P134 POSITIONAL CLONING OF A NOVEL GENE FOR PROGRESSIVE MOTOR NEURON DEGENERATION
Gopinath S, Blair IP, Kennerson ML, Nicholson GA
ANZAC Research Institute, Sydney, Australia
E‐mail address for correspondence: [email protected]
Background: Motor neuron disease and hereditary motor neuropathies (HMN) are related neurodegenerative diseases that cause the selective progressive death of motor neurons. Historically, the disorders have been classified separately based upon rate of disease progression, with MND/ALS usually rapidly progressive and fatal, while HMN is slowly progressive and non‐fatal. However, the HMNs and MNDs share clinical and pathological features and some forms share the same genetic basis, i.e. the same gene causes rapid or slowly progressive disease. As such, we anticipate that their distinction may, at least in part, be artificial. Genes identified from HMN and MND/ALS families have also been associated with disease in sporadic MND/ALS cases.
Objectives: Our aim is to use linkage analysis and positional cloning approaches to identify new disease genes for motor neuron degeneration and establish whether these are more broadly involved in familial and sporadic motor neuron degeneration, as pathogenic or susceptibility genes.
Methods: We performed a genome scan for linkage in a large HMN family comprising 25 individuals including 12 affected. A transcript map of the disease locus was generated using the human genome annotation projects of the Sanger Centre (Ensembl) and UCSC (Genome Browser). Candidate genes were selected based upon known or inferred function and expression. Mutation analysis was performed by high resolution melting (LightScanner, Idaho Technology) and direct sequencing.
Results: A genome‐wide linkage scan identified a novel HMN locus on chromosome 7q34‐q36. Significant evidence for linkage was obtained for D7S2546 with a two‐point lod score of 3.33. A multipoint lod score of 3.59 was obtained at the same marker. Additional support for linkage was obtained for two adjacent markers with multipoint lod scores of 3.50 and 3.44. Recombinant haplotype analysis defined a 21.8 cM candidate interval. We generated a transcript map of the candidate region that spans 13 Mb and contains 247 entries for known or putative protein‐coding genes. The interval encompasses large clusters of genes encoding T‐cell receptors (86), olfactory receptors (27), and taste receptors (9) as well as trypsin molecules (10). The remaining 115 transcripts were inspected to identify any obvious candidate genes based upon our knowledge of previously implicated HMN/MND genes or disease mechanisms. Candidate disease genes are currently being screened for mutations.
Discussion: We have identified a locus for progressive motor neuron degeneration on chromosome 7q34‐q36. Work is underway to positionally clone the gene in question. Once identified, we will investigate HMN, FALS, and sporadic MND/ALS cohorts to establish whether this gene, like other MND genes, is involved in the slowly progressive disorders of motor neurons (HMN) and rapidly progressive disease (ALS), as a pathogenic or susceptibility gene.
P135 SYSTEMATIC ELIMINATION OF PREVALENT MYELIN SEQUENCES FROM MOUSE SPINAL CORD cDNA PREPARATIONS
Yan Z, Lathia KB, Clapshaw PA
Solomon Park Research Institute, Kirkland, WA, USA
E‐mail address for correspondence: [email protected]
Background: We have previously demonstrated by suppression subtractive hybridization (SSH) and mirror orientation selection (MOS) that a large portion of sequences differentiating spinal cord from visual cortex in the mouse can be attributed to cDNA derived from myelin. Fully half of all up‐regulated sequences in the spinal cord subtracted against visual cortex are of glial cell origin. Proteolipid protein alone accounts for some 40% of all up‐regulated sequences in the spinal cord. It is possible, although unlikely, that these differences reflect increased numbers of glial cells in this structure or possibly the ongoing metabolic activity of those cells normally present; however, it is preferable to study the expression of mRNA in motor neurons unobstructed by the overwhelming presence of glial derived sequences.
Methods: Two approaches were designed to reduce the number of glial derived sequences in subtraction preparations before proceeding with SSH. In the first procedure, we have attached biotinylated oligonucleotides of known myelin sequences to streptavidin coated magnetic beads and, by incubating cDNA preparations from spinal cord and visual cortex to these beads with the attached capture sequences, are subtracting out specific prevalent cDNAs. In the second procedure, myelin rich cDNA from brain white matter was used as a driver for the subtraction to reduce myelin sequences present in the spinal cord.
Results: Taking the sequence derived from proteolipid protein (plp), which represents the most abundant myelin sequence up‐regulated in the spinal cord when subtracted against the visual cortex, as an example: the occurrence of this sequence was reduced by 1000‐fold by semiquantitive polymer chain reaction before and after bead subtraction from spinal cord and visual cortex. When this pre‐subtracted cDNA was subsequently used to perform SSH between spinal cortex and visual cortex, the final results showed a consistent 90% reduction of plp sequences. In the alternate procedure, where spinal cord was subtracted directly against cDNA preparations derived from white matter, we have observed no sequences that can be positively identified as originating from glial cells.
Discussion: There can be little doubt that prevalent myelin derived sequences present in spinal cord and visual cortex can be reduced precipitously by pre‐subtracting the original cDNA preparations against immobilized biotin labelled myelin sequences with streptavidin beads as well as subtracting spinal cord directly against myelin derived cDNA sequences. This will allow the removal of the glial derived cDNA sequences as well as other abundant sequences originating in the nerve cells in the spinal cord and visual cortex themselves, and provide a more in‐depth analysis of any differentially expressed genes in these structures. It is anticipated that analyses of these final motor neuron derived sequences will lend some clue as to the susceptibility of motor neurons in motor neuron disease among other pathologies affecting these cells.
P136 ALS6 REVISITED: LINKAGE ANALYSIS WITH DNA MICROARRAYS
Sreedharan J, Vance CA, Smith BN, Hu X, Al‐Chalabi A, Shaw CE
MRC Centre for Neurodegeneration Research, Institute of Psychiatry, King's College London, London, UK
E‐mail address for correspondence: [email protected]
Background: Gene hunting in familial ALS (FALS) is a difficult task due to rapid disease progression and difficulty in collecting DNA for linkage analysis. We have conducted work on a number of families with ALS using Affymetrix 10K GeneChip® DNA microarrays. These allow simultaneous genotyping of over 10,000 single nucleotide polymorphisms (SNPs). Subsequent analysis of the SNP calls results in rapid determination of regions of potential linkage.
Objectives: To identify the disease‐causing gene in a family with aggressive autosomal dominant ALS (ALS 6). A previous genome‐wide study using microsatellite markers had demonstrated strong linkage only to chromosome 16q (maximum multipoint log of the odds (LOD) score of 3.85) and a haplotype that segregated with disease.
Methods: DNA from five affected individuals was processed according to the Affymetrix protocol before being hybridized onto GeneChips®. Following staining and washing in a fluidics station, chips were then scanned. The output was formatted for linkage analysis using the Merlin linkage package. Regions of potential linkage were then fine‐mapped using microsatellite markers. Haplotype analysis was conducted to determine phase and segregation with disease.
Results: The previously linked region on chromosome 16 was verified in this study. However, regions on chromosomes 2, 9 and 11 also demonstrated significant LOD scores. Fine mapping and haplotype analysis excluded the regions on chromosomes 2 and 11, but a consistent haplotype segregating with disease was found at 9q31 (maximum LOD score of 2.3).
Discussion: Although DNA microarrays dramatically reduce the time and labour requirement for linkage analysis there are a number of inherent problems. The vast amount of data that needs to be analysed requires great computer power. This can be a significant problem when analysing large kindreds.
The discovery of two or more loci that are potentially linked with disease is a problem that is likely to be increasingly experienced as DNA microarrays become the standard approach for linkage analysis. The higher resolution they provide can pick up smaller areas that would previously have been missed by microsatellite marker sets. This can lead to an increase in the false positive rate and the problem of not knowing which region to investigate further.
P137 SCREENING FOR GENETIC VARIATION IN SEX HORMONE RECEPTOR GENES IN A DUTCH POPULATION
van Vught PWJ1, Veldink JH1, Groeneveld GJ1, van es Ma1, Wokke JHJ1, Baas F2, van den Berg LH1
1Rudolf Magnus Institute of Neuroscience, Department of Neurology, UMC Utrecht, Utrecht, Netherlands, 2Department of Neurogenetics, Academic Medical Center, Amsterdam, Netherlands
E‐mail address for correspondence: [email protected]
Background: ALS predominates in males, although this sexual dimorphism diminishes with age. Women develop ALS at an older age than men and have an earlier menopause compared to controls. These features suggest a role for sex hormones in the pathogenesis of ALS. Furthermore, both in vitro and in vivo studies have shown that oestrogen and androgens (testosterone and dihydrotestosterone) protect neurons from degeneration. The neuroprotective actions of these steroid hormones are mostly receptor‐mediated.
Objectives: To determine whether genetic variation in the coding regions of the androgen receptor (AR) and the oestrogen receptor (ER) alpha and beta are genetic susceptibility factors in ALS.
Methods: Genomic DNA from a large set of Dutch well defined ALS patients and controls was extracted from blood samples. The coding region and untranslated regions of the AR, ERα and ERβ were amplified by PCR and products were re‐sequenced to detect (novel) mutations.
Subjects were further genotyped for all known SNPs using allelic discrimination with TaqMan assays.
Allele frequencies were determined and tested for association.
Results: No novel mutations were detected in the coding and untranslated regions of the three genes. Genotyping the known polymorphisms yielded no direct association with ALS. However, we detected a lower incidence of a SNP in exon 8 of ERα in ALS patients with bulbar onset (p = 0.02).
Discussion and conclusions: We identified a relationship between an ERα single nucleotide polymorphism and bulbar ALS, suggesting a protective effect of this SNP. Our results will be validated in a larger population. Furthermore, the biological consequences of this SNP will be investigated.
P138 ADULT‐ONSET PRIMARY LATERAL SCLEROSIS IS NOT ASSOCIATED WITH MUTATIONS IN THE ALS2 GENE
Brugman F1, Eymard‐Pierre E2, van den Berg LH1, Wokke JHJ1, Boespflug‐Tanguy O2
1UMC Utrecht, Utrecht, Netherlands, 2INSERM UMR, Clermont‐Ferrand, France
E‐mail address for correspondence: [email protected]
Background: PLS is a diagnosis of exclusion in patients with an adult onset gradually progressive pure upper motor neuron (UMN) syndrome and could be part of the clinical spectrum of ALS. Adult onset PLS is almost always sporadic. Recessive mutations in ALS2 are causative for early onset upper motor neuron diseases, including infantile ascending hereditary spastic paralysis (IAHSP), juvenile amyotrophic lateral sclerosis (ALS2) and juvenile primary lateral sclerosis (JPLS).
Objective: To assess the role of ALS2 in adult onset PLS.
Methods: We screened a population of 51 adult onset PLS patients by DHPLC methods. Thirty‐nine patients had disease onset in the legs, nine in the bulbar region and three in the arms.
Results: Fourteen sequence variants were detected within ALS2, of which 12 were known single nucleotide polymorphisms (SNPs) and two were new sequence variants without pathogenic significance.
Conclusion: These results indicate that mutations of ALS2 are not a common cause of adult onset PLS.
P139 ARRAY CGH OF AMYOTROPHIC LATERAL SCLEROSIS PATIENTS: NEW CANDIDATE GENES
Waibel S1, Ludolph A1, Ropers HH2, Ullmann R2
1Department of Neurology, University, Ulm, Germany, 2Max‐Planck Institute of Molecular Genetics, Berlin, Germany
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a devastating disorder of the central nervous system that leads to progressive loss of upper and lower motor neurons.
Methods: We have used comparative genomic hybridisation array (CGH array) to screen patients with definite ALS for cryptic chromosomal aberrations in the whole genome.
Results: In 113 patients, we detected several deletions and duplications. Most interestingly, two patients showed duplications at the site of the KIF25 gene on chromosome 6q27, one patient had an alteration on chromosome 6p21.2 (heavy chain dynein polypeptide, DNAH8), and a fourth had an augmentation on chromosome 17q25.1 (intermediate dynein polypeptide, DNAI2). We also found a duplication on chromosome 1p32.2, close to the gene of the glutamate transporter EAAT5. There were several other alterations, consisting of genes of unknown function; however, their total number was not increased if compared with non‐neurological, age‐matched controls.
Conclusions: Among 113 patients with ALS, the CGH array technique was able to detect four submicroscopic chromosomal alterations in three genes relevant for motor protein function. We consider these genes candidates for ALS and related disorders. We did not find evidence for increased genomic instability in ALS.
P140 LARGE‐SCALE GENOME ASSOCIATION STUDY FOR AMYOTROPHIC LATERAL SCLEROSIS
Iida A1, Ohnishi Y2, Nakamura Y2
1RIKEN SNP Research Center, Tokyo, Japan, 2The University of Tokyo, Tokyo, Japan
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a heterogeneous group of neurodegenerative disorders characterized by progressive wasting and weakness of limb, bulbar and respiratory muscles due to the selective degeneration of motor neurons in the brain and spinal cord. Most cases (90%) of ALS are sporadic. To date, several hypotheses for mechanisms of selective motor neuron death, including glutamate‐mediated excitotoxicity, oxidative stress, cytoskeleton abnormalities, and autoimmunity, have been put forward. However, the pathogenesis of ALS remains largely unknown.
Objective: To identify genes associated with susceptibility to ALS, we performed a large‐scale case‐control study by means of single nucleotide polymorphisms (SNPs) in Japanese patients with ALS.
Subjects and methods: We obtained samples from 611 ALS cases as a part of the Japanese project of Personalized Medicine (BioBank Japan) with written informed consent. DNAs were prepared from each blood sample using standard protocols. The study was approved by the Institutional Review and Ethics Boards of the University of Tokyo and RIKEN Yokohama Institute. The SNPs used in this study were selected from the IMS‐JST Japanese SNP database. We employed the Invader assay combined with high‐throughput multiplex PCR to screen the disease associated genomic regions.
Results: We first genotyped 94 ALS patients at 50,368 SNPs and compared their allelic or genotype frequencies at these loci with those in the Japanese general population. All SNPs were successfully genotyped, and 1033 SNPs showed p‐value of 0.01 or less. We further genotyped the SNPs that had p values less than 0.01 in a larger replication panel of individuals with ALS. Finally, we found that a total of three SNPs in two unrelated genes were strongly associated with ALS (p<0.0001). We have now constructed high‐density SNP maps around these marker SNPs.
Discussion: In this study, we performed a large‐scale association study to identify the genes associated with ALS. Methodologically, our screening system has at least two advantages. First, since all SNPs used in this study were isolated around gene regions, we can efficiently investigate SNPs that are associated with susceptibility to common diseases. Secondly, using the Invader assay combined with multiplex PCR, our method allowed us low‐cost screening that requires as little as 0.1 ng of genomic DNA per single SNP. Here we identified three candidate SNPs in the two unrelated genes. Both genetic and functional analyses of these candidate genes are now ongoing.
P141 D90A SOD1 MUTATION FOUND IN A SPORADIC ALS PATIENT WITH HOMOZYGOSIS AND RECESSIVE INHERITANCE
Mendanha L1, Garcia‐Redondo A1, Garcia‐Consuegra I1, Rubio JC1, Dominguez C2, Martin‐Casanueva MA1, Esteban J2
1Neurodegenerative and Mitochondrial Diseases Laboratory, Research Center, 12 de Octubre Hospital, Madrid, Spain, 2Department of Neurology, 12 de Octubre Hospital, Madrid, Spain
E‐mail address for correspondence: [email protected]
Background: Autosomal dominant inheritance accounts for some 10% of cases of amyotrophic lateral sclerosis (ALS). A subgroup of these familial cases (around 20% of them) is linked to mutations in the gene encoding Cu/Zn superoxide dismutase (SOD1) (1). One of these mutations, D90A is the only one known to date that is consistent with both autosomal dominant and recessive inheritance (typically found in the Scandinavian population) (2).
Objective: To study the genetic characteristics of patients in both sporadic and familial ALS cases from the Spanish population.
Methods: The presence of mutations in the SOD1 gene was screened for in patients with probable or definite ALS. Methods included single strand conformational shift polymorphism (SSCP) analysis and direct sequencing. To confirm the mutation we used a PCR‐RFLP approach (2).
DNA from relatives (parents, uncles and aunts, sister and spouse) were also analysed to pinpoint the inheritance pattern of the trait.
Results: Molecular analysis showed that one patient was homozygous for the D90A mutation. The parents and the sister were asymptomatic carriers for the same mutation.
The proband had onset of weakness in legs and developed progressive tetraparesis in four years. Clinical examination revealed bilateral pyramidal signs and motor neuron involvement in the arms and legs. He failed to show bulbar or respiratory symptoms.
Discussion and conclusions: Our group documents the first case in the Spanish population of a patient with ALS associated with a homozygous D90A SOD1 mutation. This is one of the few families reported outside the Scandinavian population with the D90A SOD1 mutation associated with ALS. Current studies are trying to identify a protective factor in families with the typical recessive inheritance (3,4).
Our data could help to address studies on this protective genetic factor, since the family presented here belongs to a different ethnic background.
Acknowledgement: Funding: this study was supported by FUNDELA ‘Fundación para el Fomento de la Investigación en Esclerosis Lateral Amiotrófica’ and a Caja Madrid grant.
References
- Rosen DR, Sapp P, O’Regan J, et al. Am J Med Genet. 1994;51:61–9.
- Robberecht W, Aguirre T, van den Bosch L, et al. Neurology 1996;47:1336–9.
- Parton MJ, Broom W, Andersen PM, et al. Hum Mutat. 2002;20:473.
- Al-Chalabi A, Andersen PM, Chioza B, et al. Hum Mol Genet. 1998;7:2045–50.
P142 SCREENING OF SUPEROXIDE DISMUTASE 1 GENE MUTATIONS IN CHINESE PATIENTS WITH FAMILIAL AND SPORADIC AMYOTROPHIC LATERAL SCLEROSIS
Li X, Zhang L, Liu M, Li B, Zhao Y, Ren H, Tang X, Guo Y, Cui L
Peking Union Medical College Hospital, Chinese Academic of Medical Science, Beijing, China
E‐mail address for correspondence: [email protected]
Background: Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder involving motor neurons in the motor cortex, brainstem and spinal cord. Ninety per cent of cases are sporadic (SALS) and 10% are familial (FALS), with multiple autosomal dominant and recessive forms. Currently three major ALS genes and five additional loci have been identified. The first gene (ALS1) associated with the adult‐onset autosomal dominant form of the disease encodes for the cytoplasmic Cu/Zn superoxide dismutase (SOD1). Approximately 20% of FALS cases and 2% of overall ALS cases have identifiable mutations in this gene. At the present time more than 110 different mutations located in all five exons of the gene have been identified worldwide. However, only a few familial cases and no sporadic case with SOD1 gene mutations, have been reported in China
Objectives: We performed a retrospective study of the distribution of SOD1 gene mutations in a large cohort of Chinese ALS patients in order to assess the frequency of SOD1 gene mutations in FALS and SALS cases. Clinical features of patients with SOD1 mutations are reported.
Methods: Blood was collected from 18 individuals from two ALS families and 63 patients with sporadic ALS. The El Escorial diagnostic criteria were used. Genomic DNA was prepared from blood using standard procedures. PCR amplification of five exons and introns was performed using primers as previously described. The reaction product was sequenced on double strand with an automated DNA sequencer (ABI 377) using the Big dye terminator cycle sequencing pre‐mix kit.
Results: In five out of 16 individuals from one family we found a novel missense mutation in the SOD1 gene, which was heterozygous for the mutation, GAA to GTA, causing the substitution of valine for glutamic acid at codon 133 (Glu133Val) in exon 5. The proband has a rapid progressive disease course. The heterozygous G72C mutation was found in a suffering ‘sporadic’ son and his healthy father. Clinically, the patient exhibited early onset and rapid disease progression. According to the gene test result and the complementary family history, we identified this as an ALS family. In two out of 63 SALS patients we found two novel missense mutations. A new V29A missense mutation in exon 2 was found in a sporadic patient with a typical progressive disease course. A new N86I missense mutation in exon 4 was found in a sporadic patient with a rapid progressive disease course.
Discussion and conclusions: This study firstly reports screening of gene mutations in SOD1 in Chinese sporadic ALS patients. The frequency of SOD1 gene mutations (3.1%) in SALS cases was comparable with that found in other surveys in different cohorts all over the world. Three new mutations were found in this study.
P143 VEGF C2578A POLYMORPHISM DOES NOT CONTRIBUTE TO AMYOTROPHIC LATERAL SCLEROSIS SUSCEPTIBILITY IN SPORADIC CHINESE PATIENTS
Zhang Y, Zhang H, Fu Y, Song H, Wang L, Zhang J, Fan D
Peking University Third Hospital, Beijing, China
E‐mail address for correspondence: [email protected]
Background and objective: A linkage and association of the VEGF (vascular endothelial growth factor) C2578A polymorphism and amyotrophic lateral sclerosis (ALS) has been found in some studies (1,2). We analysed the C2578A polymorphism in sporadic ALS patients from a Chinese population.
Methods: The polymorphism was analysed in 115 patients and 200 healthy individuals by amplifying across position 2705 to 2494 of the promoter region of the VEGF gene.
Results: It was found that the frequency of the allele A was 24% in ALS patients and 28% in healthy individuals (p = 0.264). Comparing the background of this polymorphism in healthy individuals between Chinese and Caucasians, significant decreases were found in the frequencies of the A/A genotype and allele A (p<0.001).
Discussion: We concluded that VEGF C2578A polymorphism did not confer a susceptibility to sporadic Chinese ALS patients, which was in disagreement with that reported previously in Caucasian populations (2) and might be ascribed to the different genetic background between Chinese and Caucasians.
References
- Oosthuyse B, Moons L, Storkebaum E, et al. Nat Genet. 2001;28:131–8.
- Lambrechts D, Storkebaum E, Morimoto M, et al. Nat Genet. 2003;34:383–94.
P144 SCREENING FOR POTENTIAL CANDIDATE GENES AND RISK FACTORS IN A LARGE COHORT OF MND CASES: ASSESSING THE CONTRIBUTION OF ANG, CHMP2B AND HFE
Cox L, Kirby J, Namavar Y, Hartley J, Nixon H, Ince P, Shaw P
University of Sheffield, Sheffield, UK
E‐mail address for correspondence: [email protected]
Background: Although 10% of MND is familial, genetic factors are also thought to play a role in the sporadic disease. Mutations have recently been found in the angiogenin (ANG) gene in both familial and sporadic cases (1). In addition, a mutation in chromatin modifying protein 2B (CHMP2B), usually associated with frontotemporal dementia, has been found in two cases of ALS (2).
Recent evidence also supports a role for the hemochromatosis (HFE) gene in MND, with the H63D allele showing an increased frequency in MND cases, while there is no association with the C282Y mutation (3,4).
Objectives: 1) To screen ANG and CHMP2B in a large cohort of MND patients for mutations associated with the disease. 2) To determine allele frequencies of the HFE gene in the largest cohort of cases to date, compared to healthy controls.
Methods: 1) PCR and direct sequencing of the exon encoding ANG and exons 1–6 of CHMP2B were carried out on 120 DNAs extracted from CNS tissue. In addition, ANG was screened in a further 400 DNA samples extracted from blood samples.
2) The HFE H63D and C282Y mutations were detected by PCR of exons 2 and 4 followed by restriction enzyme digestion with MboI and RsaI, respectively. Five hundred and twenty MND cases were genotyped and the allele frequencies compared with 400 age‐matched healthy controls.
Results: ANG: Out of the first 100 samples sequenced, no mutations have been found, although a previously reported T to G polymorphism has been identified at nt 330.
CHMP2B: Of the 100 samples sequenced, no mutations have been identified in exons 1 or 3, although 2 novel nucleotide substitutions have been found in the 5’UTR, at positions −151 and −102, upstream of the ATG.
HFE: Screening of the H63D mutation has identified the D63 allele to be present at a frequency of 17% in our MND population, while the Y282 allele is present at a frequency of 11%.
Discussion and conclusions: The ANG gene mutations had previously been found in 1% of cases screened, so we would expect several mutations to be identified in our further cases. This suggests ANG is not a common cause of MND.
The CHMP2B gene has only been associated with MND in two cases to date. Screening of all 6 exons in over 100 cases will suggest how frequently changes in this gene are associated with MND
The role of HFE mutations in MND is not understood, although there is increasing evidence of the H63D allele being associated with the disease. This allele is much older in origin than the C282Y allele, which may explain why there is a stronger disease association with the D63 allele than the Y282.
References
- Greenway MJ, Andersen PM, Russ C, et al. Nat Genet. 2006;38:411–3.
- Parkinson N, Ince PG, Smith MO, et al. Neurology 2006;67:1074–1077.
- Wang XS, Lee S, Simmons Z, et al. J Neurol Sci. 2004;227:27–33.
- Goodall EF, Greenway MJ, van Marion I, et al. Neurology 2005;65:934–7.
P145 ELEVATED SERUM ANGIOGENIN IN AMYOTROPHIC LATERAL SCLEROSIS
Cronin S1, Greenway M3, Kieran D2, Prehn JH2, Hardiman O1
1Beaumont Hospital, Dublin, Ireland, 2Royal College of Surgeons in Ireland, Dublin, Ireland, 3The National Centre for Medical Genetics, Dublin, Ireland
E‐mail address for correspondence: [email protected]
Background: An allelic association and a series of novel mutations in the angiogenin gene suggest a role for angiogenin in the pathogenesis of amyotrophic lateral sclerosis (ALS) (1). Deletions in the hypoxia‐reponsive promoter of the related peptide vascular endothelial growth factor (VEGF) lead to an ALS phenotype in mice, and altered VEGF levels have been reported in ALS patients (2). However, the patterns of serum angiogenin expression among ALS patients have not previously been assessed.
Objective: The aim of this study was to investigate whether serum angiogenin and VEGF levels were altered in a large cohort of ALS patients.
Patients and methods: Serum angiogenin and VEGF levels were quantified in 79 patients with definite or probable ALS by the El Escorial criteria, and in 72 healthy control subjects. Among ALS patients, 57 presented with spinal onset symptoms and 22 with bulbar symptoms. Blood was drawn at diagnosis, and follow‐up 12 month sera were available in 19 cases. Serum levels were determined using a quantitative sandwich enzyme‐linked immunoassay.
Results: ALS patients exhibited significantly higher serum angiogenin (p = 0.006) but not VEGF (p = 0.55) compared to control subjects. Comparison between subgroups showed significant elevation in angiogenin for spinal (p<0.001) but not bulbar (p = 0.11) onset ALS. Angiogenin levels at 12 months did not differ from baseline. No correlation was noted between angiogenin and VEGF levels (r = −0.08, p = 0.49) in ALS patient serum.
Conclusions: These data suggest a modest elevation in serum ANG in ALS. Further investigation will be required to assess the utility of serum ANG as a biomarker for ALS and as a predictor of disease progression.
Acknowledgement: This work was supported by The Charitable Infirmary Charitable Trust, Dublin, Ireland.
References
- Greenway MJ, Andersen PM, Russ C, et al. ANG mutations segregate with familial and ‘sporadic’ amyotrophic lateral sclerosis. Nat Genet. 2006;38:411–3.
- Lambrechts D, Storkebaum E, Morimoto M, et al. VEGF is a modifier of amyotrophic lateral sclerosis in mice and humans and protects motor neurons against ischaemic death. Nat Genet. 2003;34:383–94
P146 MUTATIONS IN REGULATORY ELEMENTS OF METALLOTHIONEIN ASSOCIATED WITH ALS
Lu S, Smith AP, Lee NM
The Forbes Norris ALS Research Center, San Francisco, USA
E‐mail address for correspondence: [email protected]
Background: Several lines of evidence suggest that changes in zinc disposition could play a key role in the initiation and/or progression of ALS. Superoxide dismutase‐1 (SOD1), mutations in which are a known cause of the disease, binds zinc and mutant forms exhibit altered binding. Metallothioneins (MTs), which bind and regulate levels of cellular zinc, are up‐regulated in mutant SOD1 mice, and mutant mice with MT genes deleted reach symptom onset and death sooner than wild‐type controls. Alterations in zinc levels are also associated with all the major pathologies observed in ALS, including oxidative stress, glutamate excitotoxicity, neuroinflammation, aggregation, and others.
MTs exist in several different forms, MT‐1, 2, and 3 and 4. In humans, MT‐1 itself exists in at least seven isoforms of closely related but non‐identical sequences. It is not known whether these different isoforms have different functional properties, including zinc binding.
Objectives: We compared the gene sequences of these different MT‐1 isoforms, as well as human MT‐2 (MT‐2A) from ALS patients and controls.
Methods: DNA was isolated from white blood cells or spinal cord tissue, and PCR used to identify and analyse MT sequences in this DNA.
Results: No differences in coding region sequences were detected in any of these MTs, but in one of the MT‐1 isoforms, MT‐1E, we found a C to G change in the promoter region or 5’ UTR region of the gene. The change created a new metal response element (MRE) consensus sequence on both strands of DNA. We are currently investigating whether the frequency of this mutation differs between individuals with ALS and controls.
Conclusions: The MREs present in the promoter region of MTs allow zinc to regulate expression of this zinc‐binding protein. Our results suggest that zinc regulation of MT‐1 expression may be altered by a single nucleotide mutation in some individuals.
P147 GENOTYPE AND SERUM LEVEL OF INTERLEUKIN‐6 IN AMYOTROPHIC LATERAL SCLEROSIS PATIENTS
Partyka D1, Ostrowska M1, Tomik B1, Dziedzic T1, Slowik a1, Szczudlik A1, Figlewicz DA2
1Department of Neurology, Jagiellonian University, Medical College, Krakow, Poland, 2Department of Neurology, Ann Arbor University, Ann Arbor Michigan, USA
E‐mail address for correspondence: [email protected]
Background: The origin of motor neuron degeneration in ALS is still unknown. Experimental studies have shown that local inflammatory reaction plays a role in the pathogenesis of ALS. Interleukin‐6 (IL‐6) is a major regulator of the acute phase of inflammatory reaction and possesses neurotrophic properties. Animal models and human studies have shown that this cytokine can be involved in the process of neurodegeneration. Genes belonging to the cytokine family were also studied as candidate genes involved in the pathogenesis of SALS.
Objective: To study the G/C polymorphism of IL‐6 as well as the serum level of IL‐6, which could be potentially involved in the inflammation reaction, in sporadic ALS (SALS) patients compared to controls.
Material and methods: Genotyping of IL‐6 was performed in 127 SALS cases with definite or probable diagnosis of ALS according to WFN criteria and 251 healthy controls matched for age (±3 years) and sex. The polymorphisms were studied using PCR technique and restricted enzyme digestion.
To estimate the serum level of IL‐6 we studied 35 SALS patients fulfilling WFN criteria and 16 healthy controls. Subjects were not eligible for the study if there was medical evidence of any infection, inflammatory, immunologic or allergic processes. There was no difference in diet, age and medication between ALS and healthy subjects. Serum was consecutively separated from freshly drawn blood samples, taken in the morning from fasting patients and controls, and was stored at −70°C until assayed. An enzyme‐linked immunosorbent assay (ELISA) method with commercially available kit (R&D system) was used for serum IL‐6 analysis.
Results: The distribution of genotypes and alleles of IL‐6 G/C polymorphism between ALS cases and controls was not significantly different, i.e. genotypes: G/G 42 (33%) vs. 70 (28%), G/C 58(46%) vs. 119 (47%), C/C 27 (21%) vs. 62(25%), respectively, and alleles: G 142 (56%) vs. 259 (52%), C 112 (44%) vs. 243 (48%), respectively.
The mean (±SD) serum values of IL‐6 in ALS and control subjects were: 1.41±1.94 pg/ml vs. 2.76±3.02 pg/ml, respectively. Thus, there was a noticeable decrease of the level of serum IL‐6 in ALS patients compared to the controls; however, the difference did not reach statistical significance (p = 0.05). There was also no correlation between serum IL‐6 level and age, gender, duration of the disease, type of onset of the disease, Norris and ALSFRS scales in SALS patients (Pearson correlation).
Conclusions: These data show no correlation between genotype and serum level of IL‐6 and increased risk for developing sALS.
P148 LACK OF ASSOCIATION OF P450 1A1/1A2 POLYMORPHISMS WITH SPORADIC ALS AND ABSENCE OF INTERACTION WITH THE PON CLUSTER
Saeed M, Khan H, Siddique N, Hung WY, Driss S, Siddique T
Northwestern University, Chicago, IL, USA
E‐mail address for correspondence: [email protected]
Background: Variation in genes for xenobiotic responsive proteins may modulate susceptibility towards ALS as we recently demonstrated for the paraoxonase (PON) gene cluster (1).
Objectives: To investigate the association and interaction of CYP1A1/A2 gene polymorphisms with sporadic ALS (SALS).
Methods: We investigated the association of three CYP1A1 and CYP1A2 polymorphisms (rs4646421, rs762551, rs2470890) in a case‐control North American Caucasian cohort (n = 315/380). Genotyping was performed using TaqMan® SNP Genotyping Assays. Data were analysed using SPSS and Haploview. Gene x gene interactions were carried out using multifactor dimensionality reduction (MDR).
Results: No association with SALS was found with any of the three polymorphisms or their haplotypes. MDR showed a lack of gene x gene interactions of the three polymorphisms with each other and also with the PON cluster associated polymorphisms (rs10487132 and rs6954345) (1).
Conclusions: CYP1A1 and CYP1A2 polymorphisms are not associated with SALS. There is a lack of interaction between PON cluster and the CYP1A1/2 genes.
Acknowledgements: We would like to thank the patients with ALS and their families for their participation in this study. Without their help this work would not have been possible. This research was supported by NINDS (NS050641, NS046535), Les Turner ALS Foundation, V.E. Schaff ALS Research Fund, H. Post Research Professorship, Wenske Foundation, Falk Medical Research Trust, Les Turner ALS Foundation/Herbert C. Wenske Foundation Professorship and The David C. Asselin MD Memorial Fund.
References
- Saeed, et al. Paraoxonase gene cluster polymorphisms are associated with sporadic ALS. Neurology 2006;66 (Suppl 2). Abstract Number IN1.002.
P149 SERPINA3 SIGNAL REGION A/T POLYMORPHISM IS A RISK FACTOR OF SPORADIC AMYOTROPHIC LATERAL SCLEROSIS (SALS) IN A POLISH POPULATION
Tomik B1, Slowik A1, Partyka D1, Pera J1, Dziedzic T1, Turaj W1, Figlewicz DA2, Szczudlik A1
1Department of Neurology, Krakow, Poland, 2Department of Neurology, Ann Arbor, Michigan, USA
E‐mail address for correspondence: [email protected]
Background: Genetic factors involved in the pathogenesis of SALS still remain largely unknown. One candidate gene might be SERPINA3, a serine protease inhibitor. SERPINA3 signal region A/T polymorphism influences SERPINA3 protein expression with the highest levels in the T allele carriers. It has been shown that imbalance of serine proteases and internalized serpins may play a role in the pathogenesis of SALS. Protein complexes formed by inhibitors of serine and cysteine protease system along with damaged Nf proteins, may accumulate within the cell bodies as neuronal inclusions in SALS individuals.
Objective: To study a possible association between SERPINA3 signal region A/T polymorphism and the risk of SALS compared to healthy controls in a Polish population.
Material and methods: We have included 131 patients with SALS and 404 healthy controls matched for age and sex. The definite or probable diagnosis of SALS was established according to El Escorial criteria (1998). Familial ALS (FALS) cases were excluded based on positive ALS family history. The polymorphisms were studied by PCR and restriction enzyme digestion.
Results: The distribution of SERPINA3 signal region A/T polymorphism did not differ between the cases and the controls (cases, n = 131: CC −33, 25.1%; CT −64, 48.9%; TT −34, 26.0%; controls, n = 404: CC −106, 26.2%, CT −193, 47.8%, TT −105, 26.0%). However, when bulbar and limb sALS onset cases were studied separately, the SERPINA3 A/T polymorphism influenced the risk of bulbar SALS onset (genotype distribution in the cases, n = 43: CC −7, 16.3%, CT −19, 44.2%, TT −17, 39.5%, genotype distribution in the controls, n = 404: CC −106, 26.2%, CT −193, 47.8%, TT −105, 26.0%, p = 0.05) (allele distribution in cases: C −33, 38.4%, T −53, 61.6% vs. controls: C −405, 50.1%; T −403, 49.9%, p = 0.038).
Conclusion: SERPINA3 signal region A/T polymorphism is a risk factor of bulbar onset of SALS in a Polish population.
P150 THE PREDICTED LEVEL OF SMN PROTEIN IS ASSOCIATED WITH THE PHENOTYPE OF SPORADIC LOWER MOTOR NEURON DISEASE
Veldink JH1, Kalmijn S1, van der Hout AH2, Lemmink HH2, Scheffer H3, Wokke JHJ1, van den Berg LH1
1Rudolf Magnus Institute of Neuroscience, University Medical Center Utrecht, Utrecht, Netherlands, 2Department of Clinical Genetics, University Hospital Groningen, Groningen, Netherlands, 3Department of Medical Genetics, University Hospital St. Radboud, Nijmegen, Netherlands
E‐mail address for correspondence: [email protected]
Background: ALS can be considered to be part of a spectrum of other motor neuron disorders characterized by predominant lower motor neuron signs to predominant upper motor neuron signs. At both ends of this spectrum are adult onset lower motor neuron disease (LMND) and primary lateral sclerosis (PLS). Furthermore, the distinction between multifocal motor neuropathy (MMN), a treatable disorder, and LMND can be difficult.
The survival of motor neuron gene (SMN), the causative gene for childhood‐onset spinal muscular atrophy (SMA), was previously shown to be associated with the risk of developing LMND and ALS. In addition, copy number variation of SMN that predicts a lower smn protein expression, was shown to be associated with the severity of ALS.
Objectives: To show that copy number variation of SMN was associated with more extensive involvement of body regions in LMND, and its rate of progression.
Methods: We determined SMN1 and SMN2 copy numbers with a quantitative competitive PCR method in 47 patients with generalized LMND, 42 with non‐generalized LMND, and 175 healthy controls. We used a formula taking into account that SMN2 yields only 30% of full‐length SMN transcripts (estimated total full‐length smn protein level = SMN1 copy number+0.3 × SMN2 copy number) to estimate total smn protein levels. In addition, we determined absence of SMN2 in 34 MMN patients.
Results: We observed an over‐representation of one copy of SMN1, representing SMA carrier status, in the generalized LMND group (OR = 5.3, 95% CI 1.2–24.7). In contrast, SMA carriers were not found in the non‐generalized group. SMN2 copy numbers appeared to be lower in both groups, especially in the generalized group. A clear association was found between predicted level of smn protein in the generalized LMND group (p = 0.005) but not in the non‐generalized LMND group (p = 0.10). Although numbers were small, predicted level of smn protein was not different between rapidly progressive and slowly progressive generalized LMND (p = 0.50). A homozygous deletion of SMN2 was present in one MMN patient (2.9%; p = 0.32).
Conclusion: This study showed that phenotypic variation in MND can be partly explained by genetic variation. SMN haplotypes that predict a lower level of smn protein were associated with generalized LMND, in contrast to a lack of association with focal variants of LMND. In addition, a homozygous deletion of SMN2 was not associated with MMN in contrast to a smaller previous study.
P151 EVALUATION OF THE ROLE OF EXOGENOUS RISK FACTORS IN PATIENTS WITH AMYOTROPHIC LATERAL SCLEROSIS
Kihira T1, Kanno S1, Miwa H1, Kondo T1, Okamoto K2
1Wakayama Medical University, Wakayama City, Japan, 2Aichi Prefectural College of Nursing and Health, Nagoya City, Japan
E‐mail address for correspondence: [email protected]
Background: The incidence of amyotrophic lateral sclerosis (ALS) in Wakayama Prefecture is higher than that elsewhere in the world. Recently, however, this incidence has gradually decreased, particularly in men, and the age at onset has shifted to the elderly, indicating the possible role of exogenous factors in the development of ALS.
Objectives: The purpose of this study is to evaluate exogenous factors in ALS development.
Methods: Data of all patients (over 40 years old) consecutively admitted to our hospital between 1999 and 2004 were collected. The ALS patients were diagnosed using the El Escorial criteria. The associations of cervical spondylosis or spinal spondylotic myelopathy (CS/SSM), surgical treatment for CS/SSM, history of bone fracture, occupations at disease onset and cervical MRI findings were compared between definite ALS patients and neurological controls. Cervical MRI findings were classified into four grades (G0 to G3) according to the severity of spinal cord compression.
Results: We analysed the data of 108 definite ALS patients (male: 62%, female: 38%; age at onset 67±11.1, mean±SD years) and 302 neurological controls (male: 52.8%, female: 47.2%; age at onset 63.1±10.4 years). The frequencies of having CS/SSM, surgical therapy and bone fracture in the ALS patients were significantly higher than those in the controls (ORs: 3.7, 4.3, and 2.1, respectively). The percentage of ALS patients with G3 cervical MRI findings was significantly higher than that of the controls (OR: 4.7). The percentage of ALS patients who had secondary industrial occupations at disease onset was higher than that of the controls (OR: 2.8).
Conclusions: Continuous spinal cord compression or minor injury might be a risk factor in developing/triggering or worsening ALS.
P152 LIFESTYLE FACTORS AND RISK OF AMYOTROPHIC LATERAL SCLEROSIS: A CASE‐CONTROL STUDY IN JAPAN
Okamoto KO1, Kondo TK2, Kihira TK2
1Department of Epidemiology, Aichi Prefectural College of Nursing & Health, Nagoya, Japan, 2Department of Neurology, Wakayama Medical University, Wakayama, Japan
E‐mail address for correspondence: [email protected]
Background: Few studies have examined the relationship between lifestyle factors and the risk of ALS.
Objective: We examined associations between lifestyle factors and the risk of ALS using a population‐based case‐control study from 2003 to 2004 in Aichi Prefecture.
Methods: The study comprised 153 ALS patients diagnosed by El Escorial World Federation of Neurology criteria, and 306 gender‐ and age‐ matched controls randomly selected from the general population. Information on lifestyle was collected using a self‐administered questionnaire. The strength of association between ALS and a potential risk factor was assessed by calculating odds ratios (ORs) and 95% confidence intervals (CIs).
Results: Heavy exercise, self reported stress, type A behavior pattern, and less frequent intake of green‐yellow vegetables were significantly associated with increased risk of ALS. Smoking and drinking habits were not associated with increased risk of ALS. The greatest effect on risk for ALS was the combination of type A behavior pattern and less frequent intake of green‐yellow vegetables.
Conclusion: These data suggested that imbalance between excessive production of endogenous oxidative stress and the decrease or lack of antioxidant defence in brain nerves may increase the risk of ALS.
P153 CASE‐CONTROL STUDY FOR ALS RISK FACTORS IN A RUSSIAN POPULATION
Skvortsova VI1, Levitsky GN1, Alekhin AV1, Smirnov AP1, Spirin NN2, Kasatkina EL2, Tyaptin AA2, Gribova NP3, Motkova IV3
1Russian State Medical University, Moscow, Russia, 2Yaroslavl State Medical Academy, Yaroslavl, Russia, 3Smolensk State Medical Academy, Smolensk, Russia
E‐mail address for correspondence: [email protected]
Background: ALS is a fatal neurodegenerative disorder with an aetiology linked with a combination of genetic and environmental factors. Significant risk factors, according to foreign authors, are male gender, age over 50 years, cigarette smoking, rural living, mechanical injury, sports and physical labour. Possible factors include contact with heavy metals and some others. Little is known about plausible mechanisms and reproducibility of these factors in different populations. We conducted the first case‐control study of ALS environmental risk factors in the Russian population.
Materials and methods: We asked 100 definite ALS patients from Moscow (n = 59), Yaroslavl (n = 27) and Smolensk regions (n = 14) with diagnosis verified by EMG and MRI, as well as 100 inpatients from Moscow City Hospitals 20 and 31 who have no ALS and were matched by age and gender to ALS patients, to complete the questionnaire of 17 items relating to exposures in the six months preceding disease onset. We measured Odds ratios and performed χ2 tests to calculate frequencies.
Results: The following factors were encountered in the control group with significantly prevailing frequency: 1) surgery (OR 0.053, p = 0.0001), 2) vegetarianism (OR 0, p = 0.09), 3) contact with organic solvents (OR 0.26, p = 0.016), 4) contact with magnetic fields (OR 0.22, p = 0.028), 5) tick bites (OR 0.59, p = 0.039), 6) acute infectious disease (OR 0.29, p = 0.04), 7) physical labour (OR 0.2, p = 0.016), 8) sports (OR 0.39, p = 0.0001) and tumours in consanguine relatives (OR 0.5, p = 0.067). Only the use of more than 200 g of alcohol per week significantly prevailed in the ALS group (OR 2.68, p = 0.027). Cigarette smoking and contact with heavy metals in the ALS group had high OR (1.57 and 2.17), but low p by χ2 test (0.19 and 0.44, respectively).
Conclusions: These results are in partial conflict with results obtained in previous Western studies; risk factors such as physical activity and sports dominated in Western ALS groups and alcohol abuse was not a significant risk factor for ALS in Western studies. This controversy shows that such risk factors are dependent on different lifestyle habits in Western and Russian populations, and probably on some universal factors common to ALS patients such as high intellectual and professional facilities, that are differently estimated in different societies. However, controversies were revealed also for factors independent of life habits, such as mechanical injury, contacts with magnetic fields and organic solvents. At the same time, our findings confirm the absence of association with ALS for such factors as tick bites, acute infectious disease and vegetarianism, as well as for the presence of an association between ALS and cigarette smoking and contacts with heavy metals. Thus, it is feasible not only to verify the data obtained in a larger sample but also to perform a combined analysis of genetic and environmental risk factors of ALS in separate populations.
P154 EPIDEMIOLOGY OF FALLS IN AMYOTROPHIC LATERAL SCLEROSIS PATIENTS: ANALYSIS OF CROSS‐SECTIONAL AND PROSPECTIVE COHORTS IN THE ALS CARE REGISTRY
Brooks BR1, Lii J2, Huang W2, Anderson F2, Miller RG3, Mitsumoto H4, ALS Care Study Group1
1University of Wisconsin School of Medicine and Public Health, Madison, WI, USA, 2Center for Outcomes Research University of Massachusetts Medical School, Worcester, MA, USA, 3Forbes Norris ALS Neuromuscular Center, California Pacific Medical Center, San Francisco, CA, USA, 4Eleanor and Lou Gehrig ALS Center, Columbia University Medical Center, New York, NY, USA
E‐mail address for correspondence: [email protected]
Background: Falls in selected populations are dependent on the frequency of assessment. Annual occurrence of at least one fall increases with age from 28–35% (⩾65 years) to 32–42% (⩾75 years) in community dwellers. Institution dwellers have >50% chance of a single fall in the next year and previous fallers have a 67% chance of a second fall in the subsequent year (1). Annual occurrence of at least one fall is 62% due to postural instability in Parkinson's disease, 57% due to cardiovascular syncope in patients with syncope, 48% due to weakness in polyneuropathy patients and 12% due to seizures in epilepsy patients (2).
Objectives: To determine monthly occurrence of falls per ALS patient in ALS CARE Registry cross‐sectional and prospectively studied longitudinal cohorts.
Methods: Analysis of ALS patients entered in ALS CARE Registry during the decade 1996–2005.
Results: Falls occurred prior to enrollment in 32% (1548/4817) of evaluable ALS patients. These patients had a median age of 61 years, median time from symptom onset of 2.4 years and median time from diagnosis of 1.5 years. Site of onset in evaluable ALS patients (413) with falls was 47% lumbar, 27% cervical, 21% bulbar and 5% thoracic. Riluzole use was 50% in ALS patients with falls as in the overall ALS CARE registry.
A prospective longitudinal cohort of ALS patients demonstrated 32% (90/280) falls at six months follow‐up (5.3 falls/month per 100 patients), 27% (76/278) falls at 12 months follow‐up (4.5 falls/month per 100 patients) and 29% (37/129) falls at 18 months follow‐up (4.8 falls/month per 100 patients). These follow‐up intervals correspond to a median time post‐diagnosis of 0.8, 1.2 and 2.0 years, respectively. The annualized occurrence of falls in these ALS patients was 64%, 54% and 58%, respectively. Riluzole use in ALS patients with falls was 52% at six months follow‐up, 59% at 12 months follow‐up and 68% at 18 months follow‐up, while median age was 61, 59 and 60 years, respectively.
Discussion and conclusions: The rate of falls per month per 100 ALS patients in a cohort followed prospectively appears to be relatively stable ( 5.3 (0.8 years post diagnosis), 4.5 (1.2 years post diagnosis) and 4.8 (2.0 years post diagnosis) ). Further analysis is required to determine if this rate is stable because there is a balance between the later development of falls in ALS patients who will have lower fall rates and the removal of ALS patients with increasing fall rates or the marked reduction in fall rates in ALS patients with increasing fall rates through the introduction of adaptive devices and increased emphasis on attention to patient safety.
The observed rate of falls in ALS patients provides a benchmark for future clinical studies and safety analyses for clinical trials. The observed rate is slightly higher than the rate for polyneuropathy patients and similar to that for Parkinson's disease patients. The role of site of ALS onset, time to develop leg weakness and time to develop impaired postural reflexes in the pathophysiology of falls requires further study.
References
- Age and Ageing 2001;30:3–7.
- J. Neurol. 2004; 251:79–84.
P155 ETHNIC VARIATION IN THE INCIDENCE OF AMYOTROPHIC LATERAL SCLEROSIS: A SYSTEMATIC REVIEW
Cronin S, Hardiman O, Traynor BJ
The Irish ALS Research Group, Dublin, Ireland
E‐mail address for correspondence: [email protected]
Background: The findings of recent genetic polymorphism studies in amyotrophic lateral sclerosis suggest that the influence of genetic risk factors for the disease may vary by ethnicity. It is now widely accepted that the incidence of ALS is uniform across Caucasian populations, but whether racial variation across other ethnicities exists remains unknown.
Methods: We performed a systematic review of the known literature on the incidence, prevalence and mortality of ALS across all ethnicities, paying particular attention to sources of case finding, inclusion criteria and to studies which stratified their findings by ethnicity.
Results: The literature search identified 27 European incidence studies and 17 conducted in other ethnicities. Twenty‐two of these provided sufficient data for age‐ and sex‐adjustment to a standard population allowing direct comparison.
To date, the only well‐conducted prospective incidence study which has directly compared the frequency of ALS in those of black African descent with a Caucasian population was underpowered to report the lower incidence with certainty. Within the United States, a number of other retrospective incidence studies and mortality studies consistently support the notion of an African resistance to ALS. There are no reliable incidence or mortality studies from the African continent itself, while migration studies conducted in London and Israel provide restricted evidence of lower disease frequency in Africans.
Population‐based epidemiological studies from Asia provide evidence of a lower age‐ and sex‐adjusted incidence of ALS when directly compared to the adjusted incidence in Caucasian populations. However, a similar effect was not seen in an ethnically stratified incidence study from Hawaii.
Numerous epidemiological studies from Central and South America provide crude incidence and mortality rates for ALS in Hispanic populations that are well below non‐Hispanic Caucasian averages. None of these can be adjusted to a standard population for direct comparison. However, when compared to non‐Hispanic white subjects in the United States, lower female incidence and overall crude mortality among Hispanics has been described.
Discussion: Our findings from the limited available data, although imperfect, suggest that the incidence of ALS may be lower among African, Asian and Hispanic ethnicities than among Caucasians. We conclude that a well‐designed prospective epidemiological study concentrating on non‐Caucasian populations should be prioritized.
P156 REAL TIME STATE‐LEVEL PUBLIC HEALTH SURVEILLANCE OF AMYOTROPHIC LATERAL SCLEROSIS (ALS) AND MULTIPLE SCLEROSIS (MS): PRACTICAL GEOGRAPHIC IMPLICATIONS OF WISCONSIN'S ALS MORTALITY EXPERIENCE
Brooks BR1, Werner MA2, Bekkedal MY2, Kanarek MS1, Anderson HA2
1University of Wisconsin School of Medicine and Public Health, Madison, WI, USA, 2Wisconsin Department of Health and Family Services, Madison, WI, USA
E‐mail address for correspondence: [email protected]
Background: As electronic data systems for health outcomes and environmental contaminants have advanced, it has become increasingly apparent that governmental health and environmental data are largely not collected or aggregated in a way that supports public health surveillance efforts for chronic conditions for which environmental factors are likely contributors.
Objective: Wisconsin's Environmental Public Health Tracking (EPHT) program has undertaken a demonstration project to enhance surveillance efforts for amyotrophic lateral sclerosis (ALS) and multiple sclerosis (MS) and create a platform for generating and testing environmental hypotheses about ALS etiology by linking case information with statewide environmental hazard data. For geographical linkage, accurate geographical localization of ALS patients will be necessary.
Methods: To this end, mortality data from Wisconsin's vital records system for 1989 through 1998 were extracted and analysed in order to summarize how deaths were classified and to determine the degree to which geographically‐specific estimates of environmental hazards could be considered representative of decedent exposure.
Results: For the 1989–1998 period, a total of 1047 deaths were identified in Wisconsin for which ALS was derived as underlying cause of death (UCOD) or listed as a primary cause of death. ALS was listed as the UCOD in 508 deaths (48.5%). In cases where ALS was not specified as the UCOD, respiratory arrest, pneumonia and death due to inhalation of food or vomitus were most commonly reported as the UCOD. For the 1989–1998 period, ALS deaths increased from 72 per annum to 116 per annum. Among the 658 deaths reported as occurring outside an inpatient facility, 280 (42.6%) were classified as occurring in nursing homes rather than the decedent's place of residence.
Discussion and conclusions: From these observations, it is clear that residential history information beyond what is available from state vital records is sorely required to adequately consider environmental hypotheses about the incidence of ALS. As such, successful investigations of the role of environmental hazards in ALS incidence would be greatly aided by patient‐ and decedent‐level information about residential history, and adds to the impetus to develop national and international ALS registries for real time localization of incident ALS patients for proper environmental association studies.
P157 INTERNAL AUDIT OF ALS CARE REGISTRY: BASELINE DEMOGRAPHIC CHARACTERISTICS THAT MAY DISTINGUISH ALS PATIENTS WITH AND WITHOUT FOLLOW‐UP DATA
Brooks BR1, Lii J2, Huang W2, Mitsumoto H3, Miller RG4, Anderson F2, ALS Care Study Group2
1University of Wisconsin Hospital and Clinics, Madison, WI, USA, 2Center for Outcomes Research University of Massachusetts Medical School, Worcester, MA, USA, 3Eleanor and Lou Gehrig ALS Center Columbia University Presbyterian Medical Center, New York, NY, USA, 4Forbes Norris ALS Center, California Pacific Medical Center, San Francisco, CA, USA
E‐mail address for correspondence: [email protected]
Background: The ‘ALS CARE Registry’ (ALSCR) is currently the largest clinical outcomes database with ALS clinic‐based or neurologist‐based verification of the diagnosis of ALS. Data are available for 5171 enrolled patients. Follow‐up data are available: 2162/5171 ALS patients (41.8%) have died with death verified by Completion Form; 2430/5171 patients (47.0%) are known to be alive overall; 579/5171 patients (11.2%) are truly lost to follow‐up. However, only 1226/5171 patients (23.7%) are alive with follow‐up observations while 163/5171 patients (3.2%) left the study at enrollment as alive and 1041/5171 patients (20.1%) were enrolled as alive and have no follow‐up or verification by Completion Form. Two groups are defined as the ‘Survival Cohort’ (SC) of 2760/5171 ALS patients with follow‐up data of various intervals (53.4%) and the ‘Non‐Survival Cohort’ (NSC) of 2411/5171 ALS patients without follow‐up data (46.6%).
Objective: To compare the baseline characteristics of the SC with the baseline characteristics of the ALSCR and of the NSC to determine whether there are distinct systematic differences which might define follow‐up experience.
Methods: Group differences and distributions were compared with descriptive statistics.
Results: Male: female ratio (59% (M):41% (F)) was identical in SC and ALSCR. Other comparisons include White/Caucasian patients (93% (SC); 92% (ALSCR)), education level college and higher (42%; 38%), annual income level $60,000 or higher (19%; 19%); married (78%; 76%); spinal surgery (5%; 5%); sporadic ALS (94%; 94%); bulbar onset (30%; 30%). In the SC there was no significant baseline difference in educational level, or income level between those who died and those who did not. There was a significant difference at baseline in the M:F ratio (56%: 44% vs. 62%: 38% (p = 0.0026)); Oriental/Asian, Pacific Islander‐North American Indian, Eskimo‐Black/African American patient (3.6% vs. 6.2% (p = 0.006)); never married‐divorced‐separated patient (14% vs. 19% (p = 0.014)) characteristics of the SC, but no significant difference in the type of ALS, site of initial disease or experience of antecedent spinal or peripheral nerve surgery. Baseline characteristics that may determine poor follow‐up identified in the NSC include North American Indian, Eskimo‐Black/African American patients (5.5% vs. 7.2% (p = 0.0077)); income below $40,000 (45% vs. 51% (p<0.0001)); never married‐divorced‐separated‐widowed (22% vs. 26% (p = 0.0045)) but no significant difference in M: F ratio, education level or riluzole use at baseline.
Conclusions: In the ALSCR the SC differs from the NSC in specific baseline demographic parameters which may explain the difference in follow‐up. These baseline demographic characteristics were different from those characteristics present in the SC ALS patients who were known to survive and those who were known not to survive.
P158 A TELEPHONE SCREENING METHOD TO IDENTIFY ALS CASES IN A POPULATION: VALIDATION AGAINST MEDICAL RECORD REVIEW IN THE VETERANS AFFAIRS ALS REGISTRY
Kasarskis EJ1, Bedlack RS2, Rozear MP4, Morgenlander JC4, Sabet A1, Sams L3, Lindquist JH2, Coffman CJ2, Oddone EZ2, Allen KD2
1University of Kentucky and VA Medical Centers, Lexington KY, USA, 2Durham VA Medical Center, Durham NC, USA, 3University of Cincinnati, Cincinnati OH, USA, 4Duke University, Durham NC, USA
E‐mail address for correspondence: [email protected]
Background: A major goal of the National Registry of Veterans with ALS is to identify US military veterans with ALS and ascertain their clinical status. We identify potential ALS cases from several sources using both active and passive strategies, including surveying VA databases, patient self‐referrals to a web site, and toll‐free phone number. In order to limit the false positive rate of ALS diagnosis among Registry candidates, we devised a telephone screening method to eliminate subjects with erroneous diagnoses prior to medical record validation. We now report the effectiveness of this approach.
Objectives: The aim of this study was to ascertain the rate of false positive diagnosis of ALS using the telephone screening method compared to an established diagnosis by medical record review by a neurologist with expertise in ALS diagnosis.
Methods: A total of 3161 potential ALS registrants were identified and screened from all sources. Each was contacted by a trained interviewer. After telephone informed consent, each candidate was screened using two primary questions: 1) Did any health professional diagnose you with ALS or other MND, and 2) Have you experienced progressive muscle weakness? Other demographic variables were also obtained. Of those screened, 1356 (42.9%) were excluded. The primary reason for exclusion was not having a diagnosis of ALS or progressive muscle weakness (74.0%). Other participants were excluded for other reasons, refused participation, died prior to record review, or did not yet have enough medical evidence to determine a diagnosis. One thousand, five hundred and twelve participants proceeded to medical record review. Levels of diagnostic certainty were established using the El Escorial criteria.
Results: The population was predominantly male (97.8%) and Caucasian (93.0%) with a mean age of 63.8±11.2 years. Considering participants identified from all sources, 90.6% who passed the telephone screener proved to have ALS/MND on medical record review and 9.4% did not have ALS/MND. Seven hunred and eighty‐four subjects were self‐referred and of these, 96.4% had ALS/MND on record review and 3.6% did not, whereas of the 728 potential registrants obtained from databases, 15.6% did not have ALS/MND upon record review.
Conclusions: The results of this study indicate that a telephone‐based screening method using questions targeting the presence of progressive muscle weakness and a suggested diagnosis of ALS/MND by a health profession is associated with a low false positive rate of diagnosis using medical record review by experts as the ‘gold standard’. This was especially true when the source was self‐referral, implying that such candidates received an accurate diagnosis, and confirmed by the history of progressive weakness over the phone. Not unexpectedly, computerized data sources carried a higher false positive rate but we did not establish the source of error in this study. Our results suggest a strategy with a low false positive rate of ALS that can be used to screen large numbers of potential study candidates for population‐based research in a cost‐efficient manner.
Acknowledgements: This study was supported by the Department of Veterans Affairs VA CSP 500. Thanks are expessed to Lisa DiMartino, Karen Juntilla, Beverly McCraw, Barbara Norman, Priscilla Webster‐Williams, Laurie Marbrey, and Honore Rowe.
P159 DECREASING INCIDENCE AND RISING PREVALENCE OF ALS IN IRELAND OVER A SEVEN‐YEAR PERIOD
O'Toole O1, Traynor BJ2, Sheehan C1, Corr B1, Hardiman O1
1Beaumont Hospital, Dublin, Ireland, 2National Institutes of Health, Bethesda, MD, USA
E‐mail address for correspondence: [email protected]
Introduction: There has been a longstanding debate concerning the temporal trend of ALS incidence. However, there has been a lack of reliable, population‐based data concerning changes in ALS epidemiology over the last decade since disease‐modifying agents were introduced and access to symptomatic therapies that influence patient prognosis became more available.
Aim: The authors sought to determine the incidence and prevalence of ALS in Ireland from the three‐year period 2002 to 2004 and to compare it to the previously published findings from the three‐year period 1995 to 1997.
Design: Population‐based register of all individuals diagnosed with ALS in Ireland using multiple sources of information to ensure complete case ascertainment. To compensate for the recent shift in the demographics of the Irish population, the 2002– 2004 figures were standardized to the 1996 Irish census.
Results: Two hundred and thirty‐five residents of Ireland were diagnosed with ALS during the three‐year study period between 1 January 2002 and 31 December 2004. The average annual incidence of ALS for the time period 2002–2004 was 2.50 per 100,000 person‐years over the age of 15 years (95% CI 1.91–3.09). This represents a 10.1% decrease compared to the incidence of ALS for the 1995–1997 time period (2.78 per 100,000 person‐years over the age of 15; 95% CI 2.42–3.44), but this difference did not reach statistical significance. The drop in incidence was distributed equally among men and women: the average annual incidence among men for 2002–2004 was 10.7% lower than in 1995–1997 (2.91 per 100,000 (95% CI 2.00–3.82) compared to 3.26 (95% CI 2.71–3.81)). The average annual incidence among women was 10.3% lower (2.08 per 100,000 (95% CI 1.33–2.84) compared to 2.32 (95% CI 1.88–2.83)). In contrast to incidence, the overall prevalence of ALS in Ireland grew by 2% between 1995 and 1997 and 2002–2004: on 31 December 2003, ALS prevalence was 6.34 per 100,000 population over the age of 15 years (95% CI 5.40–7.28), whereas on 31 December 1996 prevalence was 6.20 per 100,000 (95% CI 5.3–7.1). The majority of this increase in prevalent cases was accounted for by women, in whom ALS prevalence increased by over 10% (4.99 per 100,000 in 2003 compared to 4.50 per 100,000 in 1996). In contrast, male prevalence diminished by 2.4% (7.71 per 100,000 in 2003 (95% CI 6.24–9.19) compared to 7.9 (95% CI 6.4 –9.4) in 1996.
Conclusions: The incidence rate of ALS in Ireland has decreased over the seven‐year period from 1995–1997 to 2002 –2004, whereas the prevalence rate has increased over the same time period. This temporal pattern may reflect changing practices in the diagnosis of ALS (e.g. exclusion of ‘suspected’ ALS cases). Alternatively, the advent of riluzole and the adoption of a more aggressive approach to symptomatic management may be improving survival among Irish ALS patients and may explain the rising prevalence of this fatal neurodegenerative disease in Ireland.
P160 MORTALITY AND SURVIVAL OF AMYOTROPHIC LATERAL SCLEROSIS PATIENTS IN BELGRADE, SERBIA
Stevic Z, Pekmezovic T, Kostic S, Lavrnic D, Rakocevic V, Basta I, Apostolski S
Institute of Neurology, Belgrade, Serbia
E‐mail address for correspondence: [email protected]
Background: Over the last decade increased mortality of ALS has been reported especially in European countries (1). In our first epidemiological study of ALS in Belgrade (Yugoslavia) the adjusted mortality rate was low: 0.29 per 100,000 population in a seven‐year period (1985–1991) (2). The cumulative probability of five‐year survival was 27%.
Objectives: To determine the mortality of ALS in the population of Belgrade (Serbia) during the period 1993–2005 and to estimate five‐year survival rates.
Methods: ALS cases were collected by analysing hospital inpatient and outpatient registers at the Institute of Neurology which is the national referral neurological centre and in departments of neurology in an additional three clinical centres in Belgrade. The El Escorial diagnostic criteria for ALS were applied to all cases enrolled in the register. Each patient was regularly followed up during the disease.
The mortality rates were calculated by standard procedures. The probability of survival was calculated by the Kaplan‐Meier method.
Results: During the period l993 to 2005, 218 patients were diagnosed as new cases of ALS in the District of Belgrade. Of these, nine (3.7%) had familial ALS based on detailed family history. Altogether, 175 patients (102 males and 73 females) died before 31 December 2005. The youngest patient (male) was 24 years old and the oldest patient (male) was 85 years old. Twenty‐six (11.9%) patients were under the age of 45 years. The mean age of onset was 58.5±11.4 (range 24–85) years.
The overall mortality rate was 0.8/100,000: 1.0/100,000 for males, and 0.7/100,000 for females. During the observed period the mortality rate of ALS in Belgrade showed an increasing trend (y = 0.503+0.050x, p = 0.103).
The mean survival time from the onset of disease was 3.4±2.3 years and cumulative probability of five‐year survival was 22.8±3.1%. Patients under 58 years at onset (p = 0.001) and those with the spinal onset of disease survived significantly longer (p = 0.014).
Conclusions: In comparison to our previous data the results suggest a significant increase of mortality in Belgrade (Serbia). The most important contributing factors are better diagnosis due to El Escorial criteria, more thorough registration of ALS and increased life expectancy. However, a real increase in the mortality of ALS, possibly related to environmental factors, cannot be excluded.
References
- Worms P. The epidemiology of motor neuron diseases: a review of recent studies. Journal of Neurol Sci. 2001;191:3–9.
- Alcaz S, Jerebinski M, Pekmezovic T, Stevic Z, Pavlovic S, Apostolski S. Epidemiological and clinical characteristics of ALS in Belgrade, Yugoslavia. Acta Neurol Scand. 1996;94:264–8.