Daiei Kojima

Biallelic Mutations in KDSR Disrupt Ceramide Synthesis and Result in a Spectrum of Keratinization Disorders Associated with Thrombocytopenia.

Mutations in ceramide biosynthesis pathways have been implicated in a few Mendelian disorders of keratinization, although ceramides are known to have key roles in several biological processes in skin and other tissues. Using whole-exome sequencing in four probands with undiagnosed skin hyperkeratosis/ichthyosis, we identified compound heterozygosity for mutations in KDSR, encoding an enzyme in the de novo synthesis pathway of ceramides. Two individuals had hyperkeratosis confined to palms, soles, and anogenital skin, whereas the other two had more severe, generalized harlequin ichthyosis-like skin. Thrombocytopenia was present in all patients. The mutations in KDSR were associated with reduced ceramide levels in skin and impaired platelet function. KDSR enzymatic activity was variably reduced in all patients, resulting in defective acylceramide synthesis. Mutations in KDSR have recently been reported in inherited recessive forms of progressive symmetric erythrokeratoderma, but our study shows that biallelic mutations in KDSR are implicated in an extended spectrum of disorders of keratinization in which thrombocytopenia is also part of the phenotype. Mutations in KDSR cause defective ceramide biosynthesis, underscoring the importance of ceramide and sphingosine synthesis pathways in skin and platelet biology.

Recurrent MYB rearrangement in blastic plasmacytoid dendritic cell neoplasm

Lymphoproliferative Disorders Team, Centre de Recherche des Cordeliers, Paris, France; Sorbonne Universités, UPMC Univ Paris 06, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France; Université Paris Descartes, Sorbonne Paris Cité, UMR_S 1138, Centre de Recherche des Cordeliers, Paris, France; Service d’Hématologie Biologique, GH Pitié-Salpêtrière, Paris, France; Service de Biostatistique et Informatique Médicale, Hôpital Saint Louis, Paris, France.; Département de génétique, GH Pitié-Salpêtrière, Paris, France; INSERM U1170, Institut Gustave Roussy, Villejuif, France; Département d’Hématologie, Hôpital Becquerel, Rouen, France; Pôle d’Hématologie, Hôpital Brabois, Vandoeuvre-les-Nancy, France; Service d’Hématologie Clinique, Hôpital d’Argenteuil, Argenteuil, France and Karyopharm Therapeutics, Newton, MA, USA E-mail: santos.susin@crc.jussieu.fr or florence.nguyen-khac@psl.aphp.fr These authors contributed equally to this work. Senior coauthorship.

Development of clinical paroxysmal nocturnal haemoglobinuria in children with aplastic anaemia

The clinical significance of paroxysmal nocturnal haemoglobinuria (PNH) in children with aplastic anaemia (AA) remains unclear. We retrospectively studied 57 children with AA between 1992 and 2010. During the follow‐up, five patients developed clinical PNH, in whom somatic PIGA mutations were detected by targeted sequencing. The 10‐year probability of clinical PNH development was 10·2% (95% confidence interval, 3·6–20·7%). Furthermore, the detection of minor PNH clones by flow cytometry at AA diagnosis was a risk factor for the subsequent development of clinical PNH. These patients with PNH clones at AA diagnosis should undergo periodic monitoring for potential clinical PNH development.

Efficacy of neutrophil non-muscle myosin heavy chain-IIA immunofluorescence analysis in determining the pathogenicity of MYH9 variants

Dear Editor, The recent advent of the next-generation sequencing (NGS) has revolutionized the way of genetic research and diagnosis for inherited diseases and several articles have emerged on the clinical application of targeted sequencing of hematological disorders [1, 2]. The major challenge in the application of NGS is the difficulty in determining the pathogenicity of variants of unknown significance. To address this issue, conventional biochemical analyses can be highly useful for assessing the functional consequences. We here report the usefulness of an immunofluorescence analysis of neutrophil non-muscle myosin heavy chain-IIA (NMMHC-IIA) localization in determining the pathogenicity of a novel MYH9 variant identified by NGS. A 12-year-old Japanese boy who had been clinically diagnosed with congenital thrombocytopenia (platelet count ranged between 50,000 and 100,000/μL) was referred for a genetic diagnosis of inherited bone marrow failure syndromes (IBMFS) using a targeted sequencing platform [3]. A total of 184 IBMFS-associated genes were analyzed using Agilent SureSelect custom probes and an Illumina HiSeq2500 nextgeneration sequencer [3]. The pathogenicity of identified variants was finally assessed according to the guidelines of the American College of Medical Genetics [4]. This analysis showed a novel putative causative variant in MYH9, c.1124C>T:p.S375F. The potential deleterious effect was evaluated in silico by the pathogenicity prediction programs. p.S375F was predicted to be BDisease causing^ by Mutation taster, BDamaging^ by SIFT, BNeutral^ by Provean, and BPossibly damaging^ by PolyPhen-2. However, based on the guideline, the pathogenicity was uncertain because it was a missense variant without any reported pathogenicity. Sanger sequencing verified the presence of p.S375F in the patient and the affected mother but not in the unaffected father; however, further evaluation with functional analysis was necessary. To this end, an immunofluorescence analysis of neutrophil NMMHC-IIA localization was performed [5]. All of the samples were processed in accordance with Institutional Review Boards at each institution and the principles of the Declaration of Helsinki. MYH9 encodes the NMMHC-IIA and is implicated in MYH9 disorders, which are characterized by a triad of giant platelets, thrombocytopenia, and leukocyte inclusion bodies [6, 7]. The hallmark of the disease is the presence of characteristic Döhle body-like granulocyte cytoplasmic inclusion bodies on conventionally stained peripheral blood smears. However, they are often faint or even unrecognizable. In fact, giant platelets were noted on the peripheral blood smears from the patient and the mother but granulocyte inclusion bodies were unrecognizable (Fig. 1). An immunofluorescence analysis revealed that the patient and the mother but not the father had several minute NMMHC-IIA-positive foci in the cytoplasm of their neutrophils (Fig. 1). Ser375 is located in the middle of an α-helix in the myosin head. Thus far, two mutations in the neighborhood of Ser375, p.Q372R, and p.K373 N, have been reported to result in the abnormal stabilization of the region [8, 9]. In conclusion, the * Shinji Kunishima kunishis@nnh.hosp.go.jp

Striate Palmoplantar Keratoderma Showing Transgrediens in a Patient Harbouring Heterozygous Nonsense Mutations in both DSG1 and SERPINB7.

Palmoplantar keratoderma (PPK) is a group of inherited skin disorders characterized by thickening of the skin on the palms and soles, often leading to pain. Classification has largely been clinical, but recently, causative genes for the various PPKs have been found, resulting in a need for a genetic classification of PPK (1). The most common type in the Japanese population is Nagashima-type PPK (NPPK) (2). This autosomal recessive condition is caused by a mutation in SERPINB7. The SERPINB7 protein is found in the stratum corneum of the skin, and its main function is to regulate intercellular protease activity (3). The clinical features of NPPK include diffuse hyperkeratosis associated with erythema on the palms and soles and, in addition, on the dorsal aspects, known as transgrediens (2). Another type of PPK, striate PPK (SPPK), is autosomal dominant. The clinical features include strips of hyperkeratosis running longitudinally along the hands and fingers, and generalized hyperkeratosis on the soles of the feet. A mutation in any of 3 genes is reported to be an underlying cause: desmoglein 1 (DSG1), desmoplakin (DSP) and keratin 1 (KRT1) (4). Not only are these desmosomal proteins essential in maintaining the structure and integrity of the skin, but they are also essential to cell turnover (5). DSP mutations have been shown to increase skin proliferation, leading to hyperkeratosis, and the same effect has been predicted with DSG1 mutations (1), although the exact mechanism has not been fully clarified despite DSG1 being involved in various skin disorders (6, 7). To date, there have been no reports of a patient with genetic mutations for 2 types of PPK. Here, we describe such a Japanese male.

Hematopoietic stem cell transplantation for progressive combined immunodeficiency and lymphoproliferation in patients with activated phosphatidylinositol-3-OH kinase δ syndrome type 1

Background: Activated phosphatidylinositol‐3‐OH kinase &dgr; syndrome type 1 (APDS1) is a recently described primary immunodeficiency syndrome characterized by recurrent respiratory tract infections, lymphoid hyperplasia, and Herpesviridae infections caused by germline gain‐of‐function mutations of PIK3CD. Hematopoietic stem cell transplantation (HSCT) can be considered to ameliorate progressive immunodeficiency and associated malignancy, but appropriate indications, methods, and outcomes of HSCT for APDS1 remain undefined. Objective: Our objective was to analyze the clinical manifestations, laboratory findings, prognosis, and treatment of APDS1 and explore appropriate indications and methods of HSCT. Methods: We reviewed retrospectively the medical records of cohorts undergoing HSCT at collaborating facilities. Results: Thirty‐year overall survival was 86.1%, but event‐free survival was 39.6%. Life‐threatening events, such as severe infections or lymphoproliferation, were frequent in childhood and adolescence and were common indications for HSCT. Nine patients underwent HSCT with fludarabine‐based reduced‐intensity conditioning. Seven patients survived after frequent adverse complications and engraftment failure. Most symptoms improved after HSCT. Conclusion: Patients with APDS1 showed variable clinical manifestations. Life‐threatening progressive combined immunodeficiency and massive lymphoproliferation were common indications for HSCT. Fludarabine‐based reduced‐intensity conditioning–HSCT ameliorated clinical symptoms, but transplantation‐related complications were frequent, including graft failure. GRAPHICAL ABSTRACT Figure. No caption available.

Integration Mapping of piggyBac-Mediated CD19 Chimeric Antigen Receptor T Cells Analyzed by Novel Tagmentation-Assisted PCR

Insertional mutagenesis is an important risk with all genetically modified cell therapies, including chimeric antigen receptor (CAR)-T cell therapy used for hematological malignancies. Here we describe a new tagmentation-assisted PCR (tag-PCR) system that can determine the integration sites of transgenes without using restriction enzyme digestion (which can potentially bias the detection) and allows library preparation in fewer steps than with other methods. Using this system, we compared the integration sites of CD19-specific CAR genes in final T cell products generated by retrovirus-based and lentivirus-based gene transfer and by the piggyBac transposon system. The piggyBac system demonstrated lower preference than the retroviral system for integration near transcriptional start sites and CpG islands and higher preference than the lentiviral system for integration into genomic safe harbors. Integration into or near proto-oncogenes was similar in all three systems. Tag-PCR mapping is a useful technique for assessing the risk of insertional mutagenesis.