Full Deletion Research Articles

Tyrosine kinase fusion genes in pediatric BCR-ABL1-like acute lymphoblastic leukemia.

Approximately 15% of pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL) is characterized by gene expression similar to that of BCR-ABL1-positive disease and unfavorable prognosis. This BCR-ABL1-like subtype shows a high frequency of B-cell development gene aberrations and tyrosine kinase-activating lesions. To evaluate the clinical significance of tyrosine kinase gene fusions in children with BCP-ALL, we studied the frequency of recently identified tyrosine kinase fusions, associated genetic features, and prognosis in a representative Dutch/German cohort. We identified 14 tyrosine kinase fusions among 77 BCR-ABL1-like cases (18%) and none among 76 non-BCR-ABL1-like B-other cases. Novel exon fusions were identified for RCSD1-ABL2 and TERF2-JAK2. JAK2 mutation was mutually exclusive with tyrosine kinase fusions and only occurred in cases with high CRLF2 expression. The non/late response rate and levels of minimal residual disease in the fusion-positive BCR-ABL1-like group were higher than in the non-BCR-ABL1-like B-others (p<0.01), and also higher, albeit not statistically significant, compared with the fusion-negative BCR-ABL1-like group. The 8-year cumulative incidence of relapse in the fusion-positive BCR-ABL1-like group (35%) was comparable with that in the fusion-negative BCR-ABL1-like group (35%), and worse than in the non-BCR-ABL1-like B-other group (17%, p=0.07). IKZF1 deletions, predominantly other than the dominant-negative isoform and full deletion, co-occurred with tyrosine kinase fusions. This study shows that tyrosine kinase fusion-positive cases are a high-risk subtype of BCP-ALL, which warrants further studies with specific kinase inhibitors to improve outcome.

Identification of gene-drug interactions that impact patient survival in TCGA.

BackgroundWith the advent of large scale biological data collection for various diseases, data analysis pipelines and workflows need to be established to build frameworks for integrative analysis. Here the authors present a pipeline for identifying disease specific gene-drug interactions using CNV (Copy Number Variation) and clinical data from the TCGA (The Cancer Genome Atlas) project. Two cancer types were selected for analysis, LGG (Brain lower grade glioma) and GBM (Glioblastoma multiforme), due to the possible progression from LGG to GBM in some cases. The copy number and clinical data were then used to preform survival analysis on a gene by gene basis on sub-populations of patients exposed to a given drug.ResultsSeveral gene-drug interactions are identified, where the copy number of a gene is associated to survival of a patient exposed to a certain drug. Both Irinotecan/HAS2 (Hyaluronan synthase 2) and Bevacizumab/PGAM1 (Phosphoglycerate mutase 1) are interactions found in this study with independent confirmation. Independent work in colon, breast cancer and leukemia (Györffy, Breast Cancer Res Treat 123:725-731, 2010; Mueller, Mol Cancer Ther 11:3024–3032, 2010; Hitosugi, Cancer Cell 13:585-600, 2012) showed these two interactions can lead to increased survival.ConclusionWhile the pipeline produced several possible interactions where increased survival is linked to normal or increased copy number of a given gene for patients treated with a given drug, no instance of low copy number or full deletion was linked to increased survival. The development of this pipeline shows a promising utility to identify possible beneficial gene-drug interactions that could improve patient survival and may illustrate some of the problems inherent in this kind of analysis on these data.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-016-1255-7) contains supplementary material, which is available to authorized users.

Intramolecular diffusion controls aggregation of the PAPf39 peptide

The 39-residue fragment of human prostatic acidic phosphatase (PAP) is found in high concentrations in semen and easily form fibrils. Previous work has shown that fibrillization is accelerated with a deletion of the first 8, mostly charged residues and it was hypothesized that fibrillization depended on the dynamics of these peptides. To test this hypothesis we have measured the intramolecular diffusion of the full length and 8-residue deletion peptides at two different pHs and found a correlation with fibrillization lag time. These results can be explained by a simple kinetic model of the early stages of aggregation in which oligomerization is controlled by the rate of peptide reconfiguration.

Abstract PR02: The SWI/SNF component Arid1a regulates regenerative capacity and carcinogenesis in a dose-dependent fashion

Abstract It is unclear if regenerative capacity can be dissociated from cancer risk, a question that is highly relevant for liver carcinogenesis. Frequent SWI/SNF and Arid1a loss-of-function mutations suggest tumor suppressive roles, but the functional impact of these mutations in tissue homeostasis and cancer is unclear. Mice without Arid1a possessed improved healing abilities after an array of injuries. After liver resection, chemical hepatocyte injury, and toxic bile duct injury, Arid1a deficient livers exhibited increased regeneration, reduced tissue damage, and improved organ function. Moreover, global homozygous Arid1a loss potentiated soft tissue healing after ear hole punch and hematopoietic recovery after irradiation. The chromatin state as reprogrammed by Arid1a loss restricted access to promoters by lineage-specific transcription factors that ordinarily suppress cell cycle reentry, thus increasing regeneration after injury. Intriguingly, the homozygous Arid1a deficient state protected mice from chemical injury induced hepatocellular carcinoma (HCC) and extended survival in a MYC-driven hepatoblastoma model. Since Arid1a mutations are most frequently heterozygous in human HCC genome studies, we examined the haploinsufficient state in these mouse models and observed accelerated carcinogenesis and metastasis. Transcriptome analysis identified dysregulated differentiation, proliferation, and metastasis programs. These models show that full deletion of Arid1a can enhance mammalian regeneration without increasing cancer risk, but that Arid1a’s tumor suppressor functions are highly sensitive to dose. This abstract is also presented as Poster A16. Citation Format: Xuxu Sun, Sam Wang, Thomas Maples, Lin Li, Liem Nguyen, Jen-Chieh Chuang, Mahsa Sorouri, Shuyuan Zhang, Linwei Wu, Cemre Celen, Hao Zhu. The SWI/SNF component Arid1a regulates regenerative capacity and carcinogenesis in a dose-dependent fashion. [abstract]. In: Proceedings of the AACR Special Conference: Developmental Biology and Cancer; Nov 30-Dec 3, 2015; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(4_Suppl):Abstract nr PR02.

Subcellular Trafficking and Functional Relationship of the HSV-1 Glycoproteins N and M.

The herpes simplex virus type 1 (HSV-1) glycoprotein N (gN/UL49.5) is a type I transmembrane protein conserved throughout the herpesvirus family. gN is a resident of the endoplasmic reticulum that in the presence of gM is translocated to the trans Golgi network. gM and gN are covalently linked by a single disulphide bond formed between cysteine 46 of gN and cysteine 59 of gM. Exit of gN from the endoplasmic reticulum requires the N-terminal core of gM composed of eight transmembrane domains but is independent of the C-terminal extension of gM. Co-transport of gN and gM to the trans Golgi network also occurs upon replacement of conserved cysteines in gM and gN, suggesting that their physical interaction is mediated by covalent and non-covalent forces. Deletion of gN/UL49.5 using bacterial artificial chromosome (BAC) mutagenesis generated mutant viruses with wild-type growth behaviour, while full deletion of gM/UL10 resulted in an attenuated phenotype. Deletion of gN/UL49.5 in conjunction with various gM/UL10 mutants reduced average plaque sizes to the same extent as either single gM/UL10 mutant, indicating that gN is nonessential for the function performed by gM. We propose that gN functions in gM-dependent as well as gM-independent processes during which it is complemented by other viral factors.

How to make a synaptic ribbon: RIBEYE deletion abolishes ribbons in retinal synapses and disrupts neurotransmitter release.

Synaptic ribbons are large proteinaceous scaffolds at the active zone of ribbon synapses that are specialized for rapid sustained synaptic vesicles exocytosis. A single ribbon-specific protein is known, RIBEYE, suggesting that ribbons may be constructed from RIBEYE protein. RIBEYE knockdown in zebrafish, however, only reduced but did not eliminate ribbons, indicating a more ancillary role. Here, we show in mice that full deletion of RIBEYE abolishes all presynaptic ribbons in retina synapses. Using paired recordings in acute retina slices, we demonstrate that deletion of RIBEYE severely impaired fast and sustained neurotransmitter release at bipolar neuron/AII amacrine cell synapses and rendered spontaneous miniature release sensitive to the slow Ca(2+)-buffer EGTA, suggesting that synaptic ribbons mediate nano-domain coupling of Ca(2+) channels to synaptic vesicle exocytosis. Our results show that RIBEYE is essential for synaptic ribbons as such, and may organize presynaptic nano-domains that position release-ready synaptic vesicles adjacent to Ca(2+) channels.

MUTATIONS OF THE SMARCB1 GENE IN HUMAN CANCERS

In the recent years, the full exome sequencing helped to reveal a set of mutations in the genes that are not oncogenes or tumor suppressor genes by definition, but play an important role in carcinogenesis and encode proteins involved in chromatin remodeling. Among chromatin remodeling systems, which operate through the ATP-dependent mechanism, the complex SWI/ SNF attracts the great attention. The complex consists of the catalytic ATPase (SMARCA2/4), a group of conservative core subunits (SMARCB1, SMARCC1/2), and variant subunits. Abnormalities in the genes coding for each of these components have been identified as driver mutations in various human tumors. The SMARCB1 gene is of interest for practical oncogenetics, with its typical genotype-phenotype correlations. Germinal inactivating mutations (frameshift insertions/deletions, full deletions of the gene, nonsense mutations) lead to development of rhabdoid tumors in the kidneys and the brain in children in their first years of life, or even in utero. These tumors are highly malignant (Rhabdoid Tumor Predisposition Syndrome 1 – RTPS1). If a mutation carrier survives his/hers four years of life without manifestation RTPS1 with a missense mutation or has the mutation in the hot spot of the first or the last exon, then he/she will not develop rhabdoid tumors, but after 20 years of life, shwannomatosis may develop as multiple benign tumors of peripheral nerves. Finally, some point mutations in the exons 8–9 can result in Coffin-Siris syndrome characterized by mental retardation and developmental disorders, but no neoplasms. In this regard, rational referral of patients for direct DNA diagnostics of each of the described disease entities plays an important role, based on respective minimal criteria, as well as necessity of further development of NGS technologies (full genome and full exome sequencing) that are able to sequence not only individual exons, but all candidate genes of the disorders.

A novel conditional mouse model for Nkx2-5 reveals transcriptional regulation of cardiac ion channels

Nkx2-5 is one of the master regulators of cardiac development, homeostasis and disease. This transcription factor has been previously associated with a suite of cardiac congenital malformations and impairment of electrical activity. When disease causative mutations in transcription factors are considered, NKX2-5 gene dysfunction is the most common abnormality found in patients. Here we describe a novel mouse model and subsequent implications of Nkx2-5 loss for aspects of myocardial electrical activity. In this work we have engineered a new Nkx2-5 conditional knockout mouse in which flox sites flank the entire Nkx2-5 locus, and validated this line for the study of heart development, differentiation and disease using a full deletion strategy. While our homozygous knockout mice show typical embryonic malformations previously described for the lack of the Nkx2-5 gene, hearts of heterozygous adult mice show moderate morphological and functional abnormalities that are sufficient to sustain blood supply demands under homeostatic conditions. This study further reveals intriguing aspects of Nkx2-5 function in the control of cardiac electrical activity. Using a combination of mouse genetics, biochemistry, molecular and cell biology, we demonstrate that Nkx2-5 regulates the gene encoding Kcnh2 channel and others, shedding light on potential mechanisms generating electrical abnormalities observed in patients bearing NKX2-5 dysfunction and opening opportunities to the study of novel therapeutic targets for anti-arrhythmogenic therapies.

Clinical Relevance of Tyrosine Kinase Fusion Genes in Pediatric BCR-ABL1-like Acute Lymphoblastic Leukemia

Background Patients with pediatric B-cell precursor acute lymphoblastic leukemia (BCP-ALL) with the BCR-ABL1 fusion gene form a small high-risk patient group with a poor prognosis. Approximately 15% of BCP-ALL are characterized by a gene expression signature similar to that of BCR-ABL1-positive disease and an unfavorable prognosis. This BCR-ABL1-like group shows a high frequency of B-cell development gene aberrations, especially IKZF1 deletions and tyrosine kinase-activating lesions (Den Boer et al. Lancet Oncol 2009; Mullighan et al. N Engl J Med 2009; Roberts et al. Cancer Cell 2012, N Engl J Med 2014; Van der Veer et al. Blood 2013).Aims To evaluate the clinical value of tyrosine kinase fusions in newly diagnosed children with B-cell precursor ALL, we studied their frequency, prognosis and drugability in a Dutch/German cohort.Methods This study comprised 204 children with BCP-ALL in three Dutch trials (DCOG ALL-8, 9, 10) and two German trials (COALL 06-97, 07-03) including 92 previously described BCR-ABL1-like cases identified by hierarchical clustering and 112 non-BCR-ABL1-like B-other cases. Molecular characterization included RT-PCR and FISH to detect fusions involving ABL1, PDGFRB, JAK2 and CSF1R, gene expression analysis, and copy number analysis.Results We identified 12 tyrosine kinase-activating fusion genes among 73 tested BCR-ABL1-like cases (16%) and none among 87 tested B-other cases. Eight fusions activated the ABL signaling pathway: 4 EBF1-PDGFRB, ZMIZ1-ABL1, RCSD1-ABL2, SSBP1-CSF1R, and one case with split ABL1 and an unknown fusion partner. Four fusions activated the JAK signaling pathway: 2 PAX5-JAK2, BCR-JAK2, and TERF2-JAK2. The gene fusions were confirmed by RT-PCR or targeted locus amplification. Gene expression of the involved tyrosine kinase was high in each of the fusion cases.IKZF1 deletions occurred more frequently in tyrosine kinase fusion cases compared with non-BCR-ABL1-like B-other cases (55% vs. 32%; p=0.2), and were enriched for rare, i.e. other than exon 4-7 or full deletion, variants (45% vs. 18%; p=0.05). In the remaining BCR-ABL1-like cases, the frequency of rare IKZF1 variants was similar to that in B-other (17%). Single deletion of exon 16 of EBF1 occurred in the EBF1-PDGFRB fusions and was rare among the remaining BCR-ABL1-like (0/77) and B-other cases (2/105). High CRLF2 expression co-occurred only in the BCR-JAK2 fusion case.The cumulative incidence of relapse (CIR) in the BCR-ABL1-like group with tyrosine kinase fusions (8-yr CIR 40% ± 18%) was comparable with that in the remaining BCR-ABL1-like group (8-yr CIR 36% ± 6%), and worse than in the B-other group (8-yr CIR 19% ± 4%; overall Gray p=0.04). Of the 12 tyrosine kinase fusion cases, four were late responders who only achieved remission after day 33 of induction therapy, and one was a non-responder resulting in early death. This non/late response rate was significantly higher in the tyrosine kinase fusion cases compared with non-BCR-ABL1 -like B-other (42% vs. 9%, p=0.008) and also higher compared with the remaining, fusion-negative BCR-ABL1-like cases (42% vs. 17%, p=0.06). Leukemic cells from three EBF1-PDGFRB patients were sensitive to 15 and 30 µM imatinib in ex vivo cultures, compared with lack of cytotoxic response in four EBF1-PDGFRB-negative samples, two of which even showed growth on imatinib. Combination of imatinib with 100 µg/ml prednisolone resulted in further growth inhibition in 2/3 EBF1-PDGFRB patients' ex vivo cultures.Conclusions Tyrosine kinase fusion genes were found in 16% of DCOG/COALL BCR-ABL1-like cases, representing ~3% of total BCP-ALL. BCR-ABL1-like cases with tyrosine kinase fusions were characterized by poor initial response to treatment, had an unfavorable clinical outcome compared with non-BCR-ABL1-like B-other ALL cases and a similar unfavorable outcome compared with tyrosine kinase fusion-negative BCR-ABL1-like cases. Imatinib worked additive to prednisolone in EBF1-PDGFRB patients' cells, indicating that this inhibitor may be clinically used in combination with at least prednisone. These results are in line with promising results of refractory EBF1-PDGFRB-positive and other ABL class fusion patients successfully treated with imatinib added to consolidation chemotherapy (Lengline et al. Haematologica 2013; Weston et al. J Clin Oncol 2013; Roberts et al. N Engl J Med 2014). DisclosuresNo relevant conflicts of interest to declare.

243 EFFICIENT EDITION OF THE BOVINE PRNP PRION GENE IN SOMATIC CELLS AND IVF EMBRYOS USING THE CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)/Cas9 SYSTEM

The rapid introduction of engineered nucleases technologies, such as zinc finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR), provides new opportunities for editing genes in a targeted and rather simple fashion. Few reports are available regarding CRISPR efficiency in domestic species. Here, the CRISPR/Cas9 system was employed to develop knockout and knock-in alleles of the bovine PRNP gene, responsible for bovine spongiform encephalopathy (mad cow disease), both in bovine fetal fibroblasts and in IVF embryos. Five sgRNAs were designed to target a 875-bp region within prnp exon 3; all 5 were co-delivered with hCas9 and a homologous recombination vector carrying gfp (pHRegfp). For cells, 3 transfection conditions were compared: 2 μg of hCas9 + 1 μg of sgRNAs mix ± 2 μg pHREGFP (1X) versus 4 μg of hCas9 + 2 μg of sgRNAs mix ± 4 μg of pHREGFP (2X). For IVF zygotes, cytoplasmic injection was conducted with 2 RNA concentrations: (a) 50 ng μL–1 hCas9 RNA + 25 ng μL–1 sgRNAs mix (RNA1X), ±50 ng μL–1 pHREGFP, and (b) 100 ng μL–1 hCas9 + 50 ng μL–1 sgRNAs mix (RNA2X), ±100 ng μL–1 pHREGFP, which were compared with plasmid injections with 100 ng μL–1 pCMVCas9 + 50 ng μL–1 pU6sgRNAs mix (DNA2X), ±100 ng μL–1 pHREGFP. The pHREGFP was always injected as plasmid, under the same conditions as hCas9. DNA from cells was subjected to PCR, Surveyor assay, and sequence analysis. Embryo analysis was conducted on whole-genome-amplified DNA from blastocysts, followed by PCR assays and sequencing. In cells, 2X transfection resulted in indels and amplification of PCR products of lower MW than the wild-type, indicative of the deletion of a part of the targeted PRNP region. However, it was not possible to detect an effect for 1X transfection. For the group transfected with pHREGFP, insertion of a partial EGFP sequence was detected (383 bp). Regarding embryo injection, higher blastocyst rates were obtained in all groups injected with RNA (Table 1). In 48% (21/43) of the sequenced blastocysts specific gene editing was detected (Table 1). Modifications varied among single base pair shift (3/43; 7%), high level of mismatches all over the targeted sequence and vicinity (12/43; 27.9%), full deletion of the 875-bp region (1/43; 2.3%), and partial insertion of 100–498 bp pHREGFP fragments between the HR arms (5/24; 20.8%). Most of these modifications occurred in a mosaic fashion (76%). Results demonstrate that CRISPR/Cas can be efficiently applied for site-specific edition of domestic species genomes. Table 1.In vitro development and gene editing efficiency of embryos injected with plasmids or RNA coding for CRISPR/Cas9 system targeting PRNP

Differential role of integrin beta 4 in HLA class-I induced endothelial cell activation

Aim The development of anti-donor HLA class I (HLA-I) antibodies following solid organ transplant is associated with acute and chronic rejection and graft failure. Following ligation with antibody, HLA class I molecules form a molecular partnership with integrin β 4 (ITGB4) to transduce activation and proliferative signals in endothelial cells (EC) contributing to rejection. We hypothesize that determination of the domains on the integrin β 4 molecule that interact with HLA-I will provide critical information for the development of novel therapeutics for the treatment of chronic rejection. Methods To identify the ITGB4 intracellular domain(s) that associates with HLA-I, we used the ITGB4 full length (Wild Type) construct or deletion mutants that lack the Calx- β , fibronectin type III repeat FNIII 1-4 or connecting segment (CS) domain . These constructs were sub-cloned into an adenovirus-based vector and adenovirus encoding WT or mutants were generated. Endogenous ITGB4 expression was suppressed using siRNA transfection. Primary human aortic endothelial cells (HAEC) lacking endogenous ITGB4 were infected with adenovirus and then EC activation was determined by analyzing protein phosphorylation, stress fiber formation, cell migration and proliferation. Results Our results demonstrate that HLA-I binds to ITGB4 via the CS domain. The CS domain is required for the phosphorylation of Src, Paxillin, Akt, ERK1/2 and S6RP. The FNIII 3&4 domains are required for phosphorylating Paxillin, Akt, ERK1/2 and S6RP, but not Src. HLA-I phosphorylates ITGB4 at Tyr1494 and Tyr1526 in a Src dependent manner. Furthermore, deletion of CS, FNIII3&4, Calx- β inhibited HLA-I induced EC stress fiber formation, migration and proliferation. Moreover, HLA-I induces association between ITGB4 and the adaptor protein Shc which depends on CS and FNIII3&4 domains. Suppression of endogenous Shc protein by siRNA inhibited HLA-I induced EC migration, proliferation and phosphorylation of Akt, ERK1/2 and S6RP, but not Src. Conclusion Understanding the molecular mechanisms underlying HLA antibody-induced EC proliferation and migration should enable the development of novel therapeutic strategies for the treatment of antibody-mediated chronic transplant rejection.

Genetic linkage facilitates cloning of Ert-m regulating plant architecture in barley and identified a strong candidate of Ant1 involved in anthocyanin biosynthesis.

The erectoides-m anthocyanin-less 1 (ert-m ant1) double mutants are among the very few examples of induced double mutants in barley. From phenotypic observations of mutant plants it is known that the Ert-m gene product regulates plant architecture whereas the Ant1 gene product is involved in anthocyanin biosynthesis. We used a near-isogenic line of the cultivar Bowman, BW316 (ert-m.34), to create four F2-mapping populations by crosses to the barley cultivars Barke, Morex, Bowman and Quench. We phenotyped and genotyped 460 plants, allowing the ert-m mutation to be mapped to an interval of 4.7cM on the short arm of barley chromosome 7H. Bioinformatic searches identified 21 candidate gene models in the mapped region. One gene was orthologous to a regulator of Arabidopsis thaliana plant architecture, ERECTA, encoding a leucine-rich repeat receptor-like kinase. Sequencing of HvERECTA in barley ert-m mutant accessions identified severe DNA changes in 15mutants, including full gene deletions in ert-m.40 and ert-m.64. Both deletions, additionally causing anthocyanin deficiency, were found to stretch over a large region including two putative candidate genes for the anthocyanin biosynthesis locus Ant1. Analyses of ert-m and ant1 single- and double-deletion mutants suggest Ant1 as a closely linked gene encoding a R2R3 myeloblastosis transcription factor.

The C-terminal region of the RNA helicase CshA is required for the interaction with the degradosome and turnover of bulk RNA in the opportunistic pathogen Staphylococcus aureus

Staphylococcus aureus is a versatile opportunistic pathogen that adapts readily to a variety of different growth conditions. This adaptation requires a rapid regulation of gene expression including the control of mRNA abundance. The CshA DEAD-box RNA helicase was previously shown to be required for efficient turnover of the agr quorum sensing mRNA. Here we show by transcriptome-wide RNA sequencing and microarray analyses that CshA is required for the degradation of bulk mRNA. Moreover a subset of mRNAs is significantly stabilised in absence of CshA. Deletion of the C-terminal extension affects RNA turnover similar to the full deletion of the cshA gene. In accordance with RNA decay data, the C-terminal region of CshA is required for an RNA-independent interaction with components of the RNA degradation machinery. The C-terminal truncation of CshA reduces its ATPase activity and this reduction cannot be compensated at high RNA concentrations. Finally, the deletion of the C-terminal extension does affect growth at low temperatures, but to a significantly lesser degree than the full deletion, indicating that the core of the helicase can assume a partial function and opening the possibility that CshA is involved in different cellular processes.

Genotype-phenotype analysis of von Hippel-Lindau syndrome in fifteen Indian families.

The general prevalence of the familial multi-organ tumor disorder, von Hippel-Lindau syndrome (VHL), was estimated to be 1 in 25-40,000 in western studies two decades back. Few studies were done in Indian sub-continent, amidst a surge in clinical reports on VHL specific manifestations. The syndrome is correlated with mutations of the gene VHL (located in Chr 3p25.3). We aimed to conduct a prospective case series describing phenotypic and genotypic characteristics in Indian population. The VHL-specific clinical and radiological features were collected from patients and family members. Genotypic changes such as deletion/duplication or point mutation in the VHL locus were identified using sequencing and MLPA. Thirty-one subjects, from fifteen families with diagnosed VHL, were included in the study. Multicystic pancreas was found in 71% (22/31), CNS hemangioblastoma in 68% (21/31), renal cell carcinoma and retinal angiomas in 23% (7/31) each, pheochromocytoma in 9.7% (3/31) of the population and endolymphatic sac tumor in one subject. Four families (9 subjects) had full length deletion of VHL, three families (4 subjects) had a deletion of exon 3, eight families (18 subjects) had different exonic, splice-site and intronic point mutations and one subject had a de novo in-frame indel in exon 1. Multicystic pancreas and CNS hemangioblastomas were the most common manifestations in our population. The phenotypic expression patterns in terms of tumorigenesis, tissue tropism and penetrance in comparison to the genotypic features were found to be different from previous correlative studies.

Update and Mutational Analysis of SLC20A2: A Major Cause of Primary Familial Brain Calcification.

Primary familial brain calcification (PFBC) is a heterogeneous neuropsychiatric disorder, with affected individuals presenting a wide variety of motor and cognitive impairments, such as migraine, parkinsonism, psychosis, dementia, and mood swings. Calcifications are usually symmetrical, bilateral, and found predominantly in the basal ganglia, thalamus, and cerebellum. So far, variants in three genes have been linked to PFBC: SLC20A2, PDGFRB, and PDGFB. Variants in SLC20A2 are responsible for most cases identified so far and, therefore, the present review is a comprehensive worldwide summary of all reported variants to date. SLC20A2 encodes an inorganic phosphate transporter, PiT-2, widely expressed in various tissues, including brain, and is part of a major family of solute carrier membrane transporters. Fifty variants reported in 55 unrelated patients so far have been identified in families of diverse ethnicities and only few are recurrent. Various types of variants were detected (missense, nonsense, frameshift) including full or partial SLC20A2 deletions. The recently reported SLC20A2 knockout mouse will enhance our understanding of disease mechanism and allow for screening of therapeutic compounds. In the present review, we also discuss the implications of these recent exciting findings and consider the possibility of treatments based on manipulation of inorganic phosphate homeostasis.

Fucose-Mediated Transcriptional Activation of the fcs Operon by FcsR in Streptococcus pneumoniae

In this study, we explore the impact of fucose on the transcriptome of S. pneumoniae D39. The expression of various genes and operons, including the fucose uptake PTS and utilization operon (fcs operon) was altered in the presence of fucose. By means of quantitative RT-PCR and β-galactosidase analysis, we demonstrate the role of the transcriptional regulator FcsR, present upstream of the fcs operon, as a transcriptional activator of the fcs operon. We also predict a 19-bp putative FcsR regulatory site (5′-ATTTGAACATTATTCAAGT-3′) in the promoter region of the fcs operon. The functionality of this predicted FcsR regulatory site was further confirmed by promoter-truncation experiments, where deletion of half of the FscR regulatory site or full deletion led to the abolition of expression of the fcs operon.

Contact-induced change in Jaffa Palestinian Arabic: The case of (ʕ)

One of the most salient features of Arabic is pharyngeality. It is prevalent in its phonemic inventory both in primary articulation – voiced /ʕ/ – and voiceless /ħ/ – and in secondarily articulated phonemes known commonly as “emphatic” consonants.&lt;br /&gt;&lt;p&gt;As a result of the 1948 occupation of Palestine by Zionist forces, Jaffa, and much of the Arabic-speaking population of Palestine elsewhere, has transformed from a monolingual speech community into a bilingual one, having acquired Modern Hebrew as its L2. While many Palestinian towns and villages have remained segregated, Jaffa has become a “mixed town,” in that Palestinians and Jewish Israelis have been living there side by side for the past 65 years. One of the linguistic consequences of this bilingual (or indeed multilingual) cohabitation is structural borrowing (cf. Thomason and Kaufman 1988). Of particular interest to this study are the variable lenition and/or deletion of the voiced pharyngeal fricative among native speakers of Arabic, who are also L2 speakers of Hebrew.&lt;/p&gt;&lt;p&gt;In order to test the hypothesis that structural change, especially in the phonology of Jaffa Palestinian Arabic, has been under way as a result of contact with Hebrew, sociolinguistic interviews were carried out in both Jaffa itself and in the West Bank communities of Ramallah and Jerusalem. The latter two were used as a control group, as West Bank speakers nowadays are much less exposed to Hebrew than their Jaffa counterparts.&lt;/p&gt;&lt;p&gt;Since the structural change in question has more than one possible “application” in the variable rule paradigm, various quantitative models have been applied to assess the effect of contact on language variation and change in this instance. The bar bar graph above illustrates a simple crosstabulation of speaker and the dependent variable across the two speech communities, with two different types of application: full deletion and any type of lenition of the pharyngeal. We see that both the high-contact Jaffa speech community and the low-contact West Bank community have garden varieties of lenition, but the Jaffa community, especially younger speakers, tend more to apply the full lenition rule.&lt;/p&gt;&lt;p&gt;When isolating the Jaffa community and conducting a multivariate analysis of the data from that community alone using Rbrul (Johnson 2009), having recoded the dependent variable as a continuous one on a scale from 0 (total deletion) to 4 (full pharyngeal realization), the best models emerge with several social and linguistic factors contributing the most to the application of the variable rule. Most salient among the social factors appears to be language of schooling, whereby speakers who were schooled predominantly in Hebrew tend, unsurprisingly, to lenite their pharyngeals more than those educated in Arabic. The most salient linguistic factor (though this is yet to be refined) is position of the variable in the word; coda position prefers lenition over onset and consonant clusters.&lt;/p&gt;&lt;p&gt;The paper addresses additional fine-grained explanations and hypotheses regarding language contact and its effects on phonological change both within the Jaffa community and across Palestine in general.&lt;/p&gt;&lt;p&gt;&lt;strong&gt; &lt;/strong&gt;&lt;/p&gt;

GATA-3 dose-dependent checkpoints in early T cell commitment.

GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4(-)CD8(-) (double-negative [DN]) stages 1-3. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to the DN2 stage, skewed DN2 gene regulation, and blocked appearance of the DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (quantitative PCR, RNA sequencing) showed that GATA-3-deficient DN2 cells quickly upregulated genes, including Spi1 (PU.1) and Bcl11a, and downregulated genes, including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r, and Il17rb with gene-specific kinetics and dose dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wild-type or GATA-3-deficient early T lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro-T cells to reveal B cell potential. The ability of GATA-3 to block B cell development did not require T lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B lineage exclusion, T lineage progression, and myeloid-lineage exclusion for commitment.

OR23: THE INTEGRIN β4 CONNECTING SEGMENT DOMAIN IS REQUIRED FOR HLA CLASS I-MEDIATED ENDOTHELIAL CELL ACTIVATION

Endothelial cell (EC) activation is a major consequence of HLA class I (HLA-I) antibody-mediated rejection. Previous studies have shown that the molecular association between HLA-I molecule and integrin β 4 (ITGB4) is required for EC migration and proliferation by regulating ERK1/2 signaling pathway. However, the proximal signaling pathway that regulates HLA-I and ITGB4 association remains unclear. We determined the domains of the ITGB4 cytoplasmic tail required for HLA-I mediated phosphorylation pathways and correlated these findings with functional studies. Primary human aortic endothelial cells (HAEC) were used for this study. To identify the ITGB4 intracellular domain that associates with HLA-I, we generated constructs encoding ITGB4 full length (WT) or deletion mutants that lack the Calx- β , fibronectin type III repeat (FNIII 1–4) or connecting segment (CS). These constructs were sub-cloned into adenovirus-based vector and adenovirus encoding WT or mutants were generated. Endogenous ITGB4 expression was suppressed using siRNA transfection. HAEC lacking endogenous ITGB4 were infected with adenovirus and protein phosphorylation and cell migration were further analyzed. Flow cytometry and Western blot analysis showed that both the WT and deletion mutant-infected HAEC had similar levels of cell surface or total expression of ITGB4. Coimmunoprecipitation studies showed that stimulation with the F(ab′)2 fragment of HLA-I antibody increased HLA-I association with ITGB4 in a time dependent manner in WT adenovirus infected HAEC. In contrast, HAEC infected with the CS deletion mutant significantly inhibited HLA-I association with ITGB4 compared to WT, Calx- β or FNIII mutant infected cells. In addition, the CS deletion mutant decreased HLA-I induced ERK1/2 phosphorylation as measured by Western blot. Using in vitro wound healing assay, we found that over expression of ITGB4 increased EC migration compared to LacZ infected cells, whereas CS deletion decreased HLA-I induced EC migration. Furthermore, HLA-I antibody increased the association of ITGB4 with the adaptor protein Shc. This association was decreased by CS deletion. Our results demonstrate that HLA-I binds to ITGB4 via the connecting segment domain that is required for ERK1/2 signaling and EC activation.

Molecular mimicry and clonal deletion: A fresh look

In this article, I trace the historic background of clonal deletion and molecular mimicry, two major pillars underlying our present understanding of autoimmunity and autoimmune disease. Clonal deletion originated as a critical element of the clonal selection theory of antibody formation in order to explain tolerance of self. If we did have complete clonal deletion, there would be major voids, the infamous “black holes”, in our immune repertoire. For comprehensive, protective adaptive immunity, full deletion is necessarily a rare event. Molecular mimicry, the sharing of epitopes among self and non-self antigens, is extraordinary common and provides the evidence that complete deletion of self-reactive clones is rare. If molecular mimicry were not common, protective adaptive immunity could not be all-encompassing. By taking a fresh look at these two processes together we can envision their evolutionary basis and understand the need for regulatory devices to prevent molecular mimicry from progressing to autoimmune disease.