An inherited immunoglobulin class-switch recombination deficiency associated with a defect in the INO80 chromatin remodeling complex

BACKGROUND: Immunoglobulin class-switch recombination defects (CSR-D) are rare primary immunodeficiencies characterized by impaired production of switched immunoglobulin isotypes and normal or elevated IgM levels. They are caused by impaired T:B cooperation or intrinsic B cell defects. However, many immunoglobulin CSR-Ds are still undefined at the molecular level. OBJECTIVE: This study's objective was to delineate new causes of immunoglobulin CSR-Ds and thus gain further insights into the process of immunoglobulin class-switch recombination (CSR). METHODS: Exome sequencing in 2 immunoglobulin CSR-D patients identified variations in the INO80 gene. Functional experiments were performed to assess the function of INO80 on immunoglobulin CSR. RESULTS: We identified recessive, nonsynonymous coding variations in the INO80 gene in 2 patients affected by defective immunoglobulin CSR. Expression of wild-type INO80 in patients' fibroblastic cells corrected their hypersensitivity to high doses of gamma-irradiation. In murine CH12-F3 cells, the INO80 complex accumulates at Salpha and Emu regions of the IgH locus, and downregulation of INO80 as well as its partners Reptin and Pontin impaired CSR. In addition, Reptin and Pontin were shown to interact with activation-induced cytidine deaminase. Finally, an abnormal separation of sister chromatids was observed upon INO80 downregulation in CH12-F3 cells, pinpointing its role in cohesin activity. CONCLUSION: INO80 deficiency appears to be associated with defective immunoglobulin CSR. We propose that the INO80 complex modulates cohesin function that may be required during immunoglobulin switch region synapsis

Duplication of 7q34 is specific to juvenile pilocytic astrocytomas and a hallmark of cerebellar and optic pathway tumours

BACKGROUND: Juvenile pilocytic astrocytomas (JPA), a subgroup of low-grade astrocytomas (LGA), are common, heterogeneous and poorly understood subset of brain tumours in children. Chromosomal 7q34 duplication leading to fusion genes formed between KIAA1549 and BRAF and subsequent constitutive activation of BRAF was recently identified in a proportion of LGA, and may be involved in their pathogenesis. Our aim was to investigate additional chromosomal unbalances in LGA and whether incidence of 7q34 duplication is associated with tumour type or location. METHODS AND RESULTS: Using Illumina-Human-Hap300-Duo and 610-Quad high-resolution-SNP-based arrays and quantitative PCR on genes of interest, we investigated 84 paediatric LGA. We demonstrate that 7q34 duplication is specific to sporadic JPA (35 of 53-66%) and does not occur in other LGA subtypes (0 of 27) or NFI-associated-JPA (0 of 4). We also establish that it is site specific as it occurs in the majority of cerebellar JPA (24 of 30-80%) followed by brainstem, hypothalamic/optic pathway JPA (10 of 16-62.5%) and is rare in hemispheric JPA (1 of 7-14%). The MAP-kinase pathway, assessed through ERK phosphorylation, was active in all tumours regardless of 7q34 duplication. Gain of function studies performed on hTERT-immortalised astrocytes show that overexpression of wild-type BRAF does not increase cell proliferation or baseline MAPK signalling even if it sensitises cells to EGFR stimulation. CONCLUSIONS AND INTERPRETATION: Our results suggest that variants of JPA might arise from a unique site-restricted progenitor cell where 7q34 duplication, a hallmark of this tumour-type in association to MAPK-kinase pathway activation, potentially plays a site-specific role in their pathogenesis. Importantly, gain of function abnormalities in components of MAP-Kinase signalling are potentially present in all JPA making this tumour amenable to therapeutic targeting of this pathway. British Journal of Cancer (2009) 101, 722-733. doi: 10.1038/sj.bjc.6605179 www.bjcancer.com Published online 14 July 2009 (C) 2009 Cancer Research U

Rhinobatos annandalei, Bengal Guitarfish

The Bengal Guitarfish (Rhinobatos annandalei) is a small (to 95 cm total length) guitarfish that occurs in the northern Indian Ocean from the United Arab Emirates (UAE) to Bangladesh, including Sri Lanka. The western boundary of this species distribution is uncertain due to confusion with the Spotted Guitarfish (R. punctifer). It is demersal on the inner continental shelf at depths of 5–73 m. The species is mainly a bycatch of a range of industrial and artisanal gears including demersal trawls, longlines, and gillnets. The meat is consumed locally and traded internationally, and the skins are exported from Bangladesh to Myanmar to be made into accessories (e.g. handbags). There is a high level of fisheries resource use and increasing fishing pressure across the range of this species. Severe population reduction is inferred from actual levels of exploitation, as well as several historical accounts and contemporary datasets from the UAE, Iran, Pakistan, and India. Landings data of guitarfishes and rays from the UAE, Iran, Pakistan, India, and Bangladesh indicate landings declines of 69–93% consistent with population reductions of 80% over the past three generation lengths (27 years). These levels of declines are not species-specific but are informative for understanding the broader levels of guitarfish decline in the region. It is suspected that the Bengal Guitarfish has undergone a population reduction of >80% over the past three generation lengths (27 years) due to actual levels of exploitation, and it is assessed as Critically Endangered A2d

Scoliodon laticaudus, Spadenose Shark

The Spadenose Shark (Scoliodon laticaudus) is a small (to 91 cm total length) shark that occurs in the northern Indian Ocean from the Gulf of Oman to Myanmar. It is common in coastal and estuarine waters at depths of 10-75 m, but more typically less than 50 m and prefers muddy and sandy substrates and often occurs near large freshwater outflows. It is highly productive with annual large litters of 6–20 pups, early maturation at two years and a short generation length of 4.5 years. The species is caught mainly by trawl and gillnet in industrial and artisanal fisheries and is retained for human consumption. It is the dominant shark landed in Pakistan, northern India, and Bangladesh and fishing pressure is intenseacross most of its range. The high productivity of the species and short generation length likely provide it with resilience to fishing pressure, however, the intense and ongoing fishing pressure on this species is a cause for concern. It is suspected that the Spadenose Shark has undergone a population reduction of 20–29% over the past three generation lengths (14 years) and is close to reaching the population reduction threshold due to levels of exploitation, and it is assessed as Near Threatened (nearly meeting Vulnerable A2d)

A subset of pediatric-type thalamic gliomas share a distinct DNA methylation profile, H3K27me3 loss and frequent alteration of EGFR

Background: Malignant astrocytic gliomas in children show a remarkable biological and clinical diversity. Small in-frame insertions or missense mutations in the epidermal growth factor receptor gene (EGFR) have recently been identified in a distinct subset of pediatric-type bithalamic gliomas with a unique DNA methylation pattern. Methods: Here, we investigated an epigenetically homogeneous cohort of malignant gliomas (n = 58) distinct from other subtypes and enriched for pediatric cases and thalamic location, in comparison with this recently identified subtype of pediatric bithalamic gliomas. Results EGFR gene amplification was detected in 16/58 (27%) tumors, and missense mutations or small in-frame insertions in EGFR were found in 20/30 tumors with available sequencing data (67%; 5 of them co-occurring with EGFR amplification). Additionally, 8 of the 30 tumors (27%) harbored an H3.1 or H3.3 K27M mutation (6 of them with a concomitant EGFR alteration). All tumors tested showed loss of H3K27me3 staining, with evidence of overexpression of the EZH inhibitory protein (EZHIP) in the H3 wildtype cases. Although some tumors indeed showed a bithalamic growth pattern, a significant proportion of tumors occurred in the unilateral thalamus or in other (predominantly midline) locations. Conclusions: Our findings present a distinct molecular class of pediatric-type malignant gliomas largely overlapping with the recently reported bithalamic gliomas characterized by EGFR alteration, but additionally showing a broader spectrum of EGFR alterations and tumor localization. Global H3K27me3 loss in this group appears to be mediated by either H3 K27 mutation or EZHIP overexpression. EGFR inhibition may represent a potential therapeutic strategy in these highly aggressive gliomas

Multiplex primer prediction software for divergent targets

We describe a Multiplex Primer Prediction (MPP) algorithm to build multiplex compatible primer sets to amplify all members of large, diverse and unalignable sets of target sequences. The MPP algorithm is scalable to larger target sets than other available software, and it does not require a multiple sequence alignment. We applied it to questions in viral detection, and demonstrated that there are no universally conserved priming sequences among viruses and that it could require an unfeasibly large number of primers (∼3700 18-mers or ∼2000 10-mers) to generate amplicons from all sequenced viruses. We then designed primer sets separately for each viral family, and for several diverse species such as foot-and-mouth disease virus (FMDV), hemagglutinin (HA) and neuraminidase (NA) segments of influenza A virus, Norwalk virus, and HIV-1. We empirically demonstrated the application of the software with a multiplex set of 16 short (10 nt) primers designed to amplify the Poxviridae family to produce a specific amplicon from vaccinia virus

Universal Oligonucleotide Microarray for Sub-Typing of Influenza A Virus

A universal microchip was developed for genotyping Influenza A viruses. It contains two sets of oligonucleotide probes allowing viruses to be classified by the subtypes of hemagglutinin (H1–H13, H15, H16) and neuraminidase (N1–N9). Additional sets of probes are used to detect H1N1 swine influenza viruses. Selection of probes was done in two steps. Initially, amino acid sequences specific to each subtype were identified, and then the most specific and representative oligonucleotide probes were selected. Overall, between 19 and 24 probes were used to identify each subtype of hemagglutinin (HA) and neuraminidase (NA). Genotyping included preparation of fluorescently labeled PCR amplicons of influenza virus cDNA and their hybridization to microarrays of specific oligonucleotide probes. Out of 40 samples tested, 36 unambiguously identified HA and NA subtypes of Influenza A virus

Diffuse Glioneuronal tumour with Oligodendroglioma‐like features and Nuclear Clusters (DGONC) – a molecularly‐defined glioneuronal CNS tumour class displaying recurrent monosomy 14

Aims: DNA methylation-based central nervous system (CNS) tumour classification has identified numerous molecularly distinct tumour types, and clinically relevant subgroups among known CNS tumour entities that were previously thought to represent homogeneous diseases. Our study aimed at characterizing a novel, molecularly defined variant of glioneuronal CNS tumour. Patients and methods: DNA methylation profiling was performed using the Infinium MethylationEPIC or 450 k BeadChip arrays (Illumina) and analysed using the 'conumee' package in R computing environment. Additional gene panel sequencing was also performed. Tumour samples were collected at the German Cancer Research Centre (DKFZ) and provided by multinational collaborators. Histological sections were also collected and independently reviewed. Results: Genome-wide DNA methylation data from >25 000 CNS tumours were screened for clusters separated from established DNA methylation classes, revealing a novel group comprising 31 tumours, mainly found in paediatric patients. This DNA methylation-defined variant of low-grade CNS tumours with glioneuronal differentiation displays recurrent monosomy 14, nuclear clusters within a morphology that is otherwise reminiscent of oligodendroglioma and other established entities with clear cell histology, and a lack of genetic alterations commonly observed in other (paediatric) glioneuronal entities. Conclusions: DNA methylation-based tumour classification is an objective method of assessing tumour origins, which may aid in diagnosis, especially for atypical cases. With increasing sample size, methylation analysis allows for the identification of rare, putative new tumour entities, which are currently not recognized by the WHO classification. Our study revealed the existence of a DNA methylation-defined class of low-grade glioneuronal tumours with recurrent monosomy 14, oligodendroglioma-like features and nuclear clusters

Self-assembled hydrogel fibers for sensing the multi-compartment intracellular milieu

Targeted delivery of drugs and sensors into cells is an attractive technology with both medical and scientific applications. Existing delivery vehicles are generally limited by the complexity of their design, dependence on active transport, and inability to function within cellular compartments. Here, we developed self-assembled nanofibrous hydrogel fibers using a biologically inert, low-molecular-weight amphiphile. Self-assembled nanofibrous hydrogels offer unique physical/mechanical properties and can easily be loaded with a diverse range of payloads. Unlike commercially available E. coli membrane particles covalently bound to the pH reporting dye pHrodo, pHrodo encapsulated in self-assembled hydrogel-fibers internalizes into macrophages at both physiologic (37°C) and sub-physiologic (4°C) temperatures through an energy-independent, passive process. Unlike dye alone or pHrodo complexed to E. coli, pHrodo-SAFs report pH in both the cytoplasm and phagosomes, as well the nucleus. This new class of materials should be useful for next-generation sensing of the intracellular milieu

H3.3(K27M) Cooperates with Trp53 Loss and PDGFRA Gain in Mouse Embryonic Neural Progenitor Cells to Induce Invasive High-Grade Gliomas

Gain-of-function mutations in histone 3 (H3) variants are found in a substantial proportion of pediatric high-grade gliomas (pHGG), often in association with TP53 loss and platelet-derived growth factor receptor alpha (PDGFRA) amplification. Here, we describe a somatic mouse model wherein H3.3K27M and Trp53 loss alone are sufficient for neoplastic transformation if introduced in utero. H3.3K27M-driven lesions are clonal, H3K27me3 depleted, Olig2 positive, highly proliferative, and diffusely spreading, thus recapitulating hallmark molecular and histopathological features of pHGG. Addition of wild-type PDGFRA decreases latency and increases tumor invasion, while ATRX knockdown is associated with more circumscribed tumors. H3.3K27M-tumor cells serially engraft in recipient mice, and preliminary drug screening reveals mutation-specific vulnerabilities. Overall, we provide a faithful H3.3K27M-pHGG model which enables insights into oncohistone pathogenesis and investigation of future therapies