Graphics & Design

11 pages
27 views

Large-scale pathways-based association study in amyotrophic lateral sclerosis

of 11
All materials on our website are shared by users. If you have any questions about copyright issues, please report us to resolve them. We are always happy to assist you.
Share
Description
Large-scale pathways-based association study in amyotrophic lateral sclerosis
Transcript
  See discussions, stats, and author profiles for this publication at: https://www.researchgate.net/publication/6388621 Large-scale pathways-based association study inamyotrophic lateral sclerosis  Article   in  Brain · October 2007 DOI: 10.1093/brain/awm055 · Source: PubMed CITATIONS 32 READS 34 25 authors , including: Some of the authors of this publication are also working on these related projects: Role of RNA binding proteins in neurodegenerative diseases   View projectretrograde axonal transport of signalling endosomes and their somatic sorting   View projectAmmar Al-ChalabiKing's College London 338   PUBLICATIONS   13,308   CITATIONS   SEE PROFILE Pamela J ShawThe University of Sheffield 491   PUBLICATIONS   16,494   CITATIONS   SEE PROFILE Giampietro SchiavoUniversity College London 296   PUBLICATIONS   16,007   CITATIONS   SEE PROFILE All content following this page was uploaded by Emily Goodall on 12 January 2017. The user has requested enhancement of the downloaded file. All in-text references underlined in blue are added to the srcinal documentand are linked to publications on ResearchGate, letting you access and read them immediately.  Large-scale pathways-based association study inamyotrophic lateral sclerosis Dalia Kasperavic›i  ute _ , 1 Mike E.Weale, 2 KevinV. Shianna, 3 GarethT. Banks, 1 Claire L. Simpson, 4 ValerieK. Hansen, 4 Martin R.Turner, 4 Christopher E. Shaw, 4 Ammar Al-Chalabi, 4 Hardev S. Pall, 5,6 EmilyF.Goodall, 5 Karen E. Morrison, 5,6 Richard W.Orrell, 7 Marcus Beck, 8 Sibylle Jablonka, 9 Michael Sendtner, 9 Alice Brockington, 10 Paul G. Ince, 10  Judith Hartley, 10 Hannah Nixon, 10 Pamela J. Shaw, 10 Giampietro Schiavo, 11 Nicholas W.Wood, 12 David B.Goldstein 2 and Elizabeth M.C.Fisher 1 1 Department of Neurodegenerative Disease, Institute of Neurology,University College London, London,UK, 2 IGSP Center for Population Genomics and Pharmacogenetics, Duke University,  3 IGSP Center for Applied Genomics andTechnology, Duke University, Durham, NC,USA,  4 MRC Centre for Neurodegeneration Research, King’s College London,Institute of Psychiatry, Department of Neurology, London,  5 Department of Clinical Neurosciences, Division of Neuroscience,The Medical School,University of Birmingham,  6 Neuroscience Centre,Queen Elizabeth Hospital,University HospitalsBirmingham NHS FoundationTrust, Birmingham,  7 Department of Clinical Neurosciences, Royal Free and University CollegeMedical School,University College London, London,UK,  8 Department of Neurology,University of Wuerzburg,  9 Institute of Clinical Neurobiology,Universityof Wuerzburg,Wuerzburg,Germany, 10 Academic Neurology Unit, Section of Neuroscience,University of Sheffield Medical School, Sheffield, 11 Molecular NeuroPathobiology Laboratory,Cancer Research UK LondonResearch Institute, and 12 Department of Molecular Neuroscience, Institute of Neurology, London,UKCorrespondence to: Dr D. Kasperavic›i  ute _ , Department of Neurodegenerative Disease, Institute of Neurology,University College London,Queen Square, London WC1N 3BG,UKE-mail: d.kasperaviciute@prion.ucl.ac.uk Sporadic amyotrophic lateral sclerosis (ALS), a devastating neurodegenerative disease, most likely resultsfrom complex genetic and environmental interactions. Although a number of association studies have beenperformed in an effort to find genetic components of sporadic ALS, most of them resulted in inconsistentfindings due to a small number of genes investigated in relatively small sample sizes, while the replication of results was rarely attempted. Defects in retrograde axonal transport, vesicle trafficking and xenobiotic meta-bolism have been implicated in neurodegeneration and motor neuron death both in human disease and animalmodels.To assess the role of common genetic variation in these pathways in susceptibility to sporadic ALS, weperformed a pathway-based candidate gene case-control association study with replication. Furthermore,we determined reliability of whole genome amplified DNA in a large-scale association study. In the first stageof the study, 1277 putative functional and tagging SNPs in 134 genes spanning 8.7Mb were genotyped in 822British sporadic ALS patients and 872 controls using whole genome amplified DNA. To detect variants withmodest effect size and discriminate among false positive findings19 SNPs showing a trend of association in theinitial screen were genotyped in a replication sample of 580 German sporadic ALS patients and 361 controls.We did not detect strong evidence of association with any of the genes investigated in the discovery sample(lowest uncorrected  P -value 0.00037, lowest permutation corrected  P -value 0.353). None of the suggestive asso-ciations was replicated in a second sample, further excluding variants with moderate effect size.We concludethat common variation in the investigated pathways is unlikely to have a major effect on susceptibility to spora-dic ALS.The genotyping efficiency was only slightly decreased ( » 1%) and genotyping quality was not affectedusing whole genome amplified DNA. It is reliable for large scale genotyping studies of diseases such as ALS,where DNA sample collections are limited because of low disease prevalence and short survival time.Keywords:  amyotrophic lateral sclerosis; genetic association; axonal transport; whole genome amplification Abbreviations:  ALS ¼ Amyotrophic lateral sclerosis; SMN ¼ Survival motor neuron Received December11, 2006. Revised January 25, 2007. Accepted February 26, 2007. Advance Access publication April17, 2007 doi:10.1093/brain/awm055  Brain  (2007), 130 , 2292^2301  2007 The Author(s)This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) whichpermits unrestricted non-commercialuse, distribution, andreproductionin anymedium, provided the srcinal work is properlycited.   a  t  B e i   j  i  n g N or m a l   Uni   v e r  s i   t   y on J   un e 1  ,2  0 1  3 h  t   t   p :  /   /   b r  a i  n . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   Amyotrophic lateral sclerosis (ALS) is a devastating neuro-degenerative disease characterized by progressive muscleweakness and wasting with combined upper and lowermotor neuron loss. It is the most common motor neurondisease in the developed world with a lifetime risk of 1 : 600 to1 : 1000 (Pasinelli and Brown, 2006). About 10% of ALS casesare familial mostly with autosomal dominant inheritance(Pasinelli and Brown, 2006); the remaining cases, oftenreferred to as sporadic, most likely result from complex genetic and environmental interactions. Mutations in thesuperoxide dismutase 1 ( SOD1 ) gene are identified in up to20% of familial (Rosen, 1993) and in 3–7% of sporadic ALSpatients (Jones  et al  ., 1994, 1995; Jackson  et al  ., 1997) and arethe most frequent known cause of ALS. A few other genes, forexample senataxin ( SETX  ) (Chance  et al  ., 1998; Chen  et al  .,2004), VAMP (vesicle-associated membrane protein)-associated protein B ( VAPB ) (Nishimura  et al  ., 2004), andalsin (  ALS2 ) (Yang  et al  ., 2001; Hadano  et al  ., 2001), havebeen shown to be mutated in rare forms of familial ALS andother forms of motor neuron degeneration (Pasinelli andBrown, 2006; James and Talbot, 2006); however, the aetiology of more than 98% of ALS cases remains unclear. Giventhat most of our understanding of disease pathogenesis at amolecular level comes from research on SOD1-relatedALS, which comprises only    2% of all cases, it is crucial toidentify other disease risk factors, both genetic andenvironmental.To date, no genetic risk factors have been unequivocally shown to be associated with sporadic ALS, the bestreplicated findings so far being regulatory polymorphismsin vascular endothelial growth factor ( VEGF)  (Lambrechts et al  ., 2003), copy number variation at the Survival MotorNeuron ( SMN  ) locus (Corcia  et al  ., 2002, 2006; Veldink  et al  ., 2005) and differences in tail lengths in the heavy chain neurofilament gene (  NEFH  ) (Al-Chalabi  et al  ., 1999).One reason for this lack of associations is highlighted by Simpson and Al-Chalabi (2006) in that most of theprevious genetic studies of sporadic ALS have been limitedto assessing a small number of genes within patient andcontrol groups of relatively small sample sizes—generally afew hundred patients and controls—resulting in incon-sistent findings. For the same reason, replications of association are only very rarely reported or attempted.Until recently, the difficulties in carrying out such studieshave been the lack of availability of human genomevariation data and the available platforms for highthroughput analysis of genetic variation. Within the last2 years, new data on common human genetic variationhave been published by the International HapMap project(Altshuler  et al  ., 2005) and new and more reliable androbust genotyping platforms have become available, both of which greatly enhance our ability to carry out large-scaleassociation studies. The HapMap project has facilitated highthroughput analysis of human variation by providing thecorrelational structure of single nucleotide polymorphisms(SNPs) which enables us to select small set of taggingSNPs for genotyping to capture the most common variantsin large portions of the human genome (de Bakker et al  ., 2005).Given these new developments, we set out to undertakean integrated genetic association study in ALS, for the firsttime focusing on specific pathways/protein complexesof interest, as determined by published data implicatingmembers of the pathway in ALS specifically or in otherrelated forms of neurodegeneration. The three pathways/protein complexes we studied were those involved in(i) axonal transport, specifically retrograde axonal trans-port, (ii) vesicle trafficking and (iii) xenobiotic metabolism.Axonal retrograde transport: This form of axonaltransport is driven by cytoplasmic dynein, a multi-subunitmotor complex moving towards the minus end of microtubules. Cytoplasmic dynein interacts with dynactin,which acts as a cargo adaptor and affects motor processivity (Pfister  et al  ., 2006; Duncan and Goldstein, 2006). Defectsin axonal transport have been implicated in motor neurondegeneration both in human disease and in mouse models.Transgenic mice overexpressing human mutant  SOD1  thatmodel ALS have been shown to have slower retrogradeaxonal transport (Murakami  et al  ., 2001; Kieran  et al  .,2005) and mutant SOD1 is known to disrupt dyneinlocalization (Murakami  et al  ., 2001; Ligon  et al  ., 2005).Mutations in the cytoplasmic dynein 1 heavy chain 1( Dync1h1 ) gene in mice result in slower retrograde axonaltransport and death of motor neurons (Hafezparast  et al  .,2003), while interactions of the same mutant  Dync1h1 alleles with mutant SOD1 increase lifespan of ALS mice,and these double mutant mice have been shown to haveincreased rates of retrograde axonal transport (Kieran  et al  .,2005). A mutation in the dynactin p150 subunit wasidentified in a family with a slowly progressive lower motorneuron syndrome (Puls  et al  ., 2003, 2005) and otherpossible mutations have been identified in ALS patients(Munch  et al  ., 2004), while overexpression of dynamitin,another subunit of dynactin, in mice causes disruptionof the dynein–dynactin complex and late onset motorneuron disease (LaMonte  et al  ., 2002). Thus disruption of retrograde axonal transport is implicated in specificdegeneration of motor neurons, and in some kind of interaction with mutant SOD1.Vesicle trafficking: Defects in vesicular trafficking areknown to lead to death of motor neurons. The  wobbler  mouse model for example has a mutation in the vesicularsorting protein  Vps54  (Schmitt-John  et al  ., 2005), whilea mutation in  CHMP2B , a component of the endosomalsecretory complex required for transport (ESCRTIII), may result in frontotemporal dementia in a human pedigree(Skibinski  et al  ., 2005), a disease with considerable overlapwith ALS; two possible mutations in  CHMP2B  have beenidentified in individuals with ALS (Parkinson  et al  ., 2006).Xenobiotic metabolism: Epidemiological studies haveshown association of ALS with exposure to environmentaltoxins, pesticides and heavy-metals (reviewed in Nelson and ALS association study  Brain  (2007), 130 , 2292^2301 2293   a  t  B e i   j  i  n g N or m a l   Uni   v e r  s i   t   y on J   un e 1  ,2  0 1  3 h  t   t   p :  /   /   b r  a i  n . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   McGuire, 2006), although the results of many publishedstudies are inconclusive. The exposure to such agents ismodulated by the xenobiotic metabolizing enzymes of anindividual, and enzyme activity is in part determined by genetic variation in the genes encoding these enzymes.Therefore we hypothesize that susceptibility to ALS couldbe influenced by genetic variation in genes in the xenobioticmetabolism pathways.In summary, here we elucidate the role of commongenetic variation in retrograde axonal transport, vesicletrafficking and major xenobiotic metabolism genes insusceptibility to sporadic ALS in the most powerfulassociation study of ALS so far. We are specifically studyingindividual pathways in this investigation. We comprehen-sively screen variation in 134 genes in the largest collectionof sporadic ALS patients genotyped in a single study to dateand validate the results of association analysis by genotyp-ing SNPs showing a trend of association in a replicationsample of ALS patients from Germany.In addition, by performing the initial screen entirely onwhole genome amplified DNA we validate its use on a largescale. The reduction in the amount of DNA required forgenotyping may significantly ameliorate the use of existingold DNA collections and enhance collaborations studyinglate onset neurodegenerative diseases where sample collec-tions are limited because of low disease prevalence andshort survival time. The use of whole genome amplifiedDNA until now has been limited to supplementing thestudies performed on genomic DNA with small numbers of samples for which not enough DNA was available, and tothe best of our knowledge this is the largest genome screenperformed on whole genome amplified DNA. Subjects and methods Study plan and subjects We used a two-stage study design with existing collections of sporadic ALS cases and controls from two populations. In thefirst, or discovery sample, we genotyped 822 British sporadic ALSpatients and 872 control DNA samples collected at out-patientclinics at the Motor Neurone Disease Care and Research Centre,Queen Elizabeth Hospital, Birmingham, UK (167 definite orprobable ALS patients according to El Escorial World Federationcriteria with unknown  SOD1  mutation status and 145 controls),King’s Motor Nerve Clinic, London, UK (258 definite or probableALS patients according to El Escorial criteria with no  SOD1 mutations and 245 controls), the Newcastle and Sheffield MNDCentres, UK (84 ALS patients neuropathologically confirmed atautopsy, 280 definite or probable ALS patients according toEl Escorial criteria, 33 patients with clinical variants of ALSincluding primary lateral sclerosis and progressive muscularatrophy with unknown  SOD1  mutation status and 313 controls)and National Blood Transfusion Service, UK (169 controls).The second, or replication sample, consisted of 580 Germansporadic definite or probable ALS patients according to El Escorialcriteria with unknown  SOD1  mutation status, collected at theMotor Neuron Research Clinic, Wuerzburg, Germany and 361controls, collected at the Department of Transfusion Medicine andImmunohematology, University of Wuerzburg, Germany. None of the patients had a known family history of ALS. The samplecharacteristics are given in Table 1. DNA samples were extractedfrom blood using standard methods. Participants of the study signed informed consent and the study was approved by theNational Hospital for Neurology and Neurosurgery and Instituteof Neurology Joint Research Ethics Committee.A set of SNPs covering most of the common variants incandidate gene regions was genotyped in the discovery sample andstatistical analysis performed to look for evidence for associationwith susceptibility to ALS. To detect modest effect size variantsand to discriminate among false positive findings, a subset of SNPs that showed a trend for association in the discovery samplewas genotyped in the replication sample. Candidate gene and SNP selection The candidate genes were selected following a detailed review of the literature on ALS-linked pathways and our experimental data(DK, GS and EMCF, unpublished data). We included genesencoding all known subunits of the dynein–dynactin complex,genes regulating its activity, binding to cargoes, microtubules andother interacting proteins; all known subunits of ESCRTcomplexes known to be involved in vesicle trafficking; key enzymes involved in pesticide metabolism and their targets. Thecomplete gene list is shown in Supplementary Table 1. The totalsize of genome sequence investigated was 8.7Mb.SNPs were selected in candidate gene regions based on positionsof RefSeq genes in the UCSC genome browser server(http://genome.ucsc.edu) hg16 assembly, adding 10000bp to themost 5 0 - and 3 0 -extent of the gene. As direct typing of causalvariants is more powerful in association studies, we enriched theset of markers selected to genotype with the SNPs with predictedfunctionality. First, selected regions were screened for SNPs withpredicted functionality using TAMAL software (Hemminger  et al  .,2006). The SNPs were prioritized for genotyping if they hadfrequency data in any major databases (dbSNP, HapMap, Perlegenand Affymetrix) and met one of the following criteria: were incoding regions, altered intronic splice sites, were in predictedpromoters, in regions with predicted regulatory potential, inpredicted transcription factor binding sites, in regions withconservation scores  5 99th percentile genome-wide for human-chimp-rat-mouse-chicken alignment or in miRNAs and their3 0 UTR targets. Functional SNPs for cytochrome P450 geneswere selected from the CYP450 database (http://www.imm.ki.se/CYPalleles/). Secondly, tagging SNPs were selected based onHapMap PhaseII data (release 19) using Tagger software Table1  Characteristics of genotyped ALS patient andcontrol samples British GermanALS patients 822 579Sex (males/females) 500/322 348/231Type of onset(limb/bulbar/mixed or undetermined)513/234/75 444/132/3Mean age of onset (range) 59 (20^87) 58 (16^85)Controls 872 361Sex (males/females/unknown) 430/442/0 190/136/35Mean age at sample collection (range) 52 (17^81) 32 (18^65) 2294  Brain  (2007), 130 , 2292^2301 D. Kasperavic›i  u  t e _ et al .   a  t  B e i   j  i  n g N or m a l   Uni   v e r  s i   t   y on J   un e 1  ,2  0 1  3 h  t   t   p :  /   /   b r  a i  n . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om   (de Bakker  et al  ., 2005). Since the majority of ALS patients were of ‘white’ ethnicity, only data on the HapMap CEU population wereused in selection of tagging SNPs. Where possible, putativefunctional SNPs were used as tagging SNPs, and SNPs with higherpredicted genotyping scores from Illumina were prioritized inselection—only SNPs with Illumina genotype scores  4 0.6 wereincluded in the assay. In total, 1279 tagging SNPs were selected tocapture variation of polymorphisms with minor allele frequencies 4 5% in the HapMap CEU population with mean maximumpairwise  r  2 between tagging SNP and ungenotyped SNP of 0.90.Eighty-three percent of all alleles with minor allele frequency of  4 0.05 were captured with  r  2 4 0.8 and 95.3% with  r  2 4 0.5. 157SNPs not genotyped in the HapMap project were includedbecause of predicted functionality. One hundred neutral SNPswere selected to control for population stratification. They wereselected to have minor allele frequencies of   4 5% in the HapMapCEU population and to be in more than 50kb distance from any RefSeq gene, known gene or RNA gene in UCSC genome hg17assembly and not to be in the regions with predicted functionality (as earlier). Genotyping and quality control in the discoverysample British ALS patient and control samples were genotyped by 1536-plex GoldenGate assay on an Illumina BeadArray station usingwhole genome amplified DNA. Whole genome amplification wasperformed using a Qiagen Repli-g Midi kit according tomanufacturer’s instructions using 100ng of input genomic DNAper reaction. The yields of amplified DNA were quantified using aPicogreen assay (Molecular Probes, Inc) and concentrationsadjusted to 100ng/ m l. The sex of the samples was verified usingthe amelogenin locus as a marker. We found 2% sex mismatchesand by typing additional sex-linked markers all of them wereconfirmed to be errors in labelling the original genomic DNAsamples, therefore these samples were excluded from the study.Three percent of whole genome amplified samples failed in PCRsduring sex testing and were removed from further study.Genotyping quality control was ensured by (i) mixing case andcontrol samples on the same plates and genotyping blind to theaffection status; (ii) each plate contained five duplicate samples,including one genomic DNA duplicate; (iii) three whole genomeamplified DNA samples from the Centre d’Etude duPolymorphisme Humaine (CEPH) were genotyped and resultscompared with HapMap data; (iv) SNPs that failed in more than1% of the samples were removed to avoid non-random missingdata; and (v) SNPs showing departures from Hardy–Weinbergequilibrium in controls were re-evaluated after analysis to check for possible genotyping errors and differences in genotypingperformance between patient and control samples, such asseparate clustering of patient and control samples and differencesin intensity values. Statistical analysis The association was assessed by comparing genotype and allelefrequencies between affected and unaffected individuals. For thegenotypic test, a contingency table was made up for each SNPconsisting of the three genotype categories on one axis and thephenotype categories on the other axis. The table was thenanalysed using an extension of Fisher’s Exact Test to R   C tables,using a network algorithm developed by  Mehta and Patel (1986)and implemented using R software. To assess family-wisesignificance of   P- values, permutation analysis was performedusing routines written in R. False discovery rate analyses wereperformed using the Benjamini and Hochberg linear step-upprocedure (Benjamini and Hochberg, 1995). We assumed theproportion of true null hypotheses was close to 1, and that testswere positive regression dependent (Benjamini and Yekutieli,2001). The effect of hidden population substructure on associationstatistics was assessed using the GCF Genomic Control method of Devlin  et al  . (2004). Tests of sets of   P  -values against their uniformexpectation under the null, assuming independence of tests, wereperformed using Fisher’s method for combining  P  -values. Replication analysis Permutations were performed to assess the significance of theassociation results in the discovery sample and prioritize SNPs forreplication. In addition, all SNPs with uncorrected  P- values below 0.01 in the genotypic test in the discovery sample were genotypedin the replication sample, excluding SNPs in high linkagedisequilibrium with each other and SNPs from the genomiccontrol set. We also genotyped SNPs with  P- values between 0.01and 0.05 if they had a strong prediction of functionality accordingto the following criteria: were in coding regions, within 6bp fromexon–intron boundaries in fastDB database (de la Grange  et al  .,2005), in 3’UTRs or experimentally proven promoters.Blinded genotyping was performed using Taqman on a 7900HTSequence Detection System (Applied Biosystems, Foster City, CA)using genomic DNA. As a quality control measure duplicates(100% agreement) and water blanks were used. Associationstatistics were calculated as earlier. ResultsGenotyping of whole genome amplified DNA In total 1831 samples (95.4%) were genotyped successfully on the Illumina BeadArray system, including 75 wholegenome amplified DNA blind duplicates and 17 genomicDNA—whole genome amplified DNA blind duplicates.1372 SNPs (89%) passed quality control criteria forgenotyping. We did not observe any differences betweengenomic DNA and whole genome amplified sampleduplicates in genotyping performance.In total 2510789 usable genotypes were producedincluding 126141 blind duplicates, of which 23311 weregenomic DNA—whole genome amplified DNA duplicates.Two mismatched duplicated genotypes were detected, bothof them in the same whole genome amplified duplicatesample, indicating overall error rate 1.59  10  5 . This alsosuggests that errors are sample specific, but not likely related to locus amplification bias during whole genomeamplification, as these duplicate samples were the productsof the same reaction. All genomic DNA—whole genomeamplified DNA duplicate genotypes matched exactly.Genotypes of three whole genome amplified CEPH sampleswere compared to genotypes from the HapMap project dataand one mismatch in 3706 compared genotypes was found,which indicates an error rate of 0.00027. ALS association study  Brain  (2007), 130 , 2292^2301 2295   a  t  B e i   j  i  n g N or m a l   Uni   v e r  s i   t   y on J   un e 1  ,2  0 1  3 h  t   t   p :  /   /   b r  a i  n . oxf   or  d  j   o ur n a l   s  . or  g /  D o wnl   o a  d  e  d f  r  om 
We Need Your Support
Thank you for visiting our website and your interest in our free products and services. We are nonprofit website to share and download documents. To the running of this website, we need your help to support us.

Thanks to everyone for your continued support.

No, Thanks
SAVE OUR EARTH

We need your sign to support Project to invent "SMART AND CONTROLLABLE REFLECTIVE BALLOONS" to cover the Sun and Save Our Earth.

More details...

Sign Now!

We are very appreciated for your Prompt Action!

x