Superoxide dismutase gene mutations in Italian patients with familial and sporadic amyotrophic lateral sclerosis: identification of three novel missense mutations

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder affecting motor neurons. The majority of the patients are sporadic cases (SALS), while 5-10% of the patients have a family history of ALS (familial ALS or FALS). Mutations in the gene coding for cytoplasmic Cu/Zn superoxide dismutase (SOD1) have been identified in about 20% of FALS cases. We found SOD1-gene mutations in five of 34 unrelated FALS, and in two of 44 SALS patients. Three FALS patients carried the previously described A4V (two cases) and L84F mutations (one case), while two FALS patients carried new missense mutations: a G12R substitution in exon 1, and a F45C substitution in exon 2, respectively. The newly identified mutations were both associated with a slowly progressive disease course. Two SALS patients carried the homozygous D90A and the heterozygous I113T mutation, respectively. In addition, in one SALS patient we identified an A95T amino acid substitution, that is apparently a non-pathogenic SOD1 variant. Our study increases the number of ALS-associated SOD1 gene mutations.     

Mutational analysis of the Cu/Zn superoxide dismutase gene in a Catalan ALS population: should all sporadic ALS cases also be screened for SOD1?

BACKGROUND: SOD1 gene mutations are the most common identified cause of ALS, accounting for approximately 20% of familial ALS cases and around 4% of sporadic ALS cases. However, the prevalence of SOD1 varies in different ethnic groups. No previous epidemiological studies have been carried out in Catalonia. OBJECTIVE: To determine the prevalence of SOD1 gene mutations in a Catalan ALS population, and to analyze the genotype-phenotype relationship. MATERIALS AND METHODS: 30 different FALS pedigrees and 94 sporadic ALS patients were screened for SOD1 mutations using direct sequence analysis. RESULTS: Five of the 30 FALS pedigrees (16.6%) carried a SOD1 mutant. The mutations identified in this group were G37R, D76V, S105L, I112M and N139H. Four SOD1 mutants (4.25%) were found in the sporadic ALS group (SALS). The overall frequency (FALS plus SALS) of SOD1 mutations in our series was 6.45%. In the SALS group, D90A was identified in a patient presenting the typical Scandinavian phenotype. A 53-year-old woman with no family history of ALS carried the N139H mutation. Two unrelated sporadic ALS cases carried the A140A SOD1 mutant. CONCLUSIONS: The prevalence of the SOD1 mutation in FALS in Catalonia is similar to levels in other Mediterranean countries, but lower than those in reports studying the Belgian, Japanese, and Scottish populations. The prevalence of the SOD1 mutation was 4.25% in patients with no family history of ALS. These results may have significant repercussions on genetic counseling, and screening for the SOD1 mutation in sporadic ALS cases must therefore be considered.     

SOD1 mutations in amyotrophic lateral sclerosis

Amyotrophic Lateral Sclerosis (ALS), the most common form among motoneuron diseases, is characterized by a progressive neurodegenerative process involving motor neurons in the motor cortex, brain stem and spinal cord. Sporadic (SALS) accounts for the majority of patients but in about 10% of ALS cases the disease is inherited (FALS), usually as an autosomal dominant trait.In the present study we show the results of a referred based multicenter study on the distribution of SOD1 gene mutations in the largest cohort of Italian ALS patients described so far. Two hundred and sixty-four patients (39 FALS and 225 SALS) of Italian origin were studied. In 7 out of 39 FALS patients we found the following SOD1 gene mutations: i) a new G12R missense mutation in exon 1, found in a patient with a slowly progressive disease course; ii) the G41S mutation, in four unrelated patients with rapidly progressive course complicated with cognitive decline in two of them; iii) the L114F mutation, in a patient with a slowly progressive phenotype; iv) the D90A mutation, in a heterozygous patient with atypical phenotype. In addition, in one SALS patient a previously reported synonymous variant S59S was identified. In 17 (3 FALS and 14 SALS) out of 264 patients (6.4 %) the polymorphism A-->C at position 34 of intron 3 (IVS3: + 34 A-->C) was found, and in one FALS patient a novel variant IVS3 + 62 T-->C was identified.The frequency of SOD1 gene mutations (17.9 %) in FALS cases was comparable with that found in other surveys with a similar sample size of ALS cases. No SOD1 gene mutations have been identified in SALS cases. Within FALS cases, The most frequent mutation was the G41S identified in four FALS.     

Clinical characteristics of SOD1 gene mutations in UK families with ALS.

Five to ten percent of patients with ALS have a family history of the disease, inheritance is usually autosomal dominant. Mutations of the SOD1 gene were first identified in a proportion of families with ALS by Rosen et al. The SOD1 gene encodes the enzyme copper zinc superoxide dismutase. Patients were studied from throughout the UK, where more than one individual in the family had ALS. Clinical history and examination of the individual and family were obtained, and DNA extracted from leukocytes of whole blood samples. Mutations were identified by standard sequencing methods. To date, 12 different mutations of SOD1 have been identified in 17 different families, representing around 20% of all ALS families studied. The mutations were mainly single base substitutions - H48Q, G72S, G93R, G93V, E100G, D101N, D101G, G108V, I113T, D125H, I149T - and also an insertion mutation - 132insTT - leading to a premature stop codon. The mutations were present in exons 2-5. We did not identify mutations in exon 1, although these have been identified by others in different patient samples. We have identified SOD1 mutations in around 20% of UK families with ALS studied. This is similar to that reported in other populations. Mutations have now been identified in all exons of SOD1. The individual mutations do not precisely predict disease severity, and generally it is difficult to give a specific prognosis based on the individuals' SOD1 mutations. We continue to investigate the possible pathogenic mechanisms of the SOD1 mutations. We have studied the neuropathology in patients with SOD1 mutations. We are also performing linkage studies to identify the genes involved in the 80% of families where an SOD1 mutation has not been identified.     

S100A6 overexpression within astrocytes associated with impaired axons from both ALS mouse model and human patients

Astrogliosis is one of the earliest pathological changes observed in neurodegenerative diseases in general and in amyotrophic lateral sclerosis (ALS) in particular. ALS is characterized by selective degeneration of motoneurons. There are 2 forms of the disease: sporadic ALS (SALS), comprising 90%-95% of cases, and familial ALS (FALS), comprising 5%-10% of cases. FALS is an age-dependent autosomal dominant disorder in which mutations in the homodimeric enzyme Cu/ Zn superoxide dismutase 1 (SOD1) is linked to the disease. The animal model for this disease is a transgenic mouse expressing the mutated human SOD1(G93A) gene. Here we show by immunohistochemistry and double immunofluorescence that astrocytes located near impaired axons of motoneurons that were selectively programmed to die overexpressed S100A6, a Ca2+/Zn2+ binding protein able to translocate into the nucleus. Transgenic mice overexpressing the mutated human SOD1 gene and patients suffering from SALS showed this selective astrocytic S100A6 expression. For instance, the pyramidal tract could be macroscopically detected on S100A6-labeled spinal cord and brainstem sections from SALS patients. Transgenic mice overexpressing the non-mutated SOD1 gene did not overexpress S100A6, although glial fibrillary associated protein astrogliosis was seen. Although these results do not give any clue about the beneficial or detrimental role played by S100A6, its induction may be assumed to appropriately serve some function(s).     

The physiological basis of conduction slowing in ALS patients homozygous for the D90A CuZn-SOD mutation

Familial amyotrophic lateral sclerosis (ALS) with the autosomal-recessively inherited D90A CuZn-superoxide dismutase (CuZn-SOD) mutation is characterized by a stereotypic slowly progressive, distinctive phenotype and very slow central motor conduction. To determine the basis of this slowing, we assessed corticomotoneuronal function using peristimulus time histograms (PSTHs) in 8 ALS patients homozygous for the D90A CuZn-SOD mutation. The results were compared with findings in 10 patients with multiple sclerosis (MS), in which slowing of central motor conduction is common, and 11 healthy subjects. PSTHs were constructed from 3-7 different, voluntarily recruited motor units recorded in each patient from the extensor digitorum communis muscle (EDC). In D90A and MS patients, the stimulus threshold, onset latency, number of excess bins, duration, amplitude, and synchrony of the primary peak differed significantly from controls (P < 0.0004). The mean onset latency of the primary peak in D90A patients was 35.3 ms, compared to 23.6 ms for MS patients and 19.3 ms for normal subjects (P < 0.0001). In the D90A patients, the onset latencies of the primary peak had a bimodal distribution, whereas in MS the distribution showed a continuum. Loss of synchrony was similar in D90A and MS patients, but the threshold, number of excess bins, and duration differed significantly (P < 0.0057), which suggests that either axonal loss or demyelination can result in delayed and desynchronized primary peaks. We propose that conduction slowing in the D90A homozygotes results from selective loss of fast-conducting large pyramidal cells with preservation of slow-conducting mono- or polysynaptic corticomotoneuronal connections.     

Redox system expression in the motor neurons in amyotrophic lateral sclerosis (ALS): immunohistochemical studies on sporadic ALS, superoxide dismutase 1 (SOD1)-mutated familial ALS, and SOD1-mutated ALS animal models

Peroxiredoxin-ll (Prxll) and glutathione peroxidase-l (GPxl) are regulators of the redox system that is one of the most crucial supporting systems in neurons. This system is an antioxidant enzyme defense system and is synchronously linked to other important cell supporting systems. To clarify the common self-survival mechanism of the residual motor neurons affected by amyotrophic lateral sclerosis (ALS), we examined motor neurons from 40 patients with sporadic ALS (SALS) and 5 patients with superoxide dismutase 1 (SOD1)-mutated familial ALS (FALS) from two different families (frame-shift 126 mutation and A4 V) as well as four different strains of the SOD1-mutated ALS models (H46R/G93A rats and G1H/G1L-G93A mice). We investigated the immunohistochemical expression of Prxll/GPxl in motor neurons from the viewpoint of the redox system. In normal subjects, Prxll/GPxl immunoreactivity in the anterior horns of the normal spinal cords of humans, rats and mice was primarily identified in the neurons: cytoplasmic staining was observed in almost all of the motor neurons. Histologically, the number of spinal motor neurons in ALS decreased with disease progression. Immunohistochemically, the number of neurons negative for Prxll/GPxl increased with ALS disease progression. Some residual motor neurons coexpressing Prxll/GPxl were, however, observed throughout the clinical courses in some cases of SALS patients, SOD1-mutated FALS patients, and ALS animal models. In particular, motor neurons overexpressing Prxll/GPxl, i.e., neurons showing redox system up-regulation, were commonly evident during the clinical courses in ALS. For patients with SALS, motor neurons overexpressing Prxll/GPxl were present mainly within approximately 3 years after disease onset, and these overexpressing neurons thereafter decreased in number dramatically as the disease progressed. For SOD1-mutated FALS patients, like in SALS patients, certain residual motor neurons without inclusions also overexpressed Prxll/GPxl in the short-term-surviving FALS patients. In the ALS animal models, as in the human diseases, certain residual motor neurons showed overexpression of Prxll/GPxl during their clinical courses. At the terminal stage of ALS, however, a disruption of this common Prxll/GPxl-overexpression mechanism in neurons was observed. These findings lead us to the conclusion that the residual ALS neurons showing redox system up-regulation would be less susceptible to ALS stress and protect themselves from ALS neuronal death, whereas the breakdown of this redox system at the advanced disease stage accelerates neuronal degeneration and/or the process of neuronal death.     

Phosphorylated Smad2/3 immunoreactivity in sporadic and familial amyotrophic lateral sclerosis and its mouse model

Phosphorylated Smad2/3 (pSmad2/3), the central mediators of transforming growth factor (TGF)-beta signaling, were recently identified in tau-positive inclusions in certain neurodegenerative disorders. To clarify whether the localization of pSmad2/3 is altered in amyotrophic lateral sclerosis (ALS), we immunohistochemically examined spinal cords from sporadic ALS (SALS), from familial ALS (FALS) patients with the A4V mutation in their Cu/Zn superoxide dismutase (SOD1) gene, and from G93A mutant SOD1 transgenic (mSOD1 Tg) mice. In control spinal cords, pSmad2/3 immunoreactivity was observed exclusively in neuronal and glial nuclei. In SALS and FALS patients the nuclei showed increased immunoreactivity for pSmad2/3. Noticeably, round hyaline inclusions (RHIs) and skein-like inclusions of SALS patients were immunoreactive for pSmad2/3. Double immunofluorescence staining for pSmad2/3 and transactive response-DNA-binding protein (TDP)-43 revealed co-localization of these proteins within RHIs. In contrast, Bunina bodies in SALS and Lewy body-like hyaline inclusions (LBHIs) in FALS were devoid of labeling for pSmad2/3. Similarly, in the mSOD1 Tg mice pSmad2/3 immunoreactivity was increased in the nuclei, while LBHIs were not labeled. These findings suggest increased TGF-beta-Smad signaling in SALS, FALS, and mSOD1 Tg mice, as well as impaired TGF-beta signal transduction in RHI-bearing neurons of SALS patients, presumably at the step of pSmad2/3 translocation into the nucleus. The pathomechanisms, including the process of inclusion development, appears to be different between SALS and mSOD1-related FALS or Tg mice.     

An analysis of the entire SOD1 gene in sporadic ALS

Mutations in the superoxide dismutase 1 gene (SOD1) are associated with familial ALS but the role of SOD1 in sporadic ALS (SALS) is unclear. We therefore sequenced the entire SOD1 gene in 23 patients with SALS. DNA was extracted from frozen pre-frontal cerebral cortex and from blood. The 5 exons, 4 introns and 1 kb upstream and downstream of SOD1 were sequenced. Novel genetic variants were found in 30% (7 of 23) brains and known variants in 91% (21 of 23) brains from patients with SALS. Two novel variants found in SALS patients and not controls were located in the SOD1 promoter and intron 1, with the promoter variant having potential functional implications. A previously described silent variant in exon 5 in one SALS patient appears to abolish an exonic splicing enhancer. All changes found in brain DNA were also found in blood DNA. In conclusion, sequencing the entire SOD1 gene revealed 3 variants in SALS patients that were not detected in controls. Although no unequivocal mutations were found, some of these variants have potential consequences for SALS pathogenesis.     

Limited corticospinal tract involvement in amyotrophic lateral sclerosis subjects with the A4V mutation in the copper/zinc superoxide dismutase gene

We examined 11 subjects with inherited amyotrophic lateral sclerosis (familial amyotrophic lateral sclerosis, FALS) associated with the most common copper/zinc superoxide dismutase 1 (SOD1) mutation, an alanine for valine substitution in codon 4 (A4V). Autopsies were performed on 5 subjects. The clinical and pathological findings are described and compared with those of 9 sporadic ALS (SALS) subjects. There was no clinical evidence of upper motor neuron (UMN) involvement in 10 FALS A4V subjects. All subjects had lower motor neuron (LMN) signs and electrophysiological evidence of denervation in at least three limbs. All SALS subjects had signs of both UMN and LMN involvement. Pathological studies found severe abnormalities of LMNs in all FALS and SALS subjects. UMN involvement was either absent or mild in the A4V SOD1 FALS subjects and severe in the SALS subjects. Pathological abnormalities in systems other than the motor neurons were more frequent in the FALS A4V subjects. This information suggests that current diagnostic criteria for ALS, requiring clinical evidence for both upper and lower motor neuron involvement, should be modified; ie, the diagnosis should be deemed established when there is evidence of denervation in three or more limbs and a mutation in the gene for SOD1, even without clinical signs of UMN involvement.     

Red cell superoxide dismutase activity in sporadic amyotrophic lateral sclerosis

Specific biologic markers are not available for definitive diagnosis and monitoring of disease progression in sporadic amyotrophic lateral sclerosis (SALS). Oxidative stress plays a role in ALS pathogenesis. The purpose of this study was to determine the association between Cu/Zn superoxide dismutase (SOD1) activity, diagnosis and prognosis. The present study included 25 SALS patients (SALS group; age 51+/-12 years) and 10 healthy subjects (age 45+/-5 years) as a control group. Patients were divided into groups representing four levels of diagnostic certainty of ALS in accordance with the El Escorial Revisited criteria. The disease state was determined using the modified ALS health state scale of Riviere et al. (Arch Neurol 1998:55;526-8). Red-cell SOD1 activity was determined by spectrophotometry. SOD1 activity in red cells was compared statistically with diagnostic criteria and disease state. Red cell SOD1 activity was high in all SALS patients, but there was no significant association between enzyme activity and diagnostic criteria and disease state. In this preliminary study, we did not find any correlation between SOD1 activity level and diagnosis or prognosis. Measured SOD1 activity sometimes supports ALS diagnosis, but it is neither a specific nor a prognostic factor.     

A novel central motor conduction abnormality in D90A-homozygous patients with amyotrophic lateral sclerosis

Patients with amyotrophic lateral sclerosis (ALS) who are homozygous for the D90A SOD1 mutation have been noted to have central motor abnormalities distinct from those of patients with idiopathic ALS. We stimulated the motor cortex of ten patients homozygous for the D90A SOD1 mutation, using transcranial magnetic stimulation (TMS), and recorded the response evoked in the right first dorsal interosseous muscle when the muscle was at rest and when voluntarily active. A subgroup of patients had two distinct evoked responses when the cortex was stimulated at high intensity with the muscle at rest. When the muscle was modestly contracted, the first of these responses disappeared, whereas the second response was facilitated. Both fast and slow components of the corticospinal tract were usually intact and excited by TMS in these patients. We propose that there is an abnormality of intracortical or intraspinal inhibition in a subgroup of D90A SOD1 ALS patients, which suppresses fast-conducted activity when the muscle is active. Apart from further defining the phenotype of familial ALS, these findings may have importance in understanding the pathogenesis of central motor abnormalities in these patients.     

Superoxide dismutase in amyotrophic lateral sclerosis patients homozygous for the D90A mutation

The most common of the amyotrophic lateral sclerosis (ALS)-associated superoxide dismutase-1 (SOD1) mutations, D90A, differs from others in its high structural stability and by the existence of both recessive and dominant inheritance. Here SOD1 in CNS and peripheral organs from five ALS patients homozygous for D90A were compared to controls. In most areas, including ventral horns, there were no significant differences in SOD1 activities and Western blotting patterns between controls and D90A cases. The SOD1 activities in areas vulnerable to mutant SOD1s, ventral horns and precentral gyrus were intermediate among CNS areas and much lower than in kidney and liver. Thus, the vulnerability of motor areas is not explained by high SOD1 content. The findings argue against the idea of expression-reducing protective factors being present near the D90A locus in recessive pedigrees. The similarity to wild-type SOD1 prompts speculations on the involvement of the latter in sporadic ALS.     

Comparison of the clinical and cognitive features of genetically positive ALS patients from the largest tertiary center in Serbia

Discovering novel mutations in C9orf72, FUS, ANG, and TDP-43 genes in ALS patients arises necessities for better clinical characterizations of these subjects. The aim is to determine clinical and cognitive profile of genetically positive Serbian ALS patients. 241 ALS patients were included in the study (17 familiar and 224 apparently sporadic). The following genes were analyzed: SOD1, C9orf72, ANG, FUS, and TDP-43. An extensive battery of classic neuropsychological tests was used in 27 ALS patients (22 SOD1 positive and 5 SOD1 negative) and 82 healthy controls (HCs). Overall 37 (15.4%) of 241 ALS patients carried mutations in tested genes—among 17 familiar ALS patients 16 (94.1%) were positive and among 224 apparently sporadic 21 (9.4%) had causative mutation. Mutations in SOD1 gene were the most common, representing 27 (73.0%) of all genetically positive ALS patients. The main clinical characteristics of SOD1 positive patients were: spinal onset in lower extremities, common sphincter and sensitive disturbances, and dysexecutive syndrome. Within SOD1 positive patients, we noticed somewhat earlier onset in patients with A145G, sensory and sphincter disturbances were dominant in patients with L144F, while D90A patients had significant sensory involvement. SOD1 negative group consisted of ten (27.0%) patients (six C9orf72, two ANG, one TDP-43, and one patient baring triple FUS, C9orf72 expansion, and ANG variants). Bulbar involvement and more extensive neuropsychological impairment (including executive, visuospatial, and memory difficulties) were the main features of SOD1 negative cohort. Our results suggest that meaningful clinical suspicion of certain ALS genotype might be made based on thorough clinical evaluation of patients.     

Cu/Zn superoxide dismutase activity at different ages in sporadic amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive disorder resulting from degeneration of motor neurons in the brain and spinal cord. Sporadic ALS (SALS) accounts for the majority of patients and the familial form (FALS) represents fewer than 10% of all cases. Since it was found that there are Cu/Zn superoxide dismutase (SODI) gene mutations in 20% of FALS patients and that FALS and SALS patients show similar clinical features, it has been postulated that both may share a common physiopathological mechanism. We studied Cu/Zn SOD1 activity in cytosolic extracts of erythrocytes from 125 normal individuals and 40 SALS patients. We found that enzyme activity does not change with age in control subjects and tends to decrease in most SALS patients older than 60 years. A subpopulation of five SALS patients had significantly increased SOD1 activity; four of these patients over 70 years old. There was no correlation between enzyme activity and time of onset of the disease, or clinical forms of the illness. The variation in SOD1 activity in ageing SALS patients compared with younger patients suggests that they may undergo an oxidative disbalance contributing to the development of the disease.     

二肽基肽酶-6基因多态和散发性肌萎缩侧索硬化发病风险的相关性

目的 探讨中国人二肽基肽酶-6(DPP6)基因中的单核苷酸多态位点rs10260404与散发性肌萎缩侧索硬化(SALS)易患性是否有关.方法 来自中国汉族人群的58例患者与52名健康对照分别提取样本外周血基因组DNA,采用不对称PCR方法扩增包含目标单核苷酸多态位点在内的片段,用高分辨熔解法完成非标记探针的基因分型.结果 SALS患者与健康对照之间rs10260404位点的等位基因频率(SALS组:C:12.94%,T:87.06%;健康对照组:C:10.58%,T:89.43%)差异无统计学意义(χ2=0.29,OR=1.256,95% CI 0.549～2.872,P>0.05).结论 我们未发现DPP6基因中rs10260404位点和中国人SALS的致病风险相关,这可能和ALS本身复杂的遗传异质性有关,但有必要进一步增加病例数以证实.     

Corticomotoneuronal activity in ALS: changes in the peristimulus time histogram over time.

OBJECTIVE: The primary peak in the peristimulus time histogram (PSTH) reflects the initial rising phase of the excitatory post-synaptic potential (EPSP) evoked at the anterior horn cell. In ALS the primary peak is delayed in onset. increased in duration and desynchronized. abnormalities reflecting dysfunction of the corticomotoneurons. It is not known whether these abnormalities change over time in amyotrophic lateral sclerosis (ALS). METHODS: PSTHs were constructed from changes in the firing probability of single, voluntarily activated motor units subjected to subthreshold transcranial magnetic stimuli. We studied 58 motor units in 12 patients with ALS on two separate occasions (mean time interval of 10.6 +/- 1.6 months). Results were compared with 49 motor units in 11 age matched controls. RESULTS: All the parameters except the amplitude differed significantly between normals and patients. In general the primary peak in ALS was complex, desynchronized and occasionally consisted of a double peak. The abnormalities persisted or were accentuated at tile follow up visit. This was reflected by an increase in the number of excess bins, longer duration and latency and decrease of synchrony. CONCLUSIONS: Increasing desynchronization of the primary peak over time in ALS reflects dysfunction of the monosynaptic corticomotoneuronal pathway and may also reflect activation of additional slow conducting and/or polysynaptic corticomotoneuronal connections.     

HtrA2/Omi‐immunoreactive intraneuronal inclusions in the anterior horn of patients with sporadic and Cu/Zn superoxide dismutase (SOD1) mutant amyotrophic lateral sclerosis

Y. Kawamoto, H. Ito, Y. Kobayashi, Y. Suzuki, I. Akiguchi, H. Fujimura, S. Sakoda, H. Kusaka, A. Hirano and R. Takahashi (2010) Neuropathology and Applied Neurobiology 36, 331–344
HtrA2/Omi‐immunoreactive intraneuronal inclusions in the anterior horn of patients with sporadic and Cu/Zn superoxide dismutase (SOD1) mutant amyotrophic lateral sclerosis Aims: HtrA2/Omi is a mitochondrial serine protease that promotes the apoptotic processes, but the relationship between HtrA2/Omi and amyotrophic lateral sclerosis (ALS) is still unknown. The purpose of the present study was to determine whether abnormal expression of HtrA2/Omi occurs in patients with ALS. Methods: We prepared autopsied spinal cord tissues from 7 control subjects, 11 patients with sporadic ALS (SALS) and 4 patients with Cu/Zn superoxide dismutase (SOD1)‐related familial ALS (FALS). We then performed immunohistochemical studies on HtrA2/Omi using formalin‐fixed, paraffin‐embedded sections from all of the cases. Results: In the control subjects, the anterior horn cells were mildly to moderately immunostained with HtrA2/Omi. In the patients with SALS, strong HtrA2/Omi immunoreactivity was found in some skein‐like inclusions and round hyaline inclusions as well as many spheroids, but Bunina bodies were immunonegative for HtrA2/Omi. In the patients with SOD1‐related FALS, Lewy body‐like hyaline inclusions were observed in three cases and conglomerate inclusions were observed in the remaining case, and both types of inclusions were intensely immunopositive for HtrA2/Omi. Conclusions: These results suggest that abnormal accumulations of HtrA2/Omi may occur in several types of motor neuronal inclusions in the anterior horn from SALS and SOD1‐linked FALS cases, and that HtrA2/Omi may be associated with the pathogenesis of both types of ALS.     

Identification of new ANG gene mutations in a large cohort of Italian patients with amyotrophic lateral sclerosis

Angiogenin (ANG) gene, coding for an angiogenic factor up-regulated by hypoxia and expressed in ventral horn motor neurons, is a novel candidate for the pathogenesis of amyotrophic lateral sclerosis (ALS). ALS is a fatal neurodegenerative disease characterized by the selective loss of cortical and spinal motor neurons. Missense mutations in ANG gene have been identified in two ALS populations from Northern Europe and North America, both in familial (FALS) and sporadic (SALS) patients, but they do not seem to be frequent in the Italian population. We performed a mutational screening in a large cohort of 737 Italian ALS patients, including 605 SALS and 132 FALS cases. We identified seven different mutations, five of which are novel, in nine patients (six SALS and three FALS), but not in 515 healthy controls. Three mutations are located in the signal peptide region, three in the coding sequence, and one in the 3′ untranslated region. In our ALS population, the observed mutational frequency of ANG gene accounts for about 1.2%, with an overrepresentation of FALS (2.3%) compared to SALS (1%) cases. We also found the previously described I46V substitution in six patients and four controls, suggesting that this mutation may represent a benign variant, at least in the Italian population. Our results provide further evidence of a tight link between angiogenesis and ALS pathogenesis and suggest that mutations in ANG gene are associated with an increased risk to develop ALS. Cinzia Gellera and Claudia Colombrita contributed equally to this work.