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On the aetiology of ALS: a comprehensive genetic study
Umeå University, Faculty of Medicine, Department of Pharmacology and Clinical Neuroscience. (ALS)
2013 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Introduction: Amyotrophic lateral sclerosis (ALS) is a deadly, progressive neuromuscular disease that affects individuals all over the world. About 10% of the patients have a familial predisposition (FALS) while the remainder of cases are isolated or sporadic (SALS) and of unknown cause. To date, the principal recognized risk factors for ALS are higher age, male gender, slim figure (BMI<23) and a family history of ALS. In 1993, Rosen et al. observed that some FALS cases were associated with mutations in the gene encoding the CuZn superoxide dismutase enzyme (SOD1). Since then, several mutations in the SOD1 gene have been discovered, and mutations in more than 18 other genes have been associated with causing ALS. The aim of this thesis was to identify new mutations associated with ALS pathogenesis, and by comparing patients from different countries, were we also able to identify population-specific genetic variations. The studies are referred to as I–V.

Methods: With written informed consent and adhering to the tenets of the Declaration of Helsinki, through a national network of ALS clinicians´, venous blood samples were collected from ALS patients and healthy subjects in Europe and the USA. The patients were diagnosed according to the El Escorial criteria, and as having FALS according to the criteria of Byrne et al. (2011). The DNA variations were amplified by various PCR techniques. (I, III and IV) The amplicons of ataxin 2 (ATXN2), profilin 1 (PFN1), and vesicle-associated membrane protein type B (VAPB) were characterised by direct sequencing. (II) After quantitative PCR, a genotype-phenotype correlation was performed to assess whether the survival motor neuron gene (SMN) modulates the phenotype of ALS. (V) The amplicons of the 50 base pair deletion in the SOD1 promotor (50 bp) were separated by electrophoresis on agarose.

Results: (I) We observed a significant association between CAG expansions in the ATXN2 gene and ALS in a European cohort. (II) Abnormal copy number of the SMN1 gene was identified as a risk factor in France, but not in Sweden. Homozygosity of the SMN2 deletion prolonged survival among Swedish ALS patients, compared to French patients. (III) We identified two mutations in the PFN1 gene, the novel p.Thr109Met mutation and the p.Gln117Gly mutation, in two unrelated FALS patients. (IV) In our cohort, we identified five VAPB mutations p.Asp130Glu, p.Ser160del, p.Asp162Glu, p.Met170Ile, and p.Arg184Trp, two of which are novel. (V) The 50 bp deletion upstream of the SOD1 gene was found in equal frequencies in both the patient and control cohorts. The 50 bp deletion did not affect SOD1 enzymatic activity. Furthermore, we found no differences in age of onset or disease duration in relation to the 50 bp deletion genotype.VI

Conclusions: (I) Our findings indicate that ATXN2 plays an important role in the pathogenesis of ALS, and that CAG expansions in ATXN2 are a significant risk factor for the disease. (II) We suggest that abnormal SMN1 gene copynumber cannot be considered a universal genetic susceptibility factor for ALS. We also propose that the effect of abnormal SMN2 gene copy number on ALS phenotype may differ between populations. (III) This work provides evidence that PFN1 mutations can cause ALS as a Mendelian dominant trait. The novel p.Thr109Met mutation also shows that disturbance of actin dynamics can cause motor neuron degeneration. (IV) We find it unlikely that the VAPB mutations cause ALS in our cohorts. (V) We find it unlikely that the 50 bp region contains important regulatory elements for SOD1 expression. This thesis supports the theory that ALS is a multigenetic disease, but there appears to be great genetic variation among apparently identical populations. These studies emphasise the importance of continuous genetic screening, to identify further mutations and genes involved in ALS disease, but it also highlights the importance of cooperation and comparison between countries.

Place, publisher, year, edition, pages
Umeå: Umeå Universitet , 2013. , 82 p.
Series
Umeå University medical dissertations, ISSN 0346-6612 ; 1563
Keyword [en]
ALS, risk factor, VAPB, SOD1, amyotrophic lateral sclerosis, ATXN2, SMN1, SMN2, PFN1, 50 bp deletion in SOD1 promotor, population-specific genetic variations
National Category
Neurosciences
Research subject
Neurology
Identifiers
URN: urn:nbn:se:umu:diva-67460ISBN: 978-91-7459-582-6 (print)OAI: oai:DiVA.org:umu-67460DiVA: diva2:612124
Public defence
2013-04-17, Sal Betula, By 6M, Norrlands Universitessjukhus, Umeå, 10:00 (English)
Opponent
Supervisors
Projects
On the aetiology of ALS: A comprehensive genetic study
Funder
Swedish Research Council
Available from: 2013-03-27 Created: 2013-03-19 Last updated: 2013-04-12Bibliographically approved
List of papers
1. Ataxin-2 intermediate-length polyglutamine expansions in European ALS patients
Open this publication in new window or tab >>Ataxin-2 intermediate-length polyglutamine expansions in European ALS patients
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2011 (English)In: Human Molecular Genetics, ISSN 0964-6906, E-ISSN 1460-2083, Vol. 20, no 9, 1697-1700 p.Article in journal (Refereed) Published
Abstract [en]

Amyotrophic lateral sclerosis (ALS) is a fatal adult-onset neurodegenerative disease primarily affecting motor neurons. We recently identified intermediate-length polyglutamine (polyQ) expansions (27-33 Qs) in ataxin 2 as a genetic risk factor for sporadic ALS in North American ALS patients. To extend these findings, we assessed the ataxin 2 polyQ repeat length in 1294 European ALS patients and 679 matched healthy controls. We observed a significant association between polyQ expansions and ALS (>30 Qs; P= 6.2 × 10(-3)). Thus, intermediate-length ataxin 2 polyQ repeat expansions are associated with increased risk for ALS also in the European cohort. The specific polyQ length cutoff, however, appears to vary between different populations, with longer repeat lengths showing a clear association. Our findings support the hypothesis that ataxin 2 plays an important role in predisposing to ALS and that polyQ expansions in ataxin 2 are a significant risk factor for the disease.

National Category
Neurology
Identifiers
urn:nbn:se:umu:diva-57322 (URN)10.1093/hmg/ddr045 (DOI)21292779 (PubMedID)
Available from: 2012-07-11 Created: 2012-07-11 Last updated: 2017-12-07Bibliographically approved
2. Homozygous SMN2 deletion is a protective factor in the Swedish ALS population
Open this publication in new window or tab >>Homozygous SMN2 deletion is a protective factor in the Swedish ALS population
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2012 (English)In: European Journal of Human Genetics, ISSN 1018-4813, E-ISSN 1476-5438, Vol. 20, no 5, 588-591 p.Article in journal (Refereed) Published
Abstract [en]

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. European Journal of Human Genetics (2012) 20, 588-591; doi:10.1038/ejhg.2011.255; published online 25 January 2012

Keyword
SMN1 gene, SMN2 gene, sporadic amyotrophic lateral sclerosis, association studies
National Category
Neurology
Identifiers
urn:nbn:se:umu:diva-55302 (URN)10.1038/ejhg.2011.255 (DOI)000303016800029 ()
Available from: 2012-05-19 Created: 2012-05-14 Last updated: 2017-12-07Bibliographically approved
3. A novel phosphorylation site mutation in profilin 1 revealed in a large screen of US, Nordic and German amyotrophic lateral sclerosis/frontotemporal dementia cohorts
Open this publication in new window or tab >>A novel phosphorylation site mutation in profilin 1 revealed in a large screen of US, Nordic and German amyotrophic lateral sclerosis/frontotemporal dementia cohorts
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2013 (English)In: Neurobiology of Aging, ISSN 0197-4580, E-ISSN 1558-1497, Vol. 34, no 6, 1708.e1-1708.e6 p.Article in journal (Refereed) Published
Abstract [en]

Profilin 1 is a central regulator of actin dynamics. Mutations in the gene profilin 1 (PFN1) have veryrecently been shown to be the cause of a subgroup of amyotrophic lateral sclerosis (ALS). Here, weperformed a large screen of US, Nordic, and German familial and sporadic ALS and frontotemporaldementia (FTLD) patients for PFN1 mutations to get further insight into the spectrum and pathogenicrelevance of this gene for the complete ALS/FTLD continuum. Four hundred twelve familial and 260sporadic ALS cases and 16 ALS/FTLD cases from Germany, the Nordic countries, and the United Stateswere screened for PFN1 mutations. Phenotypes of patients carrying PFN1 mutations were studied. Ina German ALS family we identified the novel heterozygous PFN1 mutation p.Thr109Met, which wasabsent in controls. This novel mutation abrogates a phosphorylation site in profilin 1. The recentlydescribed 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 overtcognitive involvement. PFN1 mutations were absent in patients with motor neuron disease anddementia, and in patients with only FTLD. We provide further evidence that PFN1 mutations can causeALS 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 proofof-principle that mutant proteins involved in the regulation of cytoskeletal dynamics can cause motorneuron degeneration. Moreover, this new mutation suggests that fine-tuning of actin polymerization byphosphorylation of profilin 1 might be necessary for motor neuron survival.

Keyword
Amyotrophic lateral sclerosis, ALS, FTLD, profilin1, Cytoskeleton, Actin
National Category
Neurosciences
Research subject
Neurology
Identifiers
urn:nbn:se:umu:diva-67457 (URN)10.1016/j.neurobiolaging.2012.10.009 (DOI)
Projects
On the aetiology of ALS: A comprehensive genetic study
Available from: 2013-03-21 Created: 2013-03-19 Last updated: 2017-12-06Bibliographically approved
4. No association between VAPB mutations and familial or sporadic ALS in Sweden, Portugal and Iceland
Open this publication in new window or tab >>No association between VAPB mutations and familial or sporadic ALS in Sweden, Portugal and Iceland
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2013 (English)In: Amyotrophic lateral sclerosis and frontotemporal degeneration, ISSN 2167-8421, Vol. 14, no 7-8, 620-627 p.Article in journal (Other academic) Published
Abstract [en]

Background. 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) co-segregates with disease.

Methods. Blood samples were studied from 973 Swedish, 126 Portuguese and 19 Icelandic ALS patients, and from 644 control subjects. 


Results. 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 co-segregate 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. 


Conclusions. 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

Place, publisher, year, edition, pages
Informa Healthcare, 2013
Keyword
ALS, risk factor, VAPB, SOD1, oligomutant
National Category
Neurosciences Neurology
Research subject
Neurology
Identifiers
urn:nbn:se:umu:diva-67455 (URN)10.3109/21678421.2013.822515 (DOI)
Projects
On the aetiology of ALS: A comprehensive genetic study
Funder
Swedish Research Council
Available from: 2013-03-19 Created: 2013-03-19 Last updated: 2013-12-12Bibliographically approved
5. Erythrocyte SOD1 enzyme activity in ALS patients is not modulated by a 50 bp deletion in the alleged SOD1 promotor
Open this publication in new window or tab >>Erythrocyte SOD1 enzyme activity in ALS patients is not modulated by a 50 bp deletion in the alleged SOD1 promotor
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(English)Manuscript (preprint) (Other academic)
Abstract [en]

Background A known cause of ALS are mutations in the SOD1 gene. There is also evidence that SOD1 may be involved in cases lacking mutations in the gene. A 50 bp deletion located 1684 bp upstream of the start codon of SOD1 has been suggested to reduce transcription of SOD1, affect enzymatic activity and to be associated with later disease onset in ALS patients. The findings have been challenged by a study of Italian ALS patients, and here we examined the 50 bp deletion in Swedish ALS patients and controls. 

Methods Blood samples from 543 Swedish ALS patients and 356 Swedish controls were analysed for the 50 bp deletion and for SOD1 enzymic activity. The results were related to the disease phenotype of the patients.

Results The frequency of the 50 bp deletion was the same in the patient and control cohorts, and both were found to be in Hardy-Weinberg equilibrium regarding the deletion. In relation to the different genotypes, no differences were detected in SOD1 enzymic activity, duration of disease, age of onset or site of onset.

Conclusions When interpreting the present results together with previous results from other populations, we find it unlikely that the 50 bp deletion region has any regulatory function for the SOD1 gene, nor any effects on the phenotype of ALS.

Keyword
ALS, SOD1, promotor, deletion, age at onset
National Category
Neurosciences
Research subject
Neurology
Identifiers
urn:nbn:se:umu:diva-67456 (URN)
Projects
On the aetiology of ALS: A comprehensive genetic study
Available from: 2013-03-21 Created: 2013-03-19 Last updated: 2013-04-09Bibliographically approved

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