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A GGGGCC-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) among Caucasians. However, little is known about the variability of the GGGGCC expansion in different tissues and whether this correlates with the observed phenotype. Here, we used Southern blotting to estimate the size of hexanucleotide expansions in C9orf72 in neural and non-neural tissues from 18 autopsied ALS and FTD patients with repeat expansion in blood. Digitalization of the Southern blot images allowed comparison of repeat number, smear distribution and expansion band intensity between tissues and between patients. We found marked intra-individual variation of repeat number between tissues, whereas there was less variation within each tissue group. In two patients, the size variation between tissues was extreme, with repeat numbers below 100 in all studied non-neural tissues, whereas expansions in neural tissues were 20-40 times greater and in the same size range observed in neural tissues of the other 16 patients. The expansion pattern in different tissues could not distinguish between diagnostic groups and no correlation was found between expansion size in frontal lobe and occurrence of cognitive impairment. In ALS patients, a less number of repeats in the cerebellum and parietal lobe correlated with earlier age of onset and a larger number of repeats in the parietal lobe correlated with a more rapid progression. In 43 other individuals without repeat expansion in blood, we find that repeat sizes up to 15 are stable, as no size variation between blood, brain and spinal cord was found.

We analyzed genotypes from ~10K single-nucleotide polymorphisms (SNPs) in two families of an F2 intercross between Red Junglefowl and White Leghorn chickens. Possible null alleles were found by patterns of incompatible and missing genotypes. We estimated that 2.6% of SNPs had null alleles compared with 2.3% with genotyping errors and that 40% of SNPs in which a parent and offspring were genotyped as different homozygotes had null alleles. Putative deletions were identified by null alleles at adjacent markers. We found two candidate deletions that were supported by fluorescence intensity data from a 60K SNP chip. One of the candidate deletions was from the Red Junglefowl, and one was present in both the Red Junglefowl and White Leghorn. Both candidate deletions spanned protein-coding regions and were close to a previously detected quantitative trait locus affecting body weight in this population. This study demonstrates that the ~50K SNP genotyping arrays now available for several agricultural species can be used to identify null alleles and deletions in data from large families. We suggest that our approach could be a useful complement to linkage analysis in experimental crosses.

Mutant human CuZn-superoxide dismutases (hSOD1s) cause amyotrophic lateral sclerosis (ALS). The most common mutation is the wild type-like D90A and to explore its properties, transgenic mice were generated and compared with mice expressing wild-type hSOD1. D90A hSOD1 was both in vivo in mice and in vitro under denaturing conditions nearly as stable as the wild-type human enzyme. It appeared less toxic than other tested mutants, but mice homozygous for the transgene insertion developed a fatal motor neuron disease. In these mice, the disease progression was slow and there were bladder disturbances similar to what is found in human ALS cases homozygous for the D90A mutation. The homozygous D90A mice accumulated detergent-resistant hSOD1 aggregates in spinal cords, and abundant hSOD1 inclusions and vacuoles were seen in the ventral horns. Mice expressing wild-type hSOD1 at a comparable rate showed similar pathologic changes but less and later. Hemizygous D90A mice showed even milder alterations. At 600 days, the wild-type hSOD1 transgenic mice had lost more ventral horn neurons than hemizygous D90A mice (38% vs 31% p < 0.01). Thus, wild-type hSOD1 shows a significant neurotoxicity in the spinal cord, that is less than equal but more than half as large as that of D90A mutant enzyme.

Mutations in CuZn-superoxide dismutase (CuZn-SOD) have been linked to familial amyotrophic lateral sclerosis (ALS), and motor neurone death is caused by the gain of a toxic property of the mutant protein. Here we determined amounts, activity and molecular forms of CuZn-SOD in CSF from ALS patients carrying the D90A and other CuZn-SOD mutations and patients without such mutations. There were no differences in amount of protein and enzymic activities of CuZn-SOD between 37 neurological controls, 54 sporadic and 12 familial ALS cases, and 10 cases homozygous for the D90A mutation. Three cases heterozygous for the A89V, S105L and G114A CuZn-SOD mutations showed low amounts of CuZn-SOD. There was no evidence for accumulation of inactive protein in any of the groups. Immunoblots showed no evidence for the presence of any precipitates or other molecular forms of CuZn-SOD with higher molecular weight in the groups. About 25% of the CuZn-SOD subunits in CSF from controls shows an N-terminal truncation. This truncated portion does not differ between controls and ALS groups not carrying CuZn-SOD mutations, but is 70% larger in samples from D90A homozygous ALS patients. The findings suggest an essentially normal amount and activity of D90A mutant CuZn-SOD in CNS tissues of ALS cases. The increased occurrence of N-terminally truncated mutant subunits may indicate a difference in degradation routes compared with the wild-type enzyme, resistance against subsequent proteolytic steps and/or a compromised downstream proteolytic machinery. Molecular fragments accumulated to a greater extent from the D90A mutant enzyme might contribute to the motor neurone degeneration. We also determined the other SOD isoenzymes: in the controls, CuZn-SOD contributed 75%, extracellular SOD 25% and Mn-SOD

Four-hundred and fifty-one blood samples from Scandinavian patients with motor neuron disease were analysed for mutations in the CuZn-superoxide dismutase gene. Forty-one (9.6%) of the 427 patients with the amyotrophic lateral sclerosis (ALS) form of the disease were found to have a disease-associated mutation, and 14 of these patients were apparently sporadic cases. A mutation was found in 12 of the 51 families with recognized familial ALS. The five different mutations found (Ala4Val, Val14Gly, Asp76Tyr, Asp90Ala, Gly127insTGGG) have different genetic characteristics and are associated with very variable phenotypes spanning from rapidly progressing disease with only lower motor neuron signs to very slowly progressing disease with both the upper and lower motor neuron systems affected. The patients showed different sites of onset, though the progressive bulbar palsy form of the disease appears to be rare among patients with a CuZn-superoxide dismutase mutation. The progression of motor signs and symptoms followed the same basic pattern in patients with different mutations. Extra-motor system symptoms were frequent among patients with a CuZn-superoxide dismutase mutation. The results suggest that patients with mutations in the CuZn-superoxide dismutase gene constitute one disease entity. The Val14Gly and Asp76Tyr mutations have not been reported before, and the latter is the first mutation to be found in exon 3 of the CuZn-superoxide dismutase gene.

We describe 36 patients (six were apparently sporadic cases and 30 were cases from nine families) with amyotrophic lateral sclerosis (ALS) characterized by a distinct phenotype associated with homozygosity for an Asp90Ala mutation in the CuZn-superoxide dismutase gene. The presenting motor manifestation in all patients was paresis in the legs, with slow progression to the upper extremities and finally to the bulbar muscles. The age of ALS onset varied from 20 to 94 years, with a mean of 44 years. Mean survival time was 13 years for the 11 deceased patients. However, this is probably biased and untypical (low) when compared with the disease duration in the surviving patients, and when considering other medical complications in the deceased patients. The rate of progression was highly variable, even within families. All patients showed signs of involvement of both upper and lower motor neurons. Other neurological features included painful muscle spasms and paraesthesiae in the lower extremities. Two-thirds of patients experienced difficulty with micturition. Electrophysiological studies confirmed the slow progression and spatial distribution of clinical symptoms in the peripheral motor system. Furthermore, [corrected] potentials evoked by transcranial magnetic stimulation (MEP) were compared with those evoked by cervical or lumbosacral electrical stimulation and often revealed marked slowing of transmission in central motor pathways. In Sweden and Finland ALS patients homozygous for the Asp90Ala mutation constitute a phenotypically characteristic subset of motor neuron disease.

Extensive preparative surgery and lengthy experimentation may lead to high rate of complications and mortality in myocardial ischaemia studies. These problems are particularly common when pigs are used as the subject as they are prone to develop lethal ventricular arrhythmias. Here, a closed-chest model is presented, in which the trauma of major preparative surgery is avoided. One-hundred and twelve pentobarbital-anaesthetized, mechanically ventilated pigs were used. Coronary occlusion was produced by injection of a 2 mm diameter ball via a modified coronary angiography catheter. Reperfusion was induced by retraction of the ball via a thin filament attached to the ball. The amount of the myocardium at risk (MAR) was 8.23 +/- 2.41% (mean +/- SD) of the left plus right ventricular weight. It was possible to carry out scheduled 24 h experiments in 87 out of 93 animals (93.5%). Preparative mortality was 1.8% and 24 h mortality 6.5%. Ventricular fibrillation (VF) occurred during preparation in 3.6%, during coronary occlusion in 7.3% and during reperfusion in 5.0% of the animals. VF was significantly related to a large zone of MAR and insufficient premedication. Catheter- or ball-induced complications were found in 10.7%. Mortality and incidence of VF are considerably lower in this closed-chest model than in a previously reported open-chest pig preparation.

Study objective – The aim of the study was to determine whether recombinant human extracellular superoxide dismutase type C (rh-EC-SOD C) has antioxidant effects similar to those of the previously studied Cu-Zn superoxide dismutase. Design – Electron paramagnetic resonance spectroscopy was used for quantification of free radical generation during reperfusion of postischaemic isolated rat hearts. The hearts were perfused with Krebs-Henseleit buffer and then subjected to 15 min global ischaemia. N- tert -butyl-α-phenylnitrone (PBN) was added as a spin trapping agent to the perfusate. Experimental material – 18 hearts from Sprague-Dawley rats (280-400 g) were used. Measurements and main results – Reperfusion resulted in a burst of radical formation. In presence of rh-EC-SOD C plus catalase, the PBN spin adduct concentration was only 18% (p

To test the possible role of superoxide radicals in the diabetogenic action of streptozotocin, blood glucose levels were measured in mice after a single high-dose (150 mg/kg body weight) or multiple low-dose (40 mg/kg for 5 days) injections of streptozotocin. Pre-treatment 6 h before streptozotocin with 250–300 mg/kg superoxide dismutase coupled to polyethylene glycol reduced the hyperglycaemic response in mice injected with a single dose of streptozotocin. The blood glucose levels after multiple low doses of streptozotocin were not affected by superoxide dismutase-polvethvlene glycol. Enzymatically inactive superoxide dismutase did not affect the development of hyperglycaemia. The results suggest that superoxide radicals may play a role in the diabetogenic action of streptozotocin injected as a high-dose single bolus.

Werner's syndrome is often regarded as a segmental progeroid syndrome. It has been suggested that free radical damage involving oxygen contributes to aging. Furthermore, lymphocytes from Werner's syndrome patients show in vitro increased chromosome breakage and are protected by exogenous superoxide dismutase and catalase. We prepared erythrocytes and lymphocytes from three patients with Werner's syndrome and determined four important enzymes protecting against oxygen toxicity; CuZn superoxide dismutase, Mn superoxide dismutase, catalase and glutathione peroxidase. All enzymic activities were found to be normal.