A Patient with Kabuki Syndrome Mutation Presenting with Very Severe Aplastic Anemia.

Kabuki syndrome (KS) is a rare congenital disorder commonly complicated by humoral immunodeficiency. Patients with KS present with mutation in the histone-lysine N-methyltransferase 2D (KMT2D) gene. Although various KMT2D mutations are often identified in lymphoma and leukemia, those encountered in aplastic anemia (AA) are limited. Herein, we present the case of a 45-year-old Japanese man who developed severe pancytopenia and hypogammaglobulinemia. He did not present with any evident malformations, intellectual disability, or detectable levels of autoantibodies. However, B-cell development was impaired. Therefore, a diagnosis of very severe AA due to a hypoplastic marrow, which did not respond to granulocyte colony-stimulating factor, was made. The patient received umbilical cord blood transplantation but died from a Pseudomonas infection before neutrophil engraftment. Trio whole-exome sequencing revealed a novel missense heterozygous mutation c.15959G >A (p.R5320H) in exon 50 of the KMT2D gene. Moreover, Sanger sequencing of peripheral blood and bone marrow mononuclear cells and a skin biopsy specimen obtained from this patient identified this heterozygous mutation, suggesting that de novo mutation associated with KS occurred in the early embryonic development. Our case showed a novel association between KS mutation and adult-onset AA.

The KMT2D Kabuki syndrome histone methylase controls neural crest cell differentiation and facial morphology.

Kabuki syndrome (KS) is a congenital craniofacial disorder resulting from mutations in the KMT2D histone methylase (KS1) or the UTX histone demethylase (KS2). With small cohorts of KS2 patients, it is not clear whether differences exist in clinical manifestations relative to KS1. We mutated KMT2D in neural crest cells (NCCs) to study cellular and molecular functions in craniofacial development with respect to UTX. Similar to UTX, KMT2D NCC knockout mice demonstrate hypoplasia with reductions in frontonasal bone lengths. We have traced the onset of KMT2D and UTX mutant NCC frontal dysfunction to a stage of altered osteochondral progenitor differentiation. KMT2D NCC loss-of-function does exhibit unique phenotypes distinct from UTX mutation, including fully penetrant cleft palate, mandible hypoplasia and deficits in cranial base ossification. KMT2D mutant NCCs lead to defective secondary palatal shelf elevation with reduced expression of extracellular matrix components. KMT2D mutant chondrocytes in the cranial base fail to properly differentiate, leading to defective endochondral ossification. We conclude that KMT2D is required for appropriate cranial NCC differentiation and KMT2D-specific phenotypes may underlie differences between Kabuki syndrome subtypes.

The histone methyltransferase KMT2D, mutated in Kabuki syndrome patients, is required for neural crest cell formation and migration.

Kabuki syndrome is an autosomal dominant developmental disorder with high similarities to CHARGE syndrome. It is characterized by a typical facial gestalt in combination with short stature, intellectual disability, skeletal findings and additional features like cardiac and urogenital malformations, cleft palate, hearing loss and ophthalmological anomalies. The major cause of Kabuki syndrome are mutations in KMT2D, a gene encoding a histone H3 lysine 4 (H3K4) methyltransferase belonging to the group of chromatin modifiers. Here we provide evidence that Kabuki syndrome is a neurocrestopathy, by showing that Kmt2d loss-of-function inhibits specific steps of neural crest (NC) development. Using the Xenopus model system, we find that Kmt2d loss-of-function recapitulates major features of Kabuki syndrome including severe craniofacial malformations. A detailed marker analysis revealed defects in NC formation as well as migration. Transplantation experiments confirm that Kmt2d function is required in NC cells. Furthermore, analyzing in vivo and in vitro NC migration behavior demonstrates that Kmt2d is necessary for cell dispersion but not protrusion formation of migrating NC cells. Importantly, Kmt2d knockdown correlates with a decrease in H3K4 monomethylation and H3K27 acetylation supporting a role of Kmt2d in the transcriptional activation of target genes. Consistently, using a candidate approach, we find that Kmt2d loss-of-function inhibits Xenopus Sema3F expression, and overexpression of Sema3F can partially rescue Kmt2d loss-of-function defects. Taken together, our data reveal novel functions of Kmt2d in multiple steps of NC development and support the hypothesis that major features of Kabuki syndrome are caused by defects in NC development.

Nuclear phospholipase C isoenzyme imbalance leads to pathologies in brain, hematologic, neuromuscular, and fertility disorders.

Phosphoinositide-specific phospholipases C (PI-PLCs) are involved in signaling pathways related to critical cellular functions, such as cell cycle regulation, cell differentiation, and gene expression. Nuclear PI-PLCs have been studied as key enzymes, molecular targets, and clinical prognostic/diagnostic factors in many physiopathologic processes. Here, we summarize the main studies about nuclear PI-PLCs, specifically, the imbalance of isozymes such as PI-PLCß1 and PI-PLCζ, in cerebral, hematologic, neuromuscular, and fertility disorders. PI-PLCß1 and PI-PLCÉ£1 affect epilepsy, depression, and bipolar disorder. In the brain, PI-PLCß1 is involved in endocannabinoid neuronal excitability and is a potentially novel signature gene for subtypes of high-grade glioma. An altered quality or quantity of PI-PLCζ contributes to sperm defects that result in infertility, and PI-PLCß1 aberrant inositide signaling contributes to both hematologic and degenerative muscle diseases. Understanding the mechanisms behind PI-PLC involvement in human pathologies may help identify new strategies for personalized therapies of these conditions.

Dissecting KMT2D missense mutations in Kabuki syndrome patients.

Kabuki syndrome is a rare autosomal dominant condition characterized by facial features, various organs malformations, postnatal growth deficiency and intellectual disability. The discovery of frequent germline mutations in the histone methyltransferase KMT2D and the demethylase KDM6A revealed a causative role for histone modifiers in this disease. However, the role of missense mutations has remained unexplored. Here, we expanded the mutation spectrum of KMT2D and KDM6A in KS by identifying 37 new KMT2D sequence variants. Moreover, we functionally dissected 14 KMT2D missense variants, by investigating their impact on the protein enzymatic activity and the binding to members of the WRAD complex. We demonstrate impaired H3K4 methyltransferase activity in 9 of the 14 mutant alleles and show that this reduced activity is due in part to disruption of protein complex formation. These findings have relevant implications for diagnostic and counseling purposes in this disease.

Telomerase and telomere biology in hematological diseases: A new therapeutic target.

Telomeres are structures confined at the ends of eukaryotic chromosomes. With each cell division, telomeric repeats are lost because DNA polymerases are incapable to fully duplicate the very ends of linear chromosomes. Loss of repeats causes cell senescence, and apoptosis. Telomerase neutralizes loss of telomeric sequences by adding telomere repeats at the 3' telomeric overhang. Telomere biology is frequently associated with human cancer and dysfunctional telomeres have been proved to participate to genetic instability. This review covers the information on telomerase expression and genetic alterations in the most relevant types of hematological diseases. Telomere erosion hampers the capability of hematopoietic stem cells to effectively replicate, clinically resulting in bone marrow failure. Furthermore, telomerase mutations are genetic risk factors for the occurrence of some hematologic cancers. New discoveries in telomere structure and telomerase functions have led to an increasing interest in targeting telomeres and telomerase in anti-cancer therapy.

Cytochrome P450 Oxidoreductase Influences CYP2B6 Activity in Cyclophosphamide Bioactivation.

INTRODUCTION: Cyclophosphamide is commonly used as an important component in conditioning prior to hematopoietic stem cell transplantation, a curative treatment for several hematological diseases. Cyclophosphamide is a prodrug activated mainly by cytochrome P450 2B6 (CYP2B6) in the liver. A high degree of inter- and intra-individual variation in cyclophosphamide kinetics has been reported in several studies. MATERIALS AND METHODS: Hydroxylation of cyclophosphamide was investigated in vitro using three microsomal batches of CYP2B6*1 with different ratios of POR/CYP expression levels. Twenty patients undergoing hematopoietic stem cell transplantation were also included in the study. All patients received an i.v. infusion of cyclophosphamide (60 mg/kg/day, for two days) as a part of their conditioning. Blood samples were collected from each patient before cyclophosphamide infusion, 6 h after the first dose and before and 6 h after the second dose. POR gene expression was measured by mRNA analysis and the pharmacokinetics of cyclophosphamide and its active metabolite were determined. RESULTS: A strong correlation between the in vitro intrinsic clearance of cyclophosphamide and the POR/CYP ratio was found. The apparent Km for CYP2B6.1 was almost constant (3-4 mM), while the CLint values were proportional to the POR/CYP ratio (3-34 µL/min/nmol CYP). In patients, the average expression of the POR gene in blood was significantly (P <0.001) up-regulated after cyclophosphamide infusion, with high inter-individual variations and significant correlation with the concentration ratio of the active metabolite 4-hydroxy-cyclophosphamide/cyclophosphamide. Nine patients were carriers for POR*28; four patients had relatively high POR expression. CONCLUSIONS: This investigation shows for the first time that POR besides CYP2B6 can influence cyclophosphamide metabolism. Our results indicate that not only CYPs are important, but also POR expression and/or activity may influence cyclophosphamide bioactivation, affecting therapeutic efficacy and treatment related toxicity and hence on clinical outcome. Thus, both POR and CYP genotype and expression levels may have to be taken into account when personalizing treatment schedules to achieve optimal therapeutic drug plasma concentrations of cyclophosphamide.

ADAMTS-18: a metalloproteinase with multiple functions.

ADAMTS-18 is a member of a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS) family of proteases, which are known to play important roles in development, angiogenesis and coagulation; dysregulation and mutation of these enzymes have been implicated in many disease processes, such as inflammation, cancer, arthritis and atherosclerosis. Mutations of ADAMTS-18 have been linked to abnormal early eye development and reduced bone mineral density. In this review, we briefly summarize the structural organization and the expression of ADAMTS-18. We will also focus on the emerging role of ADAMTS-18 in several pathophysiological conditions which include: hematological diseases, tumorgenesis, osteogenesis, eye-related diseases, central nervous system disorders, and last but not least a research perspective of ADAMTS-18 and its potential as a promising diagnostic and therapeutic target.

The impact of CDA A79C gene polymorphisms on the response and hematologic toxicity in gemcitabine-treated patients: a meta-analysis.

PURPOSE: To investigate the impact of the cytidine deaminase (CDA) A79C polymorphism on both the response to gemcitabine in non-small cell lung cancer (NSCLC) patients and the risk of hematologic toxicities in patients bearing any kind of cancer taking gemcitabine. METHODS: The PubMed and Embase databases were searched from the first available article to January 2013. Eligible studies included clinical trials that contained the keywords "gemcitabine" or "cytidine deaminase" and information about response rate of NSCLC patients or hematologic toxicities in patients with any kind of cancer. Relative risk (RR) of different genotypes and 95% confidence intervals (CI) were calculated. RESULTS: A total of 7 articles (623 patients from 6 studies) were included. The results showed that patients with wild type CDA (AA and AC) had a significantly lower rate of severe anemia than the homozygote mutant type CC (RR=0.308; 95%CI, 0.113-0.021, p=0.021). However, the rate of severe neutropenia, thrombocytopenia, and the response rate were identical between different CDA genotypes. CONCLUSION: The A79C CDA polymorphism did not show a significant impact on the response rate to gemcitabine in NSCLC patients, while the wild type CDA genotype was indeed correlated to a lower rate of incidence of severe anemia in patients taking gemcitabine.

The H3K27me3 demethylase UTX in normal development and disease.

In 2007, the Ubiquitously Transcribed Tetratricopeptide Repeat on chromosome X (UTX) was identified as a histone demethylase that specifically targets di- and tri-methyl groups on lysine 27 of histone H3 (H3K27me2/3). Since then, UTX has been proven essential during normal development, as it is critically required for correct reprogramming, embryonic development and tissue-specific differentiation. UTX is a member of the MLL2 H3K4 methyltransferase complex and its catalytic activity has been linked to regulation of HOX and RB transcriptional networks. In addition, an H3K27me2/3 demethylase independent function for UTX was uncovered in promoting general chromatin remodeling in concert with the BRG1-containing SWI/SNF remodeling complex. Constitutional inactivation of UTX causes a specific hereditary disorder called the Kabuki syndrome, whereas somatic loss of UTX has been reported in a variety of human cancers. Here, we compile the breakthrough discoveries made from the first disclosure of UTX as a histone demethylase till the identification of disease-related UTX mutations and specific UTX inhibitors.

Nuclear PI-PLCß1: an appraisal on targets and pathology.

Lipid signalling molecules are essential components of the processes that allow one extracellular signal to be transferred inside the nucleus, where specific lipid second messengers elicit reactions capable of regulating gene transcription, DNA replication or repair and DNA cleavage, eventually resulting in cell growth, differentiation, apoptosis or many other cell functions. Nuclear inositides are independently regulated, suggesting that the nucleus constitutes a functionally distinct compartment of inositol lipids metabolism. Indeed, nuclear inositol lipids themselves can modulate nuclear processes, such as transcription and pre-mRNA splicing, growth, proliferation, cell cycle regulation and differentiation. Nuclear PI-PLCß1 is a key molecule for nuclear inositide signalling, where it plays a role in cell cycle progression, proliferation and differentiation. Here we review the targets and possible involvement of nuclear PI-PLCß1 in human physiology and pathology.

Influence of acetylsalicylic acid on hematotoxicity of benzene.

OBJECTIVES: The aim of the study was to evaluate the influence of acetylsalicylic acid (ASA) on benzene hematotoxicity in rats. MATERIALS AND METHODS: The study was carried out on rats exposed for 2, 4 and 8 weeks to benzene vapour at a concentration of 1.5 or 4.5 mmol/m(3) of air (5 days per week, 6 hours per day) alone or together with ASA at the doses of 5, 150 or 300 mg/kg body weight (per os). RESULTS: Benzene at a concentration of 4.5 mmol/m(3) caused a slight lymphopenia, granulocytosis and reticulocytosis in blood. In bone marrow traits of megaloblastic renewal, presence of undifferentiated cells and giant forms of granulocytes as well as an increase in myeloperoxidase and decrease in chloroacetate esterase activity and lipids content were noted. ASA (150 and 300 mg/kg b.w.) influenced some of hematological parameters, altered by benzene intoxication. ASA limited the solvent-induced alteration in blood reticulocyte count and in the case of bone marrow in the erythroblasts count. Traits of megaloblastic renewal in bone marrow were less pronounced. Besides, higher activity of myeloperoxidase and the decrease in the level of lipids in granulocytes were noted. CONCLUSION: Our results suggest that ASA limited the benzene-induced hematotoxicity.

The arrival of JAK inhibitors: advancing the treatment of immune and hematologic disorders.

Altered production of cytokines can result in pathologies ranging from autoimmune diseases to malignancies. The Janus kinase family is a small group of receptor-associated signaling molecules that is essential to the signal cascade originating from type I and type II cytokine receptors. Inhibition of tyrosine kinase enzymatic activity using small molecules has recently become a powerful tool for treatment of several malignancies. Twenty years after the discovery of these enzymes, two inhibitors for this class of kinases have been approved for clinical use and others are currently in the final stage of development. Here we review the principles of cytokines signaling, summarize our current knowledge of the approved inhibitors, and briefly introduce some of the inhibitors that are currently under development.

Diagnosis of Pompe disease: muscle biopsy vs blood-based assays.

The diagnosis of Pompe disease (acid maltase deficiency, glycogen storage disease type II) in children and adults can be challenging because of the heterogeneous clinical presentation and considerable overlap of signs and symptoms found in other neuromuscular diseases. This review evaluates some of the methods used in the diagnosis and differential diagnosis of late-onset Pompe disease. Muscle biopsy is commonly used as an early diagnostic tool in the evaluation of muscle disease. However, experience has shown that relying solely on visualizing a periodic acid-Schiff-positive vacuolar myopathy to identify late-onset Pompe disease often leads to false-negative results and subsequent delays in identification and treatment of the disorder. Serum creatine kinase level can be normal or only mildly elevated in late-onset Pompe disease and is not very helpful alone to suggest the diagnosis, but in combination with proximal and axial weakness it may raise the suspicion for Pompe disease. A simple blood-based assay to measure the level of α-glucosidase activity is the optimal initial test for confirming or excluding Pompe disease. A timely and accurate diagnosis of late-onset Pompe disease likely will improve patient outcomes as care standards including enzyme replacement therapy can be applied and complications can be anticipated. Increased awareness of the clinical phenotype of Pompe disease is therefore warranted to expedite diagnostic screening for this condition with blood-based enzymatic assays.

Erythroid heme biosynthesis and its disorders.

Heme, which is composed of iron and the small organic molecule protoporphyrin, is an essential component of hemoglobin as well as a variety of physiologically important hemoproteins. During erythropoiesis, heme synthesis is induced before, and is essential for, globin synthesis. Although all cells possess the ability to synthesize heme, there are distinct differences between regulation of the pathway in developing erythroid cells and all other types of cells. Disorders that compromise the ability of the developing red cell to synthesize heme can have profound medical implications. The biosynthetic pathway for heme and key regulatory features are reviewed herein, along with specific human genetic disorders that arise from defective heme synthesis such as X-linked sideroblastic anemia and erythropoietic protoporphyria.

Targeting the SYK-BTK axis for the treatment of immunological and hematological disorders: recent progress and therapeutic perspectives.

Spleen Tyrosine Kinase (SYK) and Bruton's Tyrosine Kinase (BTK) are non-receptor cytoplasmic tyrosine kinases that are primarily expressed in cells of hematopoietic lineage. Both are key mediators in coupling activated immunoreceptors to downstream signaling events that affect diverse biological functions, from cellular proliferation, differentiation and adhesion to innate and adaptive immune responses. As such, pharmacological inhibitors of SYK or BTK are being actively pursued as potential immunomodulatory agents for the treatment of autoimmune and inflammatory disorders. Deregulation of SYK or BTK activity has also been implicated in certain hematological malignancies. To date, from a clinical perspective, pharmacological inhibition of SYK activity has demonstrated encouraging efficacy in patients with rheumatoid arthritis (RA), while patients with relapsed or refractory chronic lymphocytic leukemia (CLL)/small lymphocytic lymphoma (SLL) have benefited from covalent inhibitors of BTK in early clinical studies. Here, we review and discuss recent insights into the emerging role of the SYK-BTK axis in innate immune cell function as well as in the maintenance of survival and homing signals for tumor cell progression. The current progress on the clinical development of SYK and BTK inhibitors is also highlighted.

Hematologic toxicities of small molecule tyrosine kinase inhibitors.

Small molecule tyrosine kinase inhibitors (TKIs) are potent anti-cancer targeted therapies. TKIs are considered safe and efficacious therapeutic modalities, and are generally tolerated well. However, they are associated with certain side effects including hematologic toxicities such as anemia, macrocytosis, neutropenia, thrombocytopenia, hemolytic anemia, bone marrow aplasia and necrosis. Thrombotic microangiopathy, arterial thromboembolism and splenic infarction can also occur following treatment with TKIs. Cytopenias are the most common adverse effects associated with these agents, and other hematologic toxicities are not frequent. It is essential for clinicians to monitor patients closely, and recognize those side effects as early as possible, in order to improve efficacy of small molecule TKIs and optimize outcomes. This article summarizes hematologic toxicities associated with the commonly used small molecule TKIs. It also provides practical strategies for the management of these toxicities.

[m-TOR inhibitors: biology and use in the treatment of haematological diseases]. / Inhibiteurs de mTOR : de l'explication biologique à l'application thérapeutique en hématologie.

Mammalian target of rapamycyin (mTOR) is a downstream serine/threonine kinase of the PI3K/AKT pathway that integrates signals from the microenvironment such as cytokines, growth factors, and nutriments to regulate multiple cellular processes, including mRNA translation, autophagy, metabolism, growth and survival. mTOR operates in two distinct multi-protein complexes: mTORC1 and mTORC2; sharing mTOR kinase as a common catalytic subunit, mTORC1 controls cell growth and mTORC2 modulates cell survival and drug resistance. mTOR signalling pathway has been found to be deregulated in many haematological malignancies, and has been designed as an attractive anti-tumor target. Thereby, mTOR inhibition with rapamycin (sirolimus) or its derivates (rapalogs) represents promising treatments, either alone or in combination with strategies to target other pathways that may overcome resistance. At present time, numerous clinical trials with mTOR inhibitors are ongoing for treatment of haematological diseases with modest or promising results. The aim of this review is to present the rationale for using mTOR inhibitors in haematology, first via biological explanations and secondly, by focusing on each haematological malignancies with new perspective of treatment.

Relevance of JAK2V617F positivity to hematological diseases--survey of samples from a clinical genetics laboratory.

BACKGROUND: JAK2V617F is found in the majority of patients with Ph- myeloproliferative neoplasms (MPNs) and has become a valuable marker for diagnosis of MPNs. However, it has also been found in many other hematological diseases, and some studies even detected the presence of JAK2V617F in normal blood samples. This casts doubt on the primary role of JAK2V617F in the pathogenesis of MPNs and its diagnostic value. METHODS: In the present study, we analyzed JAK2V617F positivity with 232 normal blood samples and 2663 patient blood, bone marrow, and amniotic fluid specimens obtained from a clinical genetics laboratory by using a simple DNA extraction method and a sensitive nested allele-specific PCR strategy. RESULTS: We found JAK2V617F present in the majority (78%) of MPN patients and in a small fraction (1.8-8.7%) of patients with other specific hematological diseases but not at all in normal healthy donors or patients with non-hematological diseases. We also revealed associations of JAK2V617F with novel as well as known chromosomal abnormalities. CONCLUSIONS: Our study suggests that JAK2V617F positivity is associated with specific hematological malignancies and is an excellent diagnostic marker for MPNs. The data also indicate that the nested allele-specific PCR method provides clinically relevant information and should be conducted for all cases suspected of having MPNs as well as for other related diseases.