared to healthy G-CSF treated controls (mRNA expression ratio: SCN patients 0.83 ± 0.38 AU vs. healthy controls: 15.1 ± 0.4 AU; p < 0.0001). Intriguingly, LEF-1 mRNA expression levels on CD33+ cells from cyclic neutropenia patients were comparable to those of healthy controls. Immunostaining with anti-LEF-1 polyclonal antibody (kindly provided by Dr. R. Grosschedl) and confocal microscopy analysis revealed that LEF-1 protein was detectable at the expected level in CD33+ cells from healthy G-CSF treated controls. In patients with cyclic neutropenia LEF-1 protein expression in myeloid progenitor cells was comparable to healthy individuals. In contrast, in CD33+ cells from patients with SCN, LEF-1 protein was not detectable. mRNA expression of other TCFs: TCF-1, TCF-3, TCF-4 in SCN was not significantly different from healthy individuals. However, the expression level of LEF-1 binding partner in the Wnt pathway, β-catenin, was increased in SCN patients (SCN patients: 224.7 ± 42.4 AU vs. healthy controls: 107.2 ± 7.3 AU, p = 0.052). The defect in LEF-1 expression in SCN patients was further substantiated by the fact that mRNA expression of LEF-1 target genes such as c-myc, cyclin D1, survivin and neutrophil elastase were also significantly downregulated. In conclusion, our results suggest that defective LEF-1 expression might have an impact on the pathogenesis of SCN. In addition, it may help to distinguish SCN from cyclic neutropenia patients.

Incidence of SCN-Assoicated Gene Mutations in Severe Congenital Neutropenia Patients in North America

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 3553-3553
Author(s):  
Jun Xia ◽  
Audrey Anna Bolyard ◽  
Elin Rodger ◽  
Steven Stein ◽  
Andrew AG Aprikyan ◽  
...  
Keyword(s):  
North America ◽  
North American ◽  
Barth Syndrome ◽  
North American Population ◽  
Compound Heterozygous ◽  
Severe Congenital Neutropenia ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia ◽  
The North ◽  
Two Samples

Abstract Severe congenital neutropenia is a genetically heterogeneous syndrome associated with mutations in several different genes including ELA2, HAX1, GFI1, WAS, and CSF3R. The goal of this study was to define the mutation frequency of these genes in the North American SCN patient population. We also sequenced SBDS, since mutations of SBDS have been associated with congenital and acquired neutropenia. A total of 159 patients were identified in the North American Severe Chronic Neutropenia International Registry (SCNIR) for whom informed consent and genomic DNA samples adequate for sequencing were available. To accommodate our semi-automated high-throughput sequencing pipeline, 94 samples were chosen for sequencing. Since ELA2 sequencing had already been performed in most cases, preference was given to those samples without known ELA2 mutation. Among the samples, 73 were from patients with SCN, 4 with cyclic neutropenia, 10 with idiopathic neutropenia, 2 with Shwachman-Diamond Syndrome (SDS), and 3 with Barth syndrome. Two samples were excluded because of poor sequence quality. Singleton cases with validated mutations of GFI1 (N382S) and WAS (L270P) were observed. The N382S GFI1 mutation was associated with striking monocytosis. A novel nonsense mutation of GFI1 (R412X) was detected in one additional case. As expected, compound heterozygous mutations of SBDS were present in the two cases of SDS. In addition, heterozygous mutations of SBDS (84Cfs3X and Q94X) were observed in two cases of SCN. Typical truncation mutations of CSF3R were detected in 4 cases, all developing MDS or AML. Surprisingly, no mutations of HAX1 were detected. Considering only patients with a diagnosis of SCN who were from North America (125 of the total 159 cases), the incidence of ELA2 mutations was 68%. Eleven novel ELA2 mutations were identified. In 28.8% of cases, no mutation of any gene were detected. Based on these data, we recommend that ELA2 genotyping be performed in all patients with suspected SCN. In the North American population mutations in HAX1, GFI1, SBDS, and WAS are rare and routine genotyping is not indicated. Finally, the data suggest that there are yet undiscovered genetic causes of SCN.

PREGNANCY OUTCOME In GENETIC Subtypes of CONGENITAL Neutropenia

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4722-4722
Author(s):  
Cornelia Zeidler ◽  
Beate Brand ◽  
Ulrike A.H. Grote ◽  
Anna Nickel ◽  
Karl H. Welte
Keyword(s):  
Bacterial Infections ◽  
Conflicts Of Interest ◽  
Clinical Phenotype ◽  
Infectious Complications ◽  
Current Data ◽  
Congenital Neutropenia ◽  
Cyclic Neutropenia ◽  
Live Births ◽  
Genetic Subtypes ◽  
Chronic Neutropenia

Abstract Abstract 4722 Severe congenital neutropenia (CN) comprises a heterogeneous group of disorders with a common hematological and clinical phenotype characterized by a maturation arrest of myelopoiesis at the level of the promyelocyte / myelocyte stage with peripheral blood absolute neutrophil counts (ANC) below 0.5 ′ 109/l and early onset of bacterial infections. Current data on the molecular causes have demonstrated that CN is a multigene disorder with more than 10 genes described to date. Genetic analyses in autosomal dominant and sporadic cases of CN indicate that the majority of these cases are attributable to mutations in the elastase 2 (ELANE) gene encoding neutrophil elastase. However, mutations in the ELANE gene do not discriminate between patients with CN and patients with cyclic neutropenia (CyN). Since 1987, recombinant human Granulocyte-Colony stimulating factor (G-CSF) is available for the treatment of CN. Independent of the genetic subtype, more than 90% of patients respond well to G-CSF with sustained increase of absolute neutrophil counts and prolonged life expectancy. Since our first patients have reached adulthood the desire for parenthood arises. To-date there is only limited data on the infectious risk for affected mothers and their children due to G-CSF treatment during pregnancy. In this study we assessed the outcome of pregnancies reported to the SCNIR in Europe since 1994 with regard to:The neutropenia status in newborns of mothers and fathers with different genetic CN subtypes as an indicator for inheritance.The impact of G-CSF treatment on maternal and newborn complications in women of all neutropenia subtypes with or without G-CSF treatment during pregnancy. Since 1994 the SCNIR has collected long-term follow-up data of 510 patients with severe chronic neutropenia subtypes. 3 patients are diagnosed with congenital (71 ELANE-CN, 31 HAX1, 9 GC6PC3, 47 SDS, 117 unknown, 45 other), 66 with cyclic and 82 with idiopathic neutropenia. Adulthood was reached by 144 out of 304 CN patients. These include 38 ELANE-CN patients (male:14, female:24) and 11 ELA-CyN patients (male: 5, female:6). A total of 20 pregnancies in 12 mothers and 13 newborns by 7 fathers with different genetic subtypes of CN have been reported. Among them are pregnancies of 11 women with ELANE-CN, 8 with ELANE-CyN, 1 with SDS, 13 with an unknown genetic origin of CN (n=9) or CyN (n=4). No pregnancies were reported in patients with HAX1 or G6PC3 although 7 of these patients have reached adulthood. Data on neutropenia status was documented in 24 out of 31 live births. Neutropenia in newborns was diagnosed in 16 out of 30 live births from parents with genetic subtypes of CN. In 8 of the 16 affected newborns neutropenia was related to ELANE mutations. One mother registered with SDS delivered a healthy child. During pregnancy 17 women received G-CSF treatment (CN=11, CyN=4, IDN=2). Regardless of any cytokine treatment no major infectious complications were reported in our cohort. 24 of 31 reported pregnancies resulted in life births. 5 spontaneous terminations occurred in women with respectively without exposure to G-CSF. In addition, 2 still births were reported in women with idiopathic neutropenia, but G-CSF exposure remains unknown. Conclusion: The proportion of newborns with congenital neutropenia indicates the pattern of inheritance by their parents and reveals the need for genetic counseling. However, the acceptance of having affected children may reflect the high quality of life due to G-CSF treatment in affected parents. G-CSF treatment during pregnancy is well tolerated. In terms of G-CSF treatment, no differences in infectious complications during pregnancy in women with or without G-CSF administration were reported in our cohort. Interestingly, the proportion of women receiving G-CSF during pregnancy is highest among the CN subtype indicating the severe clinical phenotype. We therefore recommend the application of G-CSF in patients with severe chronic neutropenia during pregnancy. Disclosures: No relevant conflicts of interest to declare.

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