Cytosolic 5'-nucleotidase 1A autoantibody profile and clinical characteristics in inclusion body myositis.

OBJECTIVES: Autoantibodies directed against cytosolic 5'-nucleotidase 1A have been identified in many patients with inclusion body myositis. This retrospective study investigated the association between anticytosolic 5'-nucleotidase 1A antibody status and clinical, serological and histopathological features to explore the utility of this antibody to identify inclusion body myositis subgroups and to predict prognosis. MATERIALS AND METHODS: Data from various European inclusion body myositis registries were pooled. Anticytosolic 5'-nucleotidase 1A status was determined by an established ELISA technique. Cases were stratified according to antibody status and comparisons made. Survival and mobility aid requirement analyses were performed using Kaplan-Meier curves and Cox proportional hazards regression. RESULTS: Data from 311 patients were available for analysis; 102 (33%) had anticytosolic 5'-nucleotidase 1A antibodies. Antibody-positive patients had a higher adjusted mortality risk (HR 1.89, 95% CI 1.11 to 3.21, p=0.019), lower frequency of proximal upper limb weakness at disease onset (8% vs 23%, adjusted OR 0.29, 95% CI 0.12 to 0.68, p=0.005) and an increased prevalence of excess of cytochrome oxidase deficient fibres on muscle biopsy analysis (87% vs 72%, adjusted OR 2.80, 95% CI 1.17 to 6.66, p=0.020), compared with antibody-negative patients. INTERPRETATION: Differences were observed in clinical and histopathological features between anticytosolic 5'-nucleotidase 1A antibody positive and negative patients with inclusion body myositis, and antibody-positive patients had a higher adjusted mortality risk. Stratification of inclusion body myositis by anticytosolic 5'-nucleotidase 1A antibody status may be useful, potentially highlighting a distinct inclusion body myositis subtype with a more severe phenotype.

Genome-wide association study identifies HLA 8.1 ancestral haplotype alleles as major genetic risk factors for myositis phenotypes.

Autoimmune muscle diseases (myositis) comprise a group of complex phenotypes influenced by genetic and environmental factors. To identify genetic risk factors in patients of European ancestry, we conducted a genome-wide association study (GWAS) of the major myositis phenotypes in a total of 1710 cases, which included 705 adult dermatomyositis, 473 juvenile dermatomyositis, 532 polymyositis and 202 adult dermatomyositis, juvenile dermatomyositis or polymyositis patients with anti-histidyl-tRNA synthetase (anti-Jo-1) autoantibodies, and compared them with 4724 controls. Single-nucleotide polymorphisms showing strong associations (P<5×10(-8)) in GWAS were identified in the major histocompatibility complex (MHC) region for all myositis phenotypes together, as well as for the four clinical and autoantibody phenotypes studied separately. Imputation and regression analyses found that alleles comprising the human leukocyte antigen (HLA) 8.1 ancestral haplotype (AH8.1) defined essentially all the genetic risk in the phenotypes studied. Although the HLA DRB1*03:01 allele showed slightly stronger associations with adult and juvenile dermatomyositis, and HLA B*08:01 with polymyositis and anti-Jo-1 autoantibody-positive myositis, multiple alleles of AH8.1 were required for the full risk effects. Our findings establish that alleles of the AH8.1 comprise the primary genetic risk factors associated with the major myositis phenotypes in geographically diverse Caucasian populations.

Protease activated receptor-1 mediates cytotoxicity during ischemia using in vivo and in vitro models.

Protease activated receptors (PARs) populate neurons and astrocytes in the brain. The serine protease thrombin, which activates PAR-1 during the first hours after stroke, appears to be associated with the cytotoxicity. Thrombin antagonists and PAR-1 inhibitors have been correlated with reduced cell death and behavioral protection after stroke, but no data yet support a mechanistic link between PAR-1 action and benefit. We sought to establish the essential role of PAR-1 in mediating ischemic damage. Using a short hairpin mRNA packaged with green fluorescent protein in a lentivirus vector, we knocked downPAR-1 in the medial caudate nucleus prior to rat middle cerebral artery occlusion (MCAo) and in rat neurons prior to oxygen-glucose deprivation. We also compared aged PAR-1 knockout mice with aged PAR-3, PAR-4 mice and young wild-type mice in a standard MCAo model. Silencing PAR-1 significantly reduced neurological deficits, reduced endothelial barrier leakage, and decreased neuronal degeneration in vivo during MCAo. PAR-1 knock-down in the ischemic medial caudate allowed cells to survive the ischemic injury; infected cells were negative for terminal deoxynucleotidyl transferase mediated dUTP Nick End Labeling (TUNEL) and c-Fos injury markers. Primary cultured neurons infected with PAR-1 short hairpin ribonucleic acid (shRNA) showed increased neuroprotection during hypoxic/aglycemic conditions with or without added thrombin. The aged PAR-1 knockout mice showed decreased infarction and vascular disruption compared to aged controls or young wild types. We demonstrated an essential role for PAR-1 during ischemia. Silencing or removing PAR-1 significantly protected neurons and astrocytes. Further development of agents that act at PAR-1 or its downstream pathways could yield powerful stroke therapy.

High-density SNP association study and copy number variation analysis of the AUTS1 and AUTS5 loci implicate the IMMP2L-DOCK4 gene region in autism susceptibility.

Autism spectrum disorders are a group of highly heritable neurodevelopmental disorders with a complex genetic etiology. The International Molecular Genetic Study of Autism Consortium previously identified linkage loci on chromosomes 7 and 2, termed AUTS1 and AUTS5, respectively. In this study, we performed a high-density association analysis in AUTS1 and AUTS5, testing more than 3000 single nucleotide polymorphisms (SNPs) in all known genes in each region, as well as SNPs in non-genic highly conserved sequences. SNP genotype data were also used to investigate copy number variation within these regions. The study sample consisted of 127 and 126 families, showing linkage to the AUTS1 and AUTS5 regions, respectively, and 188 gender-matched controls. Further investigation of the strongest association results was conducted in an independent European family sample containing 390 affected individuals. Association and copy number variant analysis highlighted several genes that warrant further investigation, including IMMP2L and DOCK4 on chromosome 7. Evidence for the involvement of DOCK4 in autism susceptibility was supported by independent replication of association at rs2217262 and the finding of a deletion segregating in a sib-pair family.

The protein tyrosine phosphatase N22 gene is associated with juvenile and adult idiopathic inflammatory myopathy independent of the HLA 8.1 haplotype in British Caucasian patients.

OBJECTIVE: To examine single-nucleotide polymorphisms (SNPs) of the protein tyrosine phosphatase N22 gene (PTPN22) and to study the relationship between PTPN22 and the HLA region in patients with idiopathic inflammatory myopathies (IIMs). METHODS: PTPN22 SNPs were assessed in a large, cross-sectional, case-control study from the UK involving patients with adult or juvenile IIM, comprising patients with polymyositis (PM) (n=114), dermatomyositis (DM) (n=102), myositis associated with another connective tissue disease (myositis-CTD overlap syndrome) (n=64), or juvenile DM (n=101), in comparison with 748 control subjects. Seventeen PTPN22 SNPs were genotyped using the Sequenom MassArray iPLEX platform. Serotyping for myositis-specific/myositis-associated autoantibodies (MSAs/MAAs) was performed by radioimmunoprecipitation. RESULTS: A significant association was noted between the R620W variant (rs2476601) and IIM (corrected P [Pcorr]=0.0009 versus controls), and specifically with the clinical subgroup of PM (Pcorr=0.003 versus controls). A weaker association was noted with juvenile DM (Pcorr=0.009 versus controls). No significant associations were noted after stratification by serologic subgroups. The association with the R620W variant was independent of alleles forming the HLA 8.1 haplotype. No other PTPN22 SNPs were associated with IIM. The PTPN22 haplotype containing the R620W T allele was the only haplotype significantly associated with IIM. CONCLUSION: The R620W variant is a significant risk factor for IIM, independent of the HLA 8.1 haplotype. Unlike that in the HLA region, risk is not increased in individuals possessing MSAs/MAAs. These results are further evidence that the PTPN22 gene confers autoimmune susceptibility.

Analysis of IMGSAC autism susceptibility loci: evidence for sex limited and parent of origin specific effects.

BACKGROUND AND METHODS: Autism is a severe neurodevelopmental disorder, which has a complex genetic predisposition. The ratio of males to females affected by autism is approximately 4:1, suggesting that sex specific factors are involved in its development. We reported previously the results of a genomewide screen for autism susceptibility loci in 83 affected sibling pairs (ASP), and follow up analysis in 152 ASP. Here, we report analysis of an expanded sample of 219 ASP, using sex and parent of origin linkage modelling at loci on chromosomes 2, 7, 9, 15, and 16. RESULTS: The results suggest that linkage to chromosomes 7q and 16p is contributed largely by the male-male ASP (MLS = 2.55 v 0.12, and MLS = 2.48 v 0.00, for the 145 male-male and 74 male-female/female-female ASP on chromosomes 7 and 16 respectively). Conversely linkage to chromosome 15q appears to be attributable to the male-female/female-female ASP (MLS = 2.62 v 0.00, for non-male and male-male ASP respectively). On chromosomes 2 and 9, all ASP contribute to linkage. These data, supported by permutation, suggest a possible sex limited effect of susceptibility loci on chromosomes 7, 15, and 16. Parent of origin linkage modelling indicates two distinct regions of paternal and maternal identity by descent sharing on chromosome 7 (paternal MLS = 1.46 at approximately 112 cM, and maternal MLS = 1.83 at approximately 135 cM; corresponding maternal and paternal MLS = 0.53 and 0.28 respectively), and maternal specific sharing on chromosome 9 (maternal MLS = 1.99 at approximately 30 cM; paternal MLS = 0.02). CONCLUSION: These data support the possibility of two discrete loci underlying linkage of autism to chromosome 7, and implicate possible parent of origin specific effects in the aetiology of autism.

Screening of nine candidate genes for autism on chromosome 2q reveals rare nonsynonymous variants in the cAMP-GEFII gene.

The results from several genome scans indicate that chromosome 2q21-q33 is likely to contain an autism susceptibility locus. We studied the potential contribution of nine positional and functional candidate genes: TBR-1; GAD1; DLX1; DLX2; cAMP-GEFII; CHN1; ATF2; HOXD1 and NEUROD1. Screening these genes for DNA variants and association analysis using intragenic single nucleotide polymorphisms did not provide evidence for a major role in the aetiology of autism. Four rare nonsynonymous variants were identified, however, in the cAMP-GEFII gene. These variants were present in five families, where they segregate with the autistic phenotype, and were not observed in control individuals. The significance of these variants is unclear, as their low frequency in IMGSAC families does not account for the relatively strong linkage signal at the 2q locus. Further studies are needed to clarify the contribution of cAMP-GEFII gene variants to autism susceptibility.

Analysis of reelin as a candidate gene for autism.

Genetic studies indicate that chromosome 7q is likely to contain an autism susceptibility locus (AUTS1). We have followed a positional candidate gene approach to identify relevant gene(s) and report here the analysis of reelin (RELN), a gene located under our peak of linkage. Screening RELN for DNA changes identified novel missense variants absent in a large control group; however, the low frequency of these mutations does not explain the relatively strong linkage results on 7q. Furthermore, analysis of a previously reported triplet repeat polymorphism and intragenic single nucleotide polymorphisms, using the transmission disequilibrium test, provided no evidence for association with autism in IMGSAC and German singleton families. The analysis of RELN suggests that it probably does not play a major role in autism aetiology, although further analysis of several missense mutations is warranted in additional affected individuals.

FOXP2 is not a major susceptibility gene for autism or specific language impairment.

The FOXP2 gene, located on human 7q31 (at the SPCH1 locus), encodes a transcription factor containing a polyglutamine tract and a forkhead domain. FOXP2 is mutated in a severe monogenic form of speech and language impairment, segregating within a single large pedigree, and is also disrupted by a translocation in an isolated case. Several studies of autistic disorder have demonstrated linkage to a similar region of 7q (the AUTS1 locus), leading to the proposal that a single genetic factor on 7q31 contributes to both autism and language disorders. In the present study, we directly evaluate the impact of the FOXP2 gene with regard to both complex language impairments and autism, through use of association and mutation screening analyses. We conclude that coding-region variants in FOXP2 do not underlie the AUTS1 linkage and that the gene is unlikely to play a role in autism or more common forms of language impairment.

Herbicide movement and dissipation at four Midwestern sites.

This study was conducted to evaluate atrazine (2-chloro-4-ethylamino-6-isopropyl-1,3,5-triazine) and alachlor (2-chloro-N-(methoxymethyl)acetamide) dissipation and movement to shallow aquifers across the Northern Sand Plains region of the United States. Sites were located at Minnesota on a Zimmerman fine sand, North Dakota on Hecla sandy loam, South Dakota on a Brandt silty clay loam, and Wisconsin on a Sparta sand. Herbicide concentrations were determined in soil samples taken to 90 cm four times during the growing season and water samples taken from the top one m of aquifer at least once every three months. Herbicides were detected to a depth of 30 cm in Sparta sand and 90 cm in all other soils. Some aquifer samples from each site contained atrazine with the highest concentration in the aquifer beneath the Sparta sand (1.28 microg L(-1)). Alachlor was detected only once in the aquifer at the SD site. The time to 50% atrazine dissipation (DT50) in the top 15 cm of soil averaged about 21 d in Sparta and Zimmerman sands and more than 45 d for Brandt and Hecla soils. Atrazine DT50 was correlated positively with % clay and organic carbon (OC), and negatively with % fine sand. Alachlor DT50 ranged from 12 to 32 d for Zimmerman and Brandt soils, respectively, and was correlated negatively with % clay and OC and positively with % sand.

Autism: recent molecular genetic advances.

Autism (MIM 209850) is a severe neuropsychiatric disorder of unknown aetiology with profound consequences for patients and their families. Strong evidence from twin and family studies indicates the importance of genetic factors in the development of idiopathic autism, although it is clear that these influences are complex. This review focuses on recent molecular investigations to identify susceptibility loci implicated in autistic disorder.

Phosphorylation of the desmoplakin COOH terminus negatively regulates its interaction with keratin intermediate filament networks.

Desmoplakins (DPs) are the most abundant proteins in the innermost portion of the desmosomal plaque and have been proposed to play a role in the attachment of intermediate filaments (IF) to cell-cell contact sites. Our previous results suggest that the globular end domains of DP perform dual functions: first, to target DP to the desmosome via the NH2 terminus and second, to attach IF to the desmosomal plaque via the COOH terminus. When ectopically expressed in most cultured cells, the COOH terminus plus the rod domain (DP. delta N.SerC23) exhibits striking coalignment with keratin IF networks. However, in certain cell types (e.g. PtK2) or in cells treated with forskolin to activate protein kinase A, DP. delta N.SerC23 exhibits a diffuse cytoplasmic distribution. A variant molecule (DP. delta N.GlyC23) in which a serine located 23 amino acids from the COOH terminus is altered to a glycine, thereby disrupting a protein kinase A consensus phosphorylation site, co-localizes with keratin IF networks regardless of cell type or forskolin treatment. Analysis of the phosphopeptide maps of these DP variants and endogenous DP is consistent with the phosphorylation of the serine 23 residues from the COOH terminus. These results suggest that phosphorylation of a specific residue in the DP COOH terminus may negatively regulate its interaction with keratin IF networks.

Selective binding of gelsolin to actin monomers containing ADP.

The rate of reaction and the stoichiometry of binding between gelsolin and actin monomers depends on adenine nucleotides. In the presence of Ca2+ but not Mg2+, gelsolin retains the ability to sever actin filaments when incubated for more than 20 min with an excess of G-actin in the presence of ATP but loses severing activity within seconds when mixed with G-actin in ADP. Immunoprecipitation of gelsolin removes more actin from ADP than from ATP solutions. Monomeric ATP-actin in 2 mM MgCl2 and 150 mM KCl slowly destroys the filament-severing activity of gelsolin with kinetics that are first order in actin concentration and with an apparent bimolecular rate constant of 0.021 +/- 0.007 microM-1 s-1. Coincident with the slow complex formation in MgCl2, the actin bound to the calcium-sensitive actin binding domain of gelsolin hydrolyzes its ATP to ADP. These results suggest a further level of gelsolin regulation and a functional similarity between actin and GTP-binding proteins.

Modulation of gelsolin function. Activation at low pH overrides Ca2+ requirement.

The activation of gelsolin by calcium has been postulated to be involved in the receptor-mediated reorganization of the actin cytoskeleton, but cytoskeletal reorganization can also occur in cells with intracellular Ca2+ clamped at nanomolar levels. Fluorescence measurements using Fura-2 show that at pH 7.4, the Ca2+ requirement for gelsolin activation in vitro is higher than previously reported, with half-maximal activation of severing and nucleation occurring at 10 microM Ca2+. The Ca2+ requirement for gelsolin activity decreases at more acid pH and is approximately 3 microM at pH 6.5. At pH below 6.0, gelsolin no longer requires Ca2+ for activity and severs actin filaments, binds two actin monomers, and nucleates filament formation in EGTA-containing solutions. The pH-activated severing activity is inhibited by mixed lipid vesicles containing phosphatidylinositol 4,5-bisphosphate. A Ca(2+)-sensitive fragment consisting of the first 135 amino acids of human cytoplasmic gelsolin also demonstrates severing activity at pH < 6.0 in the absence of Ca2+. In contrast, the gelsolin homologs severin and villin maintain Ca2+ regulation of severing activity at low pH. These differences suggest that activation of gelsolin at low pH cannot be explained merely by destabilization of F-actin. The difference in diffusion constants of gelsolin measured at pH 5.5 and 6.5, as determined by dynamic light scattering, suggests that the molecule undergoes a shape change similar to that reported upon binding Ca2+ at neutral pH. These results suggest a mechanism by which gelsolin may be activated in vivo under conditions where Ca2+ transients do not occur.

Effects of actin filaments on fibrin clot structure and lysis.

The muscle and cytoskeletal protein actin is released from cells as a consequence of cell death and interacts with components of the hemostatic and fibrinolytic systems, including platelets, plasmin, and fibrin. We report here that incorporation of actin filaments into fibrin clots changes their viscoelastic properties by increasing their shear modulus at low deforming stresses and by nearly eliminating their tendency to become more rigid with increasing deformation (ie, exhibit strain-hardening). The viscoelastic effects depended on the length of the actin filaments as shown by the effects of the plasma filament-severing protein, gelsolin. Binding of actin to fibrin clots also varied with actin filament length. The plasma actin-binding proteins gelsolin and vitamin D-binding protein reduced, but did not eliminate, the incorporation of actin in the clot. Fluorescence microscopy showed a direct association of rhodamine-labeled actin filaments with the fibrin network. Incubation of clots containing long actin filaments in solutions containing physiologic concentrations of gelsolin (2 mumol/L) released 60% of the actin trapped in the clot. Reduction of the actin content of a fibrin clot by incubation in a gelsolin-containing solution resulted in an increased rate of clot lysis. The ability of plasma gelsolin to shorten actin filaments may therefore be of physiologic and potentially of therapeutic importance insofar as gelsolin-mediated diffusion of actin from the clot may restore the clot's rheologic properties and render it more sensitive to the lytic action of plasmin.

Attempted suicide among young adults: progress toward a meaningful estimate of prevalence.

OBJECTIVE: The results of epidemiologic surveys on attempted suicide are often difficult to interpret; they compare and provide varying estimates of the prevalence of attempted suicide. The authors sought to estimate the prevalence of attempted suicide in a young adult population and to define more precisely what respondents mean when they report a suicide attempt. METHOD: Survey respondents were a representative sample of all 18-24-year-old freshman students at a major public university. The self-administered, anonymous survey included questions about suicidal thoughts and behaviors and about any injury and need for medical care resulting from reported attempts. RESULTS: Of the 694 respondents, 374 (54%) reported having ever considered suicide and 181 (26%) had considered suicide during the preceding 12 months. Thirteen (2%) students reported having attempted suicide during the preceding 12 months, and 72 (10%) reported ever having attempted suicide. The number of students answering affirmatively to questions about injuries sustained, medical care sought, and hospitalization as a result of attempted suicide decreased progressively: only 18 (3%) students reported having ever sought medical care due to a suicide attempt, and seven (1%) were ever hospitalized. CONCLUSIONS: The prevalence of self-reported attempted suicide is not representative of the prevalence of self-injury and provides little information concerning the seriousness of the attempt. The use of specific questions similar to those used in this study should be considered in future surveys.