Diverse molecular mechanisms involved in AChR deficiency due to rapsyn mutations

Congenital myasthenic syndromes are inherited disorders of neuromuscular transmission characterized by fatigable muscle weakness. Autosomal recessive acetylcholine receptor (AChR) deficiency syndromes, in which levels of this receptor at the neuromuscular junction are severely reduced, may be caused by mutations within genes encoding the AChR or the AChR-clustering protein, rapsyn. Most patients have mutations within the rapsyn coding region and are either homozygous for N88K or heteroallelic for N88K and a second mutation. In some cases the second allele carries a null mutation but in many the mutations are missense, and are located in different functional domains. Little is known about the functional effects of these mutations, but we hypothesize that they would have an effect on AChR clustering by a variety of mechanisms that might correlate with disease severity. Here we expressed RAPSN mutations A25V, N88K, R91L, L361R and K373del in TE671 cells and in rapsyn−/− myotubes to determine their pathogenic mechanisms. The A25Vmutation impaired colocalization of rapsyn with AChR and prevented agrin-induced AChR clusters in rapsyn−/− myotubes. In TE671 cells, R91L reduced the ability of rapsyn to self-associate, and K373del-rapsyn was significantly less stable than wild-type. The effects of mutations L361R and N88K were more subtle: in TE671 cells, in comparison with wild-type rapsyn, L361R-rapsyn showed reduced expression/stability, and both N88K-rapsyn and L361R-rapsyn showed significantly reduced co-localization with AChR. N88K-rapsyn and L361R-rapsyn could effectively mediate agrin-induced AChR clusters, but these were reduced in number and were less stable than with wild-type rapsyn. The disease severity of patients harbouring the compound allelic mutations was greater than that of patients with homozygous rapsyn mutation N88K, suggesting that the second mutant allele may largely determine severit

FOXP2-positive diffuse large B-cell lymphomas exhibit a poor response to R-CHOP therapy and distinct biological signatures.

FOXP2 shares partially overlapping normal tissue expression and functionality with FOXP1; an established diffuse large B-cell lymphoma (DLBCL) oncogene and marker of poor prognosis. FOXP2 is expressed in the plasma cell malignancy multiple myeloma but has not been studied in DLBCL, where a poor prognosis activated B-cell (ABC)-like subtype display partially blocked plasma cell differentiation. FOXP2 protein expression was detected in ABC-DLBCL cell lines, and in primary DLBCL samples tumoral FOXP2 protein expression was detected in both germinal center B-cell-like (GCB) and non-GCB DLBCL. In biopsies from DLBCL patients treated with immunochemotherapy (R-CHOP), ≥ 20% nuclear tumoral FOXP2-positivity (n = 24/158) correlated with significantly inferior overall survival (OS: P = 0.0017) and progression-free survival (PFS: P = 0.0096). This remained significant in multivariate analysis against either the international prognostic index score or the non-GCB DLBCL phenotype (P < 0.05 for both OS and PFS). Expression of BLIMP1, a marker of plasmacytic differentiation that is commonly inactivated in ABC-DLBCL, did not correlate with patient outcome or FOXP2 expression in this series. Increased frequency of FOXP2 expression significantly correlated with FOXP1-positivity (P = 0.0187), and FOXP1 co-immunoprecipitated FOXP2 from ABC-DLBCL cells indicating that these proteins can co-localize in a multi-protein complex. FOXP2-positive DLBCL had reduced expression of HIP1R (P = 0.0348), which is directly repressed by FOXP1, and exhibited distinct patterns of gene expression. Specifically in ABC-DLBCL these were associated with lower expression of immune response and T-cell receptor signaling pathways. Further studies are warranted to investigate the potential functional cooperativity between FOXP1 and FOXP2 in repressing immune responses during the pathogenesis of high-risk DLBCL

Congenital myasthenic syndromes and the formation of the neuromuscular junction

The congenital myasthenic syndromes (CMS) are a heterogeneous group of disorders affecting neuromuscular transmission. Underlying mutations have been identified in at least 11 different genes. The majority of the CMS patients have disorders due to mutations in postsynaptic proteins. Initial studies focused on dysfunction of the acetylcholine receptor (AChR) itself as the major cause of CMS. However, it is becoming apparent that mutations of proteins involved in clustering the AChR and maintaining neuromuscular junction structure form important subgroups. Analysis of the mutations in the AChR-clustering protein, rapsyn, show diverse causes for defective AChR localization and suggest that the common mutation rapsyn-N88K results in AChR clusters that are less stable than those generated by wild-type rapsyn. More recently, mutations in the newly identified endplate protein Dok-7 have been shown to affect AChR clustering and the generation and maintenance of specialized structures at the endplate. Dok-7 binds MuSK and many of the mutations of DOK7 impair the MuSK signaling pathway. Components of this pathway will provide attractive gene candidates for additional forms of CMS. The phenoypic characteristics of the different CMS in which muscle groups may be differentially affected not only provide clues for targeted genetic screening, but also pose further intriguing questions about underlying molecular mechanisms

Confluent growth arrest is associated with increased FOXP2 expression.

<p>(A) Cell cycle analysis of 143B subjected to increasing confluence (approximate percentage confluence indicated top), numbers within plots from left to right indicate percentage of cells in G1/G0, S, and G2/M phase respectively, representative of three experiments; (B) Quantitation of apoptotic cell death in 143B cell populations by flow cytometric analysis of Annexin V positivity, in cultures either exponentially growing (Exp. Growth), subjected to overnight culture with 20μg/ml cyclohexmide as a positive control that induces apoptosis (cycloheximide), or subjected to 4 days growth arrest at confluence (late arrest, as per <i>A</i>). Numbers represent mean % annexin positive ± SD from three experiments; (C) Real-time PCR analyses of <i>FOXP</i> expression in MG-63, 143B and U2-OS cultured to increasing confluence, expressed as 2^-δβCT, relative to growing culture, <i>N</i> = 3 ± SD; (D) Immunoblot analyses of nuclear extracts from cells cultured as in <i>C</i>, including nucleophosmin (NPM) as a loading and transfer control, representative of two experiments, (E) Real-time PCR analyses of <i>p21</i>, <i>p27</i> and <i>IL-6</i> expression in 143B cultured to increasing confluence, expressed as 2^-δδCT, relative to growing culture, <i>N</i> = 3 ± SD.</p

Foxp2/FOXP2 expression in murine bone and association with reduced growth of osteosarcoma cell lines.

<p>(A,B) Real-time PCR analysis of gene expression in primary murine tissues from 12-week old male C57Bl/6 mice using SyBr-green, specifically whole bone marrow (BM), spleen (Spl), thymus (Thy), whole long bones after flushing and 2 rounds of collagenase digestion (Bone) and bone-associated cells from the collagenase fraction (CF). Data represent mean +/- SD of three mice. While <i>Foxp1</i> and <i>Foxp4</i> are most expressed in <i>CD45</i>+ haematopoietic tissues, <i>Foxp2</i> expression is highest in bone as are the established osteoblast genes <i>Ibsp</i> and <i>Sp7</i>. Expression normalised to <i>Hprt</i>, expressed relative to highest sample (100%); (C) Immunoblot analysis of COS-1 fibroblast-like cells transiently transfected with CMV-driven mammalian expression plasmids containing murine full-length Foxp cDNAs or pCDNA4 empty vector (control) as indicated top. All antibodies exhibited specificity for appropriate ectopically-expressed proteins, low level endogenous FOXP4 expression was detectable in all lysates; (D) Immunohistochemical detection of Foxp2 protein in murine E17.5 long bone, detail (middle panels) showing variation in Foxp2 positivity along the periosteum, staining with the anti-rabbit murine monoclonal antibody MR12 was performed on serial sections as negative control (same regions, right panels)</p

Growth arrest-induced FOXP2 transcription is upstream of the cell cycle machinery.

<p>(A, C) Cell cycle analyses of 143B grown for 6 days in reduced serum or 1 day in the CDK4/6 inhibitor Palbociclib 100 nM or 1 μM as indicated; (B, D) Real-time PCR analyses of gene expression in 143B cultured as in <i>A</i> and <i>C</i>, expressed as 2^-δδCT, relative to growing or vehicle-treated culture, <i>N</i> = 3 ± SD; (E) Real-time PCR analyses of gene expression in 143B reduced serum experiments similar to <i>A</i> over a shorter timecourse, <i>N</i> = 3 ± SD; (F) Real-time PCR analysis of <i>FOXP2</i> expression in 143B cultured at subconfluence or confluence for 24hrs in the presence of inhibitors/vehicle as indicated, (PD-98059 ERK1/2 inhibitor and LY-294002 PI3K inhibitor at 50μM, IKK inhibitor 7, Bay117082 NFκB inhibitor, and DBZ Notch pathway inhibitor at 1μM), expressed as fold change induced by confluence, inhibitors had minimal effect on subconfluent <i>FOXP2</i> expression, <i>N</i> = 5 ± SD; (G) Immunoblot analysis of nuclear extracts from 143B treated at confluence as in <i>F</i>, including nucleophosmin (NPM) as a loading and transfer control.</p

FOXP2 induction is required for efficient 143B growth arrest and p21 ^CIP1/WAF1 control.

<p>(A) Immunoblot analysis of FOXP expression in nuclear extracts from confluent 143B cells 48hr after siRNA transfection, including TATA-binding protein (TBP) as a loading and transfer control, representative of three experiments; (B) Quantitation of changes in cell cycle fractions induced by confluence as per <i>A</i>, relative to dividing cells, <i>N</i> = 3 ± SD; (C) Phase contrast images of cells as in <i>A</i>, to show frequently increased saturation density following FOXP2 depletion;; (D) Real-time PCR analysis of <i>p21/CDKN1A</i> expression in 143B cultured as in <i>A</i>, expressed as 2^-δδCT, relative to subconfluent culture (T = 0), control is mean of four different control siRNAs. <i>N</i> = at least 4 ± SD; (E) Immunoblot analyses of whole cell lysates from cells cultured as in <i>A</i>; (F) Real-time PCR analysis of <i>p21CDKN1A</i> expression in cells transfected at subconfluence (T = 0) with siRNAs as shown and harvested still at subconfluence after 48 hr culture in 1% serum, <i>N</i> = 3 ± SD; (G) Real-time PCR analysis of <i>p21CDKN1A</i> expression in and phase contrast images of cells cultured as in <i>A</i>, in the presence of either 10ng/ml hIL-6 (+ IL-6) or PBS/BSA carrier alone (—IL-6); (H) Real-time PCR analyses of cells transfected at subconfluence with p53 cDNA plasmid or empty control plasmid (vector), then treated with siLGC or each FOXP2 siRNA and harvested still at subconfluence after 48hr culture in 10% serum. Expression is shown as percentage relative to highest (100%).</p

Mutation Analysis of CHRNA1, CHRNB1, CHRND, and RAPSN Genes in Multiple Pterygium Syndrome/Fetal Akinesia Patients

Multiple pterygium syndromes (MPS) comprise a group of multiple congenital anomaly disorders characterized by webbing (pterygia) of the neck, elbows, and/or knees and joint contractures (arthrogryposis). MPS are phenotypically and genetically heterogeneous but are traditionally divided into prenatally lethal and nonlethal (Escobar) types. Previously, we and others reported that recessive mutations in the embryonal acetylcholine receptor g subunit (CHRNG) can cause both lethal and nonlethal MPS, thus demonstrating that pterygia resulted from fetal akinesia. We hypothesized that mutations in acetylcholine receptor-related genes might also result in a MPS/fetal akinesia phenotype and so we analyzed 15 cases of lethal MPS/fetal akinesia without CHRNG mutations for mutations in the CHRNA1, CHRNB1, CHRND, and rapsyn (RAPSN) genes. No CHRNA1, CHRNB1, or CHRND mutations were detected, but a homozygous RAPSN frameshift mutation, c.1177-1178delAA, was identified in a family with three children affected with lethal fetal akinesia sequence. Previously, RAPSN mutations have been reported in congenital myasthenia. Functional studies were consistent with the hypothesis that whereas incomplete loss of rapsyn function may cause congenital myasthenia, more severe loss of function can result in a lethal fetal akinesia phenotype