RESUMEN
OBJECTIVE: To characterise stabilities in erythrocytes of mutant SOD1 proteins, compare SOD1 enzymatic activities between patients with different genetic causes of ALS and search for underlying causes of deviant SOD1 activities in individuals lacking SOD1 mutations. METHODS: Blood samples from 4072 individuals, ALS patients with or without a SOD1 mutation, family members and controls were studied. Erythrocyte SOD1 enzymatic activities normalised to haemoglobin content were determined, and effects of haemoglobin disorders on dismutation assessed. Coding SOD1 sequences were analysed by Sanger sequencing, exon copy number variations by fragment length analysis and by TaqMan Assay. RESULTS: Of the 44 SOD1 mutations found, 75% caused severe destabilisation of the mutant protein but in 25% it was physically stable. Mutations producing structural changes caused halved erythrocyte SOD1 activities. There were no differences in SOD1 activities between patients without a SOD1 mutation and control individuals or carriers of TBK1 mutations and C9orf72HRE. In the low and high SOD1 activity groups no deviations were found in exon copy numbers and intron gross structures. Thalassemias and iron deficiency were associated with increased SOD1 activity/haemoglobin ratios. CONCLUSION: Adjunct erythrocyte SOD1 activity analysis reliably signals destabilising SOD1 mutations including intronic mutations that are missed by exon sequencing.
Asunto(s)
Esclerosis Amiotrófica Lateral/sangre , Esclerosis Amiotrófica Lateral/genética , Superóxido Dismutasa-1/sangre , Superóxido Dismutasa-1/genética , Anciano , Esclerosis Amiotrófica Lateral/diagnóstico , Estudios de Cohortes , Activación Enzimática/fisiología , Familia , Femenino , Humanos , Masculino , Persona de Mediana EdadRESUMEN
Mutations in the superoxide dismutase (SOD1) gene have been linked to amyotrophic lateral sclerosis (ALS). A 50 base pair (bp) deletion of SOD1 has been suggested to reduce transcription and to be associated with later disease onset in ALS. This study was aimed to reveal if the 50 bp deletion influenced SOD1 enzymatic activity, occurrence and phenotype of the disease in a Swedish ALS/control cohort. Blood samples from 512 Swedish ALS patients and 354 Swedish controls without coding SOD1 mutations were analysed for the 50 bp deletion allele. The enzymatic activity of SOD1 in erythrocytes was analysed and genotype-phenotype correlations were assessed. Results demonstrated that the genotype frequencies of the 50 bp deletion were all found to be in Hardy-Weinberg equilibrium. No significant differences were found for age of onset, disease duration or site of onset. SOD1 enzymatic activity showed a statistically significant decreasing trend in the control group, in which the allele was associated with a 5% reduction in SOD1 activity. The results suggest that the 50 bp deletion has a moderate reducing effect on SOD1 synthesis. No modulating effects, however, were found on ALS onset, phenotype and survival in the Swedish population.
Asunto(s)
Esclerosis Amiotrófica Lateral , Polimorfismo Genético/genética , Eliminación de Secuencia/genética , Superóxido Dismutasa-1/genética , Superóxido Dismutasa-1/metabolismo , Adulto , Anciano , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/epidemiología , Esclerosis Amiotrófica Lateral/genética , Análisis de Varianza , Estudios de Cohortes , Eritrocitos/enzimología , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Suecia/epidemiologíaRESUMEN
Mutations in superoxide dismutase-1 (SOD1) are a common known cause of amyotrophic lateral sclerosis (ALS). The neurotoxicity of mutant SOD1s is most likely caused by misfolded molecular species, but disease pathogenesis is still not understood. Proposed mechanisms include impaired mitochondrial function, induction of endoplasmic reticulum stress, reduction in the activities of the proteasome and autophagy, and the formation of neurotoxic aggregates. Here we examined whether perturbations in these cellular pathways in turn influence levels of misfolded SOD1 species, potentially amplifying neurotoxicity. For the study we used fibroblasts, which express SOD1 at physiological levels under regulation of the native promoter. The cells were derived from ALS patients expressing 9 different SOD1 mutants of widely variable molecular characteristics, as well as from patients carrying the GGGGCC-repeat-expansion in C9orf72 and from non-disease controls. A specific ELISA was used to quantify soluble, misfolded SOD1, and aggregated SOD1 was analysed by western blotting. Misfolded SOD1 was detected in all lines. Levels were found to be much lower in non-disease control and the non-SOD1 C9orf72 ALS lines. This enabled us to validate patient fibroblasts for use in subsequent perturbation studies. Mitochondrial inhibition, endoplasmic reticulum stress or autophagy inhibition did not affect soluble misfolded SOD1 and in most cases, detergent-resistant SOD1 aggregates were not detected. However, proteasome inhibition led to uniformly large increases in misfolded SOD1 levels in all cell lines and an increase in SOD1 aggregation in some. Thus the ubiquitin-proteasome pathway is a principal determinant of misfolded SOD1 levels in cells derived both from patients and controls and a decline in activity with aging could be one of the factors behind the mid-to late-life onset of inherited ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Fibroblastos/enzimología , Pliegue de Proteína , Superóxido Dismutasa/metabolismo , Adenina/análogos & derivados , Adenina/farmacología , Edad de Inicio , Envejecimiento/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Autofagia/efectos de los fármacos , Autofagia/fisiología , Bortezomib/farmacología , Proteína C9orf72 , Estudios de Casos y Controles , Células Cultivadas , Expansión de las Repeticiones de ADN , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Estrés del Retículo Endoplásmico , Ensayo de Inmunoadsorción Enzimática , Fibroblastos/patología , Genotipo , Humanos , Mutación , Inhibidores de Proteasas/farmacología , Complejo de la Endopetidasa Proteasomal/metabolismo , Agregación Patológica de Proteínas , Proteínas/genética , Proteolisis , Rotenona/farmacología , Solubilidad , Superóxido Dismutasa/genética , Superóxido Dismutasa-1RESUMEN
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.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genética , Proteínas/genética , Edad de Inicio , Anciano , Esclerosis Amiotrófica Lateral/patología , Secuencia de Bases , Proteína C9orf72 , Cerebelo/patología , Progresión de la Enfermedad , Femenino , Demencia Frontotemporal/patología , Estudios de Asociación Genética , Humanos , Masculino , Persona de Mediana Edad , Especificidad de Órganos , Lóbulo Parietal/patología , Secuencias Repetidas en TándemRESUMEN
Midkine (MDK) and Pleiotrophin (PTN) are small heparin-binding cytokines with closely related structures. The Drosophila genome harbours two genes encoding members of the MDK/PTN family of proteins, known as miple1 and miple2. We have investigated the role of Miple proteins in vivo, in particular with regard to their proposed role as ligands for the Alk receptor tyrosine kinase (RTK). Here we show that Miple proteins are neither required to drive Alk signaling during Drosophila embryogenesis, nor are they essential for development in the fruit fly. Additionally we show that neither MDK nor PTN can activate hALK in vivo when ectopically co-expressed in the fly. In conclusion, our data suggest that Alk is not activated by MDK/PTN related growth factors Miple1 and Miple 2 in vivo.
Asunto(s)
Citocinas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Drosophila/fisiología , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transducción de Señal , Quinasa de Linfoma Anaplásico , Animales , Proteínas Portadoras/metabolismo , Citocinas/genética , Drosophila/genética , Drosophila/crecimiento & desarrollo , Proteínas de Drosophila/genética , Femenino , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Humanos , Masculino , MidkinaRESUMEN
Sporadic amyotrophic lateral sclerosis is a multifactorial disease of environmental and genetic origin. In a previous large multicenter genome wide study, common genetic variation in the Kinesin-Associated Protein 3 (KIFAP3) gene (rs1541160) was reported to have a significant effect on survival in amyotrophic lateral sclerosis patients. However, this could not be replicated in 3 smaller independent cohorts. We conducted a large multicenter multivariate survival analysis (n = 2362) on the effect of genetic variation in rs1541160. The previously reported beneficial genotype did not show a significant improvement in survival in this patient group.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/mortalidad , Proteínas del Citoesqueleto/genética , Variación Genética/genética , Estudio de Asociación del Genoma Completo , Anciano , Estudios de Cohortes , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Polimorfismo de Nucleótido Simple , Análisis de SupervivenciaRESUMEN
BACKGROUND: The GGGGCC-repeat expansion in C9orf72 is the most frequent mutation found in patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Most of the studies on C9orf72 have relied on repeat-primed PCR (RP-PCR) methods for detection of the expansions. To investigate the inherent limitations of this technique, we compared methods and results of 14 laboratories. METHODS: The 14 laboratories genotyped DNA from 78 individuals (diagnosed with ALS or FTD) in a blinded fashion. Eleven laboratories used a combination of amplicon-length analysis and RP-PCR, whereas three laboratories used RP-PCR alone; Southern blotting techniques were used as a reference. RESULTS: Using PCR-based techniques, 5 of the 14 laboratories got results in full accordance with the Southern blotting results. Only 50 of the 78 DNA samples got the same genotype result in all 14 laboratories. There was a high degree of false positive and false negative results, and at least one sample could not be genotyped at all in 9 of the 14 laboratories. The mean sensitivity of a combination of amplicon-length analysis and RP-PCR was 95.0% (73.9-100%), and the mean specificity was 98.0% (87.5-100%). Overall, a sensitivity and specificity of more than 95% was observed in only seven laboratories. CONCLUSIONS: Because of the wide range seen in genotyping results, we recommend using a combination of amplicon-length analysis and RP-PCR as a minimum in a research setting. We propose that Southern blotting techniques should be the gold standard, and be made obligatory in a clinical diagnostic setting.
Asunto(s)
Servicios de Laboratorio Clínico/normas , Pruebas Genéticas/métodos , Pruebas Genéticas/normas , Proteínas/genética , Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72 , Femenino , Demencia Frontotemporal/genética , Humanos , Masculino , Reproducibilidad de los ResultadosRESUMEN
Linkage analysis in Brazilian families with amyotrophic lateral sclerosis (ALS) revealed that a missense mutation p.Pro56Ser in a conserved gene VAMP-associated protein type B and C (VAPB) cosegregates with disease. Blood samples were studied from 973 Swedish, 126 Portuguese and 19 Icelandic ALS patients, and from 644 control subjects. We identified five VAPB mutations, two of which are novel, in 14 Swedish ALS patients and in nine control individuals from Sweden and Portugal. The 14 patients with VAPB mutations all carried a diagnosis of sporadic ALS. Mutations were also found in healthy adult relatives. The p.Asp130Glu VAPB mutation was also found in two patients from an Icelandic ALS family, but the mutation did not cosegregate with disease. All patients were instead found to be heterozygous for a p.Gly93Ser SOD1 mutation. There were no clinical differences between them, suggesting that the p.Asp130Glu VAPB mutation is unrelated to the disease process. In conclusion, the VAPB mutations were as frequent in control individuals as in patients. This observation, in combination with the finding of several healthy relatives carrying the VAPB mutations and no ancestors with ALS disease, suggests that it is unlikely that these VAPB mutations are pathogenic.
Asunto(s)
Esclerosis Amiotrófica Lateral/diagnóstico , Esclerosis Amiotrófica Lateral/genética , Mutación Missense/genética , Proteínas de Transporte Vesicular/genética , Adulto , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/epidemiología , Estudios de Cohortes , Femenino , Humanos , Islandia/epidemiología , Masculino , Persona de Mediana Edad , Linaje , Portugal/epidemiología , Suecia/epidemiologíaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating, adult-onset neurodegenerative disorder of the upper and lower motor systems. It leads to paresis, muscle wasting and inevitably to death, typically within 3-5 years. However, disease onset and survival vary considerably ranging in extreme cases from a few months to several decades. The genetic and environmental factors underlying this variability are of great interest as potential therapeutic targets. In ALS, men are affected more often and have an earlier age of onset than women. This gender difference is recapitulated in transgenic rodent models, but no underlying mechanism has been elucidated. Here we report that SNPs in the brain-specific promoter region of the transcriptional co-activator PGC-1α, a master regulator of metabolism, modulate age of onset and survival in two large and independent ALS populations and this occurs in a strictly male-specific manner. In complementary animal studies, we show that deficiency of full-length (FL) Pgc-1α leads to a significantly earlier age of onset and a borderline shortened survival in male, but not in female ALS-transgenic mice. In the animal model, FL Pgc-1α-loss is associated with reduced mRNA levels of the trophic factor Vegf-A in males, but not in females. In summary, we indentify PGC-1α as a novel and clinically relevant disease modifier of human and experimental ALS and report a sex-dependent effect of PGC-1α in this neurodegenerative disorder.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Adulto , Edad de Inicio , Anciano , Animales , Modelos Animales de Enfermedad , Femenino , Humanos , Masculino , Ratones , Ratones Transgénicos , Persona de Mediana Edad , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Polimorfismo de Nucleótido Simple , Caracteres Sexuales , Factor A de Crecimiento Endotelial Vascular/genética , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
A common cause of amyotrophic lateral sclerosis (ALS) is mutations in the gene encoding superoxide dismutase-1. There is evolving circumstantial evidence that the wild-type protein can also be neurotoxic and that it may more generally be involved in the pathogenesis of ALS. To test this proposition more directly, we generated mice that express wild-type human superoxide dismutase-1 at a rate close to that of mutant superoxide dismutase-1 in the commonly studied G93A transgenic model. These mice developed an ALS-like syndrome and became terminally ill after around 370 days. The loss of spinal ventral neurons was similar to that in the G93A and other mutant superoxide dismutase-1 models, and large amounts of aggregated superoxide dismutase-1 were found in spinal cords, but also in the brain. The findings show that wild-type human superoxide dismutase-1 has the ability to cause ALS in mice, and they support the hypothesis of a more general involvement of the protein in the disease in humans.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/enzimología , Animales , Secuencia de Bases , Northern Blotting , Western Blotting , Cartilla de ADN , Humanos , Ratones , Ratones Transgénicos , Reacción en Cadena de la Polimerasa , Médula Espinal/enzimología , Superóxido Dismutasa-1RESUMEN
An intronic GGGGCC-hexanucleotide repeat expansion in C9ORF72 was recently identified as a major cause of amyotrophic lateral sclerosis and frontotemporal dementia. Some amyotrophic lateral sclerosis patients have signs of parkinsonism, and many parkinsonism patients develop dementia. In this study we examined if the hexanucleotide repeat expansion was present in parkinsonism patients, to clarify if there could be a relationship between the repeat expansion and disease. We studied the size of the hexanucleotide repeat expansion in a well defined population-based cohort of 135 Parkinson's disease patients and 39 patients with atypical parkinsonism and compared with 645 Swedish control subjects. We found no correlation between Parkinson's disease or atypical parkinsonism and the size of the GGGGCC repeat expansion in C9ORF72. In conclusion, this GGGGCC-repeat expansion in C9ORF72 is not a cause of parkinsonism in the Swedish population.
Asunto(s)
Expansión de las Repeticiones de ADN/genética , Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/genética , Enfermedad de Parkinson/epidemiología , Enfermedad de Parkinson/genética , Polimorfismo de Nucleótido Simple/genética , Proteínas/genética , Anciano , Secuencia de Bases , Proteína C9orf72 , Femenino , Marcadores Genéticos/genética , Humanos , Desequilibrio de Ligamiento/genética , Masculino , Persona de Mediana Edad , Datos de Secuencia Molecular , Prevalencia , Factores de Riesgo , Suecia/epidemiologíaRESUMEN
Mutations in the optineurin (OPTN) gene have been associated with normal tension glaucoma and with amyotrophic lateral sclerosis (ALS). Here, we screened German familial ALS cases for OPTN mutations to gain additional insight into the spectrum and pathogenic relevance of this gene for ALS. One hundred familial German ALS cases and 148 control subjects were screened for OPTN mutations by sequence analysis of the complete OPTN coding sequence, and phenotypes of OPTN mutant patients were described. We identified a novel heterozygous truncating OPTN mutation p.Lys440Asnfs*8 in 1 ALS family with an aggressive ALS disease phenotype. This mutation abolishes protein domains crucial for nuclear factor κB signaling. Moreover, we detected 3 different nonsynonymous sequence variants, which have been described previously as risk factors for primary retinal ganglion cell degeneration in normal tension glaucoma. Two of them were detected on the same allele in a family that also carries a p.Asn352Ser disease mutation in the ALS gene TARDBP. All OPTN mutant patients presented with typical spinal onset ALS. Taken together, we detected a novel truncating OPTN mutation associated with an aggressive form of ALS and confirmed that OPTN mutations are a rare cause of ALS. In addition our data suggest that in some cases plausibly more than 1 mutation in OPTN or another ALS gene might be needed to cause ALS. Finally, our findings show that motoneurons and retinal ganglion cells, which are both projecting central nervous system neurons, might share common susceptibility factors.
Asunto(s)
Esclerosis Amiotrófica Lateral/epidemiología , Esclerosis Amiotrófica Lateral/genética , Glaucoma/epidemiología , Glaucoma/genética , Mutación Missense/genética , Polimorfismo de Nucleótido Simple/genética , Factor de Transcripción TFIIIA/genética , Proteínas de Ciclo Celular , Comorbilidad , Femenino , Marcadores Genéticos/genética , Alemania/epidemiología , Humanos , Masculino , Proteínas de Transporte de Membrana , Persona de Mediana Edad , Mutación/genética , Prevalencia , Factores de RiesgoRESUMEN
Profilin 1 is a central regulator of actin dynamics. Mutations in the gene profilin 1 (PFN1) have very recently been shown to be the cause of a subgroup of amyotrophic lateral sclerosis (ALS). Here, we performed a large screen of US, Nordic, and German familial and sporadic ALS and frontotemporal dementia (FTLD) patients for PFN1 mutations to get further insight into the spectrum and pathogenic relevance of this gene for the complete ALS/FTLD continuum. Four hundred twelve familial and 260 sporadic ALS cases and 16 ALS/FTLD cases from Germany, the Nordic countries, and the United States were screened for PFN1 mutations. Phenotypes of patients carrying PFN1 mutations were studied. In a German ALS family we identified the novel heterozygous PFN1 mutation p.Thr109Met, which was absent in controls. This novel mutation abrogates a phosphorylation site in profilin 1. The recently described p.Gln117Gly sequence variant was found in another familial ALS patient from the United States. The ALS patients with mutations in PFN1 displayed spinal onset motor neuron disease without overt cognitive involvement. PFN1 mutations were absent in patients with motor neuron disease and dementia, and in patients with only FTLD. We provide further evidence that PFN1 mutations can cause ALS as a Mendelian dominant trait. Patients carrying PFN1 mutations reported so far represent the "classic" ALS end of the ALS-FTLD spectrum. The novel p.Thr109Met mutation provides additional proof-of-principle that mutant proteins involved in the regulation of cytoskeletal dynamics can cause motor neuron degeneration. Moreover, this new mutation suggests that fine-tuning of actin polymerization by phosphorylation of profilin 1 might be necessary for motor neuron survival.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Demencia Frontotemporal/genética , Tamizaje Masivo , Mutación Puntual/genética , Profilinas/genética , Adulto , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/epidemiología , Esclerosis Amiotrófica Lateral/metabolismo , Estudios de Cohortes , Femenino , Demencia Frontotemporal/epidemiología , Demencia Frontotemporal/metabolismo , Alemania/epidemiología , Humanos , Masculino , Tamizaje Masivo/métodos , Persona de Mediana Edad , Linaje , Fosforilación/genética , Profilinas/metabolismo , Suecia/epidemiología , Estados Unidos/epidemiología , Adulto JovenRESUMEN
A reason for screening amyotrophic lateral sclerosis (ALS) patients for mutations in the superoxide dismutase-1 (SOD1) gene is the opportunity to find novel mutations with properties that can give information on pathogenesis. A novel c.352C>G (L117V) SOD1 mutation was found in two Syrian ALS families living in Europe. The disease showed unusually low penetrance and slow progression. In erythrocytes, the total SOD1 activity, as well as specific activity of the mutant protein, was equal in carriers of the mutation and family controls lacking SOD1 mutations. The structural stabilities of the L117V mutant and wild-type SOD1 under denaturing conditions were likewise equal, but considerably lower than that of murine SOD1. As analyzed with an ELISA specific for misfolded SOD1 species, no differences were found in the content of misfolded SOD1 protein between extracts of fibroblasts from wild-type controls and from an L117V patient. In contrast, elevated levels of misfolded SOD1 protein were found in fibroblasts from ALS patients carrying seven other mutations in the SOD1 gene. We conclude that mutations in SOD1 that result in a fully stable protein are associated with low disease penetrance for ALS and may be found in cases of apparently sporadic ALS. Wild-type human SOD1 is moderately stable, and was found here to be within the stability range of ALS-causing SOD1 variants, lending support to the hypothesis that wild-type SOD1 could be more generally involved in ALS pathogenesis.
Asunto(s)
Esclerosis Amiotrófica Lateral/enzimología , Mutación , Superóxido Dismutasa/genética , Adulto , Edad de Inicio , Esclerosis Amiotrófica Lateral/genética , Animales , Estudios de Casos y Controles , Línea Celular , Estabilidad de Enzimas , Femenino , Fibroblastos/enzimología , Heterocigoto , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Linaje , Pliegue de Proteína , Superóxido Dismutasa/química , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1RESUMEN
Excitotoxicity is thought to play a pathogenic role in amyotrophic lateral sclerosis (ALS). Excitotoxic motor neuron death is mediated through the Ca(2+)-permeable α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA)-type of glutamate receptors and Ca(2+) permeability is determined by the GluR2 subunit. We investigated whether polymorphisms or mutations in the GluR2 gene (GRIA2) predispose patients to ALS. Upon sequencing 24 patients and 24 controls no nonsynonymous coding variants were observed but 24 polymorphisms were identified, 9 of which were novel. In a screening set of 310 Belgian ALS cases and 794 healthy controls and a replication set of 3157 cases and 5397 controls from 6 additional populations no association with susceptibility, age at onset, or disease duration was observed. We conclude that polymorphisms in the GluR2 gene (GRIA2) are not a major contributory factor in the pathogenesis of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Predisposición Genética a la Enfermedad/epidemiología , Predisposición Genética a la Enfermedad/genética , Mutación/genética , Polimorfismo de Nucleótido Simple/genética , Receptores AMPA/genética , Anciano , Anciano de 80 o más Años , Esclerosis Amiotrófica Lateral/epidemiología , Bélgica/epidemiología , Estudios de Asociación Genética , Humanos , Persona de Mediana Edad , Prevalencia , Medición de Riesgo , Factores de RiesgoRESUMEN
OBJECTIVE: Several studies have suggested an increased frequency of variants in the gene encoding angiogenin (ANG) in patients with amyotrophic lateral sclerosis (ALS). Interestingly, a few ALS patients carrying ANG variants also showed signs of Parkinson disease (PD). Furthermore, relatives of ALS patients have an increased risk to develop PD, and the prevalence of concomitant motor neuron disease in PD is higher than expected based on chance occurrence. We therefore investigated whether ANG variants could predispose to both ALS and PD. METHODS: We reviewed all previous studies on ANG in ALS and performed sequence experiments on additional samples, which allowed us to analyze data from 6,471 ALS patients and 7,668 controls from 15 centers (13 from Europe and 2 from the USA). We sequenced DNA samples from 3,146 PD patients from 6 centers (5 from Europe and 1 from the USA). Statistical analysis was performed using the variable threshold test, and the Mantel-Haenszel procedure was used to estimate odds ratios. RESULTS: Analysis of sequence data from 17,258 individuals demonstrated a significantly higher frequency of ANG variants in both ALS and PD patients compared to control subjects (p = 9.3 × 10(-6) for ALS and p = 4.3 × 10(-5) for PD). The odds ratio for any ANG variant in patients versus controls was 9.2 for ALS and 6.7 for PD. INTERPRETATION: The data from this multicenter study demonstrate that there is a strong association between PD, ALS, and ANG variants. ANG is a genetic link between ALS and PD.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Predisposición Genética a la Enfermedad , Variación Genética/genética , Enfermedad de Parkinson/genética , Ribonucleasa Pancreática/genética , Bases de Datos Factuales/estadística & datos numéricos , Europa (Continente) , Femenino , Humanos , Masculino , Estudios Multicéntricos como Asunto , Estados UnidosRESUMEN
In this report we describe an ALS family with a novel missense SOD1 mutation with substitution of serine for cysteine at the sixth amino acid (C6S). This mutation has interesting implications for the role of disulfides in causing disease. After identification of the ALS proband, we examined 17 members of an extended family and performed DNA mutation analysis on 21 family members. The level and activity of SOD1 in C6S carriers and wild-type family members was analyzed in erythrocytes. We found that the C6S mutation results in disease with an autosomal dominant mode of inheritance and markedly reduced penetrance. The S6 mutated protein demonstrates high stability relative to the C6 wild-type protein. The specific dismutation activity of S6 SOD1 is normal. In conclusion, C6S is a novel FALS associated mutation with reduced disease penetrance, long survival time and a phenotype very different from the other SOD1 mutations reported in codon C6. This mutation may provide insight into the role of SOD1 structural changes in disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Asesoramiento Genético , Mutación , Superóxido Dismutasa/genética , Adulto , Anciano , Esclerosis Amiotrófica Lateral/enzimología , Esclerosis Amiotrófica Lateral/fisiopatología , Cisteína/química , Cisteína/genética , Análisis Mutacional de ADN , Disulfuros/química , Femenino , Pruebas Genéticas , Genotipo , Humanos , Masculino , Linaje , Serina/genética , Superóxido Dismutasa-1RESUMEN
More than 145 mutations have been found in the gene CuZn-Superoxide dismutase (SOD1) in patients with amyotrophic lateral sclerosis (ALS). The vast majority are easily detected nucleotide mutations in the coding region. In a patient from a Swiss ALS family with half-normal erythrocyte SOD1 activity, exon flanking sequence analysis revealed a novel thymine to guanine mutation 7 bp upstream of exon 4 (c.240-7T>G). The results of splicing algorithm analyses were ambiguous, but five out of seven analysis tools suggested a potential novel splice site that would add six new base pairs to the mRNA. If translated, this mRNA would insert Ser and Ile between Glu78 and Arg79 in the SOD1 protein. In fibroblasts from the patient, the predicted mutant transcript and the mutant protein were both highly expressed, and despite the location of the insertion into the metal ion-binding loop IV, the SOD1 activity appeared high. In erythrocytes, which lack protein synthesis and are old compared with cultured fibroblasts, both SOD1 protein and enzymic activity was 50% of controls. Thus, the usage of the novel splice site is near 100%, and the mutant SOD1 shows the reduced stability typical of ALS-associated mutant SOD1s. The findings suggests that this novel intronic mutation is causing the disease and highlights the importance of wide exon-flanking sequencing and transcript analysis combined with erythrocyte SOD1 activity analysis in comprehensive search for SOD1 mutations in ALS. We find that there are potentially more SOD1 mutations than previously reported.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Empalme del ARN , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/enzimología , Western Blotting , Exones , Humanos , Intrones , Mutación , ARN Mensajero/genética , Superóxido Dismutasa/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease selectively affecting motor neurons in the brain and spinal cord. Recent genome-wide association studies (GWASs) have identified several common variants which increase disease susceptibility. In contrast, rare copy-number variants (CNVs), which have been associated with several neuropsychiatric traits, have not been studied for ALS in well-powered study populations. To examine the role of rare CNVs in ALS susceptibility, we conducted a CNV association study including over 19,000 individuals. In a genome-wide screen of 1875 cases and 8731 controls, we did not find evidence for a difference in global CNV burden between cases and controls. In our association analyses, we identified two loci that met our criteria for follow-up: the DPP6 locus (OR = 3.59, P = 6.6 × 10(-3)), which has already been implicated in ALS pathogenesis, and the 15q11.2 locus, containing NIPA1 (OR = 12.46, P = 9.3 × 10(-5)), the gene causing hereditary spastic paraparesis type 6 (HSP 6). We tested these loci in a replication cohort of 2559 cases and 5887 controls. Again, results were suggestive of association, but did not meet our criteria for independent replication: DPP6 locus: OR = 1.92, P = 0.097, pooled results: OR = 2.64, P = 1.4 × 10(-3); NIPA1: OR = 3.23, P = 0.041, pooled results: OR = 6.20, P = 2.2 × 10(-5)). Our results highlight DPP6 and NIPA1 as candidates for more in-depth studies. Unlike other complex neurological and psychiatric traits, rare CNVs with high effect size do not play a major role in ALS pathogenesis.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Variaciones en el Número de Copia de ADN , Dipeptidil-Peptidasas y Tripeptidil-Peptidasas/genética , Estudio de Asociación del Genoma Completo , Proteínas de la Membrana/genética , Proteínas del Tejido Nervioso/genética , Canales de Potasio/genética , Estudios de Casos y Controles , Predisposición Genética a la Enfermedad , Variación Genética , Genoma Humano , Humanos , Neuronas Motoras , Reacción en Cadena de la Polimerasa , Polimorfismo de Nucleótido Simple , Factores de Riesgo , Paraplejía Espástica Hereditaria/genéticaRESUMEN
OBJECTIVE: To estimate the frequency of SOD1 mutations in a large referral cohort of familial amyotrophic lateral sclerosis (FALS) and sporadic amyotrophic lateral sclerosis (SALS) patients from The Netherlands and to compare this frequency with that of other developed countries. METHODS: A total of 451 sporadic and 55 FALS patients were screened for SOD1 mutations. The authors performed PCR amplification of all five coding exons of SOD1 followed by direct DNA sequencing using forward and reverse primers. RESULTS: One novel mutation (p.I99V) and a homozygous p.D90A mutation were identified in SALS patients. In a pedigree with Mendelian dominant FALS, one patient was found to be heterozygous for the p.D90A mutation. SOD1 mutation frequency was found to be significantly lower in The Netherlands compared with other countries with p=0.0004 for FALS (21.9% vs 2.5%) and p=0.005 for SALS (2.5% vs 0.44%). CONCLUSIONS: The authors demonstrate that SOD1 mutations are rare in The Netherlands in familial and SALS. This observation suggests that the genetic background of amyotrophic lateral sclerosis differs between different populations, countries and regions. This may have consequences for the interpretation of association studies and explain why replication of association studies has proven difficult in amyotrophic lateral sclerosis.