The Src family kinase (SFK) member SRC is a major target in drug development because it is activated in many human cancers, yet deleterious SRC germline mutations have not been reported. We used ...genome sequencing and Human Phenotype Ontology patient coding to identify a gain-of-function mutation in SRC causing thrombocytopenia, myelofibrosis, bleeding, and bone pathologies in nine cases. Modeling of the E527K substitution predicts loss of SRC's self-inhibitory capacity, which we confirmed with in vitro studies showing increased SRC kinase activity and enhanced Tyr(419) phosphorylation in COS-7 cells overexpressing E527K SRC. The active form of SRC predominates in patients' platelets, resulting in enhanced overall tyrosine phosphorylation. Patients with myelofibrosis have hypercellular bone marrow with trilineage dysplasia, and their stem cells grown in vitro form more myeloid and megakaryocyte (MK) colonies than control cells. These MKs generate platelets that are dysmorphic, low in number, highly variable in size, and have a paucity of α-granules. Overactive SRC in patient-derived MKs causes a reduction in proplatelet formation, which can be rescued by SRC kinase inhibition. Stem cells transduced with lentiviral E527K SRC form MKs with a similar defect and enhanced tyrosine phosphorylation levels. Patient-derived and E527K-transduced MKs show Y419 SRC-positive stained podosomes that induce altered actin organization. Expression of mutated src in zebrafish recapitulates patients' blood and bone phenotypes. Similar studies of platelets and MKs may reveal the mechanism underlying the severe bleeding frequently observed in cancer patients treated with next-generation SFK inhibitors.
Roifman syndrome is a rare inherited disorder characterized by spondyloepiphyseal dysplasia, growth retardation, cognitive delay, hypogammaglobulinemia, and, in some patients, thrombocytopenia. ...Compound heterozygous variants in the small nuclear RNA gene RNU4ATAC, which is necessary for U12-type intron splicing, were identified recently as driving Roifman syndrome.
We studied 3 patients from 2 unrelated kindreds harboring compound heterozygous or homozygous stem II variants in RNU4ATAC to gain insight into the mechanisms behind this disorder.
We systematically profiled the immunologic and hematologic compartments of the 3 patients with Roifman syndrome and performed RNA sequencing to unravel important splicing defects in both cell lineages.
The patients exhibited a dramatic reduction in B-cell numbers, with differentiation halted at the transitional B-cell stage. Despite abundant B-cell activating factor availability, development past this B-cell activating factor–dependent stage was crippled, with disturbed minor splicing of the critical mitogen-activated protein kinase 1 signaling component. In the hematologic compartment patients with Roifman syndrome demonstrated defects in megakaryocyte differentiation, with inadequate generation of proplatelets. Platelets from patients with Roifman syndrome were rounder, with increased tubulin and actin levels, and contained increased α-granule and dense granule markers. Significant minor intron retention in 354 megakaryocyte genes was observed, including DIAPH1 and HPS1, genes known to regulate platelet and dense granule formation, respectively.
Together, our results provide novel molecular and cellular data toward understanding the immunologic and hematologic features of Roifman syndrome.
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Each year, blood transfusions save millions of lives. However, under current blood-matching practices, sensitization to non–self-antigens is an unavoidable adverse side effect of transfusion. We ...describe a universal donor typing platform that could be adopted by blood services worldwide to facilitate a universal extended blood-matching policy and reduce sensitization rates. This DNA-based test is capable of simultaneously typing most clinically relevant red blood cell (RBC), human platelet (HPA), and human leukocyte (HLA) antigens. Validation was performed, using samples from 7927 European, 27 South Asian, 21 East Asian, and 9 African blood donors enrolled in 2 national biobanks. We illustrated the usefulness of the platform by analyzing antibody data from patients sensitized with multiple RBC alloantibodies. Genotyping results demonstrated concordance of 99.91%, 99.97%, and 99.03% with RBC, HPA, and HLA clinically validated typing results in 89 371, 3016, and 9289 comparisons, respectively. Genotyping increased the total number of antigen typing results available from 110 980 to >1 200 000. Dense donor typing allowed identification of 2 to 6 times more compatible donors to serve 3146 patients with multiple RBC alloantibodies, providing at least 1 match for 176 individuals for whom previously no blood could be found among the same donors. This genotyping technology is already being used to type thousands of donors taking part in national genotyping studies. Extraction of dense antigen-typing data from these cohorts provides blood supply organizations with the opportunity to implement a policy of genomics-based precision matching of blood.
•Red blood cell, platelet, and white cell antigens can be typed accurately with a single, unified, DNA-based test.•As this test will be embedded in national genotyping studies, full blood cell typing data will be available for millions of individuals.
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Dimethylation of histone H3 Arg2 (H3R2me2) maintains transcriptional silencing by inhibiting Set1 mediated trimethylation of H3K4. Here we demonstrate that Arg2 is also monomethylated (H3R2me1) in ...yeast but that its functional characteristics are distinct from H3R2me2: (i) H3R2me1 does not inhibit histone H3 Lys4 (H3K4) methylation; (ii) it is present throughout the coding region of genes; and (iii) it correlates with active transcription. Collectively, these results indicate that different H3R2 methylation states have defined roles in gene expression.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
We present YOGY a web-based resource for orthologous proteins from nine eukaryotic organisms: Homo sapiens, Mus musculus, Rattus norvegicus, Arabidopsis thaliana, Drosophila melanogaster, ...Caenorhabditis elegans, Plasmodium falciparum, Schizosaccharomyces pombe and Saccharomyces cerevisiae. Using a gene name from any of these organisms as a query, this database provides comprehensive, combined information on orthologs in other species using data from five independent resources: KOGs, Inparanoid, HomoloGene, OrthoMCL and a table of curated fission and budding yeast orthologs. Associated Gene Ontology (GO) terms of orthologs can also be retrieved for functional inference. Integrating these different and complementary datasets provides a straightforward tool to identify known and predicted orthologs of proteins from a variety of species. This resource should be useful for bench scientists looking for functional clues for their genes of interest as well as for curators looking for information that can be transferred based on orthology and for rapidly identifying the relevant GO terms as an aid to literature curation. YOGY is accessible online at http://www.sanger.ac.uk/PostGenomics/S_pombe/YOGY/.
Several pilot experiments have indicated that improvements in older NMR structures can be expected by applying modern software and new protocols (Nabuurs et al. in Proteins 55:483-186, 2004; ...Nederveen et al. in Proteins 59:662-672, 2005; Saccenti and Rosato in J Biomol NMR 40:251-261, 2008). A recent large scale X-ray study also has shown that modern software can significantly improve the quality of X-ray structures that were deposited more than a few years ago (Joosten et al. in J. Appl Crystallogr 42:376-384, 2009; Sanderson in Nature 459:1038-1039, 2009). Recalculation of three-dimensional coordinates requires that the original experimental data are available and complete, and are semantically and syntactically correct, or are at least correct enough to be reconstructed. For multiple reasons, including a lack of standards, the heterogeneity of the experimental data and the many NMR experiment types, it has not been practical to parse a large proportion of the originally deposited NMR experimental data files related to protein NMR structures. This has made impractical the automatic recalculation, and thus improvement, of the three dimensional coordinates of these structures. We here describe a large-scale international collaborative effort to make all deposited experimental NMR data semantically and syntactically homogeneous, and thus useful for further research. A total of 4,014 out of 5,266 entries were ‘cleaned' in this process. For 1,387 entries, human intervention was needed. Continuous efforts in automating the parsing of both old, and newly deposited files is steadily decreasing this fraction. The cleaned data files are available from the NMR restraints grid at http://restraintsgrid.bmrb.wisc.edu.
Inherited bleeding, thrombotic, and platelet disorders (BPDs) are diseases that affect ∼300 individuals per million births. With the exception of hemophilia and von Willebrand disease patients, a ...molecular analysis for patients with a BPD is often unavailable. Many specialized tests are usually required to reach a putative diagnosis and they are typically performed in a step-wise manner to control costs. This approach causes delays and a conclusive molecular diagnosis is often never reached, which can compromise treatment and impede rapid identification of affected relatives. To address this unmet diagnostic need, we designed a high-throughput sequencing platform targeting 63 genes relevant for BPDs. The platform can call single nucleotide variants, short insertions/deletions, and large copy number variants (though not inversions) which are subjected to automated filtering for diagnostic prioritization, resulting in an average of 5.34 candidate variants per individual. We sequenced 159 and 137 samples, respectively, from cases with and without previously known causal variants. Among the latter group, 61 cases had clinical and laboratory phenotypes indicative of a particular molecular etiology, whereas the remainder had an a priori highly uncertain etiology. All previously detected variants were recapitulated and, when the etiology was suspected but unknown or uncertain, a molecular diagnosis was reached in 56 of 61 and only 8 of 76 cases, respectively. The latter category highlights the need for further research into novel causes of BPDs. The ThromboGenomics platform thus provides an affordable DNA-based test to diagnose patients suspected of having a known inherited BPD.
•Developed a targeted sequencing platform covering 63 genes linked to heritable bleeding, thrombotic, and platelet disorders.•The ThromboGenomics platform provides a sensitive genetic test to obtain molecular diagnoses in patients with a suspected etiology.
Pulmonary arterial hypertension (PAH) is a rare disease with an emerging genetic basis. Heterozygous mutations in the gene encoding the bone morphogenetic protein receptor type 2 (
) are the ...commonest genetic cause of PAH, whereas biallelic mutations in the eukaryotic translation initiation factor 2 alpha kinase 4 gene (
) are described in pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Here, we determine the frequency of these mutations and define the genotype-phenotype characteristics in a large cohort of patients diagnosed clinically with PAH.
Whole-genome sequencing was performed on DNA from patients with idiopathic and heritable PAH and with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis recruited to the National Institute of Health Research BioResource-Rare Diseases study. Heterozygous variants in
and biallelic
variants with a minor allele frequency of <1:10 000 in control data sets and predicted to be deleterious (by combined annotation-dependent depletion, PolyPhen-2, and
predictions) were identified as potentially causal. Phenotype data from the time of diagnosis were also captured.
Eight hundred sixty-four patients with idiopathic or heritable PAH and 16 with pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis were recruited. Mutations in
were identified in 130 patients (14.8%). Biallelic mutations in
were identified in 5 patients with a clinical diagnosis of pulmonary veno-occlusive disease/pulmonary capillary hemangiomatosis. Furthermore, 9 patients with a clinical diagnosis of PAH carried biallelic
mutations. These patients had a reduced transfer coefficient for carbon monoxide (Kco; 33% interquartile range, 30%-35% predicted) and younger age at diagnosis (29 years; interquartile range, 23-38 years) and more interlobular septal thickening and mediastinal lymphadenopathy on computed tomography of the chest compared with patients with PAH without
mutations. However, radiological assessment alone could not accurately identify biallelic
mutation carriers. Patients with PAH with biallelic
mutations had a shorter survival.
Biallelic
mutations are found in patients classified clinically as having idiopathic and heritable PAH. These patients cannot be identified reliably by computed tomography, but a low Kco and a young age at diagnosis suggests the underlying molecular diagnosis. Genetic testing can identify these misclassified patients, allowing appropriate management and early referral for lung transplantation.
We present a suite of software for the complete and easy deposition of NMR data to the PDB and BMRB. This suite uses the CCPN framework and introduces a freely downloadable, graphical desktop ...application called CcpNmr Entry Completion Interface (ECI) for the secure editing of experimental information and associated datasets through the lifetime of an NMR project. CCPN projects can be created within the CcpNmr Analysis software or by importing existing NMR data files using the CcpNmr FormatConverter. After further data entry and checking with the ECI, the project can then be rapidly deposited to the PDBe using AutoDep, or exported as a complete deposition NMR-STAR file. In full CCPN projects created with ECI, it is straightforward to select chemical shift lists, restraint data sets, structural ensembles and all relevant associated experimental collection details, which all are or will become mandatory when depositing to the PDB. Instructions and download information for the ECI are available from the PDBe web site at http://www.ebi.ac.uk/pdbe/nmr/deposition/eci.html.
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