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Objective: To characterise stabilities in erythrocytes of mutant SOD1 proteins, compare SOD1 enzymatic activities between patients with different genetic causes of ALS and search for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. Methods: Blood samples from 4072 individuals, ALS patients with or without a SOD1 mutation, family members and controls were studied. Erythrocyte SOD1 enzymatic activities normalised to haemoglobin content were determined, and effects of haemoglobin disorders on dismutation assessed. Coding SOD1 sequences were analysed by Sanger sequencing, exon copy number variations by fragment length analysis and by TaqMan Assay. Results: Of the 44 SOD1 mutations found, 75% caused severe destabilisation of the mutant protein but in 25% it was physically stable. Mutations producing structural changes caused halved erythrocyte SOD1 activities. There were no differences in SOD1 activities between patients without a SOD1 mutation and control individuals or carriers of TBK1 mutations and C9orf72HRE. In the low and high SOD1 activity groups no deviations were found in exon copy numbers and intron gross structures. Thalassemias and iron deficiency were associated with increased SOD1 activity/haemoglobin ratios. Conclusion: Adjunct erythrocyte SOD1 activity analysis reliably signals destabilising SOD1 mutations including intronic mutations that are missed by exon sequencing. [ABSTRACT FROM AUTHOR]

Mutations in the superoxide dismutase (SOD1) gene have been linked to amyotrophic lateral sclerosis (ALS). A 50 base pair (bp) deletion of SOD1 has been suggested to reduce transcription and to be associated with later disease onset in ALS. This study was aimed to reveal if the 50 bp deletion influenced SOD1 enzymatic activity, occurrence and phenotype of the disease in a Swedish ALS/control cohort. Blood samples from 512 Swedish ALS patients and 354 Swedish controls without coding SOD1 mutations were analysed for the 50 bp deletion allele. The enzymatic activity of SOD1 in erythrocytes was analysed and genotype-phenotype correlations were assessed. Results demonstrated that the genotype frequencies of the 50 bp deletion were all found to be in Hardy-Weinberg equilibrium. No significant differences were found for age of onset, disease duration or site of onset. SOD1 enzymatic activity showed a statistically significant decreasing trend in the control group, in which the allele was associated with a 5% reduction in SOD1 activity. The results suggest that the 50 bp deletion has a moderate reducing effect on SOD1 synthesis. No modulating effects, however, were found on ALS onset, phenotype and survival in the Swedish population. [ABSTRACT FROM PUBLISHER]

Mutations in superoxide dismutase-1 (SOD1) are a common known cause of amyotrophic lateral sclerosis (ALS). The neurotoxicity of mutant SOD1s is most likely caused by misfolded molecular species, but disease pathogenesis is still not understood. Proposed mechanisms include impaired mitochondrial function, induction of endoplasmic reticulum stress, reduction in the activities of the proteasome and autophagy, and the formation of neurotoxic aggregates. Here we examined whether perturbations in these cellular pathways in turn influence levels of misfolded SOD1 species, potentially amplifying neurotoxicity. For the study we used fibroblasts, which express SOD1 at physiological levels under regulation of the native promoter. The cells were derived from ALS patients expressing 9 different SOD1 mutants of widely variable molecular characteristics, as well as from patients carrying the GGGGCC-repeat-expansion in C9orf72 and from non-disease controls. A specific ELISA was used to quantify soluble, misfolded SOD1, and aggregated SOD1 was analysed by western blotting. Misfolded SOD1 was detected in all lines. Levels were found to be much lower in non-disease control and the non-SOD1 C9orf72 ALS lines. This enabled us to validate patient fibroblasts for use in subsequent perturbation studies. Mitochondrial inhibition, endoplasmic reticulum stress or autophagy inhibition did not affect soluble misfolded SOD1 and in most cases, detergent-resistant SOD1 aggregates were not detected. However, proteasome inhibition led to uniformly large increases in misfolded SOD1 levels in all cell lines and an increase in SOD1 aggregation in some. Thus the ubiquitin-proteasome pathway is a principal determinant of misfolded SOD1 levels in cells derived both from patients and controls and a decline in activity with aging could be one of the factors behind the mid-to late-life onset of inherited ALS. [ABSTRACT FROM AUTHOR]

Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo. [ABSTRACT FROM AUTHOR]

Linkage analysis in Brazilian families with amyotrophic lateral sclerosis (ALS) revealed that a missense mutation p.Pro56Ser in a conserved gene VAMP-associated protein type B and C ( VAPB) cosegregates with disease. Blood samples were studied from 973 Swedish, 126 Portuguese and 19 Icelandic ALS patients, and from 644 control subjects. We identified five VAPB mutations, two of which are novel, in 14 Swedish ALS patients and in nine control individuals from Sweden and Portugal. The 14 patients with VAPB mutations all carried a diagnosis of sporadic ALS. Mutations were also found in healthy adult relatives. The p.Asp130Glu VAPB mutation was also found in two patients from an Icelandic ALS family, but the mutation did not cosegregate with disease. All patients were instead found to be heterozygous for a p.Gly93Ser SOD1 mutation. There were no clinical differences between them, suggesting that the p.Asp130Glu VAPB mutation is unrelated to the disease process. In conclusion, the VAPB mutations were as frequent in control individuals as in patients. This observation, in combination with the finding of several healthy relatives carrying the VAPB mutations and no ancestors with ALS disease, suggests that it is unlikely that these VAPB mutations are pathogenic. [ABSTRACT FROM AUTHOR]

An intronic GGGGCC-hexanucleotide repeat expansion in C9ORF72 was recently identified as a major cause of amyotrophic lateral sclerosis and frontotemporal dementia. Some amyotrophic lateral sclerosis patients have signs of parkinsonism, and many parkinsonism patients develop dementia. In this study we examined if the hexanucleotide repeat expansion was present in parkinsonism patients, to clarify if there could be a relationship between the repeat expansion and disease. We studied the size of the hexanucleotide repeat expansion in a well defined population-based cohort of 135 Parkinson's disease patients and 39 patients with atypical parkinsonism and compared with 645 Swedish control subjects. We found no correlation between Parkinson's disease or atypical parkinsonism and the size of the GGGGCC repeat expansion in C9ORF72. In conclusion, this GGGGCC-repeat expansion in C9ORF72 is not a cause of parkinsonism in the Swedish population. [ABSTRACT FROM AUTHOR]

In this report we describe an ALS family with a novel missense SOD1 mutation with substitution of serine for cysteine at the sixth amino acid (C6S). This mutation has interesting implications for the role of disulfides in causing disease. After identification of the ALS proband, we examined 17 members of an extended family and performed DNA mutation analysis on 21 family members. The level and activity of SOD1 in C6S carriers and wild-type family members was analyzed in erythrocytes. We found that the C6S mutation results in disease with an autosomal dominant mode of inheritance and markedly reduced penetrance. The S6 mutated protein demonstrates high stability relative to the C6 wild-type protein. The specific dismutation activity of S6 SOD1 is normal. In conclusion, C6S is a novel FALS associated mutation with reduced disease penetrance, long survival time and a phenotype very different from the other SOD1 mutations reported in codon C6. This mutation may provide insight into the role of SOD1 structural changes in disease. [ABSTRACT FROM AUTHOR]

We conducted a genome-wide association study among 2,323 individuals with sporadic amyotrophic lateral sclerosis (ALS) and 9,013 control subjects and evaluated all SNPs with P < 1.0 × 10−4 in a second, independent cohort of 2,532 affected individuals and 5,940 controls. Analysis of the genome-wide data revealed genome-wide significance for one SNP, rs12608932, with P = 1.30 × 10−9. This SNP showed robust replication in the second cohort (P = 1.86 × 10−6), and a combined analysis over the two stages yielded P = 2.53 × 10−14. The rs12608932 SNP is located at 19p13.3 and maps to a haplotype block within the boundaries of UNC13A, which regulates the release of neurotransmitters such as glutamate at neuromuscular synapses. Follow-up of additional SNPs showed genome-wide significance for two further SNPs (rs2814707, with P = 7.45 × 10−9, and rs3849942, with P = 1.01 × 10−8) in the combined analysis of both stages. These SNPs are located at chromosome 9p21.2, in a linkage region for familial ALS with frontotemporal dementia found previously in several large pedigrees. [ABSTRACT FROM AUTHOR]

A genome-wide association study (GWAS) using pooled DNA samples from 386 sporadic ALS patients and 542 controls from the USA, identified genetic variation in FGGY ( FLJ10986) as a risk factor, as well as 66 additional candidate SNPs. Considering the large number of hypotheses that are tested in GWAS, independent replication of associations is crucial for identifying true-positive genetic risk factors for disease. The primary aim of this study was to study the association between FGGY and sporadic ALS in large, homogeneous populations from northern Europe. Genotyping experiments were performed using Illumina Beadchips, Sequenom iPLEX assays and Taqman technology on large case-control series from The Netherlands, Belgium, Sweden and Ireland (total: 1883 sporadic ALS patients and 2063 controls). No significant association between sporadic ALS and the six previously reported associated SNPs in FGGY was observed: rs6700125 ( p =0.56), rs6690993 ( p =0 .30), rs10493256 ( p =0 .68), rs6587852 ( p =0 .64), rs1470407 ( p =0 .28) and rs333662 ( p =0 .44). Screening of the additional candidate loci did not yield significant associations either, with the lowest p-value in joint analysis for rs7772593 ( p =0.14). We concluded that common genetic variation in FGGY is not associated with susceptibility to sporadic ALS in genetically homogeneous populations from northern Europe. [ABSTRACT FROM AUTHOR]

Midkine (MK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. To date, this family of proteins has been implicated in multiple processes, such as growth, survival, and migration of various cells, and has roles in neurogenesis and epithelial–mesenchymal interaction during organogenesis. In this report, we have characterized two members of the MK/PTN family of proteins in Drosophila, named Miple1 and Miple2, from Midkine and Pleiotrophin. Drosophila miple1 and miple2 encode secreted proteins which are expressed in spatially restricted, nonoverlapping patterns during embryogenesis. Expression of miple1 can be found at high levels in the central nervous system, while miple2 is strongly expressed in the developing midgut endoderm. The identification of homologues of the MK/PTN family in this genetically tractable model organism should allow an analysis of their function during complex developmental processes. [ABSTRACT FROM AUTHOR]

Polycomb group (PEG) proteins are required to maintain stable repression of the homeotic genes and others throughout development. The PcG proteins ESC and E(Z) are present in a prominent 600-kDa complex as well as in a number of higher-molecular-mass complexes. Here we identify and characterize a 1-MDa ESC/E(Z) complex that is distinguished from the 600-kDa complex by the presence of the PeG protein Polycomb-like (PCL) and the histone deacetylase RPD3. In addition, the 1-MDa complex shares with the 600-kDa complex the histone binding protein p55 and the PeG protein SU(Z)12. Coimmunoprecipitation assays performed on embryo extracts and gel filtration column fractions indicate that, during embryogenesis E(Z), SU(Z)12, and p55 are present in all ESC complexes, while PCL and RPD3 are associated with ESC, E(Z), SU(Z)12, and p55 only in the 1-MDa complex. Glutathione transferase pulldown assays demonstrate that RPD3 binds directly to PCL via the conserved PHD fingers of PCL and the N terminus of RPD3. PCL and E(Z) colocalize virtually completely on polytene chromosomes and are associated with a subset of RPD3 sites. As previously shown for E(Z) and RPD3, PCL and SU (Z)12 are also recruited to the insertion site of a minimal Ubx Polycomb response element transgene in vivo. Consistent with these biochemical and cytological results, Rpd3 mutations enhance the phenotypes of Pcl mutants, further indicating that RPD3 is required for PcG silencing and possibly for PCL function. These results suggest that there may be multiple ESC/E(Z) complexes with distinct functions in vivo. [ABSTRACT FROM AUTHOR]

We identified a SNP in the DPP6 gene that is consistently strongly associated with susceptibility to amyotrophic lateral sclerosis (ALS) in different populations of European ancestry, with an overall P value of 5.04 × 10−8 in 1,767 cases and 1,916 healthy controls and with an odds ratio of 1.30 (95% confidence interval (CI) of 1.18–1.43). Our finding is the first report of a genome-wide significant association with sporadic ALS and may be a target for future functional studies. [ABSTRACT FROM AUTHOR]