Q8IYL2 is a candidate gene for the familial epilepsy syndrome of Partial Epilepsy with Pericentral Spikes (PEPS)

ARTICLE IN PRESS+ModelEPIRES-4531 No of Pages 7

Epilepsy Research (2011) xxx xxxmdashxxx

jou rna l h omepa g e wwwelsev ier com locate ep i lepsyres

Q8IYL2 is a candidate gene for the familial epilepsysyndrome of Partial Epilepsy with Pericentral Spikes(PEPS)

Guy D Leschzinerablowast Alison J Coffeyb Toby Andrewb Sheila P GregoriogEmmanuel Dias-Netoc Maria Calafatob David R Bentleyb Lucy KintondJosemir W Sanderef Michael R Johnsona

a Division of Neuroscience Imperial College London UKb Wellcome Trust Sanger Institute Cambridge UKc Faculty of Medicine University of Sao Paulo and Hospital AC Camargo Sao Paulo Brazild Southampton University Hospital NHS Trust UKe UCL Institute of Neurology Queen Square London UKf SEIN mdash Epilepsy Institutes in the Netherlands Foundation Heemstede The Netherlandsg Agilent Technologies Santa Clara California USA

Received 9 October 2010 received in revised form 3 May 2011 accepted 11 May 2011

KEYWORDSMendelian epilepsyQ8IYL2Partial Epilepsy withPericentral SpikesPEPS

SummaryPurpose Partial Epilepsy with Pericentral Spikes (PEPS) is a novel Mendelian idiopathic epilepsywith evidence of linkage to Chromosome 4p15 Our aim was to identify the causative mutationin this epilepsy syndromeMethods We re-annotated all 42 genes in the linked chromosomal region and sequenced allgenes within the linked interval All exons intronmdashexon boundaries and untranslated regionswere sequenced in the original pedigree and novel changes segregating correctly were sub-jected to bioinformatic analysis Quantitative polymerase chain reaction was performed toexamine for potential copy number variation (CNV)Results 29 previously undescribed variants correctly segregating with the linked haplotype wereidentified Bioinformatic analysis demonstrated that six variants were non-synonymous codingsequence polymorphisms one of which in Q8IYL2 (Gly400Ala) was found in neither Caucasian(n = 243) and ancestry-matched Brazilian (n = 180) control samples nor subjects from the 1000

Genome Project No gene duplications or deletions were identified in the linked region

Please cite this article in press as Leschziner GD et al Q8IYof Partial Epilepsy with Pericentral Spikes (PEPS) Epilepsy Res

Discussion We postulate that Q8and bioinformatic analysis Thetissuecopy 2011 Elsevier BV All rights re

lowast Corresponding author at Room 10E06 Charing Cross Hospital ImperE-mail addresses guyleschgmailcom (GD Leschziner) mjohnson

0920-1211$ mdash see front matter copy 2011 Elsevier BV All rights reserveddoi101016jeplepsyres201105010

L2 is a candidate gene for the familial epilepsy syndrome (2011) doi101016jeplepsyres201105010

IYL2 is a causative gene for PEPS after exhaustive resequencing function of this gene is unknown but it is expressed in brain

served

ial College London London W6 8RF UK Tel +44 0 208 846 1194imperialacuk (MR Johnson)


2 GD Leschziner et al

Figure 1 Limited PEPS pedigree adapted from Kinton et al (2002) Five digit numbers are DNA codes illustrating the fifteen sub-jects utilised for this study in red for homozygote affecteds blue for heterozygote affecteds and black for homozygote unaffected( nd t

I

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pciebeaeltwa

cdafdhppotlata

tasre

M

G

T3g(

ws(sMaia

S

FpmdashhltTmatched with the pedigree both for ancestral history (Portuguese

For interpretation of the references to color in this figure lege

ntroduction

pilepsy is a heterogeneous disorder with a variety of aeti-logies ranging from the purely genetic to environmentallycquired A large proportion of cases may have a geneticasis (Annegers et al 1996) of whom a small proportionisplay monogenic inheritance

To date the majority of genes accounting for idio-athic Mendelian epilepsies are ligand- or voltage-gated ionhannels (Helbig et al 2008) although recent discover-es of non-ion channel genes causing Mendelian idiopathicpilepsy have extended our knowledge of epilepsy geneseyond solely ion channel subunit gene mutations (Nobilet al 2009 Johnson 2011) The observation that rare vari-nts occurring in genes previously identified as Mendelianpilepsy genes are associated with sporadic epilepsy high-ights the potential for family studies in epilepsy to informhe spectrum of candidate genes for common epilepsy asell as confirm causal associations with detected rare vari-nts (eg Stogmann et al 2006)

A previously undocumented familial epilepsy syndromealled Partial Epilepsy with Pericentral Spikes (PEPS) wasescribed in 2002 (Kinton et al 2002) Affected individu-ls in the large family Brazilian of Portuguese extractionrom Paraiba state in the Northeast region exhibited fouristinct partial seizure types including hemiclonic andemitonic simple partial seizures temporal lobe complexartial seizures and stereotyped episodes of epigastricain Onset was in the first two decades of life with a benignutcome and affected individuals exhibited a characteris-ic EEG of pericentral epileptiform activity Genome-wideinkage analysis demonstrated linkage to region 4p15 with

Please cite this article in press as Leschziner GD et al Q8Iof Partial Epilepsy with Pericentral Spikes (PEPS) Epilepsy Res

maximum LOD score of 33 at marker D4S2311 Haplo-ype analysis defined the limits of the region to be D4S2983nd D4S2926 To identify the causative gene we under-

nNp

he reader is referred to the web version of the article)

ook a study involving several techniques including manualnnotation of the entire region to identify all transcribedequences sequencing of all transcribed sequences andeal-time quantitative polymerase chain reaction (PCR) toxclude copy number variation

ethods

ene annotation

he interval was examined in the Ensembl genome browser Build4 (wwwensemblorg) and candidate genes were identified Uni-ene entries were examined to determine expression patternswwwncbinlmnihgovunigene)

Gene structures were reanalysed and manually annotatedithin an internal Sanger Institute platform Otterlace Genomic

equence was analysed with ab initio gene prediction softwareFgenesH GENSCAN) as well as software predicting transcriptiontart sites (Eponine) CpG islands and repeat regions (Repeat-asker) All known mRNAs proteins and cDNAs from all species wereligned against the genomic sequence and using this as support-ng evidence gene structures were manually annotated includinglternative transcripts

ubjects

ive samples of subjects were used for this project The first sam-le consisted of fifteen members of the original pedigree (Fig 1)

one homozygote unaffected two homozygote affecteds and 12eterozygote affecteds The second was a sample of 47 normal unre-ated Caucasians was derived from the CEPH Utah collection Thehird was a collection of 243 unrelated normal Caucasian subjectshe fourth sample was a collection of normal Brazilian controls

YL2 is a candidate gene for the familial epilepsy syndrome (2011) doi101016jeplepsyres201105010

= 95) and for region of Brazil and ancestral history (Paraiba stateorth-East region and Portuguese n = 85) Informed consent wasrovided by all patients and controls and the DNA analysis and


Q8IYL2 as Candidate Gene for PEPS 3

Figure 2 Diagrammatic representation of the region of Chromosome 4p15 linked to PEPS Known genes are represented in redwhile novel transcripts as characterised in Ensembl are represented in black (For interpretation of the references to color in this

rticl

R

PgdvtbPtSapptpPosaDompb

pndt

G

UtBmo

R

D

figure legend the reader is referred to the web version of the a

storage was approved by the statutory Research Ethics Commit-tees DNA from subjects and controls was extracted using a standardphenolmdashchloroform extraction method and purity and concentra-tion confirmed by spectrophotometry

Sequencing

Primers were designed to sequence all coding and untrans-lated flanking sequence for the annotated genes bycutting the exon sequence from AceDB an internal SangerInstitute database specifically designed for gene annota-tion and pasting it into Primer3 primer design software(httpfrodowimiteducgi-binprimer3primer3cgi) Param-eters were set to produce primers with at least 60 bases eitherside of the intronmdashexon boundary Primer sequences werechecked for unique site alignment using the BLASTN facilityprovided by Ensembl (httpwwwensemblorgMultiblastviewspecies=Homo sapiens) Primers were designed successfully forall genes using this method except DRD5 This gene has twopseudogenes (Nguyen et al 1991) and primers designed usingthe methods above resulted in three 100 alignments on thegenome To circumvent this problem the transcript sequences forthe DRD5 gene and two pseudogenes were multiply aligned usingClustalw web-based software (httpwwwebiacukclustalw)DRD5 is a single exon gene with a transcript of 2031 bp and so fiveoverlapping primer pairs were designed manually by identifyingareas of unique sequence within the DRD5 gene and aiming toplace the 3prime ends of primer sequence over these areas

Primers were ordered from Sigma Genosys and were initiallytested with genomic DNA using the HS60 protocol comprising theThermostart DNA Polymerase protocol (ABgene) with an anneal-ing temperature of 60 C Failing primers were retested at 55and 65 C and subsequently with modified protocols with 7-Deaza-dGTP-enriched dNTPs (Roche 0988537) andor 5 DMSOPCR fragments were purified with Shrimp Alkaline Phosphatase(USB 70092X) and Exonuclease I (USB 70073X) Amplified frag-ments were sequenced in forward and reverse directions usingthe Sanger dideoxyterminator sequence reaction with fluores-cently labelled terminators from ABI on the ABI 3730 or 3700capillary sequencer machines DNA from four subjects in the pedi-gree mdash heterozygous affected 13858 homozygous affected 14844homozygous affected 14924 unaffected 14885 mdash and one unrelatednormal female control were amplified and sequenced Sequencewas viewed and analysed with Mutation Surveyor (SoftGenetics mdash


wwwsoftgeneticscommutationsurveyor) Initially polymorphismswere detected using the automated default settings and werethen examined manually In addition all coding sequence was re-examined by eye

EDoW

e)

eal-time PCR

rimers for exonic regions in the forty-two known and putativeenes in the linked region were designed Forty primer pairs wereesigned for exons or fragments of exons for genes within the inter-al Two primer pairs were utilised for controls an exonic region ofhe beta-actin (ACTB) gene on Chr7 and an exonic region of theeta-2 microglobulin (B2 M) gene on Chr15 Real-time quantitativeCR was performed using the SYBR Green protocol with an annealingemperature of 60 C and the ABI Prism 7000 Sequence Detectionystem on an unrelated female normal control and a homozygoteffected individual from the pedigree 14858 Quantitative PCR waserformed with both control primer pairs and ten test primer pairser plate Each primer pair was amplified six times mdash twice withhe normal template DNA twice with homozygous affected tem-late DNA and twice with water controls At the end of each fullCR process a dissociation protocol was performed to verify purityf product in each well Automated analysis of the real-time PCRelected a DNA concentration in the exponential phase of the PCRnd determined the number of cycles or cycle time (CT) to reach theNA concentration for each well For each primer pair an averagef the CT was calculated for each DNA Mean CT was then nor-alised by subtraction from the mean CT for both the control primerairs Normalised mean CTs for all primer pairs were then comparedetween the normal and homozygous affected DNAs

In total thirty-one primer pairs with an average density of onerimer pair every 313 kb were successfully amplified against bothormal and homozygote affected DNAs with consistent CTs betweenuplicate wells and a well-defined peak with the dissociation pro-ocol

enotyping

K samples were genotyped for polymorphisms identified as poten-ial causative mutations on the Sequenom MALDI-TOF platformrazilian samples were screened for polymorphisms with a Taq-an assay One polymorphism (CPZ-37) was assayed by sequencing

wing to problematic Taqman primer design

esults

etermination of candidate genes


xamination of the interval between markers D4S2983 and4S2926 determined the interval to be 97 Mb long withne 150 kb gap in the defined genomic sequence (Fig 2)ithin this region 42 genes were identified in Ensembl



Table 1 Genes in interval listed from telomeric to centromeric limit Gene names with are those not known to be expressedin the central nervous system

Gene Ensembl gene ID Aliases Description

NP067651 196526 AFAP Actin filament associated proteinQ9BT26 178589 MGC10981 proteinNP981968 178571 Q8N5S0NP115808 163995 ABLIM2 Actin binding LIM protein family member 2SH3TC1 125089 Q8TEM9 SH3 domain and tetratricopeptide repeats containing protein 1HTRA3 170801 Probable serine protease HTRA3 precursorACOX3 87008 Acyl-coenzyme A oxidase 3 peroxisomalQ9H9A4 155275 Q8IYL2 NP689757GPR78 155269 Probable G-protein coupled receptor 78CPZ 109625 Carboxypeptidase ZENST344638 188438DB131 186146 Beta-defensin 131 precursorXP211908 184795 ENST332844ENST340703 186234 ENST347726ENST355821 197004 NPR1DRD5 169676 ENST304374 D5 dopamine receptorSLC2A9 109667 Solute carrier family 2 facilitated glucose transporter member 9ENST264785 71127 WDR1 WD repeat domain 1NP444270 178163 Q9C0D4NP443196 109684 Q9P2U9 Mast cell immunoreceptor signal transducerHS3ST1 2587 Heparan sulphate D-glucosaminyl 3-O-sulphotransferase 1 precursorRAB28 157869 Ras-related protein 28BAPX1 109705 Bagpipe homeobox protein homologue 1Q9H6G0 38219 Q9P2L9FAM44A 145133 NM148894 Family with sequence similarity 44 member ACPEB2 137449 Cytoplasmic polyadenylation element binding protein 2 isoform AC1QTNF7 163145 Complement C1q tumour necrosis factor-related protein 7 precursorQ9H8A7 48342 Q9P2K1FBXL5 118564 F-box and leucine-rich repeat protein 5NP004325 109743 BST1 Bone marrow stromal cell antigen 1 precursorCD38 4468 ADP-ribosyl cyclase 1FGFBP1 137440 HBP17 Fibroblast growth factor binding protein 1NP114156 137441 KSP37PROM1 7062 Prominin 1 precursorQ9NZK9 169762 NM153365NP689897 173244 NM152684LDB2 169744 LIM binding domain 2QDPR 151552 Dihydropteridine reductaseENST342925 188022LAP3 2549 Cytosol aminopeptidaseMED28 118579 EG1 Tumour angiogenesis marker EG-1

Bbgmi5ciwwapa

tnbdwdt

Q9ULE4 47662 KIAA1276

uild 34 (Table 1) All but 11 genes were demonstrated toe expressed in brain (Table 1 mdash highlighted) Among thoseenes with a known function several were identified thatight have a potential role in epilepsy aetiology These

ncluded the ligand-gated ion channel dopamine receptor (DRD5) ABLIM2 and LDB2 which have a role in neuralell differentiation CPZ and LAP3 which may be involvedn neurotransmitter synthesis and degradation and HBP17hich may have a role in neurite outgrowth These genes


ere initially targeted for sequencing but subsequentlyll known genes in the interval were sequenced In totalrimers were designed to successfully amplify and sequencell transcribed and translated regions in the 42 genes with

I

Afi

he exception of two short sequences the first 150 bp ofovel transcript E332844 and a 20 bp region of Q9C0D4etween base positions 5145 and 5165 The former provedifficult to amplify due to a large repeat region extendingell into the first exon preventing suitable primer designespite numerous attempts The latter proved problematico sequence owing to a large polyA repeat


dentification of potential causative variants

ll amplimers totalling 540 were sequenced across theve pedigree subject DNAs and were analysed within



Table 2 Summary of variants not previously described in dbSNP Build 124 Wildtype sequence followed by mutant sequence isprovided in brackets Public database ID refers to re-analysis in dbSNP Build 130 and 1000 Genomes Project (1000G) December2009 release date

Gene Location PositionChange NCBI35 Chr 4base position

Sequence Publicdatabase ID

ABLIM2 5primeUTR C33T 8191767 GCCGC(CT)GCCGC 1000GABLIM2 Intron 23 115 bp upstream of exon 24 8087024 GCACG(CG)TGAGG rs28409515AFAP Intron 4 160 bp downstream of exon 4 7975103 GTTTT(GC)AGAAG rs28701532AFAP 3primeUTR G2273A 7881346 AGCAC(GA)CAAAG Not foundC1QTNF7 Intron 2 17 bp downstream of exon 2 15106016 ACTCT(TA)TTTTT 1000GCPEB2 Intron 3 62 bp downstream of exon 3 14686381 TATGA(GA)TAGGA Not foundCPZ 5primeflanking 37 bp upstream of 5primeUTR 8712421 GGCAG(GA)GGCCT 1000GCPZ 5primeflanking 33 bp upstream of 5primeUTR 8712425 AGGGC(CA)TTTAT rs28535544CPZ Exon 4 Asp207Asp 8723931 TTCGA(CT)GGCAG rs28421391CPZ Intron 9 6 bp downstream of exon 9 8731966 GTGCG(GT)CTTCC rs28417411HTRA3 Intron 5 35 bp upstream of exon 6 8413852 TTCCC(CT)CAACT 1000GQ8IYL2 5primeflanking 9 bp upstream of 5primeUTR of alt transcript 8574205 GTAAC(AC)ACCAT rs73211401Q8IYL2 Exon 2 A253T Met59Leu 8569630 CCATT(AT)TGAAG rs73211375Q8IYL2 Exon 8 A718G Ile214Val 8585228 ACTTC(AG)TTGGA rs34873641Q8IYL2 Exon 9 G1277C Gly400Ala 8588139 GAATG(GC)GGGAG Not foundQ8N5S0 5primeflanking 75 bp upstream of 5primeUTR 8058723 CAGCT(CA)CGGGC 1000GQ8N5S0 Exon 1 T591C Ala142Ala 8059390 GGAGC(TC)TCCAC rs60272249Q8N5S0 Exon 1 G619C Gly152Arg 8059417 CCGCC(GC)GGTCC rs58349148Q8N5S0 3primeUTR G1061A 8059859 GAGCC(GA)GCCCG rs66479458Q9C0D4 3primeUTR T5548G 10118945 CCCCT(TG)TGGAT rs28553891Q9C0D4 3primeUTR 5620mdash5624indel 10118869-74 TTCAC(mdashGTAAA)GTAGAQ9C0D4 3primeUTR T5689C 10118804 AGATA(TC)AGAGA rs28519672Q9C0D4 3primeUTR 5871mdash5872indel 10118621 TCAAC(mdashTG)TTCAAQ9C0D4 3primeUTR 6216mdash6218indel 10118277mdash79 TCAAC(TTAmdash)TTTTTQ9C0D4 3primeUTR C6328G 10118165 GTATA(CG)TCTGA rs28427256Q9H9A4 5primeUTR G1001T 8596441 CAAAG(GT)TAAGA Not foundSH3TC1 Exon 12 G1889A Ala608Thr 8347314 ACCTG(GA)CCAGC 1000GSH3TC1 Exon 14 C3476T Arg1137Trp 8356079 AGGAC(CT)GGGCC rs61740112

aQclvsiofcsSMcwebi

o

SLC2A9 Exon 1 T184C Ser40Ser

Mutation Surveyor Sixty-nine variants (not shown) wereidentified in the sequence that segregated according to link-age data across all available samples Flanking sequencefor each variant was aligned against genomic sequencewithin Ensembl and 40 were found to have been previ-ously described within dbSNP Build 124 Therefore a totalof 29 previously undescribed variants segregating correctlywith the linked haplotype were identified (Table 2) Theseconsisted of 26 single nucleotide substitutions (six intronicSNPs four 5primeflanking sequence SNPs two 5primeUTR SNPs five3primeUTR SNPs three silent coding sequence SNPs and sixnon-synonymous coding SNPs) one 5-base and one 2-baseinsertion in a 3primeUTR and a 3-base deletion in a 3primeUTRNo previously unidentified correctly segregating variant wasidentified in DRD5 the only channel subunit gene in theinterval

The six non-synonymous coding SNPs were prioritisedfor further study Polymorphisms were screened initiallythrough analysis of peptide residue interspecies conserva-tion Only one change the Arg1137Trp change in SH3TC1


was found to be highly conserved between species althoughQ8IYL2 Gly400Ala was completely conserved in rat mouseand human Assessment of interspecies conservation was notpossible for the change in Q8N5S0 as no orthologs were

(aap

9699200 AGGAG(TC)GGGGT Not found

vailable for alignment The remainder mdash Q8IYL2 Met59Leu8IYL2 Ile214Val and SH3TC1 Ala608Thr mdash were less wellonserved These six changes segregated according to hap-otype in the eleven remaining members of the pedigree aserified by sequencing All six changes were subsequentlycreened for in the CEPH panel (n = 47) by sequencing andn the Caucasian controls (n = 243) through genotyping Nonef the 6 variants were identified in the CEPH panel Of thoseour changes successfully genotyped in the CEPH and UKontrol samples Q8IYL2 Met59Leu was found on 5 chromo-omes Q8IYL2 Ile214Val was found on 14 chromosomes andH3TC1 Arg1137Trp was found on one chromosome Q8IYL2et59Leu and Q8IYL2 Ile214Val were excluded as potentialandidates in view of the frequency at which the variantsere seen in normal subjects SH3TC1 Arg1137Trp was notxcluded on the basis that the residue is highly conservedetween species and that its detection on one chromosomen the UK normals might represent reduced penetrance

Thus four variants were screened for in the samplef 180 Brazilian controls matched for ancestral history


Q8IYL2 Gly400Ala SH3TC1 Ala608Thr SH3TC1 Arg1137Trpnd Q8N5S0 G619C) Q8IYL2 Gly400Ala SH3TC1 Ala608Thrnd SH3TC1 Arg1137Trp were successfully genotyped butrimer design was unsuccessful for Q8N5S0 G619C Genotyp-

IN PRESS+ModelE


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243

Cauc

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SH3T

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Gly

400A

la

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d

to

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omor

phic

in

all c

ontr

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sam

ples

(sha

ded)

WT

wild

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M

mut

ant

Inte

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cons

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tion

CEPH

(n

=

47)

UK

cont

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(n

=

243)

Braz

ilian

Cauc

asia

ns

ofPo

rtug

uese

ance

stry

(n

=

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Nor

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Braz

ilian

Cauc

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Succ

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M

Hom

Succ

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Med

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400A

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Med

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0

0

94

94

0

0

85

85

0

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152A

rg

NA

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Fail

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ARTICLEPIRES-4531 No of Pages 7

ng in the Brazilian controls demonstrated six chromosomesarrying the SH3TC1 Arg1137Trp change but neither theH3TC1 Ala608Thr nor Q8IYL2 Gly400Ala variants were iden-ified in any control population (see Table 3 for a summary)

Thus three non-synonymous coding polymorphisms couldot be excluded as causative variants mdash SH3TC1 Ala608Thr8IYL2 Gly400Ala and because of failure to genotype8N5S0 Gly152Arg

All twenty-nine previously identified variants were re-xamined in the public databases and in the 1000 Genomesroject (www1000genomesorg mdash release date December009) (Table 2) Re-analysis demonstrated that all but fiveingle nucleotide variants had been described in normalubjects and indeed only one of these Q8IYL2 Gly400Alaas a coding sequence variant Q8N5S0 Gly152Arg andH3TC1 Ala608Thr were identified in public SNP databasesrs58349148) and in the 1000 Genomes Project (4 chromo-omes in Yoruban controls) respectively

The function of Q8IYL2 is unclear but it has beeneported as a likely tRNA (uracil-O(2)-) methyltransferasewwwhttpwwwncbinlmnihgovproteinQ8IYL2) ands expressed in brain No functional domains were identifiedn the hypothetical protein by Ensembl and a PIX analy-is (wwwhmgpmrcacuk mdash no longer available) revealedhree very marginal hits one with biopolymer transport pro-ein ExbDTolR one with geminivirus AL2 coat protein andne with PAZ domain IPB003180A a domain seen in purineNA glycosylases The change was not located in one of theseomains

The Gly400 residue is conserved in rat and mouse butot in other mammals

uantitative PCR

eletions of exons or genes could be ruled out by the suc-essful sequencing of all exons in the homozygous affectedndividual However quantitative PCR was performed toxamine for duplications

For each of the 31 primer pairs mean CTs of the two reac-ions against normal and affected DNA were corrected byubtraction from the mean CT for the control primer pairsorrected mean CT values in the affected DNA were sub-racted from those in the normal DNA Differences betweenormal and affected ranged between minus229 and 0486 (dataot shown) In the event of a duplication in the affectedNA the CT should be shorter in the affected DNA than theormal as the pre-amplification concentration of that exonn the affected DNA should be two-fold that of the normalNA Therefore in the event of a single duplication subtrac-ion of the CT of the affected DNA from that of the normalNA should result in a positive value of approximately 1hus there was no evidence of such a duplication involvingny of the exonic regions screened

iscussion

revious linkage data and haplotype analysis in a large

Please cite this article in press as Leschziner GD et al Q8IYL2 is a candidate gene for the familial epilepsy syndromeof Partial Epilepsy with Pericentral Spikes (PEPS) Epilepsy Res (2011) doi101016jeplepsyres201105010

ortheast Brazilian pedigree of Portuguese extraction hasocated the causative locus for a novel Mendelian epilepsyalled PEPS to a 97 Mb region of 4p15 Manual annota-ion of genes in this interval and the sequencing of all

Tabl

e

3

colle

ctio

nan

d

Q8I

YL

Vari

ant

Q8I

YL2

MQ

8IYL

2

IleQ

8IYL

2

GQ

8N5S

0

GSH

3TC1

ASH

3TC1

Aa

No

ort

bSc

reen

IN+Model

A

GS

ir

R

A

H

J

K

L

N

N

P

S

ARTICLEEPIRES-4531 No of Pages 7

Q8IYL2 as Candidate Gene for PEPS

genes in the linked interval resulted in the identification oftwo non-synonymous coding changes that segregate accord-ing to haplotype data have not been previously identifiedas polymorphisms and are not present in normal controlsmatched for both ancestry and region of Brazil Of the twochanges only one substitution Gly400Ala (G1277C changein exon 9) of the Q8IYL2 gene was also not identified inthe 1000 Genomes Project While the Gly400Ala substitutionresults in a change to a residue that is not highly con-served in mammalian species this would not be the firstneurological Mendelian syndrome for which this is the case(Polymeropoulos et al 1997) The Q8IYL2 gene has unknownfunction but is expressed in brain tissue It has been iden-tified as a likely tRNA uracil methyltransferase This class ofenzyme modifies uracil residues in the tRNA molecule alter-ing the codon which the tRNA recognises and binds to (Limand Curran 2001) Therefore disruption of the modificationprocess may ultimately result in a change in the amino acidresidue incorporated into a protein In humans mutations ofanother tRNA methyltransferase have been associated withepilepsy through their effects on codonmdashanticodon interac-tions (Yasukawa et al 2001)

In conclusion an exhaustive mutation analysis for PEPSsuggests that a mutation in the Q8IYL2 gene is causative ofthe phenotype Of course we cannot exclude the possibilitythat the causative mutation is a non-coding sequence vari-ant and that the change identified is a benign rare variantDefinitive proof of disease causation remains problematicand would require exclusion of other candidate changesthrough sequencing of the entire 97 Mb region and func-tional analysis of the gene or confirmation of its role inadditional epilepsy families showing linkage to 4p15 sincethe PEPS phenotype remains undescribed in any other familyto date

Since genes previously implicated in monogenic epilepsyhave established the principle that rare variants inMendelian epilepsy genes may be causal for sporadicepilepsy Q8IYL2 is thus a candidate gene for future rarevariant research in epilepsy as well as in epilepsy familiesshowing linkage to 4p15


Conflicts of interest

None of the authors has any conflict of interest to discloseDRB is currently an employee of Illumina

Y

PRESS7

cknowledgements

L was in part supported by the Neurology EntryExitcholarship from the Guarantors of Brain

We confirm that we have read the Journalrsquos position onssues involved in ethical publication and affirm that thiseport is consistent with those guidelines

eferences

nnegers JF Rocca WA Hauser WA 1996 Causes of epilepsycontributions of the Rochester epidemiology project Mayo ClinProc 71 570mdash575

elbig I Scheffer IE Mulley JC Berkovic SF 2008 Navi-gating the channels and beyond unravelling the genetics of theepilepsies Lancet Neurol 7 (3) 231mdash245

ohnson MR 2011 The genetic contribution to epilepsy theknown and missing heritability In Shorvon S Andermann FGuerrini R (Eds) The Causes of Epilepsy Cambridge UniversityPress pp 63mdash67

inton L Johnson MR Smith SJ Farrell F Stevens JRance JB Claudino AM Duncan JS Davis MB WoodNW Sander JW 2002 Partial epilepsy with pericentralspikes a new familial epilepsy syndrome with evidence for link-age to chromosome 4p15 Ann Neurol 51 740mdash749

im VI Curran JF 2001 Analysis of codonanticodon inter-actions within the ribosome provides new insights into codonreading and the genetic code structure RNA 7 942mdash957

obile C Michelucci R Andreazza S Pasini E Tosatto SCStriano P 2009 LGI1 mutations in autosomal dominant and spo-radic lateral temporal epilepsy Hum Mutat 30 (4) 530mdash536

guyen T Bard J Jin H Taruscio D Ward DC KennedyJL Weinshank R Seeman P OrsquoDowd BF 1991 Humandopamine D5 receptor pseudogenes Gene (109) 211mdash218

olymeropoulos MH Lavedan C Leroy E Ide SE Dehe-jia A Dutra A Pike B Root H Rubenstein J BoyerR Stenroos ES Chandrasekharappa S Athanassiadou APapapetropoulos T Johnson WG Lazzarini AM DuvoisinRC Di IG Golbe LI Nussbaum RL 1997 Mutation inthe alpha-synuclein gene identified in families with Parkinsonrsquosdisease Science 276 2045mdash2047

togmann E Lichtner P Baumgartner C Bonelli S Assem-Hilger E Leutmezer F Schmied M Hotzy C StromTM Meitinger T Zimprich F Zimprich A 2006 Dec 12Idiopathic generalized epilepsy phenotypes associated with dif-


ferent EFHC1 mutations Neurology 67 (11) 2029mdash2031asukawa T Suzuki T Ishii N Ohta S Watanabe K 2001

Wobble modification defect in tRNA disturbs codonmdashanticodoninteraction in a mitochondrial disease EMBO J 20 4794mdash4802



Figure 1 Limited PEPS pedigree adapted from Kinton et al (2002) Five digit numbers are DNA codes illustrating the fifteen sub-jects utilised for this study in red for homozygote affecteds blue for heterozygote affecteds and black for homozygote unaffected( nd t

I

Eoabd

pciebeaeltwa

cdafdhppotlata

tasre

M

G

T3g(

ws(sMaia

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FpmdashhltTmatched with the pedigree both for ancestral history (Portuguese

For interpretation of the references to color in this figure lege

ntroduction

pilepsy is a heterogeneous disorder with a variety of aeti-logies ranging from the purely genetic to environmentallycquired A large proportion of cases may have a geneticasis (Annegers et al 1996) of whom a small proportionisplay monogenic inheritance

To date the majority of genes accounting for idio-athic Mendelian epilepsies are ligand- or voltage-gated ionhannels (Helbig et al 2008) although recent discover-es of non-ion channel genes causing Mendelian idiopathicpilepsy have extended our knowledge of epilepsy geneseyond solely ion channel subunit gene mutations (Nobilet al 2009 Johnson 2011) The observation that rare vari-nts occurring in genes previously identified as Mendelianpilepsy genes are associated with sporadic epilepsy high-ights the potential for family studies in epilepsy to informhe spectrum of candidate genes for common epilepsy asell as confirm causal associations with detected rare vari-nts (eg Stogmann et al 2006)

A previously undocumented familial epilepsy syndromealled Partial Epilepsy with Pericentral Spikes (PEPS) wasescribed in 2002 (Kinton et al 2002) Affected individu-ls in the large family Brazilian of Portuguese extractionrom Paraiba state in the Northeast region exhibited fouristinct partial seizure types including hemiclonic andemitonic simple partial seizures temporal lobe complexartial seizures and stereotyped episodes of epigastricain Onset was in the first two decades of life with a benignutcome and affected individuals exhibited a characteris-ic EEG of pericentral epileptiform activity Genome-wideinkage analysis demonstrated linkage to region 4p15 with


maximum LOD score of 33 at marker D4S2311 Haplo-ype analysis defined the limits of the region to be D4S2983nd D4S2926 To identify the causative gene we under-

nNp

he reader is referred to the web version of the article)

ook a study involving several techniques including manualnnotation of the entire region to identify all transcribedequences sequencing of all transcribed sequences andeal-time quantitative polymerase chain reaction (PCR) toxclude copy number variation

ethods

ene annotation

he interval was examined in the Ensembl genome browser Build4 (wwwensemblorg) and candidate genes were identified Uni-ene entries were examined to determine expression patternswwwncbinlmnihgovunigene)

Gene structures were reanalysed and manually annotatedithin an internal Sanger Institute platform Otterlace Genomic

equence was analysed with ab initio gene prediction softwareFgenesH GENSCAN) as well as software predicting transcriptiontart sites (Eponine) CpG islands and repeat regions (Repeat-asker) All known mRNAs proteins and cDNAs from all species wereligned against the genomic sequence and using this as support-ng evidence gene structures were manually annotated includinglternative transcripts

ubjects

ive samples of subjects were used for this project The first sam-le consisted of fifteen members of the original pedigree (Fig 1)

one homozygote unaffected two homozygote affecteds and 12eterozygote affecteds The second was a sample of 47 normal unre-ated Caucasians was derived from the CEPH Utah collection Thehird was a collection of 243 unrelated normal Caucasian subjectshe fourth sample was a collection of normal Brazilian controls


= 95) and for region of Brazil and ancestral history (Paraiba stateorth-East region and Portuguese n = 85) Informed consent wasrovided by all patients and controls and the DNA analysis and




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(n

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Nor

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Tabl

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3

colle

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YL

Vari

ant

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MQ

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IleQ

8IYL

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GSH

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3TC1

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A

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ir

R

A

H

J

K

L

N

N

P

S









Y

PRESS7

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IN PRESS+ModelE


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Q

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Cont

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asia

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M

mut

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Inte

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cies

cons

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tion

CEPH

(n

=

47)

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cont

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(n

=

243)

Braz

ilian

Cauc

asia

ns

ofPo

rtug

uese

ance

stry

(n

=

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Nor

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Braz

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asia

ns

(n

=

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Succ

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ully

geno

type

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Hom

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Hom

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geno

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Hom

Het

M

Hom

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Leu

Med

ium

0

521

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Med

ium

0

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la

Med

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0

0

94

94

0

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85

85

0

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ium

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0

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Tabl

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3

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YL

Vari

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No

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R

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H

J

K

L

N

N

P

S









Y

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IN+Model

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H

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P

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Y

PRESS7

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eferences













Documents

Q8IYL2 is a candidate gene for the familial epilepsy syndrome of Partial Epilepsy with Pericentral Spikes (PEPS)