Deleted 4977-bp mitochondrial DNA mutation is associated with sporadic amyotrophic lateral sclerosis: a hospital-based case-control study

We investigated the relationship between the most common 4977-bp deleted mitochondrial DNA (mtDNA) mutations and the occurrence of sporadic amyotrophic lateral sclerosis (ALS). Primer-shift and quantitative polymerase chain reaction (PCR) were used to determine the 4977-bp deleted mtDNA in the muscle specimens from 36 patients with sporadic ALS and 69 age-matched controls with other neuromuscular disorders. We found that the 4977-bp deleted mtDNA mutations were significantly higher in the ALS patients than controls in both frequency (50.0% vs. 8.7%, P < 0.01) and amount (0.35 +/- 0.53% vs. 0.085 +/- 0.35%, P < 0.05). Subjects with, rather than without, deleted mtDNA were at a significantly higher risk for having ALS after adjustment for age and sex. Moreover, male subjects had a higher risk than female subjects of having sporadic ALS. This study suggested that 4977-bp deleted mtDNA is significantly associated with the occurrence of sporadic ALS.     

Needle electromyography of the rectus abdominis in patients with amyotrophic lateral sclerosis

We examined the role of needle electromyography (EMG) of the rectus abdominis (RA) in assessing thoracic involvement in amyotrophic lateral sclerosis (ALS). Needle EMG of the RA was performed in 67 patients with sporadic ALS and 110 healthy controls. The presence of abnormal spontaneous activity, configuration of motor unit action potentials (MUAPs), and recruitment pattern of motor unit potentials were examined. In ALS patients, MUAPs in the RA were of prolonged duration, large amplitude, and showed increased prevalence of polyphasic waveforms compared to controls. Significant differences in MUAP parameters, presence of abnormal spontaneous potentials, and interference patterns were noted between ALS patients and controls. Additionally, we found that active denervation was more frequent in the RA of ALS patients with dyspnea than those without dyspnea. Thus, conventional needle EMG of the RA is a valuable electrophysiological method to assess clinical and subclinical involvement of thoracic lower motor neurons in patients with suspected ALS.     

Molecular genetic analysis of the APEX nuclease gene in amyotrophic lateral sclerosis.

We analyzed genomic DNA from ALS patients for mutations in the apurinic/apyrimidinic endonuclease (APEX nuclease) gene. We identified three rare polymorphisms in the untranslated region of the gene and one common two-allele polymorphism (D148E). The allelic frequency D148E was significantly different in sporadic ALS patients compared with controls. A conserved amino acid change and a 4-base pair deletion were also identified in sporadic ALS patients. These data suggest that APEX nuclease may contribute to the etiology of ALS.     

Cerebrospinal fluid neurofilament light levels in amyotrophic lateral sclerosis: impact of SOD1 genotype

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative syndrome with familial and sporadic forms. Most ALS-associated mutations are found in the superoxide dismutase 1 ( SOD1 ) gene. We conducted a study including 60 sporadic and 19 familial ALS patients, 206 reference patients with other neurological disorders and 40 age- and sex-matched healthy controls to test the hypothesis that cerebrospinal fluid (CSF) levels of neurofilament light (NF-L) protein, a marker of axonal degeneration, might provide diagnostic and prognostic information on the disease. All ALS patients were screened for SOD1 mutations. Ten of the familial and five of the sporadic cases carried SOD1 mutations. NF-L concentration [median (range)] was strongly elevated in ALS [2110 (255–10 800) ng/l] compared with reference patients and healthy controls [277 (<125–15 506) and 175 (<125–710) ng/l, respectively, P  < 0.001] and correlated inversely with disease duration (Spearman R  = −0.518, P  = 0.001). NF-L levels were lower in SOD1 mutation-associated ALS compared with SOD1 wild-type (wt) ALS ( P  = 0.03). In conclusion, CSF NF-L levels may provide both diagnostic and prognostic information, particularly in SOD1 wt ALS.     

Glial cells of the spinal cord and subcortical white matter up-regulate neuronal nitric oxide synthase in sporadic amyotrophic lateral sclerosis

Several studies have suggested that excessive generation of nitric oxide (NO) may contribute to the pathogenesis of amyotrophic lateral sclerosis (ALS). Recently, a selective induction of the neuronal isoform of nitric oxide synthase (nNOS) in glial cells has been reported in an animal model of familial ALS. We therefore examined in postmortem tissue the expression of nNOS in patients with sporadic ALS and patients without any history of neurological disease. Using immunohistochemistry, we found an up-regulation of nNOS in glial cells of the spinal cord and subcortical white matter in ALS patients compared to controls. The enhanced glial nNOS expression seen in ALS patients could conceivably contribute to motoneuronal degeneration through NO-mediated cytotoxic effects.     

Copy-number variation in sporadic amyotrophic lateral sclerosis: a genome-wide screen

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterised by the selective death of motor neurons in the brain and spinal cord. Genetic risk factors have been implicated in susceptibility to ALS. Like single nucleotide polymorphisms (SNPs), copy-number variants (CNVs) are a source of genetic variation that have important effects on gene expression and disease phenotypes, and our aim was to identify CNVs that predispose to sporadic ALS. METHODS: We did a genome-wide screen for CNVs by analysis of Illumina 317K SNP arrays for 406 patients with sporadic ALS and 404 controls. We examined CNVs for association with ALS, and used the Kyoto Encyclopedia of Genes and Genomes database and the Gene Ontology database to investigate the functionality of genes that were deleted exclusively in patients with ALS. FINDINGS: We detected 2328 CNVs in 810 individuals. No CNV locus was significantly associated with sporadic ALS. 406 genes were duplicated or deleted exclusively in patients with ALS and have not been reported in previous studies of CNVs. Of the 390 genes heterozygously deleted in patients with sporadic ALS, 155 (40%) deletions were recorded exclusively in patients. By contrast, of the 323 genes heterozygously deleted in control participants, only 51 (16%) were exclusive to the controls (p=2.15 x 10(-12) for difference between groups). Products of the genes deleted specifically in patients with sporadic ALS include proteins involved in oxidative phosphorylation, regulation of the actin cytoskeleton, and interactions between cytokines and their receptors. INTERPRETATION: Common CNVs in the regions of the genome represented on the SNP array are unlikely to be associated with sporadic ALS. However, the high number of genes deleted specifically in patients with ALS strongly suggests that multiple rare deletions might have an important role in ALS pathogenesis.     

Genome-wide genotyping in amyotrophic lateral sclerosis and neurologically normal controls: first stage analysis and public release of data

BACKGROUND: The cause of sporadic ALS is currently unknown. Despite evidence for a role for genetics, no common genetic variants have been unequivocally linked to sporadic ALS. We sought to identify genetic variants associated with an increased or decreased risk for developing ALS in a cohort of American sporadic cases. METHODS: We undertook a genome-wide association study using publicly available samples from 276 patients with sporadic ALS and 271 neurologically normal controls. 555 352 unique SNPs were assayed in each sample using the Illumina Infinium II HumanHap550 SNP chip. FINDINGS: More than 300 million genotypes were produced in 547 participants. These raw genotype data are freely available on the internet and represent the first publicly accessible SNP data for ALS cases. 34 SNPs with a p value less than 0.0001 (two degrees of freedom) were found, although none of these reached significance after Bonferroni correction. INTERPRETATION: We generated publicly available genotype data for sporadic ALS patients and controls. No single locus was definitively associated with increased risk of developing disease, although potentially associated candidate SNPs were identified.     

Coexistence of dominant and recessive familial amyotrophic lateral sclerosis with the D90A Cu,Zn superoxide dismutase mutation within the same country

The Cu,Zn superoxide dismutase (Cu,Zn SOD) mutations described in amyotrophic lateral sclerosis (ALS) have, for the most part, a dominant influence. However, while a few cases with a heterozygous D90A mutation have been described in different countries, D90A has been recently proven to be recessively inherited with a common founder effect in Scandinavia. We screened French ALS families for Cu,Zn SOD mutations. The presence of the D90A allele was found in two index-cases, and their families were subsequently studied. In the first family the ALS patients were homozygotes for D90A, while in the second, all ALS patients were heterozygotes. In both families the disease was found to initially involve the lower limbs with slower progression than in sporadic cases, and frequent atypical signs such as paresthesia and urgency of micturition. We determined the D90A allele frequency in controls (n = 200) and sporadic ALS patients (n = 408). No D90A allele was found. This is the first report of coexistence of dominant and recessive families with the D90A Cu,Zn SOD mutation within the same country.     

No association of the SOD1 locus and disease susceptibility or phenotype in sporadic ALS

Mutations in the copper zinc superoxide dismutase gene (SOD1) are found in 20% of familial and 3% of sporadic ALS patients. SOD1 protein aggregation can be detected in motor neurons of mutation-negative sporadic cases but a pathogenic role for wild-type SOD1 in ALS has not been demonstrated. In this study of 233 ALS cases and 248 controls the authors found no significant association between four individual single nucleotide polymorphisms and a deletion spanning the SOD1 locus (or their combined haplotypes), and disease susceptibility, or phenotype.     

Blood Superoxide Dismutase,Catalase and Glutathione Peroxidase Activities in Familial and Sporadic Amyotrophic Lateral Sclerosis

Recent studies have implicated free radicals in the pathogenesis of amyotrophic lateral sclerosis (ALS), a fatal, paralytic disorder of motor neurons. Herein we report on measurements of erythrocyte activity of the three main free radical scavenging enzymes: copper/zinc superoxide dismutase (Cu/Zn-SOD), catalase, and glutathione peroxidase. We studied 31 patients with sporadic ALS, 18 with familial ALS, and 24 controls, Mean Cu/Zn-SOD activity was reduced in eight familial ALS patients with mutations of Cu/Zn-SOD but was normal in patients with both familial ALS without identified Cu/Zn-SOD mutations and sporadic ALS. Glutathione peroxidase activity was significantly reduced only in sporadic ALS patients treated with insulin-like growth factor I (100 μg/kg). Catalase activity was normal in sporadic and familial ALS. Neither glutathione peroxidase nor catalase activities correlated significantly with duration of symptoms or age at onset. Vitamin E, vitamin C, and β-carotene did not affect any of the three enzyme activities. These observations indicate that disturbances of catalase and glutathione peroxidase function are not likely to be central factors in the pathogenesis of ALS.     

ITPR2 as a susceptibility gene in sporadic amyotrophic lateral sclerosis: a genome-wide association study

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a devastating disease characterised by progressive degeneration of motor neurons in the brain and spinal cord. ALS is thought to be multifactorial, with both environmental and genetic causes. Our aim was to identify genetic variants that predispose for sporadic ALS. METHODS: We did a three-stage genome-wide association study in 461 patients with ALS and 450 controls from The Netherlands, using Illumina 300K single-nucleotide polymorphism (SNP) chips. The SNPs that were most strongly associated with ALS were analysed in a further 876 patients and 906 controls in independent sample series from The Netherlands, Belgium, and Sweden. We also investigated the possible pathological functions of associated genes using expression data from whole blood of patients with sporadic ALS and of control individuals who were included in the genome-wide association study. FINDINGS: A genetic variant in the inositol 1,4,5-triphosphate receptor 2 gene (ITPR2) was associated with ALS (p=0.012 after Bonferroni correction). Combined analysis of all samples (1337 patients and 1356 controls) confirmed this association (p=3.28x10(-6), odds ratio 1.58, 95% CI 1.30-1.91). ITPR2 expression was greater in the peripheral blood of 126 ALS patients than in that of 126 healthy controls (p=0.00016). INTERPRETATION: Genetic variation in ITPR2 is a susceptibility factor for ALS. ITPR2 is a strong candidate susceptibility gene for ALS because it is involved in glutamate-mediated neurotransmission, is one of the main regulators of intracellular calcium concentrations, and has an important role in apoptosis.     

Molecular analysis of the superoxide dismutase 1 gene in Spanish patients with sporadic or familial amyotrophic lateral sclerosis

We performed a genetic analysis of the Cu/Zn superoxide dismutase gene (SOD1) in Spanish patients with sporadic or familial amyotrophic lateral sclerosis (ALS). We found mutations in 2 of 11 families (18%) with ALS. In addition, 1 of the 87 sporadic ALS patients studied harbored a mutation in the same gene. We identified G37R in exon 2 of the SOD1 gene in 1 family. Another patient, with sporadic ALS, showed a novel N65S in exon 3. In addition, we found a novel I112M in exon 4 in another family. Our data highlight the genetic heterogeneity of patients with ALS harboring mutations in the SOD1 gene and confirm that families with autosomal dominant inheritance of the trait, regardless of their ethnic background, are more likely to carry mutations in such a gene.     

Alterations of the blood‐spinal cord barrier in sporadic amyotrophic lateral sclerosis

The blood‐spinal cord barrier (BSCB) of the spinal cord capillary consists of non‐fenestrated endothelial cells with tight junctions, basal laminae, pericytes and astrocyte feet processes, referred to as a “neurovascular unit.” The primary function of the BSCB is the maintenance and control of homeostasis of the spinal cord parenchyma by the selective transport of molecules and cells from the systemic compartment. Dysfunction of the BSCB shows important function in the etiology or progression of several pathological conditions of the spinal cord, including amyotrophic lateral sclerosis (ALS). However, the role of BSCB in the pathogenesis of ALS is still unclear. Here the changes of BSCB in sporadic ALS patients were studied by electron microscopy to determine whether the BSCB is disrupted and involved in the pathogenesis of motor neuron degeneration. A total of 358 and 366 cross‐sectioned capillaries were quantitatively examined in controls and ALS patients, respectively. The frequency of degenerated endothelia and pericytes, vacuolar changes of the cytoplasm in the endothelia and pericytes, and the replication of basement membranes was significantly higher in ALS patients than in the controls (P = 0.0175). The areas of the capillaries with diameters of ≤ 5 µm in the ALS patients were significantly smaller than those in the controls (P = 0.0124). The frequency of collagen fiber content of more than a moderate degree around the perivascular space was significantly higher in the ALS patients compared to the controls (P = 0.048), although there was no significant difference in the mild degree of accumulation of collagen fibers. Thus, the BSCB may be disrupted in sporadic ALS patients due to increased permeability and reduced microcirculation, leading to motor neuron degeneration and to the progression of the disease.     

Immunohistochemical expression of IGF‐I and GSK in the spinal cord of Kii and Guamanian ALS patients

Insulin‐like growth factor‐I (IGF‐I) is a potent survival factor for motor neurons in animals, and glycogen synthase kinase‐3β (GSK‐3β) is suspected to play roles in apoptosis and tau phosphorylation. Here we report the immunological expression of IGF‐I, GSK‐3β, phosphorylated‐GSK‐3α/β (p‐GSK‐3α/β) and phosphorylated‐tau in the spinal cord and hippocampus of Kii and Guam amyotrophic lateral sclerosis (ALS) patients. Sixteen ALS patients (10 Japanese sporadic, 3 Kii and 3 Guam ALS) and 14 neurological controls (10 Japanese and 4 Guamanian) were examined. The immunoreactivity for each antibody was rated by the percentages of positive neurons to total anterior horn neurons in each patient and was analyzed statistically. Many normal‐looking neurons from Japanese sporadic ALS, Kii ALS and Guam ALS patients, as well as from Japanese and Guam controls, were positive for anti‐IGF‐I antibody. A positive correlation between IR scores for anti‐IGF‐I antibody and clinical durations of Japanese sporadic ALS patients was found in this study (P < 0.0001). This suggested that IGF‐I might have a protective effect against ALS degeneration. In Japanese sporadic ALS patients, abnormal as well as normal‐looking neurons showed significant high IR scores for anti‐GSK‐3β antibody than those of controls. Anterior horn neurons from Guam and Kii ALS patients characteristically showed weak staining for anti‐GSK‐3β antibody but were markedly positive for anti‐pGSK‐3α/β antibody compared to those from both Japanese controls and Japanese sporadic ALS patients, and showed the co‐localization of IGF‐I and p‐GSK‐3α/β. This suggested that the IGF‐I signaling pathway in Guam and Kii ALS patients might function to phosphorylate GSK‐3β to protect neurons from ALS degeneration. Neurofibrillary tangles (NFTs) in the hippocampus and spinal cord from Kii and Guam ALS patients showed the co‐localization of PHF‐tau and p‐GSK‐3α/β by a confocal laser scanning technique. The predominant expression of p‐GSK‐3α/β compared to GSK‐3β in spinal motor neurons and the co‐localization of p‐GSK‐3α/β and PHF‐tau in NFT‐laden neurons in the hippocampus and spinal cord were characteristic findings of Kii and Guam ALS patients.     

Homozygous deletion of the survival motor neuron 2 gene is a prognostic factor in sporadic ALS

BACKGROUND: Spinal muscular atrophy (SMA) results from mutations of the survival motor neuron (SMN) gene on chromosome 5. The SMN gene exists in two highly homologous copies, telomeric (SMN1) and centromeric (SMN2). SMA is caused by mutations in SMN1 but not SMN2. The clinical phenotype of SMA appears to be related to the expression of SMN2. Patients suffering from the milder forms of SMA carry more copies of the SMN2 gene compared with patients with more severe SMA. It is suggested that the SMN2 gene is translated into an at least partially functional protein that protects against loss of motor neurons. OBJECTIVE: To investigate whether genetic mechanisms implicated in motor neuron death in SMA have a role in ALS. METHODS: The presence of deletions of exons 7 and 8 of SMN1 and SMN2 was determined in 110 patients with sporadic ALS and compared with 100 unaffected controls. RESULTS: The presence of a homozygous SMN2 deletion was overrepresented in patients with ALS compared with controls (16% versus 4%; OR, 4.4; 95% CI, 1.4 to 13.5). Patients with a homozygous SMN2 deletion had a shorter median time of survival (p < 0.009). Furthermore, multivariate regression analysis showed that the presence of an SMN2 deletion was independently associated with survival time (p < 0.02). No homozygous deletions in SMN1 were found. Carrier status of SMA appeared to be equally present in patients and controls (1 in 20). CONCLUSION: These results indicate that, similar to SMA, the SMN2 gene can act as a prognostic factor and may therefore be a phenotypic modifier in sporadic ALS. Increasing the expression of the SMN2 gene may provide a strategy for treatment of motor neuron disease.     

Mitochondrial alterations in the spinal cord of patients with sporadic amyotrophic lateral sclerosis

Little information is available about morphologic changes of mitochondria in sporadic amyotrophic lateral sclerosis (ALS). We examined the anterior horns of the lumbar spinal cord in 14 patients with sporadic ALS and 15 age-matched controls by electron microscopy to illuminate the subject. In the controls, one patient showed occasional swollen mitochondria with markedly increased cristae and marked accumulation of mitochondria in the somata of anterior horn neurons. Another patient had periodic, stubby protrusions on the outer membrane. Among the patients with ALS, 7 showed filamentous structures in the inner compartment of the mitochondria mainly of the somata and only occasionally of the axons. The structures were composed of a stack of multilayered cristae consisting of linear structures on a longitudinal section. Other abnormal structures were periodic transverse processes like rungs of a ladder predominantly in somata and only occasionally in the axons, marked accumulation of mitochondria in the somata, dendrites or proximal axons (axon hillock and initial segment), stubby protrusions on the outer membrane, and swollen mitochondria with markedly increased cristae in the somata. The findings in this study may reflect the metabolic disturbance of mitochondria, probably associated with the pathomechanism of degenerative processes of anterior horn neurons in sporadic ALS.     

Neuronal nitric oxide synthase immunoreactivity in the spinal cord in amyotrophic lateral sclerosis

We investigated the spinal cords of 15 patients with sporadic amyotrophic lateral sclerosis (ALS) immunohistochemically using an anti-human neuronal nitric oxide synthase (nNOS) antibody to examine whether there is increased nNOS immunoreactivity in anterior horn neurons. Specimens from 16 patients without any neurological disease served as controls. In the controls, nNOS immunoreactivity of large anterior horn neurons was detected in 10 out of 16 cases. However, there were few nNOS-positive neurons, and most of large anterior horn neurons were spared. In the ALS patients, the mean number of nNOS-positive anterior horn neurons per transverse section of L4 and L5 was significantly larger (16.2 +/- 10.9) than that in the controls (7.0 +/- 9.2) (P < 0.0001). Moreover, 41.4% of large anterior horn neurons in ALS showed nNOS immunoreactivity in remarkable contrast to 7.6% in the controls. All ALS patients, whether showing mild, moderate or severe depletion of anterior horn neurons, displayed a higher percentage of nNOS-positive anterior horn neurons than the control patients showing nNOS immunoreactivity (P < 0.01). Most of the remaining anterior horn neurons in ALS showed more intense nNOS immunoreactivity on the surface of the neurons and their neuronal processes compared with the controls. Degenerated anterior horn neurons frequently demonstrated more intense nNOS immunoreactivity on the surface of the neurons than normal-appearing neurons. Some anterior horn cells displayed nNOS immunoreactivity in the somata. Dot-like nNOS deposits on anterior horn neurons were also positively immunoreactive with anti-synaptophysin antibody. Thus, increased nNOS expression is located mainly at the synaptic sites on the anterior horn neurons in sporadic ALS, which may be related to the degeneration of anterior horn neurons in this disease. Further studies are needed to determine whether the increased nNOS immunoreactivity plays a neuroprotective or neurotoxic role in the anterior horn neurons, and to show nitric oxide production in ALS.     

Lack of association between VEGF polymorphisms and ALS in a Dutch population

Sequence alterations in the promoter region of the vascular endothelial growth factor (VEGF) gene have been implicated in increasing the risk of developing ALS. VEGF promoter haplotypes were determined in 373 patients with sporadic ALS and 615 matched healthy controls in The Netherlands. No significant association between the previously reported at-risk haplotypes and ALS was found. Pooling our results with the previously studied population still showed a significant association with the AAG haplotype.     

Autophagy in spinal cord motor neurons in sporadic amyotrophic lateral sclerosis

To assess the potential role of autophagy in amyotrophic lateral sclerosis (ALS), lumbar spinal cords in a total of 19 sporadic ALS cases and 27 age-matched controls were investigated. Immunohistochemical analysis using antibodies to the markers of autophagy microtubule-associated protein light chain 3 (LC3) and p62 was performed on samples from 12 ALS and 15 controls. Electron microscopy was performed on samples from 16 ALS and 15 controls, including overlapping cases. In the ALS cases, the somata of normal-appearing and degenerated motor neurons and round bodies were occasionally immunostained for LC3; round bodies and skein-like inclusions were immunostained for p62. By electron microscopy, all 16 ALS patients showed features of autophagy in the cytoplasm of normal-appearing motor neurons and, more frequently, in degenerated motor neurons. Autophagosomes surrounded by a double-membrane and autolysosomes isolated by a single membrane contained sequestered cytoplasmic organelles, such as mitochondria and ribosome-like structures. These autophagy features were also found in close association with the characteristic inclusions of ALS(i.e. round bodies, skein-like inclusions, and Bunina bodies); honeycomb-like structures also occasionally showed autophagy-associated features. Normal-appearing anterior horn neurons in control patients showed no autophagy features. Thus, autophagy seems to be activated and upregulated in the cytoplasm of motor neurons and may be involved in the mechanisms of neurodegeneration of motor neurons in sporadic ALS.     

Increased cerebrospinal fluid levels of substance P in patients with amyotrophic lateral sclerosis

To clarify the mechanism of brain and spinal cord impairment in amyotrophic lateral sclerosis (ALS), we measured the cerebrospinal fluid (CSF) levels of substance P (SP) in 11 patients with sporadic ALS. Findings were compared with those obtained in controls and diseased controls. The CSF SP levels of patients with ALS, and particularly in patients with a disease duration of less than 2.5 years, were significantly higher than those in controls. These findings strongly suggested that SP may play an important role in the pathophysiology of ALS.