Elodie Lainey

Proper desensitization of CXCR4 is required for lymphocyte development and peripheral compartmentalization in mice

Desensitization controls G protein-dependent signaling of chemokine receptors. We investigate the physiologic implication of this process for CXCR4 in a mouse model harboring a heterozygous mutation of the Cxcr4 gene, which engenders a desensitization-resistant receptor. Such anomaly is linked to the warts, hypogammaglobulinemia, infections, myelokathexis (WHIM) syndrome, a human rare combined immunodeficiency. Cxcr4(+/mutant(1013)) mice display leukocytes with enhanced responses to Cxcl12 and exhibit leukopenia as reported in patients. Treatment with CXCL12/CXCR4 antagonists transiently reverses blood anomalies, further demonstrating the causal role of the mutant receptor in the leukopenia. Strikingly, neutropenia occurs in a context of normal bone marrow architecture and granulocyte lineage maturation, indicating a minor role for Cxcr4-dependent signaling in those processes. In contrast, Cxcr4(+/1013) mice show defective thymopoiesis and B-cell development, accounting for circulating lymphopenia. Concomitantly, mature T and B cells are abnormally compartmentalized in the periphery, with a reduction of primary follicles in the spleen and their absence in lymph nodes mirrored by an unfurling of the T-cell zone. These mice provide a model to decipher the role of CXCR4 desensitization in the homeostasis of B and T cells and to investigate which manifestations of patients with WHIM syndrome may be overcome by dampening the gain of CXCR4 function.

Juvenile myelomonocytic leukemia displays mutations in components of the RAS pathway and the PRC2 network

Juvenile myelomonocytic leukemia (JMML) is a rare and severe myelodysplastic and myeloproliferative neoplasm of early childhood initiated by germline or somatic RAS-activating mutations. Genetic profiling and whole-exome sequencing of a large JMML cohort (118 and 30 cases, respectively) uncovered additional genetic abnormalities in 56 cases (47%). Somatic events were rare (0.38 events/Mb/case) and restricted to sporadic (49/78; 63%) or neurofibromatosis type 1 (NF1)-associated (8/8; 100%) JMML cases. Multiple concomitant genetic hits targeting the RAS pathway were identified in 13 of 78 cases (17%), disproving the concept of mutually exclusive RAS pathway mutations and defining new pathways activated in JMML involving phosphoinositide 3-kinase (PI3K) and the mTORC2 complex through RAC2 mutation. Furthermore, this study highlights PRC2 loss (26/78; 33% of sporadic JMML cases) that switches the methylation/acetylation status of lysine 27 of histone H3 in JMML cases with altered RAS and PRC2 pathways. Finally, the association between JMML outcome and mutational profile suggests a dose-dependent effect for RAS pathway activation, distinguishing very aggressive JMML rapidly progressing to acute myeloid leukemia.

Additional erythrocytic and reticulocytic parameters helpful for diagnosis of hereditary spherocytosis: results of a multicentre study.

Hereditary spherocytosis (HS) is characterised by weakened vertical linkages between the membrane skeleton and the red blood cell’s lipid bilayer, leading to the release of microparticles. All the reference tests suffer from specific limitations. The aim of this study was to develop easy to use diagnostic tool for screening of hereditary spherocytosis based on routinely acquired haematological parameters like percentage of microcytes, percentage of hypochromic cells, reticulocyte counts, and percentage of immature reticulocytes. The levels of haemoglobin, mean cell volume, mean corpuscular haemoglobin concentration, reticulocytes (Ret), immature reticulocytes fraction (IRF), hypochromic erythrocytes (Hypo-He) and microcytic erythrocytes (MicroR) were determined on EDTA samples on Sysmex instruments from a cohort of 45 confirmed SH. The HS group was then compared with haemolytical disorders, microcytic anaemia, healthy individuals and routine samples (n = 1,488). HS is characterised by a high Ret count without an equally elevated IRF. All 45 HS have Ret >80,000/μl and Ret(109/L)/IRF (%) greater than 7.7 (rule 1). Trait and mild HS had a Ret/IRF ratio greater than 19. Moderate and severe HS had increased MicroR and MicroR/Hypo-He (rule 2). Combination of both rules gave predictive positive value and negative predictive value of respectively 75% and 100% (n = 1,488), which is much greater than single parameters or existing rules. This simple and fast diagnostic method could be used as an excellent screening tool for HS. It is also valid for mild HS, neonates and ABO incompatibilities and overcomes the lack of sensitivity of electrophoresis in ankyrin deficiencies.

A landscape of germline mutations in a cohort of inherited bone marrow failure patients

Bone marrow (BM) failure (BMF) in children and young adults is often suspected to be inherited, but in many cases diagnosis remains uncertain. We studied a cohort of 179 patients (from 173 families) with BMF of suspected inherited origin but unresolved diagnosis after medical evaluation and Fanconi anemia exclusion. All patients had cytopenias, and 12.0% presented ≥5% BM blast cells. Median age at genetic evaluation was 11 years; 20.7% of patients were aged ≤2 years and 36.9% were ≥18 years. We analyzed genomic DNA from skin fibroblasts using whole-exome sequencing, and were able to assign a causal or likely causal germ line mutation in 86 patients (48.0%), involving a total of 28 genes. These included genes in familial hematopoietic disorders (GATA2, RUNX1), telomeropathies (TERC, TERT, RTEL1), ribosome disorders (SBDS, DNAJC21, RPL5), and DNA repair deficiency (LIG4). Many patients had an atypical presentation, and the mutated gene was often not clinically suspected. We also found mutations in genes seldom reported in inherited BMF (IBMF), such as SAMD9 and SAMD9L (N = 16 of the 86 patients, 18.6%), MECOM/EVI1 (N = 6, 7.0%), and ERCC6L2 (N = 7, 8.1%), each of which was associated with a distinct natural history; SAMD9 and SAMD9L patients often experienced transient aplasia and monosomy 7, whereas MECOM patients presented early-onset severe aplastic anemia, and ERCC6L2 patients, mild pancytopenia with myelodysplasia. This study broadens the molecular and clinical portrait of IBMF syndromes and sheds light on newly recognized disease entities. Using a high-throughput sequencing screen to implement precision medicine at diagnosis can improve patient management and family counseling.

ORIGINAL ARTICLE: Evaluation of the CellaVision™ DM automated microscope in pediatrics

The DM is an automated microscope, which performs WBC differential counts and monitors red cell morphology. The user either validates the cell recognition if the DM has correctly identified the WBCs or reclassifies the WBCs in the good category in case of a DM mis‐assignment. Morphological anomalies of leukocytes, red blood cells or platelets are analyzed and registered. We studied 521 newborns and infants sorted by age and pathology. The results correlated well with those using conventional microscopy except for samples containing blasts, in which the percentage of malignant cells was underestimated. Newborns had the lowest rates of overall accuracy and postclassification agreement. For red cell analysis, 10% of the selected areas were considered unreadable. However, the DM diagnosed faithfully all studied red cell pathologies. The DM was also very useful in analyzing samples of storage diseases. Timesavings ranging from 1 up to 10 min (for vacuolated lymphocyte screening) were observed when performing analysis with the DM. The DM represents a useful diagnostic and training tool. However, conventional microscopy remains essential, in particular when the image quality is poor, such as in the case of lymphoblasts, and in the screening of platelet clusters.

Hémogramme en pédiatrie : variations physiologiques

Resume Les donnees de l’hemogramme se modifient profondement au cours des premieres annees de vie et sont le reflet des differents stades du developpement. Nous detaillerons successivement les parametres pre-analytiques influencant l’interpretation des resultats (faible volume disponible, hyperviscosite, site de prelevement,…), les caracteristiques de l’hematopoiese du fœtus et du nouveau-ne et les normes de la numeration globulaire et de la formule leucocytaire en fonction de l’âge. Les particularites morphologiques en periode neonatale seront egalement abordees dans cet article. La connaissance de ces variations physiologiques est en effet un pre-requis a l’identification de situations pathologiques en pediatrie.

Coordinated epigenetic regulation of autophagy and apoptosis

Comment on: Cluzeau T, et al. Cell Cycle 2011; 10; 2339-43.

Vacuolation of neutrophils and acanthocytosis in child with medium chain acyl‐CoA dehydrogenase deficiency

A three-day-old female neonate was hospitalized for emesis and lethargy. On admission she was comatose, hypotonic and dehydrated. She was found to have an increased white cell count (25 · 10/l), mild hypoglycaemia (glucose 2Æ3 mmol/l), ventilatory alkalosis and hyperammonaemia (196 lmol/l). The presence of dicarboxylic acids in urine samples and of abnormal peaks of medium chain acylcarnitines in blood, were indicative of a medium chain acyl-CoA dehydrogenase deficiency. This diagnosis was confirmed by enzymatic assay in lymphocytes and by molecular analysis. Peculiar and transient haematological features were first noted on the evaluation performed before starting specific treatment with high glucose provision and l-carnitine supplementation. On light microscopy, the peripheral blood film showed prominent neutrophils; almost all showed cytoplasmic vacuolation, as did the neutrophil precursors. Septicaemia and inflammatory disease, the most common causes of neutrophil vacuolation, were excluded (absence of toxic granulation and Döhle bodies, Creactive protein <10 mg/l). The neutral lipid content of these vacuoles was confirmed by their staining with Oil-Red-O. In addition, an abnormally high percentage of acanthocytes (nearly 50%) was present. Characteristically, most of the red cells had spicules, which varied in size and number. The acanthocytosis in this condition is thought to be a result of abnormal repartition of cholesterol and phospholipids in the membrane of erythrocytes, resulting in structural change and reduced fluidity of the membrane. Vacuolation of neutrophils in an appropriate clinical context is suggestive of this condition but is not uniformly present. Medium chain acyl-CoA dehydrogenase deficiency is an autosomal recessive disorder that is responsible for fasting hypoketotic hypoglycaemia. If undiagnosed, this condition can be responsible for unexpected death, while rapid diagnosis and treatment are sufficient to maintain health and good quality of life. The frequency of this highly treatable disorder has lead many countries to organize a neonatal screening programme.

Down-syndrome-like acute megakaryoblastic leukemia in a patient with Cornelia de Lange syndrome

Cornelia de Lange Syndrome (CdLS) is a rare autosomal dominant developmental disorder characterized by a distinctive facial dysmorphism and variable developmental anomalies including prenatal and postnatal growth delay, microcephaly, intellectual disability, upper limb anomalies and hirsutism. CdLS

Pneumocystosis revealing immunodeficiency secondary to TERC mutation

Telomerase-related gene (TRG) mutations are evidenced in about 25% of patients with familial pulmonary fibrosis, and less frequently in sporadic interstitial lung disease (ILD) [1]. Even though TRG mutations can be associated with haematological, hepatic and cutaneous manifestations, most adult ILD patients with TRG mutations only present asymptomatic extrapulmonary involvement such as thrombocytopenia or premature hair greying [2]. However, some specific extrapulmonary manifestations such as immunodeficiency or portal hypertension can be severe and need early detection. We report herein the case of a patient with primary immunodeficiency revealed by pneumocystosis, which could have been misdiagnosed as an idiopathic acute exacerbation of ILD. TERC mutation may be associated with a primary immunodeficiency http://ow.ly/zY7b30fWbD9