The C-terminus of p63 contains multiple regulatory elements with different functions

The transcription factor p63 is expressed as at least six different isoforms, of which two have been assigned critical biological roles within ectodermal development and skin stem cell biology on the one hand and supervision of the genetic stability of oocytes on the other hand. These two isoforms contain a C-terminal inhibitory domain that negatively regulates their transcriptional activity. This inhibitory domain contains two individual components: one that uses an internal binding mechanism to interact with and mask the transactivation domain and one that is based on sumoylation. We have carried out an extensive alanine scanning study to identify critical regions within the inhibitory domain. These experiments show that a stretch of ~13 amino acids is crucial for the binding function. Further, investigation of transcriptional activity and the intracellular level of mutants that cannot be sumoylated suggests that sumoylation reduces the concentration of p63. We therefore propose that the inhibitory function of the C-terminal domain is in part due to direct inhibition of the transcriptional activity of the protein and in part due to indirect inhibition by controlling the concentration of p63. Keywords: p63, transcriptional regulation, auto-inhibition, sumoylatio

Expression and localization of nuclear proteins in autosomal-dominant Emery-Dreifuss muscular dystrophy with LMNA R377H mutation

BACKGROUND: The autosomal dominant form of Emery-Dreifuss muscular dystrophy (AD-EDMD) is caused by mutations in the gene encoding for the lamins A and C (LMNA). Lamins are intermediate filament proteins which form the nuclear lamina underlying the inner nuclear membrane. We have studied the expression and the localization of nuclear envelope proteins in three different cell types and muscle tissue of an AD-EDMD patient carrying a point mutation R377H in the lamin A/C gene. RESULTS: Lymphoblastoid cells, skin fibroblasts, primary myoblasts and muscle thin sections were studied by immunocytochemistry and electron microscopy. Cellular levels of A-type lamins were reduced compared to control cells. In contrast, the amount of emerin and lamin B appeared unaltered. Cell synchronization experiments showed that the reduction of the cellular level of A-type lamin was due to instability of lamin A. By electron microscopy, we identified a proportion of nuclei with morphological alterations in lymphoblastoid cells, fibroblasts and mature muscle fibres. Immunofluorescence microscopy showed that a major population of the lamin B receptor (LBR), an inner nuclear membrane protein, was recovered in the cytoplasm in association with the ER. In addition, the intranuclear organization of the active form of RNA polymerase II was markedly different in cells of this AD-EDMD patient. This aberrant intranuclear distribution was specifically observed in muscle cells where the pathology of EDMD predominates. CONCLUSIONS: From our results we conclude: Firstly, that structural alterations of the nuclei which are found only in a minor fraction of lymphoblastoid cells and mature muscle fibres are not sufficient to explain the clinical pathology of EDMD; Secondly, that wild type lamin A is required not only for the retention of LBR in the inner nuclear membrane but also for a correct localization of the transcriptionally active RNA pol II in muscle cells. We speculate that a rearrangement of the internal chromatin could lead to muscle-specific disease symptoms by interference with proper mRNA transcription

Ultra-deep amplicon sequencing indicates absence of low-grade mosaicism with normal cells in patients with type-1 NF1 deletions

Different types of large NF1 deletion are distinguishable by breakpoint location and potentially also by the frequency of mosaicism with normal cells lacking the deletion. However, low-grade mosaicism with fewer than 10% normal cells has not yet been excluded for all NF1 deletion types since it is impossible to assess by the standard techniques used to identify such deletions, including MLPA and array analysis. Here, we used ultra-deep amplicon sequencing to investigate the presence of normal cells in the blood of 20 patients with type-1 NF1 deletions lacking mosaicism according to MLPA. The ultra-deep sequencing entailed the screening of 96 amplicons for heterozygous SNVs located within the NF1 deletion region. DNA samples from three previously identified patients with type-2 NF1 deletions and low-grade mosaicism with normal cells as determined by FISH or microsatellite marker analysis were used to validate our methodology. In these type-2 NF1 deletion samples, proportions of 5.3%, 6.6% and 15.0% normal cells, respectively, were detected by ultra-deep amplicon sequencing. However, using this highly sensitive method, none of the 20 patients with type-1 NF1 deletions included in our analysis exhibited low-grade mosaicism with normal cells in blood, thereby supporting the view that the vast majority of type-1 deletions are germline deletions

Phenotypic and genotypic overlap between mosaic NF2 and schwannomatosis in patients with multiple non-intradermal schwannomas

Schwannomatosis and neurofibromatosis type 2 (NF2) are both characterized by the development of multiple schwannomas but represent different genetic entities. Whereas NF2 is caused by mutations of the NF2 gene, schwannomatosis is associated with germline mutations of SMARCB1 or LZTR1. Here, we studied 15 sporadic patients with multiple non-intradermal schwannomas, but lacking vestibular schwannomas and ophthalmological abnormalities, who fulfilled the clinical diagnostic criteria for schwannomatosis. None of them harboured germline NF2 or SMARCB1 mutations as determined by the analysis of blood samples but seven had germline LZTR1 variants predicted to be pathogenic. At least two independent schwannomas from each patient were subjected to NF2 mutation testing. In five of the 15 patients, identical somatic NF2 mutations were identified (33%). If only those patients without germline LZTR1 variants are considered (n = 8), three of them (37.5%) had mosaic NF2 as concluded from identical NF2 mutations identified in independent schwannomas from the same patient. These findings imply that a sizeable proportion of patients who fulfil the diagnostic criteria for schwannomatosis, are actually examples of mosaic NF2. Hence, the molecular characterization of tumours in patients with a clinical diagnosis of schwannomatosis is very important. Remarkably, two of the patients with germline LZTR1 variants also had identical NF2 mutations in independent schwannomas from each patient which renders differential diagnosis of LZTR1-associated schwannomatosis versus mosaic NF2 in these patients very difficult

Genomic landscape of rat strain and substrain variation

Background: Since the completion of the rat reference genome in 2003, whole-genome sequencing data from more than 40 rat strains have become available. These data represent the broad range of strains that are used in rat research including commonly used substrains. Currently, this wealth of information cannot be used to its full extent, because the variety of different variant calling algorithms employed by different groups impairs comparison between strains. In addition, all rat whole genome sequencing studies to date used an outdated reference genome for analysis (RGSC3.4 released in 2004). Results: Here we present a comprehensive, multi-sample and uniformly called set of genetic variants in 40 rat strains, including 19 substrains. We reanalyzed all primary data using a recent version of the rat reference assembly (RGSC5.0 released in 2012) and identified over 12 million genomic variants (SNVs, indels and structural variants) among the 40 strains. 28,318 SNVs are specific to individual substrains, which may be explained by introgression from other unsequenced strains and ongoing evolution by genetic drift. Substrain SNVs may have a larger predicted functional impact compared to older shared SNVs. Conclusions: In summary we present a comprehensive catalog of uniformly analyzed genetic variants among 40 widely used rat inbred strains based on the RGSC5.0 assembly. This represents a valuable resource, which will facilitate rat functional genomic research. In line with previous observations, our genome-wide analyses do not show evidence for contribution of multiple ancestral founder rat subspecies to the currently used rat inbred strains, as is the case for mouse. In addition, we find that the degree of substrain variation is highly variable between strains, which is of importance for the correct interpretation of experimental data from different labs

Acute Muscular Sarcocystosis: An International Investigation Among Ill Travelers Returning From Tioman Island, Malaysia, 2011-2012

A large outbreak of acute muscular sarcocystosis (AMS) among international tourists who visited Tioman Island, Malaysia, is described. Clinicians evaluating travelers returning ill from Malaysia with myalgia, with or without fever, should consider AMS in their differential diagnosi

Combined fit to the spectrum and composition data measured by the Pierre Auger Observatory including magnetic horizon effects

The measurements by the Pierre Auger Observatory of the energy spectrum and mass composition of cosmic rays can be interpreted assuming the presence of two extragalactic source populations, one dominating the flux at energies above a few EeV and the other below. To fit the data ignoring magnetic field effects, the high-energy population needs to accelerate a mixture of nuclei with very hard spectra, at odds with the approximate E$^{-2}$ shape expected from diffusive shock acceleration. The presence of turbulent extragalactic magnetic fields in the region between the closest sources and the Earth can significantly modify the observed CR spectrum with respect to that emitted by the sources, reducing the flux of low-rigidity particles that reach the Earth. We here take into account this magnetic horizon effect in the combined fit of the spectrum and shower depth distributions, exploring the possibility that a spectrum for the high-energy population sources with a shape closer to E$^{-2}$ be able to explain the observations