Myelin basic protein mRNA levels affect myelin sheath dimensions, architecture, plasticity, and density of resident glial cells.

Myelin basic protein (Mbp) is essential for both elaboration and maintenance of CNS myelin, and its reduced accumulation results in hypomyelination. How different Mbp mRNA levels affect myelin dimensions across the lifespan and how resident glial cells may respond to such changes are unknown. Here, to investigate these questions, we used enhancer-edited mouse lines that accumulate Mbp mRNA levels ranging from 8% to 160% of wild type. In young mice, reduced Mbp mRNA levels resulted in corresponding decreases in Mbp protein accumulation and myelin sheath thickness, confirming the previously demonstrated rate-limiting role of Mbp transcription in the control of initial myelin synthesis. However, despite maintaining lower line specific Mbp mRNA levels into old age, both Mbp protein levels and myelin thickness improved or fully normalized at rates defined by the relative Mbp mRNA level. Sheath length, in contrast, was affected only when mRNA levels were very low, demonstrating that sheath thickness and length are not equally coupled to Mbp mRNA level. Striking abnormalities in sheath structure also emerged with reduced mRNA levels. Unexpectedly, an increase in the density of all glial cell types arose in response to reduced Mbp mRNA levels. This investigation extends understanding of the role Mbp plays in myelin sheath elaboration, architecture, and plasticity across the mouse lifespan and illuminates a novel axis of glial cell crosstalk.

Transcriptional regulators of the Golli/myelin basic protein locus integrate additive and stealth activities.

Myelin is composed of plasma membrane spirally wrapped around axons and compacted into dense sheaths by myelin-associated proteins. Myelin is elaborated by neuroepithelial derived oligodendrocytes in the central nervous system (CNS) and by neural crest derived Schwann cells in the peripheral nervous system (PNS). While some myelin proteins accumulate in only one lineage, myelin basic protein (Mbp) is expressed in both. Overlapping the Mbp gene is Golli, a transcriptional unit that is expressed widely both within and beyond the nervous system. A super-enhancer domain within the Golli/Mbp locus contains multiple enhancers shown previously to drive reporter construct expression specifically in oligodendrocytes or Schwann cells. In order to determine the contribution of each enhancer to the Golli/Mbp expression program, and to reveal if functional interactions occur among them, we derived mouse lines in which they were deleted, either singly or in different combinations, and relative mRNA accumulation was measured at key stages of early development and at maturity. Although super-enhancers have been shown previously to facilitate interaction among their component enhancers, the enhancers investigated here demonstrated largely additive relationships. However, enhancers demonstrating autonomous activity strictly in one lineage, when missing, were found to significantly reduce output in the other, thus revealing cryptic "stealth" activity. Further, in the absence of a key oligodendrocyte enhancer, Golli accumulation was markedly and uniformly attenuated in all cell types investigated. Our observations suggest a model in which enhancer-mediated DNA-looping and potential super-enhancer properties underlie Golli/Mbp regulatory organization.

Self-labelled encoder-decoder (SLED) for multi-echo gradient echo-based myelin water imaging.

PURPOSE: Reconstruction of high quality myelin water imaging (MWI) maps is challenging, particularly for data acquired using multi-echo gradient echo (mGRE) sequences. A non-linear least squares fitting (NLLS) approach has often been applied for MWI. However, this approach may produce maps with limited detail and, in some cases, sub-optimal signal to noise ratio (SNR), due to the nature of the voxel-wise fitting. In this study, we developed a novel, unsupervised learning method called self-labelled encoder-decoder (SLED) to improve gradient echo-based MWI data fitting. METHODS: Ultra-high resolution, MWI data was collected from five mouse brains with variable levels of myelination, using a mGRE sequence. Imaging data was acquired using a 7T preclinical MRI system. A self-labelled, encoder-decoder network was implemented in TensorFlow for calculation of myelin water fraction (MWF) based on the mGRE signal decay. A simulated MWI phantom was also created to evaluate the performance of MWF estimation. RESULTS: Compared to NLLS, SLED demonstrated improved MWF estimation, in terms of both stability and accuracy in phantom tests. In addition, SLED produced less noisy MWF maps from high resolution MR microscopy images of mouse brain tissue. It specifically resulted in lower noise amplification for all mouse genotypes that were imaged and yielded mean MWF values in white matter ROIs that were highly correlated with those derived from standard NLLS fitting. Lastly, SLED also exhibited higher tolerance to low SNR data. CONCLUSION: Due to its unsupervised and self-labeling nature, SLED offers a unique alternative to analyze gradient echo-based MWI data, providing accurate and stable MWF estimations.

X Chromosome Contribution to the Genetic Architecture of Primary Biliary Cholangitis.

BACKGROUND & AIMS: Genome-wide association studies in primary biliary cholangitis (PBC) have failed to find X chromosome (chrX) variants associated with the disease. Here, we specifically explore the chrX contribution to PBC, a sexually dimorphic complex autoimmune disease. METHODS: We performed a chrX-wide association study, including genotype data from 5 genome-wide association studies (from Italy, United Kingdom, Canada, China, and Japan; 5244 case patients and 11,875 control individuals). RESULTS: Single-marker association analyses found approximately 100 loci displaying P < 5 × 10-4, with the most significant being a signal within the OTUD5 gene (rs3027490; P = 4.80 × 10-6; odds ratio [OR], 1.39; 95% confidence interval [CI], 1.028-1.88; Japanese cohort). Although the transethnic meta-analysis evidenced only a suggestive signal (rs2239452, mapping within the PIM2 gene; OR, 1.17; 95% CI, 1.09-1.26; P = 9.93 × 10-8), the population-specific meta-analysis showed a genome-wide significant locus in East Asian individuals pointing to the same region (rs7059064, mapping within the GRIPAP1 gene; P = 6.2 × 10-9; OR, 1.33; 95% CI, 1.21-1.46). Indeed, rs7059064 tags a unique linkage disequilibrium block including 7 genes: TIMM17B, PQBP1, PIM2, SLC35A2, OTUD5, KCND1, and GRIPAP1, as well as a superenhancer (GH0XJ048933 within OTUD5) targeting all these genes. GH0XJ048933 is also predicted to target FOXP3, the main T-regulatory cell lineage specification factor. Consistently, OTUD5 and FOXP3 RNA levels were up-regulated in PBC case patients (1.75- and 1.64-fold, respectively). CONCLUSIONS: This work represents the first comprehensive study, to our knowledge, of the chrX contribution to the genetics of an autoimmune liver disease and shows a novel PBC-related genome-wide significant locus.

An international genome-wide meta-analysis of primary biliary cholangitis: Novel risk loci and candidate drugs.

BACKGROUNDS & AIMS: Primary biliary cholangitis (PBC) is a chronic liver disease in which autoimmune destruction of the small intrahepatic bile ducts eventually leads to cirrhosis. Many patients have inadequate response to licensed medications, motivating the search for novel therapies. Previous genome-wide association studies (GWAS) and meta-analyses (GWMA) of PBC have identified numerous risk loci for this condition, providing insight into its aetiology. We undertook the largest GWMA of PBC to date, aiming to identify additional risk loci and prioritise candidate genes for in silico drug efficacy screening. METHODS: We combined new and existing genotype data for 10,516 cases and 20,772 controls from 5 European and 2 East Asian cohorts. RESULTS: We identified 56 genome-wide significant loci (20 novel) including 46 in European, 13 in Asian, and 41 in combined cohorts; and a 57th genome-wide significant locus (also novel) in conditional analysis of the European cohorts. Candidate genes at newly identified loci include FCRL3, INAVA, PRDM1, IRF7, CCR6, CD226, and IL12RB1, which each play key roles in immunity. Pathway analysis reiterated the likely importance of pattern recognition receptor and TNF signalling, JAK-STAT signalling, and differentiation of T helper (TH)1 and TH17 cells in the pathogenesis of this disease. Drug efficacy screening identified several medications predicted to be therapeutic in PBC, some of which are well-established in the treatment of other autoimmune disorders. CONCLUSIONS: This study has identified additional risk loci for PBC, provided a hierarchy of agents that could be trialled in this condition, and emphasised the value of genetic and genomic approaches to drug discovery in complex disorders. LAY SUMMARY: Primary biliary cholangitis (PBC) is a chronic liver disease that eventually leads to cirrhosis. In this study, we analysed genetic information from 10,516 people with PBC and 20,772 healthy individuals recruited in Canada, China, Italy, Japan, the UK, or the USA. We identified several genetic regions associated with PBC. Each of these regions contains several genes. For each region, we used diverse sources of evidence to help us choose the gene most likely to be involved in causing PBC. We used these 'candidate genes' to help us identify medications that are currently used for treatment of other conditions, which might also be useful for treatment of PBC.

A modifier in the 129S2/SvPasCrl genome is responsible for the viability of Notch1[12f/12f] mice.

BACKGROUND: Mouse NOTCH1 carries a highly conserved O-fucose glycan at Thr466 in epidermal growth factor-like repeat 12 (EGF12) of the extracellular domain. O-Fucose at this site has been shown by X-ray crystallography to be recognized by both DLL4 and JAG1 Notch ligands. We previously showed that a Notch1 Thr466Ala mutant exhibits very little ligand-induced NOTCH1 signaling in a reporter assay, whereas a Thr466Ser mutation enables the transfer of O-fucose and reverts the NOTCH1 signaling defect. We subsequently generated a mutant mouse with the Thr466Ala mutation termed Notch1[12f](Notch1tm2Pst). Surprisingly, homozygous Notch1[12f/12f] mutants on a mixed background were viable and fertile. RESULTS: We now report that after backcrossing to C57BL/6 J mice for 11-15 generations, few homozygous Notch1[12f/12f] embryos were born. Timed mating showed that embryonic lethality occurred by embryonic day (E) ~E11.5, somewhat delayed compared to mice lacking Notch1 or Pofut1 (the O-fucosyltransferase that adds O-fucose to Notch receptors), which die at ~E9.5. The phenotype of C57BL/6 J Notch1[12f/12f] embryos was milder than mutants affected by loss of a canonical Notch pathway member, but disorganized vasculogenesis in the yolk sac, delayed somitogenesis and development were characteristic. In situ hybridization of Notch target genes Uncx4.1 and Dll3 or western blot analysis of NOTCH1 cleavage did not reveal significant differences at E9.5. However, qRT-PCR of head cDNA showed increased expression of Dll3, Uncx4.1 and Notch1 in E9.5 Notch1[12f/12f] embryos. Sequencing of cDNA from Notch1[12f/12f] embryo heads and Southern analysis showed that the Notch1[12f] locus was intact following backcrossing. We therefore looked for evidence of modifying gene(s) by crossing C57BL/6 J Notch1 [12f/+] mice to 129S2/SvPasCrl mice. Intercrosses of the F1 progeny gave viable F2 Notch1[12f/12f] mice. CONCLUSION: We conclude that the 129S2/SvPasCrl genome contains a dominant modifying gene that rescues the functions of NOTCH1[12f] in signaling. Identification of the modifying gene has the potential to illuminate novel factor(s) that promote Notch signaling when an O-fucose glycan is absent from EGF12 of NOTCH1.

Variation at DENND1B and Asthma on the Island of Tristan da Cunha.

A high prevalence of asthma has been documented among the inhabitants of Tristan da Cunha, an isolated island in the South Atlantic. The population derives from just 28 founders. We performed lung function testing, including methacholine inhalation challenge, allergen skin prick testing, and collected DNA from essentially all of the current island population (269 individuals), and genotyped a panel of 43 single-nucleotide polymorphisms (SNPs) reported as associated with asthma and atopy. We carried out a mixed-model association analysis using the known pedigree. There were 96 individuals diagnosed as asthmatic (36%), and heritability estimates were similar to those from nonisolated population samples (multifactorial threshold model, h2 = 48%). The first component from a genetic principal components analysis using the entire SNP panel was nonlinearly associated with asthma, with the maximum risk to those intermediate to reference (Human Genome Diversity Project) European and African samples means. The single most strongly associated SNP was rs2786098 (p = 5.5 × 10-5), known to regulate the gene DENND1B. This explained approximately one-third of the trait heritability, with an allelic odds ratio for the A allele of 2.6. Among A/A carriers, 10 out of 12 individuals were asthmatic. The rs2786098*A variant was initially reported to decrease the risk of childhood (atopic) asthma in European but slightly increase the risk in African-descended populations, and does significantly alter Th2 cell function. Despite an absence of overall association with this variant in recent asthma genome wide association studies meta-analyses, an effect may exist on the particular genetic background of the Tristan da Cunha population.

The Arabidopsis SWI2/SNF2 chromatin Remodeler BRAHMA regulates polycomb function during vegetative development and directly activates the flowering repressor gene SVP.

The chromatin remodeler BRAHMA (BRM) is a Trithorax Group (TrxG) protein that antagonizes the functions of Polycomb Group (PcG) proteins in fly and mammals. Recent studies also implicate such a role for Arabidopsis (Arabidopsis thaliana) BRM but the molecular mechanisms underlying the antagonism are unclear. To understand the interplay between BRM and PcG during plant development, we performed a genome-wide analysis of trimethylated histone H3 lysine 27 (H3K27me3) in brm mutant seedlings by chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq). Increased H3K27me3 deposition at several hundred genes was observed in brm mutants and this increase was partially supressed by removal of the H3K27 methyltransferase CURLY LEAF (CLF) or SWINGER (SWN). ChIP experiments demonstrated that BRM directly binds to a subset of the genes and prevents the inappropriate association and/or activity of PcG proteins at these loci. Together, these results indicate a crucial role of BRM in restricting the inappropriate activity of PcG during plant development. The key flowering repressor gene SHORT VEGETATIVE PHASE (SVP) is such a BRM target. In brm mutants, elevated PcG occupancy at SVP accompanies a dramatic increase in H3K27me3 levels at this locus and a concomitant reduction of SVP expression. Further, our gain- and loss-of-function genetic evidence establishes that BRM controls flowering time by directly activating SVP expression. This work reveals a genome-wide functional interplay between BRM and PcG and provides new insights into the impacts of these proteins in plant growth and development.

Klinefelter's syndrome (47,XXY) is in excess among men with Sjögren's syndrome.

Primary Sjögren's syndrome (pSS) has a strong female bias. We evaluated an X chromosome dose effect by analyzing 47,XXY (Klinefelter's syndrome, 1 in 500 live male births) among subjects with pSS. 47,XXY was determined by examination of fluorescence intensity of single nucleotide polymorphisms from the X and Y chromosomes. Among 136 pSS men there were 4 with 47,XXY. This was significantly different from healthy controls (1 of 1254 had 47,XXY, p=0.0012 by Fisher's exact test) as well men with rheumatoid arthritis (0 of 363 with 47,XXY), but not different compared to men with systemic lupus erythematosus (SLE) (4 of 136 versus 8 of 306, Fisher's exact test p=NS). These results are consistent with the hypothesis that the number of X chromosomes is critical for the female bias of pSS, a property that may be shared with SLE but not RA.

Rare, low-frequency, and common variants in the protein-coding sequence of biological candidate genes from GWASs contribute to risk of rheumatoid arthritis.

The extent to which variants in the protein-coding sequence of genes contribute to risk of rheumatoid arthritis (RA) is unknown. In this study, we addressed this issue by deep exon sequencing and large-scale genotyping of 25 biological candidate genes located within RA risk loci discovered by genome-wide association studies (GWASs). First, we assessed the contribution of rare coding variants in the 25 genes to the risk of RA in a pooled sequencing study of 500 RA cases and 650 controls of European ancestry. We observed an accumulation of rare nonsynonymous variants exclusive to RA cases in IL2RA and IL2RB (burden test: p = 0.007 and p = 0.018, respectively). Next, we assessed the aggregate contribution of low-frequency and common coding variants to the risk of RA by dense genotyping of the 25 gene loci in 10,609 RA cases and 35,605 controls. We observed a strong enrichment of coding variants with a nominal signal of association with RA (p < 0.05) after adjusting for the best signal of association at the loci (p(enrichment) = 6.4 × 10(-4)). For one locus containing CD2, we found that a missense variant, rs699738 (c.798C>A [p.His266Gln]), and a noncoding variant, rs624988, reside on distinct haplotypes and independently contribute to the risk of RA (p = 4.6 × 10(-6)). Overall, our results indicate that variants (distributed across the allele-frequency spectrum) within the protein-coding portion of a subset of biological candidate genes identified by GWASs contribute to the risk of RA. Further, we have demonstrated that very large sample sizes will be required for comprehensively identifying the independent alleles contributing to the missing heritability of RA.

Human genetics in rheumatoid arthritis guides a high-throughput drug screen of the CD40 signaling pathway.

Although genetic and non-genetic studies in mouse and human implicate the CD40 pathway in rheumatoid arthritis (RA), there are no approved drugs that inhibit CD40 signaling for clinical care in RA or any other disease. Here, we sought to understand the biological consequences of a CD40 risk variant in RA discovered by a previous genome-wide association study (GWAS) and to perform a high-throughput drug screen for modulators of CD40 signaling based on human genetic findings. First, we fine-map the CD40 risk locus in 7,222 seropositive RA patients and 15,870 controls, together with deep sequencing of CD40 coding exons in 500 RA cases and 650 controls, to identify a single SNP that explains the entire signal of association (rs4810485, P = 1.4×10(-9)). Second, we demonstrate that subjects homozygous for the RA risk allele have ∼33% more CD40 on the surface of primary human CD19+ B lymphocytes than subjects homozygous for the non-risk allele (P = 10(-9)), a finding corroborated by expression quantitative trait loci (eQTL) analysis in peripheral blood mononuclear cells from 1,469 healthy control individuals. Third, we use retroviral shRNA infection to perturb the amount of CD40 on the surface of a human B lymphocyte cell line (BL2) and observe a direct correlation between amount of CD40 protein and phosphorylation of RelA (p65), a subunit of the NF-κB transcription factor. Finally, we develop a high-throughput NF-κB luciferase reporter assay in BL2 cells activated with trimerized CD40 ligand (tCD40L) and conduct an HTS of 1,982 chemical compounds and FDA-approved drugs. After a series of counter-screens and testing in primary human CD19+ B cells, we identify 2 novel chemical inhibitors not previously implicated in inflammation or CD40-mediated NF-κB signaling. Our study demonstrates proof-of-concept that human genetics can be used to guide the development of phenotype-based, high-throughput small-molecule screens to identify potential novel therapies in complex traits such as RA.

Identification of secreted phosphoprotein 1 gene as a new rheumatoid arthritis susceptibility gene.

OBJECTIVE: To evaluate the contribution of the SPP1 rs11439060 and rs9138 polymorphisms, previously reported as autoimmune risk variants, in the rheumatoid arthritis (RA) genetic background according to anti-citrullinated protein antibodies (ACPAs) status of RA individuals. METHODS: We analysed a total of 11,715 RA cases and 26,493 controls from nine independent cohorts; all individuals were genotyped or had imputed genotypes for SPP1 rs11439060 and rs9138. The effect of the SPP1 rs11439060 and rs9138 risk-allele combination on osteopontin (OPN) expression in macrophages and OPN serum levels was investigated. RESULTS: We provide evidence for a distinct contribution of SPP1 to RA susceptibility according to ACPA status: the combination of ≥3 SPP1 rs11439060 and rs9138 common alleles was associated mainly with ACPA negativity (p=1.29×10(-5), ORACPA-negative 1.257 (1.135 to 1.394)) and less with ACPA positivity (p=0.0148, ORACPA-positive 1.072 (1.014 to 1.134)). The ORs between these subgroups (ie, ACPA-positive and ACPA-negative) significantly differed (p=7.33×10(-3)). Expression quantitative trait locus analysis revealed an association of the SPP1 risk-allele combination with decreased SPP1 expression in peripheral macrophages from 599 individuals. To corroborate these findings, we found an association of the SPP1 risk-allele combination and low serum level of secreted OPN (p=0.0157), as well as serum level of secreted OPN correlated positively with ACPA production (p=0.005; r=0.483). CONCLUSIONS: We demonstrate a significant contribution of the combination of SPP1 rs11439060 and rs9138 frequent alleles to risk of RA, the magnitude of the association being greater in patients negative for ACPAs.

Hepatocyte-specific Ptpn6 deletion promotes hepatic lipid accretion, but reduces NAFLD in diet-induced obesity: potential role of PPARγ.

UNLABELLED: Hepatocyte-specific Shp1 knockout mice (Ptpn6(H-KO)) are protected from hepatic insulin resistance evoked by high-fat diet (HFD) feeding for 8 weeks. Unexpectedly, we report herein that Ptpn6(H-KO) mice fed an HFD for up to 16 weeks are still protected from insulin resistance, but are more prone to hepatic steatosis, as compared with their HFD-fed Ptpn6(f/f) counterparts. The livers from HFD-fed Ptpn6(H-KO) mice displayed 1) augmented lipogenesis, marked by increased expression of several hepatic genes involved in fatty acid biosynthesis, 2) elevated postprandial fatty acid uptake, and 3) significantly reduced lipid export with enhanced degradation of apolipoprotein B (ApoB). Despite more extensive hepatic steatosis, the inflammatory profile of the HFD-fed Ptpn6(H-KO) liver was similar (8 weeks) or even improved (16 weeks) as compared to their HFD-fed Ptpn6(f/f) littermates, along with reduced hepatocellular damage as revealed by serum levels of hepatic enzymes. Interestingly, comparative microarray analysis revealed a significant up-regulation of peroxisome proliferator-activated receptor gamma (PPARγ) gene expression, confirmed by quantitative polymerase chain reaction. Elevated PPARγ nuclear activity also was observed and found to be directly regulated by Shp1 in a cell-autonomous manner. CONCLUSION: These findings highlight a novel role for hepatocyte Shp1 in the regulation of PPARγ and hepatic lipid metabolism. Shp1 deficiency prevents the development of severe hepatic inflammation and hepatocellular damage in steatotic livers, presenting hepatocyte Shp1 as a potential novel mediator of nonalcoholic fatty liver diseases in obesity.

Significance of left ventricular apical-basal muscle bundle identified by cardiovascular magnetic resonance imaging in patients with hypertrophic cardiomyopathy.

AIMS: Cardiovascular magnetic resonance (CMR) has improved diagnostic and management strategies in hypertrophic cardiomyopathy (HCM) by expanding our appreciation for the diverse phenotypic expression. We sought to characterize the prevalence and clinical significance of a recently identified accessory left ventricular (LV) muscle bundle extending from the apex to the basal septum or anterior wall (i.e. apical-basal). METHODS AND RESULTS: CMR was performed in 230 genotyped HCM patients (48 ± 15 years, 69% male), 30 genotype-positive/phenotype-negative (G+/P-) family members (32 ± 15 years, 30% male), and 126 controls. Left ventricular apical-basal muscle bundle was identified in 145 of 230 (63%) HCM patients, 18 of 30 (60%) G+/P- family members, and 12 of 126 (10%) controls (G+/P- vs. controls; P < 0.01). In HCM patients, the prevalence of an apical-basal muscle bundle was similar among those with disease-causing sarcomere mutations compared with patients without mutation (64 vs. 62%; P = 0.88). The presence of an LV apical-basal muscle bundle was not associated with LV outflow tract obstruction (P = 0.61). In follow-up, 33 patients underwent surgical myectomy of whom 22 (67%) were identified to have an accessory LV apical-basal muscle bundle, which was resected in all patients. CONCLUSION: Apical-basal muscle bundles are a unique myocardial structure commonly present in HCM patients as well as in G+/P- family members and may represent an additional morphologic marker for HCM diagnosis in genotype-positive status.

Immunochip analyses identify a novel risk locus for primary biliary cirrhosis at 13q14, multiple independent associations at four established risk loci and epistasis between 1p31 and 7q32 risk variants.

To further characterize the genetic basis of primary biliary cirrhosis (PBC), we genotyped 2426 PBC patients and 5731 unaffected controls from three independent cohorts using a single nucleotide polymorphism (SNP) array (Immunochip) enriched for autoimmune disease risk loci. Meta-analysis of the genotype data sets identified a novel disease-associated locus near the TNFSF11 gene at 13q14, provided evidence for association at six additional immune-related loci not previously implicated in PBC and confirmed associations at 19 of 22 established risk loci. Results of conditional analyses also provided evidence for multiple independent association signals at four risk loci, with haplotype analyses suggesting independent SNP effects at the 2q32 and 16p13 loci, but complex haplotype driven effects at the 3q25 and 6p21 loci. By imputing classical HLA alleles from this data set, four class II alleles independently contributing to the association signal from this region were identified. Imputation of genotypes at the non-HLA loci also provided additional associations, but none with stronger effects than the genotyped variants. An epistatic interaction between the IL12RB2 risk locus at 1p31and the IRF5 risk locus at 7q32 was also identified and suggests a complementary effect of these loci in predisposing to disease. These data expand the repertoire of genes with potential roles in PBC pathogenesis that need to be explored by follow-up biological studies.

N-WASp is required for Schwann cell cytoskeletal dynamics, normal myelin gene expression and peripheral nerve myelination.

Schwann cells elaborate myelin sheaths around axons by spirally wrapping and compacting their plasma membranes. Although actin remodeling plays a crucial role in this process, the effectors that modulate the Schwann cell cytoskeleton are poorly defined. Here, we show that the actin cytoskeletal regulator, neural Wiskott-Aldrich syndrome protein (N-WASp), is upregulated in myelinating Schwann cells coincident with myelin elaboration. When N-WASp is conditionally deleted in Schwann cells at the onset of myelination, the cells continue to ensheath axons but fail to extend processes circumferentially to elaborate myelin. Myelin-related gene expression is also severely reduced in the N-WASp-deficient cells and in vitro process and lamellipodia formation are disrupted. Although affected mice demonstrate obvious motor deficits these do not appear to progress, the mutant animals achieving normal body weights and living to advanced age. Our observations demonstrate that N-WASp plays an essential role in Schwann cell maturation and myelin formation.

Association of granulomatosis with polyangiitis (Wegener's) with HLA-DPB1*04 and SEMA6A gene variants: evidence from genome-wide analysis.

OBJECTIVE: To identify genetic determinants of granulomatosis with polyangiitis (Wegener's) (GPA). METHODS: We carried out a genome-wide association study (GWAS) of 492 GPA cases and 1,506 healthy controls (white subjects of European descent), followed by replication analysis of the most strongly associated signals in an independent cohort of 528 GPA cases and 1,228 controls. RESULTS: Genome-wide significant associations were identified in 32 single-nucleotide polymorphic (SNP) markers across the HLA region, the majority of which were located in the HLA-DPB1 and HLA-DPA1 genes encoding the class II major histocompatibility complex (MHC) DPß chain 1 and DPα chain 1 proteins, respectively. Peak association signals in these 2 genes, emanating from SNPs rs9277554 (for DPß chain 1) and rs9277341 (DPα chain 1) were strongly replicated in an independent cohort (in the combined analysis of the initial cohort and the replication cohort, P = 1.92 × 10(-50) and 2.18 × 10(-39) , respectively). Imputation of classic HLA alleles and conditional analyses revealed that the SNP association signal was fully accounted for by the classic HLA-DPB1*04 allele. An independent single SNP, rs26595, near SEMA6A (the gene for semaphorin 6A) on chromosome 5, was also associated with GPA, reaching genome-wide significance in a combined analysis of the GWAS and replication cohorts (P = 2.09 × 10(-8) ). CONCLUSION: We identified the SEMA6A and HLA-DP loci as significant contributors to risk for GPA, with the HLA-DPB1*04 allele almost completely accounting for the MHC association. These two associations confirm the critical role of immunogenetic factors in the development of GPA.

Ras-guanine-nucleotide-releasing factors 1 and 2 interact with PLCγ at focal adhesions to enable IL-1-induced Ca(2+) signalling, ERK activation and MMP-3 expression.

IL (interleukin)-1 signalling in anchorage-dependent cells involves focal-adhesion-restricted and Ca2+-dependent Ras and ERK (extracellular-signal-regulated kinase) activation that leads to MMP (matrix metalloproteinase) release and extracellular matrix remodelling. Ras activity is regulated, in part, by the Ca2+-responsive Ras GRFs (guanine-nucleotide-releasing factors) 1 and 2, but the mechanisms that link and localize IL-1-induced Ca2+ signalling to focal adhesions are not defined. In the present study we characterized the role of Ras-GRF1/2 in Ca2+ and Ras→ERK signalling after IL-1 stimulation. By immunoprecipitation we found that Ras-GRF1/2 associates with PLCγ1 (phospholipase Cγ1). This association enables PLCγ1 recruitment to focal adhesions and is required for Ras signalling, ERK activation and MMP-3 release downstream of IL-1 stimulation. Depletion of PLCγ1 by siRNA (small interfering RNA) abolished IL-1-induced Ras activation and MMP-3 expression. Buffering of cytosolic Ca2+ reduced Ras interactions with Ras-GRF1/2 and blocked MMP-3 release. The results of the present study show that, in addition to their functions as Ras-exchange factors, Ras-GRF1 and -GRF2 may act as adaptors that bind PLCγ1 and restrict Ca2+ signalling to the vicinity of focal adhesions, indicating a new role for these GRFs that is required for IL-1 induction of the Ras→ERK pathway and MMP-3 expression.