De novo variants of CSNK2B cause a new intellectual disability-craniodigital syndrome by disrupting the canonical Wnt signaling pathway

CSNK2B encodes for casein kinase II subunit beta (CK2b), the regulatory subunit of casein kinase II (CK2), which is known to mediate diverse cellular pathways. Variants in this gene have been recently identified as a cause of Poirier-Bienvenu neurodevelopmental syndrome (POBINDS), but functional evidence is sparse. Here, we report five unrelated individuals: two of them manifesting POBINDS, while three are identified to segregate a new intellectual disability-craniodigital syndrome (IDCS), distinct from POBINDS. The three IDCS individuals carried two different de novo missense variants affecting the same codon of CSNK2B. Both variants, NP_001311.3; p.Asp32His and NP_001311.3; p.Asp32Asn, lead to an upregulation of CSNK2B expression at transcript and protein level, along with global dysregulation of canonical Wnt signaling. We found impaired interaction of the two key players DVL3 and b-catenin with mutated CK2b. The variants compromise the kinase activity of CK2 as evident by a marked reduction of phosphorylated b-catenin and consequent absence of active b-catenin inside nuclei of the patient-derived lymphoblastoid cell lines (LCLs). In line with these findings, whole-transcriptome profiling of patient-derived LCLs harboring the NP_001311.3; p.Asp32His variant confirmed a marked difference in expression of genes involved in the Wnt signaling pathway. In addition, whole-phosphoproteome analysis of the LCLs of the same subject showed absence of phosphorylation for 313 putative CK2 substrates, enriched in the regulation of nuclear b-catenin and transcription of the target genes. Our findings suggest that discrete variants in CSNK2B cause dominant-negative perturbation of the canonical Wnt signaling pathway, leading to a new craniodigital syndrome distinguishable from POBINDS

Deficiency of cyclase-associated protein 2 promotes arrhythmias associated with connexin43 maldistribution and fibrosis

Introduction: Cyclase-associated protein 2 (CAP2) plays a major role in regulating the actin cytoskeleton. Since inactivation of CAP2 in a mouse model by a gene trap approach (Cap2(gt/gt)) results in cardiomyopathy and increased mortality, we hypothesized that CAP2 has a major impact on arrhythmias and electrophysiological parameters. Material and methods: We performed long-term-ECG recordings in transgenic CAP2 deficient mice (C57BL/6) to detect spontaneous arrhythmias. In vivo electrophysiological studies by right heart catheterization and ex vivo epicardial mapping were used to analyze electrophysiological parameters, the inducibility of arrhythmias, and conduction velocities. Expression and distribution of cardiac connexins and the amount of cardiac fibrosis were evaluated. Results: Spontaneous ventricular arrhythmias could be detected in Cap2(gt/gt) during the long-term-ECG recording. Cap2(gt/gt) showed marked conduction delays at atrial and ventricular levels, including a reduced heart rate (421.0 +/- 40.6 bpm vs. 450.8 +/- 27.9 bpm; p < 0.01), and prolongations of PQ (46.3 +/- 4.1 ms vs. 38.6 +/- 6.5 ms; p < 0.01), QRS (16.2 +/- 2.6 ms vs. 12.6 +/- 1.4 ms; p < 0.01), and QTc interval (55.8 +/- 6.0 ms vs. 45.2 +/- 3.3 ms; p = 0.02) in comparison to wild type mice. The PQ prolongation was due to an infra-Hisian conduction delay (HV: 9.7 +/- 2.1 ms vs. 6.5 +/- 3.1 ms; p = 0.02). The inducibility of ventricular tachycardias during the electrophysiological studies was significantly elevated in the mutant mice (inducible animals: 88% vs. 33%; p = 0.04). Cap2(gt/gt) showed more abnormal distribution of connexin43 compared to WT (23.0 +/- 4.7% vs. 2.9 +/- 0.8%; p < 0.01). Myocardial fibrosis was elevated in Cap2(gt/gt) hearts (9.1 +/- 6.7% vs. 5.5 +/- 3.3%; p < 0.01). Conclusions: Loss of CAP2 results in marked electrophysiological disturbances including impaired sinus node function, conduction delays, and susceptibility to malignant arrhythmias. Structural changes in Cap2(gt/gt) are associated with alterations in myocardial connexins and fibrosis

Jos kaikki olisi hyvin : Utajärveläisten lapsiperheiden vanhempien kuvauksia arjen hyvinvoinnistaan ja sitä mahdollistavista asioista

Opinnäytetyömme tarkoituksena on kuvailla, mistä asioista utajärveläisten lapsiperheiden vanhempien arjen hyvinvointi koostuu ja mikä vahvistaa ja mahdollistaa tätä arjen hyvinvointia. Kuvaukset muodostuvat vanhempien kokemuksista, toiveista ja ilmaisemista tarpeista. Niiden avulla haetaan vastausta siihen, mistä muodostuu sujuva arki ja mikä auttaa vanhempia jaksamaan niin, että myös lapset sitä kautta voivat hyvin. Opinnäytetyössä on käytetty aineistona alun perin Sohvi-hankkeelle tehtyjä utajärveläisten lapsiperheiden vanhempien haastatteluja. Sohvi-hankkeen päätavoitteena oli kerätä kokemustietoa hyvinvoinnista ja hyödyntää hyvinvointia tuottavien alojen yhteistyötä uusien palvelukokonaisuuksien ja työntekijöiden osaamisen kehittämisessä. Tulkitsemme aineistoa ennaltaehkäisevän lastensuojelutyön ja aikuissosiaalityön rajapinnassa. Opinnäytetyön tietoperusta painottuu hyvinvoinnin ja arjen käsitteisiin. Hyvinvointia käsitellään yleisellä tasolla sekä osana sosiaalipolitiikan strategioita. Lisäksi käsitellään aikuisen arkea ja Utajärveä asuinkuntana. Opinnäytetyö on laadullinen tutkimus, joka pohjautuu fenomenologis-hermeneuttiseen ajatteluun. Aineisto kerättiin käyttämällä Tulevaisuuden muistelu – haastattelumenetelmää, jossa avointen kysymysten avulla pohdittiin niin hyvää tulevaisuutta kuin nykyhetkeäkin. Haastateltavat valikoi-tuivat lastenneuvolan, päivähoidon, lastensuojelun ja perhetyön työntekijöiden toimesta. Haastateltavia oli 11. Haastattelut toteutettiin yksilöhaastatteluina. Aineisto analysoitiin teoriaohjaavaa sisällönanalyysiä käyttäen. Tulosten mukaan vanhempien arjen hyvinvointia vahvistaa omaan työhön, toimeentuloon, asumiseen ja ihmissuhteisiin liittyvien asioiden lisäksi omien läheisten ja erityisesti lasten hyvinvointi. Lapsiperheen hyvinvointi on muodoltaan kehällinen, jossa eri perheenjäsenten oma hyvinvointi tuottaa hyvinvointia myös toisille perheenjäsenille. Arki näyttäytyy kiireisenä ja työläänä. Arjen pyöritykseen kaivataan ulkopuolista, konkreettista, käytännön apua ilman että siitä tehdään ongelmaa. Auttavan käsiparin saaminen siivoukseen, pyykinpesuun, ruuanlaittoon ja lastenhoitoon kiperissä tilanteissa ilman vanhemman leimautumista lastensuojeluasiakkaaksi olisi parasta ennaltaehkäisevää lastensuojelua. Asiasanat: hyvinvointi, lapsiperheet, vanhemmat, arki, perhetyöThis study uses the data collected by another project. The project was about getting new knowledge in Finland based on people´s own experiences of their well-being. The aim of the study is to describe, what are the most valuable elements of well-being and good everyday life according to the parents in Utajärvi. The purpose of our study is to clarify, what makes the parents withstand the pressures of everyday life. The descriptions by the parents re-sult from their experiences, needs and hopes. The thesis assignment was a qualitative study. The data was collected using open-ended inter-views. There were 11 parents interviewed. All the interviewees were selected by the profession-als working at Health and Social Services. The data gathered was analyzed by using the method of theory-driven content analysis. Results indicated that the well-being of the relatives and especially the well-being of the children strengthens the well-being of the parents. In addition to this there are many other things having an effect on good everyday life: elements in work, subsistence, housing, and personal relationships. Everyday life seems busy and laborious. Running daily routines requires the helping hand and substantial aid from the third party people. This aid should be considered as a normal procedure without stigma of firstly having been treated as a client of child welfare. Keywords: well-being, families with children, parents, everyday life, family wor

Nesprin-2 mediated nuclear trafficking and its clinical implications

Nuclear translocation of proteins has a crucial role in the pathogenesis of cancer, Alzheimer disease and viral infections. A complete understanding of nuclear trafficking mechanisms is therefore necessary in order to establish effective intervention strategies. Here we elucidate the role of Nesprin-2 in Ca2+/Calmodulin mediated nuclear transport. Nesprin-2 is an actin-binding nuclear envelope (NE) protein with roles in maintaining nuclear structure and location, regulation of transcription and mechanotransduction. Upon depletion of Nesprin-2 using shRNA, HaCaT cells show abnormal localization of the shuttling proteins BRCA1 and NF-B. We show that their nuclear transport is unlikely due to the canonical RAN mediated nuclear import, but rather to a RAN independent Ca2+/Calmodulin driven mechanism involving Nesprin-2. We report novel interactions between the actin-binding domain of Nesprin-2 and Calmodulin and between the NLS containing region of BRCA1 and Calmodulin. Strikingly, displacing Nesprins from the NE resulted in increased steady state Ca2+ concentrations in the cytoplasm suggesting a previously unidentified role of Nesprins in Ca2+ regulation. On comparing Nesprin-2 and BRCA1 localization in the ovarian cancer cell lines SKOV-3 and Caov-3, Nesprin-2 and BRCA1 were localized to the NE envelope and the nucleus in SKOV-3, respectively, and to the cytoplasm in Caov-3 cells. Fibroblasts obtained from EDMD5 (Emery Dreifuss muscular dystrophy) patients showed loss of Nesprin-2 from the nuclear envelope, corresponding reduced nuclear localization of BRCA1 and enhanced cytoplasmic Ca2+. Taken together, the data suggests a novel role of Nesprin-2 in Ca2+/Calmodulin mediated nuclear trafficking and provides new insights which can guide future therapies

Experimental research Deficiency of cyclase-associated protein 2 promotes arrhythmias associated with connexin43 maldistribution and fibrosis

A b s t r a c t Introduction: Cyclase-associated protein 2 (CAP2) plays a major role in regulating the actin cytoskeleton. Since inactivation of CAP2 in a mouse model by a gene trap approach (Cap2 gt/gt ) results in cardiomyopathy and increased mortality, we hypothesized that CAP2 has a major impact on arrhythmias and electrophysiological parameters. Material and methods: We performed long-term-ECG recordings in transgenic CAP2 deficient mice (C57BL/6) to detect spontaneous arrhythmias. In vivo electrophysiological studies by right heart catheterization and ex vivo epicardial mapping were used to analyze electrophysiological parameters, the inducibility of arrhythmias, and conduction velocities. Expression and distribution of cardiac connexins and the amount of cardiac fibrosis were evaluated. Results: Spontaneous ventricular arrhythmias could be detected in Cap2 gt/gt during the long-term-ECG recording. Cap2 gt/gt showed marked conduction delays at atrial and ventricular levels, including a reduced heart rate (421.0 ±40.6 bpm vs. 450.8 ±27.9 bpm; p &lt; 0.01), and prolongations of PQ (46.3 ±4.1 ms vs. 38.6 ±6.5 ms; p &lt; 0.01), QRS (16.2 ±2.6 ms vs. 12.6 ±1.4 ms; p &lt; 0.01), and QTc interval (55.8 ±6.0 ms vs. 45.2 ±3.3 ms; p = 0.02) in comparison to wild type mice. The PQ prolongation was due to an infra-Hisian conduction delay (HV: 9.7 ±2.1 ms vs. 6.5 ±3.1 ms; p = 0.02). The inducibility of ventricular tachycardias during the electrophysiological studies was significantly elevated in the mutant mice (inducible animals: 88% vs. 33%; p = 0.04). Cap2 gt/gt showed more abnormal distribution of connexin43 compared to WT (23.0 ±4.7% vs. 2.9 ±0.8%; p &lt; 0.01). Myocardial fibrosis was elevated in Cap2 gt/gt hearts (9.1 ±6.7% vs. 5.5 ±3.3%; p &lt; 0.01). Conclusions: Loss of CAP2 results in marked electrophysiological disturbances including impaired sinus node function, conduction delays, and susceptibility to malignant arrhythmias. Structural changes in Cap2 gt/gt are associated with alterations in myocardial connexins and fibrosis

Lack of a Retinal Phenotype in a Syne-2/Nesprin-2 Knockout Mouse Model

Syne-2 (also known as Nesprin-2) is a member of a family of proteins that are found primarily in the outer nuclear membrane, as well as other subcellular compartments. Syne-2 contains a C-terminal KASH transmembrane domain and is part of a protein network that associates the nuclear envelope to the cytoskeleton via the binding to actin filaments. Syne-2 plays a role in nuclear migration, nuclear positioning during retinal development, and in ciliogenesis. In a previous study, we showed a connection between Syne-2 and the multifunctional scaffold protein Pericentrin (Pcnt). The elimination of the interaction of Syne-2 and Pcnt showed defects in nuclear migration and the formation of outer segments during retinal development, as well as disturbances in centrosomal migration at the beginning of ciliogenesis in general. In this study, the Syne-2 KO mouse model Nesprin-2oABD (Syne-2(tm1Ngl), MGI) with special attention to Pcnt and ciliogenesis was analyzed. We show reduced expression of Syne-2 in the retina of the Syne-2 KO mouse but found no significant structural-and only a minor functional-phenotype. For the first time, detailed expression analyses showed an expression of a Syne-2 protein larger than 400 kDa (similar to 750 kDa) in the Syne-2/Nesprin-2 KO mouse. In conclusion, the lack of an overt phenotype in Syne-2/Nesprin-2 KO mice suggests the usage of alternative translational start sites, producing Syne-2 splice variants with an intact Pcnt interaction site. Nevertheless, deletion of the actin-binding site in the Syne-2/Nesprin-2 KO mouse revealed a high variability in scotopic oscillatory potentials assuming a novel function of Syne-2 in synchronizing inner retinal processes

NKAP is a novel RS-related protein that interacts with RNA and RNA binding proteins

NKAP is a highly conserved protein with roles in transcriptional repression, T-cell development, maturation and acquisition of functional competency and maintenance and survival of adult hematopoietic stem cells. Here we report the novel role of NKAP in splicing. With NKAP-specific antibodies we found that NKAP localizes to nuclear speckles. NKAP has an RS motif at the N-terminus followed by a highly basic domain and a DUF 926 domain at the C-terminal region. Deletion analysis showed that the basic domain is important for speckle localization. In pull-down experiments, we identified RNA-binding proteins, RNA helicases and splicing factors as interaction partners of NKAP, among them FUS/TLS. The FUS/TLS-NKAP interaction takes place through the RS domain of NKAP and the RGG1 and RGG3 domains of FUS/TLS. We analyzed the ability of NKAP to interact with RNA using in vitro splicing assays and found that NKAP bound both spliced messenger RNA (mRNA) and unspliced pre-mRNA. Genome-wide analysis using crosslinking and immunoprecipitation-seq revealed NKAP association with U1, U4 and U5 small nuclear RNA, and we also demonstrated that knockdown of NKAP led to an increase in pre-mRNA percentage. Our results reveal NKAP as nuclear speckle protein with roles in RNA splicing and processing

Nonsteroidal anti-inflammatory drug sulindac sulfide suppresses structural protein Nesprin-2 expression in colorectal cancer cells

Background: Nonsteroidal anti-inflammatory drugs (NSAIDs) are well known for treating inflammatory disease and have been reported to have anti-tumorigenic effects. Their mechanisms are not fully understood, but both cyclooxygenase (COX) dependent and independent pathways are involved. Our goal was to shed further light on COX-independent activity. Methods: Human colorectal cancer cells were observed under differential interference contrast microscopy (DICM), fluorescent microscopy, and micro-impedance measurement. Microarray analysis was performed using HCT-116 cells treated with sulindac sulfide (SS). PCR and Western blots were performed to confirm the microarray data and immunohistochemisty was performed to screen for Nesprin-2 expression. Micro-impedance was repeating including Nesprin-2 knock-down by siRNA. Results: HCT-116 cells treated with SS showed dramatic morphological changes under DICM and fluorescent microscopy, as well as weakened cellular adhesion as measured by micro-impedance. Nesprin-2 was selected from two independent microarrays, based on its novelty in relation to cancer and its role in cell organization. SS diminished Nesprin-2 mRNA expression as assessed by reverse transcriptase and real time PCR. Various other NSAIDs were also tested and demonstrated that inhibition of Nesprin-2 mRNA was not unique to SS. Additionally, immunohistochernistry showed higher levels of Nesprin-2 in many tumors in comparison with normal tissues. Further micro-impedance experiments on cells with reduced Nesprin-2 expression showed a proportional loss of cellular adhesion. Conclusions: Nesprin-2 is down-regulated by NSAIDs and highly expressed in many cancers. General significance: Our data suggest that Nesprin-2 may be a potential novel oncogene in human cancer cells and NSAIDs could decrease its expression. (C) 2013 Elsevier B.V. All rights reserved

A novel homozygous splicing mutation of CASC5 causes primary microcephaly in a large Pakistani family

Primary microcephaly is a disorder characterized by a small head and brain associated with impaired cognitive capabilities. Mutations in 13 different genes encoding centrosomal proteins and cell cycle regulators have been reported to cause the disease. CASC5, a gene encoding a protein important for kinetochore formation and proper chromosome segregation during mitosis, has been suggested to be associated with primary microcephaly-4 (MCPH4). This was based on one mutation only and circumstantial functional evidence. By combining homozygosity mapping and whole-exome sequencing in an MCPH family from Pakistan, we identified a second mutation (NM_170589.4;c.6673-19T > A) in CASC5. This mutation induced skipping of exon 25 of CASC5 resulting in a frameshift and the introduction of a premature stop codon (p.Met2225Ilefs*7). The C-terminally truncated protein lacks 118 amino acids that encompass the region responsible for the interaction with the hMIS12 complex, which is essential for proper chromosome alignment and segregation. Furthermore, we showed a down-regulation of CASC5 mRNA and reduction of the amount of CASC5 protein by quantitative RT-PCR and western blot analysis, respectively. As a further sign of functional deficits, we observed dispersed dots of CASC5 immunoreactive material outside the metaphase plate of dividing patient fibroblasts. Normally, CASC5 is a component of the kinetochore of metaphase chromosomes. A higher mitotic index in patient cells indicated a mitotic arrest in the cells carrying the mutation. We also observed lobulated and fragmented nuclei as well as micronuclei in the patient cells. Moreover, we detected an altered DNA damage response with higher levels of gamma H2AX and 53BP1 in mutant as compared to control fibroblasts. Our findings substantiate the proposed role of CASC5 for primary microcephaly and suggest that it also might be relevant for genome stability