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AmplideX™

PCR Kit CE IVD

FMR1

Fragile X Syndrome and Associated Disorders

• Fragile X Syndrome (FXS)iscausedbyexpansionofCGGrepeatsintheFMR1gene,andisthemostcommonknowngeneticcauseofautism.

•FragileXcarriersmaybeatriskforfragileX-associatedtremor/ataxiasyndrome(FXTAS),aformofage-relatedcognitiveandmotordysfunction,orprimaryovarianinsufficiency(FXPOI),acauseofprematuremenopause.

•FragileXSyndromeisthemostcommoncauseofinheritedmentalretardationwithanestimatedincidenceof1in4000-9000malesand1in7000-15,000females.

•NewtherapiesandtargeteddrugsfortreatingFXSareindevelopment.

The Challenge of Fragile X Molecular Analyses

•Iscostly,laborious,andtime-consuming(~1week)toperform

•RequireslargeamountsofinputgDNA(5-10µg)

•ProvidesinaccurateCGGrepeatquantification

•Mayhavepoorsensitivitytodetectmosaicalleles

ExpansionofCGGrepeatsandmethylationstatusoftheFMR1geneareusedtostudyFragileXSyndromeandrelateddisorders.

(CGG)n

>200

59

50-58

<50

Full mutation Methylation

Premutation

Intermediate

Normal

5´ -UTR FMR1

Translation

None

Defect

Low RNALow Protein

High RNALow Protein

Normal RNANormal Protein

Consequence Outcomes

FXS

Normal

FXTASFXPOI

Current FMR1 molecular analyses relies heavily on Southern blot analysis, because laboratory PCR methods have been inadequate. However,Southernblotanalysis:

•TheAmplideX™FMR1PCRKitisaninvitrodiagnosticdeviceforprofessionaluseinclinicallaboratoriestoamplifyanddetectthecytosine-guanine-guanine(CGG)repeatregioninthe5'-untranslatedregionofthefragileXmentalretardation-1(FMR1)gene.

•ThedeviceisintendedasanaidindiagnosisoffragileXsyndromeandfragileXassociateddisorders,e.g.tremorandataxiasyndrome(FX-TAS)andprimaryovarianinsufficiency(FX-POI),throughdeterminationofCGGrepeatlengthupto200CGGanddetectionofallelesgreaterthan200CGG.

•Thetestconsistsofapolymerasechainreaction(PCR)ofgenomicDNApurifiedfromwholebloodfollowedbyfragmentsizingonanAppliedBiosystemsGeneticAnalyzerandconversionofproductsizetothenumberofCGGrepeats.

AmplideX™

PCR Kit CE IVD

FMR1

• FulllengthandCGGpeaksforeachallele.ExpansionsaredetectedirrespectiveofCGGlength

• Allexpansionsdetectedtodate(>200fullmutations)

• MoresensitivethanSouthernblotting

• Resolvesfemalezygosity• DetectsinterruptingAGGsequences

• CompatiblewithbloodinEDTA

CGGRepeatPrimed(RP)FMR1PCR

CGG Repeat PrimerG Repeat Prim

Electropherogramoffemalefullmutation

AmplideX™

PCR Kit CE IVD

FMR1

Supports a Simple PCR Mastermix Setup and Analysis Workflow

CGGRPorGene-SpecificPCR

PCR~30 min setup

(CE)~30 min setup

GS PCR ~3.5 hrs

or

CGG RP PCR ~5.5 hrs

13μLPCRmastermix+2μLsample(10-40ng/mL)

CE

PCR

GSPCRPlateorCGGRPPCRPlate

CEPlate

2μLofPCRproduct

+ROXLadder

AnyABImulti-capillaryinstrumentrunningPOP-7liquidpolymer

AmplideX™

PCR Kit CE IVD

FMR1

Components

Component Volume Description

Human FMR1 F,R FAM‑Primers 50µLAproprietaryFAM-labeledprimerpairforhumanFMR1geneamplificationbyPCR

FMR1 CGG Primer 50µLAnunlabeledCGGprimerforprobingthenumberofCGGrepeatsbyPCRamplification

Amp GC‑Rich AMP Buffer 1.2mLAbufferedsolutioncontainingPCRamplificationcomponentsoptimizedforGC-richDNA

GC‑Rich Polymerase Mix 5µLAnoptimizedmixtureofpolymerasesfortheamplificationofGC-richDNA

Diluent 1mL AproprietarysolutionusedinPCRmastermixpreparation

ROX 1000 Size Ladder 200µL ROXlabeledDNAfragmentsforsizingfrom79to1007bp

AmplideX™protocols,CEinstrumentmodulesandGeneMapperconfigurationfilesareprovidedintheIFU.Additionaltechnicalinformationcanbeprovideduponspecificrequest.

AmplideX™

PCR Kit CE IVD

FMR1

PCR Reagents have been Validated in Peer‑ reviewed Publications

PCR Categorically Detects Full Mutation Alleles up to at least 1300 CGG

SizinginredprovidedfromSouthernBlotAnalysis

PCR Analytical Sensitivity EnablesDetection to as Little as 1% Full Mutation Allele

% FM Allele

0%

1%400pgFM+39.6ngNOR

3%1.2ngFM+38.8ngNOR

5%2ngFM+38ngNOR

CGG Repeat PrimerG Repeat Prim

Thedetectionofalowabundantfullmutationallelewasdeterminedat40nginputusingananalyticaltitrationoftwoclinicalsamples.Amalefullmutationclinicalsamplewasadmixedwitha31CGGmalenormal(NOR)alleletogeneratedifferentpercentageinputsofthefullmutationallelebetween0to100%keepingthetotalinputofDNAconstantat40ng.TheCGGrepeatpeaksandfulllengthgene-specificproductpeaksweredetectedinaslowasa1%fullmutationallelewhichequatedto400pgofafullmutationalleleinabackgroundof39.6ngnormalallele.ThesignalintensityofthefulllengthproductpeakandCGGRPpeaksincreasedwithincreasingrelativeinputofthefullmutationallele.Thedetectionlimitforrobustandreproducibledetectionofafullmutationallelewas5%.

*These2samplespresentedpremutationallelesbybothmethodsandlowintensityfullmutationallelesdetectedonlybytheAmplideX™FMR1PCRKit.

1Chen,L.,A.Hadd,S.Sah,S.Filipovic-Sadic,J.Krosting,etal.:Aninformation-richCGGrepeatprimedPCRthatdetectsthefullrangeoffragileXexpandedallelesandminimizestheneedforsouthernblotanalysis.JMolDiagn,2010.12(5):589-600.

2EllesBoon,PatrickvanBunderen,BertBakker,ValidationoftwonewPCRtechnologiesfordiagnostictestingoffragileXCGGrepeataswellasFMR1methylationstatus,presentedatthe2010EuropeanSocietyforHumanGeneticsMeeting,Vienna,May2010

Demonstrates 100% Clinical Sensitivity to Southern blot Analysis

AmplideX™ PCR1,2

Sou

ther

n

Blo

t

Positive Negative TotalPositive 72 0 72Negative 2* 122 124Total 74 122 196

AmplideX™

PCR Kit CE IVD

FMR1

CGG Repeat Primed PCR Resolves Zygosity in Female Samples

TheabsenceofCGGprimedampliconsbeyondthesizeofthedetectedalleleindicatesthelackofalonger,heterozygousallele

ThepresenceofCGGprimedampliconsbeyondthe30CGGalleleindicatesthedetectionofalonger,heterozygousallele

CGG Repeat Primed PCR Detects Interrupting AGG Sequences That can Stabilize the CGG Repeat Region

ThesequencecontextofAGGinterruptionscanbedeterminedinmanysamples

AGGinterruptionscanbedetectedinFMR1alleleswithcomplexgenotypes

FMR1 PCR Fragment Sizing Accurately Determines CGG Repeat Length up to 200 CGG

• Samplesweretestedbetweentwosites• GenotypingresultswereobtainedviaSouthernblotanalysisandscoredforpremutationalleles(59-200CGG)priortoAmplideX™FMR1PCR.

• ThegenotypingresultswereblindedtothetechniciansperformingtheAmplideX™FMR1PCR.

• Thespecificityforpremutationalleleswas100%[95%CI:91.8-100%]andthesensitivityforpremutationalleleswas100%[95%CI:91.8-100%].

• TheaccuracyforpremutationallelesrelevanttoFX-TASandFX-POIwas100%.

Determines Premutation Alleles Relevant to FX‑TAS and FX‑POI

PMAmplideX™ FMR1 PCR

Positive Negative Total

Sou

th‑

ern

Blo

t Positive 43 0 43Negative 0 153 153Total 43 153 196

1Chen,L.,A.Hadd,S.Sah,S.Filipovic-Sadic,J.Krosting,etal.:Aninformation-richCGGrepeatprimedPCRthatdetectsthefullrangeoffragileXexpandedallelesandminimizestheneedforsouthernblotanalysis.JMolDiagn,2010.12(5):589-600.

2EllesBoon,PatrickvanBunderen,BertBakker,ValidationoftwonewPCRtechnologiesfordiagnostictestingoffragileXCGGrepeataswellasFMR1methylationstatus,presentedatthe2010EuropeanSocietyforHumanGeneticsMeeting,Vienna,May2010

AmplideX™

PCR Kit CE IVD

FMR1

Summary

•AmplideX™FMR1PCRtechnologycanreportthepresenceoffullmutationallelesirrespectiveoflength,andindependentlyrevealsfulllengthampliconsthatspantheentireCGGrepeatregion.Alleleswithuptoatleast1300CGGarereproduciblydetected.

•CEfragmentsizingresolvesFMR1allelesthatdifferbyasingleCGGrepeat,andprovidesaccuraterepeatquantificationacrossallallelecategoriesupto250CGG.

•AsinglePCRreactionprovidesdetectionofallalleleexpansions,enumerationofCGGrepeats,resolutionoffemalezygosity,anddetectionofinterruptingAGGsequencesinsupportofcomprehensiveFMR1genotyping.ThiscapabilitycanreducetheburdenofSouthernblotanalysisbyasmuchas10-to50-fold.

•TheAmplideX™FMR1PCRkithasbeenusedtosuccessfullyprofileover2500clinicalsamples,including>200fullmutationexpansionswithconcordancetoSouthernblotanalysis.ThehighsensitivityoftheAmplideX™FMR1PCRkitcanreportlowabundanceexpansionsthatmaybeundetectablebySouthernblot.

•Twopeer-reviewedpublicationsdescribethevalidationofthetechnology,whichhasbeenevaluatedatmorethantwodozenlaboratoriesworldwide.

AmplideX™

PCR Kit CE IVD

FMR1