Screening of the PFN1 gene in sporadic amyotrophic lateral sclerosis and in frontotemporal dementia

Mutations in the profilin 1 (PFN1) gene, encoding a protein regulating filamentous actin growth through its binding to monomeric G-actin, have been recently identified in familial amyotrophic lateral sclerosis (ALS). Functional studies performed on ALS-associated PFN1 mutants demonstrated aggregation propensity, alterations in growth cone, and cytoskeletal dynamics. Previous screening of PFN1 gene in sporadic ALS (SALS) cases led to the identification of the p.E117G mutation, which is likely to represent a less pathogenic variant according to both frequency data in control subjects and cases, and functional experiments. To determine the effective contribution of PFN1 mutations in SALS, we analyzed a large cohort of 1168 Italian SALS patients and also included 203 frontotemporal dementia (FTD) cases because of the great overlap between these 2 neurodegenerative diseases. We detected the p.E117G variant in 1 SALS patient and the novel synonymous change p.G15G in another patient, but none in a panel of 1512 control subjects. Our results suggest that PFN1 mutations in sporadic ALS and in FTD are rare, at least in the Italian population.     

ATAXIN2 CAG-repeat length in Italian patients with amyotrophic lateral sclerosis: risk factor or variant phenotype? Implication for genetic testing and counseling

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease mainly involving cortical and spinal motor neurons. Several studies indicated that intermediate CAG expansions in ataxin-2 gene (ATXN2) are associated with increased risk of ALS. We analyzed ATXN2 CAG repeats in 658 sporadic ALS patients (SALS), 143 familial ALS cases (FALS), 231 sporadic ataxic subjects, and 551 control subjects. The frequency of ATXN2 alleles with 27-30 repeats was similar in SALS and control subjects. Fifteen SALS subjects carried ≥ 31 CAG repeats. This difference was statistically significant (p = 0.0014). No alleles with ≥ 34 CAG were found. In FALS, the distribution of ATXN2 alleles was similar to control subjects. Our results further contributed in refining CAG-repeat range significantly associated with sporadic ALS. Literature data and our findings indicate that only alleles with ≥ 31 CAG may represent low-penetrance disease/susceptibility alleles associated with variable neurodegenerative phenotypes, including cerebellar ataxia, parkinsonism, and ALS. Overlapping phenotypes should be considered in genetic testing and counseling, both for patients and at-risk family members.     

Vesicle associated membrane protein B (VAPB) is decreased in ALS spinal cord

The aim of this study was to quantify spinal cord expression of genes known to cause familial amyotrophic lateral sclerosis (FALS) or influence survival in a large cohort of sporadic cases of ALS (SALS), in order to determine their relevance to pathogenic mechanisms occurring in SALS. The expression of superoxide dismutase 1 (SOD1), vesicle associated membrane protein (VAPB), senataxin (SETX), dynactin (DCTN1), vascular endothelial growth factor (VEGF), insulin-like growth factor-1 (IGF1), the small heat shock proteins, HSPB1 and HSPB8, and three genes activated during disease progression, caspases-1 and -3 and glial fibrillary acidic protein (GFAP), were quantified. Robust changes in the expression of four genes were found, VAPB mRNA levels were decreased in the spinal cord of ALS patients compared to controls (p<0.006), whilst HSPB1, HSPB8 and caspase-1 showed significant increases (1.5-2.3-fold). Expression of VAPB mRNA and protein was predominantly localised to large motor neurones further supporting the relevance of this finding to disease progression occurring in SALS.     

De novo FUS gene mutations are associated with juvenile-onset sporadic amyotrophic lateral sclerosis in China

Juvenile amyotrophic lateral sclerosis (ALS) is a rare form of motor neuron disease and occurs before 25 years of age. Only very few sporadic cases of juvenile-onset ALS have been reported. Rare SOD1 mutations and several FUS mutations have been identified in juvenile-onset ALS patients. To define the genetics of juvenile-onset sporadic ALS (SALS) of Chinese origin, we sequenced all 5 exons of SOD1, exons 3–6 and 12–15 of FUS in 11 juvenile-onset SALS patients, 105 adult-onset ALS patients (including 6 familial ALS [FALS] pedigrees), and 245 healthy controls. For the 11 juvenile-onset SALS and 6 FALS cases, the other 7 exons of FUS were also screened. A heterozygous de novo missense mutation c.1574C>T (p.P525L), a heterozygous de novo 2-base pair deletion c.1509_1510delAG (p.G504Wfs*12), and a nonsense mutation c.1483C>T (p.R495X) was each identified in 1 juvenile SALS patient. A heterozygous missense mutation c.1561C>G (p.R521G) was identified in a FALS proband. In the Chinese population, the frequency of FUS mutation in FALS is 11.4% (95% confidence interval [CI], 0.9%–22.0%), higher than the Japanese (10%; 95% CI, 0.7%–19.3%), and Caucasians (4.9%; 95% CI, 3.9%–6.0%). The frequency of FUS mutation in SALS patients is 1.5% (95% CI, 0.2%–2.9%), which is similar to Koreans (1.6%; 95% CI, 0%–3.2%), but higher than in Caucasians (0.6%; 95% CI, 0.4%–0.8%). Our findings suggest that de novo FUS mutations are associated with juvenile-onset SALS of Chinese origin and that this gene should be screened in ALS patients with a young age of onset, aggressive progression, and sporadic occurrence.     

Increase in oxidized NO products and reduction in oxidized glutathione in cerebrospinal fluid from patients with sporadic form of amyotrophic lateral sclerosis

To determine the role of free radical mechanisms in the pathogenesis of amyotrophic lateral sclerosis (ALS), cerebrospinal fluid concentrations of oxidized nitric oxide (NO) products (nitrite and nitrate) and reduced and oxidized forms of glutathione (GSH and GSSG, respectively) were compared between patients with the sporadic form of ALS (SALS) and controls. In the SALS patients, the nitrate levels were significantly higher (by 73%) in contrast to remarkably lower GSSG/GSH ratio, approximately 3-fold, compared to controls. These results suggest that NO production or oxidation is activated in SALS patients, leading to a decrease in superoxide radicals to oxidize GSH. The subsequent generation of a highly reactive anion, peroxynitrite, may play a causal role in the pathogenesis of SALS.     

A novel peripherin gene (PRPH) mutation identified in one sporadic amyotrophic lateral sclerosis patient

Motor neurons in amyotrophic lateral sclerosis (ALS) are characterized by the presence of inclusion bodies composed of intermediate filament (IF) proteins. Peripherin protein is as components of these inclusions and rare mutations in peripherin gene (PRPH) were identified in sporadic ALS cases. The aim of this study was to further define the spectrum of PRPH mutations in a cohort of 122 Italian ALS patients. We screened the coding sequence, the exon/intron boundaries, and the 5′-3′ un-translated regions (UTRs) in 122 ALS patients. Eighteen sequence variations were detected. Seven variants were not identified in a panel of at least 245 matched controls, including 2 missense variations, namely p.R133P and p.D141Y, each identified in one heterozygous patient. p.R133P was newly identified whereas p.D141Y was previously described in one homozygous sporadic ALS patient. These 2 variants were predicted to have a deleterious effect on protein structure or function. This work contributes to determine the role of PRPH gene variants in ALS. Further studies are necessary to define the mechanisms through which the mutant peripherin could cause ALS phenotype.     

Is erythropoietin gene a modifier factor in amyotrophic lateral sclerosis?

To investigate the role of erythropoietin (EPO) as genetic determinant in the susceptibility to sporadic amyotrophic lateral sclerosis (SALS). We sequenced a 259-bp region spanning the 3'hypoxia-responsive element of the EPO gene in 222 Italian SALS patients and 204 healthy subjects, matched for age and ethnic origin. No potentially causative variation was detected in SALS subjects; in addition, two polymorphic variants (namely C3434T and G3544T) showed the same genotype and haplotype frequencies in patients and controls. Conversely, a weak but significant association between G3544T and age of disease onset was observed (p=0.04). Overall, our data argue against the hypothesis of EPO as a genetic risk factor for motor neuron dysfunction, at least in Italian population. However, further studies on larger cohort of patients are needed to confirm the evidence of EPO gene as modifier factor.     

C9ORF72 repeat expansion in a large Italian ALS cohort: evidence of a founder effect

A hexanucleotide repeat expansion (RE) in C9ORF72 gene was recently reported as the main cause of amyotrophic lateral sclerosis (ALS) and cases with frontotemporal dementia. We screened C9ORF72 in a large cohort of 259 familial ALS, 1275 sporadic ALS, and 862 control individuals of Italian descent. We found RE in 23.9% familial ALS, 5.1% sporadic ALS, and 0.2% controls. Two cases carried the RE together with mutations in other ALS-associated genes. The phenotype of RE carriers was characterized by bulbar-onset, shorter survival, and association with cognitive and behavioral impairment. Extrapyramidal and cerebellar signs were also observed in few patients. Genotype data revealed that 95% of RE carriers shared a restricted 10-single nucleotide polymorphism haplotype within the previously reported 20-single nucleotide polymorphism risk haplotype, detectable in only 27% of nonexpanded ALS cases and in 28% of controls, suggesting a common founder with cohorts of North European ancestry. Although C9ORF72 RE segregates with disease, the identification of RE both in controls and in patients carrying additional pathogenic mutations suggests that penetrance and phenotypic expression of C9ORF72 RE may depend on additional genetic risk factors.     

Screening of the TARDBP gene in familial and sporadic amyotrophic lateral sclerosis patients of Chinese origin

TAR DNA-binding protein (TARDBP) mutations have been reported in patients with amyotrophic lateral sclerosis (ALS) in different populations. Only a few studies have screened for TARDBP mutations in Chinese populations. Here, we sequenced the coding region of all five TARDBP exons for mutations in 13 familial ALS (FALS) pedigrees and 312 sporadic ALS (SALS) patients of Chinese origin, as well as 245 healthy control subjects. Two heterozygous missense mutations, c.875G>A (p.S292N) and c.1043G>T (p.G348V), were identified in two and one SALS patients, respectively. One synonymous substitution, c.1098C>G (p.A366A), was identified in two SALS patients. None of the substitutions were found in healthy control subjects. In Chinese populations, the estimated frequency of TARDBP mutations in SALS patients (0.73%) is higher than Japanese and lower than White populations, whereas the estimated mutation frequency in superoxide dismutase 1 (SOD1)-negative FALS patients (15.2%) is higher than both Japanese and White populations. Our findings provide an overview of the occurrence of TARDBP mutations in Chinese ALS patients and highlight the importance of TARDBP mutation screening in Chinese ALS patients.     

Homozygous SMN2 deletion is a protective factor in the Swedish ALS population

Abnormal survival motor neuron 1 (SMN1)-copy number has been associated with an increased risk of amyotrophic lateral sclerosis (ALS) in French and Dutch population studies. The aim of this study was to determine whether SMN gene copy number increases the risk of ALS or modulates its phenotype in a cohort of Swedish sporadic ALS (SALS) patients. In all, 502 Swedes with SALS and 502 Swedish controls matched for gender and age were enrolled. SMN1 and SMN2 gene copy numbers were studied by a semi-quantitative PCR method. A genotype-phenotype comparison was performed in order to determine whether SMN genes modulate the phenotype of ALS. The results were also compared with our previously reported French cohort of ALS patients. There was no difference between Swedish patients and controls in the frequency of SMN1 and SMN2 copy numbers. The frequency of SMN1 gene copies differed significantly between the French and Swedish ALS populations. The duration of the disease was significantly longer in the Swedish cohort with homozygous deletions of SMN2 when compared with the French cohort. Abnormal SMN1 gene copy number cannot be considered as a universal genetic susceptibility factor for SALS and this result underlines the importance of reproducing association gene studies in groups from different origins. We also suggest that SMN2 gene copy number might have different effects on ALS progression in disparate human populations.     

Novel heterozygous nonsense mutation of the OPTN gene segregating in a Danish family with ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder. About 10% of ALS cases are familial (FALS) and the genetic defect is known only in approximately 20%-30% of these cases. The most common genetic cause of ALS is SOD1 (superoxide dismutase 1) mutation. Very recently, mutations of the optineurin gene (OPTN), which is involved in open-angle glaucoma, were identified in 3 Japanese patients/families with ALS, and subsequently in a few FALS patients of European descent. We found a heterozygous nonsense mutation (c.493C>T, p.Gln165X, exon 6) in the OPTN gene in a Danish patient with ALS, and the mutation segregated from his affected father. The p.Gln165X mutation could not be detected in 1070 healthy Danish controls, in 1000 Danish individuals with metabolic phenotypes or in 64 sporadic ALS (SALS) cases. The p.Gln165X mutation described in this study is the first mutation reported in a Danish family and is likely involved in disease pathogenesis. Until now, only few OPTN mutations have been associated with ALS. As the underlying genetic defect is known only in approximately 20%-30% of FALS families, further screening of these cases is necessary for establishing the contribution of OPTN mutations in disease pathogenesis.     

Analysis of OPTN as a causative gene for amyotrophic lateral sclerosis

Mutations in the OPTN gene are well known to be associated with the development of glaucoma. Recently, unique variations in the same gene have been reported in familial and sporadic Japanese cases of amyotrophic lateral sclerosis (ALS). We set out to evaluate the frequency of OPTN mutations in a sample of our familial and sporadic ALS cohorts. All coding exons of the OPTN gene were amplified and sequenced in 95 unrelated familial ALS (FALS) and 95 sporadic ALS (SALS) cases of European descent. Two variants were newly identified in 2 individual FALS cases. Unique variations in the OPTN gene are rare in FALS cases and were not identified in any SALS patients, all of European descent.     

Progranulin does not affect motor neuron degeneration in mutant SOD1 mice and rats

Motor neuron degeneration in amyotrophic lateral sclerosis (ALS) is familial in 10% of patients, with mutations in SOD1 and C9orf72 being the most frequent cause. There is convincing evidence for overlap between ALS and frontotemporal lobar degeneration at the genetic, pathological, and clinical level. Null mutations in progranulin (PGRN) are a frequent cause of familial frontotemporal lobar degeneration. PGRN exerts neurotrophic properties on motor neurons in vitro and in vivo. We therefore examined whether PGRN could affect disease progression in mutant SOD1 mice and rats, both established models for ALS. Overexpression of PGRN in mice and intracerebroventricular delivery of PGRN in rats did not affect onset or progression of motor neuron degeneration.     

Mutational analysis of the Cu/Zn superoxide dismutase gene in 23 familial and 69 sporadic cases of amyotrophic lateral sclerosis in Belgium.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder caused by degeneration of motor neurons of the spinal cord and brain. The majority of ALS cases are sporadic (SALS). However, in 10-15% of ALS cases the disease is inherited as an autosomal dominant trait (familial ALS or FALS). We used a non-radioactive SSCP method, in combination with solid phase sequencing, to screen the entire SOD1 (Cu/Zn superoxide dismutase) coding region and flanking intronic sequences for mutations. Twenty-three patients from 11 ALS families and 69 SALS patients of Belgian origin were studied. Three different mutations were identified (L38V, D90A and G93C) in seven families. Importantly, the D90A was only found in the heterozygous state. In addition two single base pair variants (IVS1 + 19G > A and AAC139 AAT) were identified in two SALS patients. These results suggest that the SOD1 analysis is useful in FALS but less so in SALS cases. The SSCP analysis has proved fast and reliable for this purpose.     

A novel heterozygous nonsense mutation of the OPTN gene segregating in a Danish family with ALS

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder. About 10% of ALS cases are familial (FALS) and the genetic defect is known only in approximately 20%-30% of these cases. The most common genetic cause of ALS is SOD1 (superoxide dismutase 1) mutation. Very recently, mutations of the optineurin gene (OPTN), which is involved in open-angle glaucoma, were identified in 3 Japanese patients/families with ALS, and subsequently in a few FALS patients of European descent. We found a heterozygous nonsense mutation (c.493C>T, p.Gln165X, exon 6) in the OPTN gene in a Danish patient with ALS, and the mutation segregated from his affected father. The p.Gln165X mutation could not be detected in 1070 healthy Danish controls, in 1000 Danish individuals with metabolic phenotypes or in 64 sporadic ALS (SALS) cases. The p.Gln165X mutation described in this study is the first mutation reported in a Danish family and is likely involved in disease pathogenesis. Until now, only few OPTN mutations have been associated with ALS. As the underlying genetic defect is known only in approximately 20%-30% of FALS families, further screening of these cases is necessary for establishing the contribution of OPTN mutations in disease pathogenesis.     

Absence of angiogenic genes modification in Italian ALS patients

To investigate the role of vascular endothelial growth factor (VEGF) and angiogenin (ANG) as genetic determinants in the susceptibility to sporadic ALS in Italian patients. VEGF genotype and haplotype analysis revealed no association between any variants and the risk of ALS. Regarding ANG gene, no mutation was detected and the rs11701 polymorphism, previously described as associated with ALS, was not differently distributed between patients and controls. Overall, our data argue against the hypothesis of both genes as risk factors for motoneuron neurodegeneration, at least in an Italian population.     

Alteration in cell cycle-related proteins in lymphoblasts from carriers of the c.709-1G>A PGRN mutation associated with FTLD-TDP dementia

Frontotemporal lobar degeneration with neuronal inclusions containing TAR DNA binding protein 43 (TDP-43) is associated in most cases with null-mutations in the progranulin gene (PGRN). While the mechanisms by which PGRN haploinsufficiency leads to neurodegeneration remained speculative, increasing evidence support the hypothesis that cell cycle reentry of postmitotic neurons precedes many instances of neuronal death. Based in the mitogenic and neurotrophic activities of PGRN, we hypothesized that PGRN deficit may induce cell cycle disturbances and alterations in neuronal vulnerability. Because cell cycle dysfunction is not restricted to neurons, we studied the influence of PGRN haploinsufficiency, on cell cycle control in peripheral cells from patients suffering from frontotemporal dementia, bearing the PGRN mutation c.709-1G>A. Here we show that progranulin deficit increased cell cycle activity in immortalized lymphocytes. This effect was associated with increased levels of cyclin-dependent kinase 6 (CDK6) and phosphorylation of retinoblastoma protein (pRb), resulting in a G₁/S regulatory failure. A loss of function of TDP-43 repressing CDK6 expression may result from altered subcellular TDP-43 distribution. The distinct functional features of lymphoblastoid cells from c.709-1 G>A carriers offer an invaluable, noninvasive tool to investigate the etiopathogenesis of frontotemporal lobar degeneration.     

High frequency of TARDBP gene mutations in Italian patients with amyotrophic lateral sclerosis

Recent studies identified rare missense mutations in amyotrophic lateral sclerosis (ALS) patients in the TARDBP gene encoding TAR DNA binding protein (TDP)‐43, the major protein of the ubiquitinated inclusions (UBIs) found in affected motor neurons (MNs). The aim of this study was to further define the spectrum of TARDBP mutations in a large cohort of 666 Italian ALS patients (125 familial and 541 sporadic cases). The entire coding region was sequenced in 281 patients, while in the remaining 385 cases only exon 6 was sequenced. In 18 patients, of which six are familial, we identified 12 different heterozygous missense mutations (nine novel) all locating to exon 6, which were absent in 771 matched controls. The c.1144G>A (p.A382T) variation was observed in seven patients, thus representing the most frequent TARDBP mutation in ALS. Analysis of microsatellites surrounding the TARDBP gene indicated that p.A382T was inherited from a common ancestor in 5 of the 7 patients. Altogether, the frequency of TARDBP gene mutations appears to be particularly high in Italian ALS patients compared to individuals of mainly Northern European origin (2.7% vs. 1%). Western blot analysis of lymphocyte extracts from two patients carrying the p.A382T and p.S393L TARDBP mutations showed the presence of lower molecular weight TDP‐43 bands, which were more abundant than observed in healthy controls and patients negative for TARDBP mutations. In conclusion, this report contributes to the demonstration of the causative role of the TARDBP gene in ALS pathogenesis and indicates that mutations may affect the stability of the protein even in nonneuronal tissues. Hum Mutat 0, 1–7, 2009. © 2009 Wiley‐Liss, Inc.     

Evidence for an oligogenic basis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder with a substantial heritable component. In pedigrees affected by its familial form, incomplete penetrance is often observed. We hypothesized that this could be caused by a complex inheritance of risk variants in multiple genes. Therefore, we screened 111 familial ALS (FALS) patients from 97 families, and large cohorts of sporadic ALS (SALS) patients and control subjects for mutations in TAR DNA-binding protein (TARDBP), fused in sarcoma/translated in liposarcoma (FUS/TLS), superoxide dismutase-1 (SOD1), angiogenin (ANG) and chromosome 9 open reading frame 72 (C9orf72). Mutations were identified in 48% of FALS families, 8% of SALS patients and 0.5% of control subjects. In five of the FALS families, we identified multiple mutations in ALS-associated genes. We detected FUS/TLS and TARDBP mutations in combination with ANG mutations, and C9orf72 repeat expansions with TARDBP, SOD1 and FUS/TLS mutations. Statistical analysis demonstrated that the presence of multiple mutations in FALS is in excess of what is to be expected by chance (P = 1.57 × 10(-7)). The most compelling evidence for an oligogenic basis was found in individuals with a p.N352S mutation in TARDBP, detected in five FALS families and three apparently SALS patients. Genealogical and haplotype analyses revealed that these individuals shared a common ancestor. We obtained DNA of 14 patients with this TARDBP mutation, 50% of whom had an additional mutation (ANG, C9orf72 or homozygous TARDBP). Hereby, we provide evidence for an oligogenic aetiology of ALS. This may have important implications for the interpretation of whole exome/genome experiments designed to identify new ALS-associated genes and for genetic counselling, especially of unaffected family members.