http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.

0019095

4-Aminobutyrate Aminotransferase (ABAT): Genetic and Pharmacological Evidence for an nvolvement in Gastro Eso!hageal "eflu# $isease
ohan! irholt1"#!$engt!%sling1"&#!'aul!(a))ond2#!*eoffre+!,a-idson2#!.i/ael!0nutsson1#! Anna!1alentinsson1#! 2rgen!..! ensen1#!Anders!3eh)ann1#!3ars!Agreus3#!.aria! 3agerstr2)4Fer)er1 1!Astra5eneca!67,#!.2lndal#!8weden#!2!*astroenterolog+!9nit#!1o)en:s!7!;hildren:s! (ospital#!<orth!Adelaide#!Australia#!3!;enter!for!Fa)il+!and!;o))unit+!.edicine#! 0arolins/a!=nstitutet#!(uddinge#!8weden

Abstract Top
*astro4esophageal!reflu>!disease!?*@6,A!is!partl+!caused!B+!genetic!factors.!Che! underl+ing!susceptiBilit+!genes!are!currentl+!un/nown#!with!the!e>ception!of!;D33A1.! 1e!used!three!independent!*@6,!patient!cohorts!to!identif+!*@6,!susceptiBilit+!genes.! Chirt+4si>!fa)ilies#!de)onstrating!do)inant!trans)ission!of!*@6,!were!suBjected!to! whole!geno)e!)icrosatellite!genot+ping!and!lin/age!anal+sis.!Fi-e!lin/ed!regions!were! identified.!Cwo!fa)ilies!shared!a!lin/ed!region!?3D,!3.9!and!2.0A!on!chro)oso)e!1E.! 1e!used!two!additional!independent!*@6,!patient!cohorts#!one!consisting!of!219!trios! ?affected!child!with!parentsA!and!the!other!an!adult!*@6,!case!control!cohort!consisting! of!25E!cases!and!FG5!controls#!to!-alidate!indi-idual!genes!in!the!lin/ed!region!through! association!anal+sis.!8i>t+!si>!single!nucleotide!pol+)orphis)!?8<'A!)ar/ers!distriButed! o-er!the!nine!genes!present!in!the!lin/ed!region!were!genot+ped!in!the!independent! *@6,!trio!cohort.!Crans)ission!diseHuiliBriu)!test!anal+sis!followed!B+!)ultiple!testing! adjust)ents!re-ealed!a!significant!genetic!association!for!one!8<'!located!in!an!intron!of! the!gene!F4a)inoBut+rate!a)inotransferase!?A$ACA!?'adj!I!0.027A.!Chis!association!did! not!replicate!in!the!adult!case4control!cohort#!possiBl+!due!to!the!differences!in!ethnicit+! Between!the!cohorts.!Finall+#!using!the!selecti-e!A$AC!inhiBitor!-igaBatrin!?J4-in+l! *A$AA!in!a!dog!stud+#!we!were!aBle!to!show!a!reduction!of!transient!lower!esophageal! sphincter!rela>ations!?C3@86sA!B+!57.3K11.F!%!?p!I!0.007A!and!the!reflu>!e-ents!fro)! 3.1K0.F!to!0.GK0.F!?p!I!0.007A.!Dur!results!de)onstrate!the!direct!in-ol-e)ent!of!A$AC! in!pathwa+s!affecting!lower!esophageal!sphincter!?3@8A!control!and!identifies!A$AC!as!a! genetic!ris/!factor!for!*@6,. Citation: irholt! #!%sling!$#!(a))ond!'#!,a-idson!*#!0nutsson!.#!et!al.!?2011A!F4 A)inoBut+rate!A)inotransferase!?A$ACA:!*enetic!and!'har)acological!@-idence!for!an!

=n-ol-e)ent!in!*astro!@sophageal!6eflu>!,isease.!'3o8!D<@!E?FA:!e19095.! doi:10.1371/journal.pone.0019095 Editor: .arie4'ierre!,uBL#!9ni-ersite!de!.ontreal#!;anada Received:!<o-e)Ber!G#!2010M!Accepted:!.arch!2G#!2011M!Published:!April!2G#!2011 Copyright:!N!2011! irholt!et!al.!Chis!is!an!open4access!article!distriButed!under!the!ter)s! of!the!;reati-e!;o))ons!AttriBution!3icense#!which!per)its!unrestricted!use#! distriBution#!and!reproduction!in!an+!)ediu)#!pro-ided!the!original!author!and!source!are! credited. Funding:!Che!stud+!was!funded!B+!Astra5eneca!67,!and!the!stud+!design#!generation! of!data#!anal+sis!of!data#!decision!to!puBlish!as!well!as!preparation!of!the!)anuscript!was! perfor)ed!B+!the!authors!of!which!se-eral!are!e)plo+ed!B+!Astra5eneca!67,#!8weden. Competing interests:!Chis!stud+!was!funded!and!perfor)ed!B+!Astra5eneca!67,! .2lndal#!8weden!in!collaBoration!with!1(;#!Adelaide#!Australia!and!0=#!8toc/hol)#! 8weden.!8e-eral!authors!are!currentl+!e)plo+ed!at!Astra5eneca!67,!.2lndal#!8weden.! <o!other!conflicts!of!interest!e>ist. &!@4)ail:!$engt.AslingOastraPeneca.co) "!Chese!authors!contriButed!eHuall+!to!this!wor/.!

ntroduction Top
*astroesophageal!reflu>!disease!?*@6,A!is!an!increasingl+!co))on!gastrointestinal! disorder!with!a!pre-alence!of!25QF0%!in!the!western!world!R1S#!R2S.!*@6,!s+)pto)s#! such!as!heartBurn!and!regurgitation#!lea-e!affected!indi-iduals!with!a!se-erel+!i)paired! Hualit+!of!life!and!place!a!consideraBle!health!econo)ic!Burden!on!societ+!R3S#!RFS. ,iagnosis!relies!on!clinical!assess)ent!and!)ultidi)ensional!Huestionnaires!co)Bined! with!ph+sical!e>a)inations!such!as!endoscopic!e>a)ination!and!2F!hour!p(! )easure)ents!of!the!esophagus.!Che!disease!has!Been!suBdi-ided!into!erosi-e!reflu>! disease!?@6,A!and!non4erosi-e!reflu>!disease!?<@6,A!R5S.!Che!patient!classification! helps!predict!li/elihood!of!response!to!proton!pu)p!inhiBitors#!@6,!indi-iduals! e>hiBiting!a!Better!response!rate!than!<@6,!indi-iduals.!@6,!is!thought!to!Be!caused! )ainl+!B+!acid!reflu>!into!the!esophagus!while!<@6,!)a+!include!other!factors!such!as! wea/l+!acidic!or!non4acidic!reflu>.!3ess!than!half!of!affected!indi-iduals!ha-e!the!erosi-e! for)!of!the!disease!where!the!esophageal!)ucosa!is!da)aged!B+!acidic!reflu>.!Che! )ajorit+!suffer!fro)!<@6,#!where!affected!indi-iduals!e>perience!t+pical!*@6,! s+)pto)s!without!-isiBle!esophageal!injuries!RES. Dnset!of!the!disease!is!-ariaBle!and!occurs!a)ong!children!as!well!as!in!adults.!=t!has! Been!suggested!that!*@6,!)a+!originate!in!childhood!R1S#!R7SQR10S.!=n!fact#!*@6,!is!the!

)ost!co))on!esophageal!disorder!in!children!and!11%!of!infants!are!affected!during! their!first!+ear!of!life!R11S.!(owe-er#!there!are!differences!Between!pediatric!and!adult! *@6,!such!that!the!erosi-e!for)!of!the!disease!is!rarel+!seen!in!the!+oung!R12S. $oth!en-iron)ental!and!genetic!factors!contriBute!to!disease!susceptiBilit+.!Chere!is!a! clear!genetic!co)ponent!to!*@6,#!as!has!Been!shown!in!se-eral!studies!through!fa)ilial! clustering!and!twin!concordance!rates!R13SQR1ES.!=n!the!large!twin!stud+!B+!;a)eron!et! al.!?2002A#!the!genetic!heritaBilit+!was!esti)ated!at!31%.!(owe-er#!with!the!one! e>ception!of!;D33A1#!the!identities!of!the!causati-e!genes!are!presentl+!un/nown.! ;D33A1!encodes!collagen!t+pe!===!alpha!1!pro-iding!)olecular!support!for!the!notion! that!there!is!a!connecti-e!tissue!wea/ness!co)ponent!to!*@6,!R17S. A!nu)Ber!of!lifest+le!and!en-iron)ental!factors!such!as!oBesit+!and!alcohol!consu)ption! has!also!Been!shown!to!Be!associated!with!an!increased!ris/!of!acHuiring!the!disease!R2S.! =t!is!proBaBle!that!so)e!of!these!en-iron)ental!factors!are!not!present!in!+oung!*@6,! patients#!suggesting!that!the!genetic!co)ponent!)a+!Be!)ore!penetrant!in!the!+oung. Creat)ent!of!*@6,!is!pri)aril+!pro-ided!through!proton!pu)p!inhiBitors!?''=sA!that! reduce!the!acidit+!of!the!sto)ach:s!gastric!acid.!(owe-er#!a!large!proportion!of!*@6,! patients!do!not!Benefit!fro)!this!treat)ent!lea-ing!a!large!un)et!need!for!new! phar)acological!inter-entions.!A!large!effort!has!Been!de-oted!to!de-eloping!drugs!that! inhiBit!transient!lower!esophageal!sphincter!rela>ations!?C3@86sA#!the!)ajor!underl+ing! )echanis)!for!gastroesophageal!reflu>!R1GSQR20S#!ai)ing!to!reduce!the!nu)Ber!of!reflu>! e-ents!in!affected!indi-iduals.!Chis!research!has!to!so)e!e>tent!Been!dependent!on!ani)al! )odels#!where!dogs!and!ferrets!are!the!Best!estaBlished!)odels!R21S#!R22S.!Future!anti4 reflu>!drugs!hold!the!hope!of!Being!effecti-e!for!the!treat)ent!of!patients!who!e>perience! *@6,!s+)pto)s!despite!Being!on!''=s!R23S. *enetics!can!Be!used!to!address!the!underl+ing!disease!)echanis)s!and!pro-ide!potential! new!targets!for!drug!treat)ent!as!well!as!Bio)ar/ers!for!patient!stratification.!1e!ha-e! used!a!co)Bination!of!hu)an!genetic!approaches!coupled!with!-alidation!in!the!C3@86! dog!)odel!to!identif+!F4a)inoBut+rate!a)inotransferase!?A$ACA!as!a!potential!*@6,! susceptiBilit+!gene!affecting!control!of!the!3@8.

!ethods Top
Ethics statement 1ritten!infor)ed!consent!was!oBtained!Before!enrol)ent.!All!data!and!,<A/tissue! sa)ples!were!coded.!@thical!appro-al!was!oBtained!for!all!patient!collections!and!hu)an! research!fro)!the!1o)en:s!and!;hildren:s!(ospital!?AdelaideA!6esearch!@thics! ;o))ittee!with!reference!nu)Bers!6@;12E2/11/200F!7!6@;13F0/E/2005. Che!adult!case!control!cohort!was!appro-ed!B+!the!8wedish!regional!ethics!re-iew!Board! in!9)eT!with!reference!nu)Ber!321/03#!diar+!nr!0342G5.

All!procedures!in-ol-ing!laBorator+!ani)als!were!appro-ed!B+!the!regional!ani)al!ethics! re-iew!Board!in!*othenBurg#!8weden!with!reference!nu)Ber!3EE42005. Patient collections Chree!groups!of!*@6,!patients!ha-e!Been!used!in!this!stud+.!Che!first!is!a!cohort!of! suBjects!of!all!ages!in!who)!there!is!a!con-incing!fa)il+!histor+!of!*@6,!?referred!to!as! UFa)iliesVA.!Che!second!is!a!cohort!of!children!with!*@6,!without!a!strong!fa)il+! histor+!of!*@6,#!along!with!their!parents!?referred!to!as!UCriosVA.!Che!third!is!a! collection!of!adult!indi-iduals!suffering!fro)!*@6,!together!with!health+!control! indi-iduals!?referred!to!as!Uadult!case!control!cohortVA. Ethnicity Che!fa)il+!and!trio!cohorts!were!collected!fro)!the!di-erse!)ulti4ethnic!Australian! population!R2FS.!Che!Uethnic!strengthV!descriBes!how!)uch!a!specific!ethnicit+! contriButes!to!a!specific!population.!For!the!Australian!population#!it!is!as!follows:! appro>i)atel+!70%!Anglo4;eltic#!1G%!@uropean!?including!0.2G%!8wedish!and!0.13%! FinnishA#!E%!Asian#!1.5%!ABoriginal/Correns!8trait!=slander!and!F.5%!African#! ewish#! 'acific!and!=ndigenous!A)erican!collecti-el+.!Che!adult!case!control!cohort!was! collected!fro)!north4eastern!8weden.!Chis!population!is!a!)i>ture!of!largel+!three!ethnic! groups!where!the!Finnish!and!8wedish!contriButions!are!roughl+!eHual!with!an! appro>i)atel+!10%!8aa)i!ad)i>ture!R25S. Families @nrol)ent!of!patients!was!carried!out!in!the!*astroenterolog+!9nit!at!1o)en:s!and! ;hildren:s!(ospital!?1;(#!Adelaide#!AustraliaA#!fro)!2001!to!2005!B+!identif+ing! children!?age!up!to!1G!+earsA!diagnosed!with!*@6,!and!displa+ing!at!least!one!of!the! following!criteria!within!the!last!fi-e!+ears:!an!aBnor)al!finding!fro)!endoscopic! e>a)ination!or!2F!hours!esophageal!p(!test!or!ha-ing!Been!suBjected!to!a!fundoplication.! 'roBands!were!identified!B+!e>a)ination!of!1;(!patient!records!and!B+!contacting!the! local!*@6,!support!association!?F=68AM!Friends!of!=nfant!6eflu>!8upport!AssociationA.! 'arents!of!the!proBand!were!contacted!and!the!fa)il+!was!enrolled!if!a!positi-e!fa)il+! histor+!of!*@6,!could!Be!shown.!,isease!status!was!assessed!-ia!ph+sician!diagnosis! Based!on!results!of!pre-iousl+!perfor)ed!in-estigations.!'atients!with!a!)edical!condition! /nown!to!predispose!to!*@6,!were!e>cluded. Trios Che!trio!cohort!consisted!of!pediatric!patients!diagnosed!with!*@6,!Between!three! )onths!and!17!+ears!of!age!without!conditions!predisposing!to!*@6,#!and!their! corresponding!parents.!Che!diagnosis!of!*@6,!was!Based!on!the!following:!a!pediatric! gastroenterologist:s!e-aluation!of!s+)pto)s!to!Be!consistent!with!*@6,#!in!addition!to! either!p(!proBe!deter)ined!acid!e>posure!consistent!with!*@6,#!endoscopic!findings! consistent!with!*@6,!and/or!a!definite!and!significant!i)pro-e)ent!in!s+)pto)s!of!

*@6,!shortl+!after!co))encing!anti4reflu>!treat)ent.!'re-ious!anti4reflu>!surger+!was! also!considered!as!e-idence!of!*@6,.!'atients!were!identified!fro)!dataBases!of! endoscop+#!p(!proBe!and!outpatient!diagnoses!fro)!the!*astroenterolog+!9nit!at!1;(.! All!the!proBands!that!were!endoscopicall+!e-aluated!also!had!a!histological!e>a)ination! of!esophageal!Biopsies.!*rading!of!esophageal!injuries!found!at!endoscopic!e>a)inations! was!Based!on!the!C+tgat!classification!s+ste)!R2ES#!the!)ost!appropriate!endoscopic! grading!s+ste)!for!pediatric!*@6,!R27S.!Che!classification!of!the!histological! e>a)inations!of!esophageal!Biopsies!was!Based!on!0nuff!7!3eape!R2GS#!R29S.!Chere!is!a! poor!correlation!Between!endoscopic!e-aluation!and!histological!e>a)inations!in!*@6,! diagnosis!R27S.!1e!therefore!used!Both!)ethods!as!inclusion!criteriaM!indi-iduals!with! positi-e!findings!fro)!either!endoscop+!or!histolog+!e-aluation!were!included!in!the!trio! cohort. 'atients!were!e>cluded!if!the+!had!Been!diagnosed!with!a!condition!predisposing!to! *@6,#!such!as!se-ere!?non4a)BulantA!cereBral!pals+#!c+stic!fiBrosis#!)ajor!congenital! aBnor)alities!of!the!gastrointestinal!tract!?e.g.!esophageal!atresia/tracheo4esophageal! fistulaA#!and!se-ere!chronic!lung!disease!?for!e>a)ple!oral!steroid!dependent!asth)atics! and!Bronchopul)onar+!d+splasiaA.!Che!total!nu)Ber!of!indi-iduals!)eeting!these!criteria! was!219.!Che!a-erage!age!was!E!W!+ears!and!these!patients!had!an!a-erage!$.=!of!1G.2! /g/)2.!Che!cohort!consisted!of!E5%!)ales.!Che!proBands!)eeting!the!inclusion!criteria! were!endoscopicall+!classified!into!grade!0!through!5!with!the!freHuencies!20%#!5G%#! 1E%#!F%#!1%#!and!1%#!respecti-el+.!;orrespondingl+#!histopathological!grading!resulted! in!22%#!FF%#!30%#!3%#!1%#!and!1%!of!patients!classified!with!grades!0!through!5#! respecti-el+. Adult "ER# case control cohort Che!0ali>anda!cohort!is!a!collection!of!patients!fro)!northern!8weden!where! epide)iological!factors!related!to!*@6,!ha-e!Been!in-estigated!R30S#!R31S.!A!careful! description!of!this!cohort!has!pre-iousl+!Been!puBlished!R31S!including!confounding! factors.!=n!Brief#!all!indi-iduals!went!through!an!endoscopic!e>a)ination!that!together! with!a!written!Huestionnaire!was!used!to!diagnose!*@6,!B+!professional! gastroenterologists.!9sing!the!sa)e!criteria!and!the!sa)e!ph+sicians#!we!e>tended!this! cohort!with!100!additional!*@6,!patients!fro)!the!sa)e!de)ographic!area#!resulting!in! the!adult!case!control!cohort!used!in!this!stud+.!Che!cohort!consists!of!25E!adult!*@6,! cases!?39%!)alesA!and!FG5!health+!control!indi-iduals!?FE%!)alesA.!Che!)edian!age!was! 5E!and!5F!+ears!in!the!case!and!control!groups!respecti-el+. Animals Cen!adult!3aBrador!retrie-ers!were!used!in!the!stud+.!;er-ical!esophagosto)ies!R32S! were!)ade.!After!reco-er+!fro)!surger+!the!dogs!were!accusto)ed!to!rest!in!a!'a-lo-! stand.!$efore!the!e>peri)ents#!the!dogs!were!fasted!for!appro>i)atel+!1EQ1G!h#!But!with! free!access!to!water. Techni$ues

#%A e&traction'

,<A!was!e>tracted!fro)!Blood!or!Buccal!swaBs!using!Xiagen!?Xiagen#!Yalencia#!;A#! 98AA!or!*eno)'hi!?A)ersha)!$iosciences!A$#!9ppsala#!8wedenA!according!to! )anufacturer:s!protocol.!=n!cases!where!,<A!a)ount!was!li)ited#!whole!geno)es!were! a)plified!B+!.olecular!8taging!=nc!?,en-er#!;D#!98AA!through!their!6epli4g!ser-ice.

"enotyping'

.icrosatellite!genot+ping!was!perfor)ed!in!F19!,<A!sa)ples!using!A$=!'ris)!3in/age! .apping!set!-2.5!(,5!for!,<A!frag)ent!anal+sis!?Applied!$ios+ste)s#!Foster!;it+#! ;A#!98AA.!Additional!)icrosatellites!were!a)plified!using!puBlic!pri)er!seHuences.!Che! forward!pri)er!was!ordered!5Z4fluorophore!laBeled!?8ig)a4*enos+s#!;a)Bridge#!9nited! 0ingdo)A!while!the!re-erse!pri)er!was!opti)iPed!R33S.!8iPe!fractioning!of!,<A! frag)ents!was!done!in!A$=!3700!or!3730!,<A!anal+Pers!?Applied!$ios+ste)sA.!Alleles! were!called!using!the!*enot+per!-3.0!software!?Applied!$ios+ste)sA.!Che!)ean!distance! Between!adjacent!)ar/ers!was!F.32!centi)organ!?8,!K!2.E5#!range!0Q1F.7GA.!8ingle! nucleotide!pol+)orphis)!?8<'A!genot+ping!was!perfor)ed!using!the!CaH.an!assa+! ?Applied!$ios+ste)sA!and!detection!in!A$=!7900(C!?Applied!$ios+ste)sA!according!to! )anufacturers!reco))endations#!apart!fro)!reducing!the!total!reaction!-olu)e!to!2.5![l.! All!data!were!anal+Ped!with!8eHuence!,etection!8+ste)s!N!software!-2.1!?Applied! $ios+ste)sA.
(e$uencing'

8eHuencing!was!perfor)ed!fro)!Both!directions!using!the!Applied!$ios+ste)s!3700!or! 3730!auto)ated!,<A!seHuencers!?Applied!$ios+ste)s!=ncA!according!to!the! )anufacturer:s!reco))endation.!8eHuence!anal+sis!was!done!using!?8eHuencher!F.E#! *enecodes#!Ann!ArBor#!.=#!98AA!and!co)pared!to!the!puBlic!geno)ic!seHuence! ?@nse)Bl!release!F9A.!8eHuence!differences!were!)anuall+!chec/ed!and!re)aining! inconsistencies!were!reseHuenced.

!easurement o) T*E(R'

Che!)ethod!applied!for!the!studies!ha-e!Been!descriBed!pre-iousl+!R3FS.!$riefl+#!the!dogs! were!intuBated!with!a!water4perfused!,entslee-e!)ultilu)en!asse)Bl+!to!record!the! gastric#!3@8!and!esophageal!pressures.!An!anti)on+!p(!electrode!was!placed!3!c)!aBo-e! the!3@8!for!)easure)ent!of!acid!reflu>!episodes!and!a!water4perfused!catheter!was! placed!in!the!h+pophar+n>!to!)easure!swallows.!C3@86s!were!sti)ulated!B+!gastric! infusion!of!an!acidified!liHuid!nutrient!?30!)l//gM!100!)l/)inA!followed!B+!air! insufflation!?500!)l/)inA!to!)aintain!a!gastric!pressure!of!10K1!))(g!during!the! e>peri)ent.!Che!nu)Ber!of!C3@86s!was!)easured!during!a!F5!)in!period!starting!fro)! infusion!of!the!liHuid.!C3@86s!were!defined!as!a!rapid!decrease!in!3@8!pressure!?\1! ))(g/sA#!to!a!pressure!]2!))(g!aBo-e!gastric!pressure!and!a!duration!\1!s#!without!an+! phar+ngeal!signal!]2!s!Before!onset.!6eflu>!episodes!were!defined!as!a!drop!in!p(\1!unit! within!5!seconds!with!an!a-erage!p(!of!]F!in!the!15!seconds!following!nadir!p(.! YigaBatrin!?8aBrile>^A!was!dissol-ed!in!sterile!water!and!ad)inistered!intragastricall+!

?100!)g//gM!0.5!)l//gA!2!h!Before!start!of!)easure)ent!or!2!h!and!1E!h!?100!)g//g!at! each!occasionA!Before!start!of!)easure)ent. (tatistics

+uality Control o) )amilial genotype data'

.endelian!inconsistencies!were!detected!using!'ed;hec/!?-.1.1A!R35S.!.ar/ers!with!a! large!nu)Ber!of!errors!were!either!reanal+Ped!or!e>cluded.!=ndi-idual!erroneous! genot+pes!were!either!rechec/ed!or!discarded.!(ard+41einBerg!testing!was!perfor)ed!in! 'edstats!?-.0.E.FA!R3ES.!Finall+#!.erlin!?-.0.10.2A!R37S!and#!in!the!case!of!fa)il+!1FE#! 8i)1al/2!?-.2.91A!R3GSQRF0S!were!used!to!find!unli/el+!genot+pes.!Although!)ar/ers! de-iating!fro)!(ard+41einBerg!eHuiliBriu)!and!unli/el+!genot+pes!were!not! auto)aticall+!discarded#!care!was!ta/en!to!ensure!that!the+!did!not!influence!the!lin/age! pea/s. +uality control o) the trio cohort and the adult case control cohort genotype data .endelian!inconsistencies!in!trio!genot+pes!were!detected!using!'ed;hec/!?-.1.1A.! (ard+41einBerg!testing!as!well!as!calculation!of!descripti-e!statistics!was!perfor)ed!in! Both!'edstats!?-.0.E.FA!and!(aplo-iew!-.3.32!RF1S!for!Both!the!Crios!and!the!adult!case! control!cohort.!(ard+41einBerg!testing!was!used!as!a!tool!to!find!8<'s!to!reanal+Pe! rather!than!to!discard!8<'s!fro)!the!association!anal+sis.!(owe-er#!it!was!)ade!sure!that! positi-e!associations!could!not!Be!e>plained!B+!de-iations!fro)!(ard+41einBerg! eHuiliBriu). *in,age analysis *enetic!)ap!positions!used!in!the!lin/age!anal+sis!were!oBtained!fro)!the!,ecode!)ap! RF2S.!.ar/ers!that!did!not!ha-e!an!assigned!position!were!e>trapolated!into!the!)ap!Based! on!ph+sical!distance!to!the!flan/ing!)ar/ers.!Fa)il+!wise!)ultipoint!3D,!score!cur-es! were!calculated!for!all!autoso)al!chro)oso)es!and!the!_!chro)oso)e!using!*enehunter! -.2.1!RF3S#!RFFS!and#!in!the!case!of!fa)il+!1FE#!8i)1al/2!?-.2.91A#!assu)ing!a!do)inant! )ode!of!inheritance.!8ingle!point!3D,!scores!were!calculated!and!co)pared!to!the! )ultipoint!3D,!scores!for!data!consistenc+.!3in/ages!are!reported!according!to! estaBlished!guidelines#!that!is#!suggesti-e!and!significant!lin/age!corresponding!to!3D,! scores!aBo-e!1.9!and!3.3!respecti-el+!RF5S.!Che!core!lin/ed!region!is!defined!as!the!region! where!the!3D,!score!is!larger!than!the!)a>i)u)!3D,!score!suBtracted!with!1!3D,!unit! ?id est!5.541!I!F.5A.!8ensiti-it+!anal+sis!was!perfor)ed!B+!?iA!-ar+ing!the!para)eters!in! the!do)inant!)odel#!?iiA!assessing!an!additi-e!)ode!of!inheritance#!and!?iiiA!perfor)ing! nonpara)etric!anal+sis.!Chese!anal+ses!pro-ided!no!further!infor)ation!and!the!results! were!thus!o)itted. Association analysis trios

Crans)ission!diseHuiliBriu)!test!?C,CA!anal+sis!Based!on!single!8<'s!was!perfor)ed!in! *enehunter!?-.2.1A!and!(aplo-iew!?-.3.32A.!p4-alues!adjusted!for!)ultiple!testing!?padjA! were!assessed!B+!per)utations!Between!trans)itted!and!untrans)itted!alleles!using! (aplo-iew!?-.3.32A.!Che!significance!le-els!were!also!chec/ed!using!the!$onferroni! correction!?no)inal!p!>!EE!8<'sA!for!consistenc+!and!no!changes!in!significant! association!was!found.!3in/age!diseHuiliBriu)!calculations!?,ZA!and!plots!were!generated! fro)!genot+pe!data!in!the!trio!cohort!using!(aplo-iew. Association analysis adult case control cohort Allelic!case4control!anal+sis!Based!on!single!8<'s#!co)paring!the!allelic!distriBution! Between!cases!and!controls!in!a!2`2!taBle#!was!perfor)ed!in!8A8!?-.G.2A!using!Fisher:s! e>act!test.!8ince!none!of!the!8<'s!reached!no)inal!significance!?no)inal!p4-alue!Below! 0.05#!see!the!6esults!sectionA#!no!adjust)ent!for!)ultiple!testing!was!perfor)ed.!Che! anal+sis!was!also!repeated!co)paring!genot+pes!in!a!3`2!taBle. T*E(R Calculations in #ogs @ach!dog!ser-ed!as!its!own!control.!;hanges!in!the!nu)Ber!of!C3@86s!were!calculated! with!regard!to!the!)ean!of!fi-e!preceding!control!e>peri)ents!for!each!dog.!,ata!is! presented!as!)eanK8@..!8tudent:s!paired!t4test!was!used!for!statistical!anal+sis.!A!p4 -alue!]0.05!was!regarded!as!statisticall+!significant.

Results Top
*in,age analysis Chirt+4si>!fa)ilies!displa+ing!do)inant!inheritance!of!*@6,!were!used!for!whole! geno)e!)icrosatellite!genot+ping!and!suBseHuent!lin/age!anal+sis.!Che!outco)e!of!the! fa)il+!wise!lin/age!anal+sis!was!the!identification!of!one!region!reaching!the!threshold! for!significant!lin/age!and!four!regions!reaching!the!threshold!for!suggesti-e!lin/age! ?CaBle!1A.!A!lin/ed!region!on!chro)oso)e!2!co))on!for!a!suBset!of!the!fa)ilies!has! Been!puBlished!pre-iousl+!R17S.!.ost!of!the!identified!lin/ed!regions!were!large!in!siPe! due!to!the!li)ited!nu)Ber!of!indi-iduals!of!the!fa)ilies!used.!(owe-er#!two!of!the! fa)ilies!displa+ed!an!o-erlapping!lin/age!to!a!relati-el+!s)all!region!located!on! chro)oso)e!1Ep.!Further)ore#!the!lin/age!for!one!of!these!fa)ilies!was!significant#! reaching!a!)a>i)u)!3D,!of!3.9.!Che!other!fa)il+!displa+ed!a!co)pletel+!o-erlapping! lin/age!cur-e!and!reached!a!suggesti-e!lin/age!of!2.0!?Figure!1A.!Additional! )icrosatellite!genot+ping!reduced!the!e>tent!of!the!region.!(+pothesiPing!that!the!sa)e! genetic!perturBation!was!causati-e!in!these!two!fa)ilies#!we!reached!a!co)Bined!3D,! score!of!5.5!?Figure!1A.!Che!siPe!of!the!region#!as!defined!B+!the!closest!unlin/ed!)ar/ers#! co))on!to!Both!fa)ilies#!was!5.1!)ega!Base!pairs!?.BpA.!1hile!the!core!lin/ed!region! spanned!onl+!3.E!.Bp#!we!were!not!aBle!to!reduce!the!lin/age!region!an+!further!through! lin/age!anal+sis!due!to!aBsence!of!reco)Bination!e-ents!in!the!core!lin/ed!region.

Figure 1' -verlapping lin,age pea, at chromosome 1.p )or )amilies 122 and 1/.' 8trongest!lin/age!was!oBtained!for!fa)il+!1FE!showing!a! 3D,!of!3.9.!Che!other!fa)il+#!122#!showed!a!lin/age!of! 3D,!2.0.!Che!co)Bined!lin/age!cur-e!reaches!a!)a>i)al! 3D,!score!of!5.5. doi:10.1371/journal.pone.0019095.g001 #o0nload: 'ower'oint!slide!a!larger!i)age!?5F0$!'<*A!a!original!i)age!?1130$! C=FFA!

Table 1' A summary o) the )ive lin,ed regions identi)ied in the "ER# )amily collection' doi:10.1371/journal.pone.0019095.t001 #o0nload: 'ower'oint!slide!a!larger!i)age!?E30$!'<*A!a!original!i)age!?23F0$! C=FFA! "enetic association analysis o) the trio cohort Che!identified!chro)oso)al!region!defined!B+!the!co)Bined!lin/age!cur-es!was! relati-el+!s)all!and!contained!9!genes!?Figure!2A.!1e!argued!that!the!causati-e!gene!)ost! li/el+!would!Be!a!co))on!*@6,!gene#!present!in!other!*@6,!patients.!Co!test!this!we! decided!to!in-estigate!if!an+!of!the!genes!located!in!the!lin/ed!region!were!geneticall+! associated!with!*@6,!in!an!independent!pediatric!*@6,!trio!cohort.!1e!selected!and! genot+ped!EE!different!8<'!)ar/ers!located!in#!and!around#!the!9!genes.!Crans)ission! diseHuiliBriu)!test!anal+sis#!followed!B+!)ultiple!testing!adjust)ents!for!all!EE!8<'s! genot+ped#!re-ealed!significant!genetic!association!for!the!8<'!rs1EF1021.!Chis!8<'! reaches!an!adjusted!p4-alue!of!0.027!?calculated!as!the!no)inal!p4-alue!0.000F!)ultiplied! with!EEA.!Che!data!for!this!8<'#!together!with!the!1E!other!8<'s!located!in!the!A$AC! region#!are!shown!in!taBle!2.!Che!associated!8<'#!rs1EF1021#!is!located!within!intron!1E! of!A$AC.

Figure 2' The lin,ed region at chromosome 1.p' Che!Bo>!represents!the!region!of!the!co)Bined!)a>i)u)!3D,41.!Chis!appro>i)ates!a! 95%!confidence!inter-al!for!the!location!of!the!lin/ed!candidate!gene!and!co-ers!

appro>i)atel+!5!.Bp.!Che!genes!located!within!the!region! areM!Ata>in!$inding!'rotein!1!?A2$'1A#!chro)oso)e!1E! open!reading!fra)e!EG!?;1ED6FEGA#!F4a)inoBut+rate! a)inotransferase!?A$ACA#!trans)e)Brane!protein!1GE! ?C.@.1GEA#!phospho)anno)utase!2!?'..2A#!calciu)! regulated!heat!staBle!protein!1#!2F!/,a!?;A6(8'1A#! uBiHuitin!specific!peptidase!7!?98'7A#!chro)oso)e!1E! open!reading!fra)e!72!?;1ED6F72A!and!gluta)ate!receptor#!ionotropic#!<4)eth+l!,4 aspartate!2A!?*6=<2AA. doi:10.1371/journal.pone.0019095.g002 #o0nload: 'ower'oint!slide!a!larger!i)age!?FE0$! '<*A!a!original!i)age!?1050$!C=FFA!

Table 2' "enetic association in the trio cohort' doi:10.1371/journal.pone.0019095.t002 #o0nload: 'ower'oint!slide!a!larger!i)age! ?1250$!'<*A!a!original!i)age!?F720$!C=FFA! Che!genetic!association!we!identified!for!rs1EF1021!could! originate!fro)!A$AC#!or!depending!on!the!3in/age!,iseHuiliBriu)!?3,A!properties!of! the!region#!fro)!a!gene!located!nearB+.!1e!therefore!constructed!a!3,!)ap!fro)!the!trios! used!in!this!stud+!?Figure!3A.!Chis!)ap!shows!that!rs1EF1021!lies!outside!an!3,!Bloc/! which!co-ers!the!3Z!end!of!A$AC!together!with!the!neighBoring!genes!C.@.1GE#! '..2!and!;A6(8'1.!Che!3,!structure#!surrounding!the!associated!8<'!rs1EF1021#! i)plies!that!this!)ar/er!is!lin/ed!with!a!nearB+!located!true!disease!associated!)utation.! 1e!therefore!conclude!that!the!association!seen!for!rs1EF1021!originates!fro)!A$AC.

Figure 3' *# map o) the region containing A1AT' Che!associated!8<'#!rs1EF1021!is!located!adjacent!to!a!3,!Bloc/!co-ering!the!3Z!part!of! A$AC!together!with!C.@.1GE#!'..2!and!;A6(8'1. doi:10.1371/journal.pone.0019095.g003 #o0nload: 'ower'oint!slide!a!larger!i)age!?2290$!'<*A!a!original!i)age! ?F270$!C=FFA! "enetic association analysis o) the adult case control cohort

=n!an!effort!to!replicate!the!association!in!the!trio!cohort#!nine!8<'s!distriButed!o-er!the! A$AC!3,!Bloc/!were!genot+ped!in!the!adult!case!control! cohort!?CaBle!2A.!Che!associated!8<'!in!the!trio!cohort! ?rs1EF1021A!reached!a!no)inal!p!I!0.3F!?no)inal!p!-alue! Between!0.2F!and!0.9F!for!the!other!8<'sA.!<one!of!the! genot+ped!8<'s!showed!an!indication!for!association.! Che!allele!freHuenc+!of!rs1EF1021!did!not!differ!Between! the!trio!cohort!?F1%A#!the!controls!in!the!case4control! cohort!?F5%A!or!the!freHuenc+!puBlished!for!;aucasians!in! the!(ap)ap!project!?F2.F%A!RFES.!Che!statistical!power! was!calculated!in!this!cohort!B+!esti)ating!the!ris/!ratio!in! trios!for!the!)inor!-ersus!)ajor!allele!to!1.E7!?122/73A.!Assu)ing!a!)inor!allele! freHuenc+!of!0.F!in!controls#!a!ris/!ratio!of!1.5#!and!that!250!cases!and!F75!controls!ha-e! Been!successfull+!genot+ped#!the!power!to!replicate!the!rs1EF1021!association!in!the! adult!case4control!cohort!was!95%.!Che!power!was!G0%!to!detect!an!association!at!the! le-el!of!significance!0.0055!?$onferroni!correction!for!9!8<'sA. (e$uencing 1e!suBseHuentl+!seHuenced!the!entire!coding!seHuence!of!A$AC#!including!intron4e>on! splicing!Boundaries#!in!three!affected!and!three!health+!indi-iduals!fro)!each!of!the!two! fa)ilies!showing!lin/age!to!chro)oso)e!1Ep.!<o!a)ino!acid!changing!)utation!was! identified. T*E(R measurements in dogs =n!an!atte)pt!to!Better!understand!the!role!of!A$AC!in!*@6,#!we!assessed!the!effect!of! -igaBatrin#!a!selecti-e!irre-ersiBle!inhiBitor!of!A$AC#!on!C3@86s!in!dogs.!A!single!dose! of!-igaBatrin#!ad)inistered!2!h!?100!)g//gA!Before!the!start!of!the!e>peri)ents#!produced! a!statisticall+!significant!inhiBition!of!C3@86s!B+!30%K9.G%.!1hen!-igaBatrin!?100! )g//gA!was!ad)inistered!twice!?at!2!h!and!1E!hA!Before!the!e>peri)ent#!the!inhiBitor+! effect!was!further!increased!to!57.3%K11.F%!?Figure!FAA.!Che!a-erage!nu)Ber!of! C3@86s!in!all!control!e>peri)ents!was!9.9K0.5.!=n!addition#!-igaBatrin!produced!a! statisticall+!significant!reduction!of!the!nu)Ber!of!acid!reflu>!episodes!fro)!3.1K0.F! ?controlA!to!0.GK0.F!?100!)g//g!dosed!2!h!and!1E!hA!during!the!F5!)in!)easure)ent! period.!A!single!dose!of!-igaBatrin!produced!no!significant!reduction!of!reflu>!episodes! ?3K0.F!and!2K0.5!in!control!and!-igaBatrin!treated!ani)als#!respecti-el+A!?Figure!F$A.

Figure /' Analysis o) A1AT inhibition in dogs' =nhiBition!of!C3@86s!?AA!and!reflu>!episodes!?$A!in!dogs!after!ad)inistration!of!the! A$AC!specific!inhiBitor!YigaBatrin.!Ad)inistration!was!perfor)ed!2!hours!or!2!and!1E! hours!Before!)easure)ents.!@ach!group!consisted!of!si>!dogs.!C3@86!and!reflu>!e-ents!

are!presented!as!nu)Ber!of!e-ents/F5!)inutes.!'4-alues!were!calculated!using!paired!t4 test.!Dpen!Bo>es!represent!the!control!e>peri)ent#!filled!Bo>es!are!after!the! ad)inistration!of!YigaBatrin#!n.s.!denotes!not!significant. doi:10.1371/journal.pone.0019095.g00F #o0nload: 'ower'oint!slide!a!larger!i)age!?F50$!'<*A!a!original!i)age!?1150$! C=FFA!

#iscussion Top
1e!ha-e!in-estigated!the!genetic!predisposition!to!*@6,!using!three!different!patient! cohorts.!1e!started!B+!perfor)ing!a!lin/age!anal+sis!in!a!collection!of!3E!fa)ilies!with! the!ai)!to!identif+!Both!general!and!fa)il+!specific!lin/ed!chro)oso)al!regions.!Chese! fa)ilies!were!selected!for!the!presence!of!*@6,!in!e-er+!generation#!in!a!do)inant! fashion#!to!)a>i)iPe!the!genetic!signal.!1e!anal+Ped!the!lin/age!data!Both!collecti-el+! ?as!pre-iousl+!puBlished!in!R17SA!and!fa)il+wise!and!it!Beca)e!clear!to!us!fro)!this! anal+sis#!where!we!de)onstrate!lin/age!to!fi-e!different!chro)oso)al!regions#!that! *@6,!is!a!disease!with!)an+!different!genetic!predispositions. =n!the!re)ainder!of!this!wor/!we!focused!on!the!two!fa)ilies!that!showed!lin/age!to!the! sa)e!region!on!chro)oso)e!1E.!Chese!lin/ages!were!identical!to!the!resolution!pro-ided! B+!)icrosatellite!)apping!and!although!the!region!was!relati-el+!s)all#!it!contained!nine! genes. Co!in-estigate!if!an+!of!these!nine!genes!were!geneticall+!associated!with!*@6,!we!used! a!separate!patient!collection!with!sufficient!resolution!to!allow!)apping!of!single!genes.! 1e!genot+ped!EE!8<'s!distriButed!o-er!the!nine!genes!in!a!pediatric!*@6,!trio!cohort.! Appl+ing!)ultiple!testing!adjust)ents!using!per)utations!anal+sis!we!identified!a! significant!association!in!A$AC#!But!not!in!an+!of!the!other!genes#!showing!that!A$AC!is! a!*@6,!associated!gene!in!our!pediatric!*@6,!population.!Che!cohort!consists!of!)ore! )ales!than!fe)ales!But!we!cannot!detect!a!gender!Bias!in!the!association.!1e!atte)pted!to! replicate!the!genetic!association!in!A$AC!using!an!adult!8wedish!*@6,!case4control! cohort!B+!testing!the!associated!A$AC!8<'!together!with!surrounding!8<'s!But!found! no!e-idence!for!genetic!association.!8tatistical!power!esti)ates!do!not!e>plain!the!lac/!of! replication!in!this!cohort. Chis!aBsence!of!association!)a+!Be!due!to!the!fact!that!the!proBand!in!the!Australian! fa)ilies!were!pediatric!cases#!the!trio!cohort!is!also!pediatric!and!collected!in!Australia! But!the!8wedish!case4control!cohort!consists!of!adult!*@6,!cases.!Further)ore#!the! population!histor+!clearl+!differs!Between!the!two!cohorts.!Che!adult!case4control!cohort! is!a!)i>ed!8wedish!and!Finnish!)aterial!with!8aa)i!influence!while!the!trio!cohort!is! )ainl+!Anglo4;eltic.!=n!our!pre-ious!puBlication#!we!de)onstrated!that!;D33A1!alleles! were!reciprocall+!associated!with!*@6,!?see!taBle!2!in!R17SA!i)plicating!that!*@6,! )utations!ha-e!arisen!separatel+!in!these!two!different!patient!cohorts.!Dur!conclusion!is! that!A$AC!alleles!are!not!present!as!genetic!ris/!factors!for!*@6,!in!the!8wedish!adult! case!control!cohort.!(owe-er#!the!statisticall+!significant!association!in!the!trio!cohort!

clearl+!shows!that!one!pol+)orphic!-ariant!within!A$AC!is!associated!to!*@6,.!Fro)! our!seHuencing!results!we!de)onstrate!that!the!open!reading!fra)e!of!the!A$AC!gene!is! intact.!Che!few!/nown!disease!causing!)utations!in!the!A$AC!protein!cause! ps+cho)otor!retardation!and!earl+!death!RF7S#!RFGS.!'erhaps!se-ere!)utations!are!not!to! Be!e>pected!in!a!disease!such!as!*@6,.!.ore!li/el+#!a!*@6,!associated!)utation!in! A$AC!)a+!Be!e>pected!to!Be!located!in!non4coding!seHuences!such!as!regulator+! ele)ents!or!non!coding!6<As#!as!has!recentl+!Been!discussed!in!the!literature!regarding! co)ple>!diseases!RF9S. ,ue!to!A$ACs!predo)inant!e>pression!in!neuronal!tissues!it!was!not!possiBle!to! in-estigate!the!function!of!A$AC!in!*@6,!patients!Because!suitaBle!sa)ples!were! unoBtainaBle!to!us.!=nstead#!we!in-estigated!how!A$AC!perturBation!)a+!lead!to!the! de-elop)ent!of!*@6,!through!inhiBition!of!A$AC!function!in vivo!in!dogs.! Ad)inistration!of!the!A$AC!specific!inhiBitor!-igaBatrin!significantl+!inhiBited!C3@86s! and!reflu>!episodes!in!dogs.!Chis!result!i)plies!that!hu)ans!carr+ing!disease!associated! A$AC!alleles!)a+!suffer!fro)!an!increased!nu)Ber!of!reflu>!episodes!due!to! d+sregulation!of!the!3@8. A$AC!catal+ses!the!first!step!in!the!degradation!of!*A$A.!=nhiBition!of!A$AC!leads!to! ele-ated!le-els!of!*A$A!in!the!s+naptic!junctions!causing!increased!*A$A4)ediated! signaling!through!the!*A$A!receptors!R50S.!Che!neurotrans)itter!*A$A!is!in-ol-ed!in! the!control!of!the!3@8!R51S.!8ignaling!through!Both!*A$AA!R52S!and!especiall+!*A$A$! receptors!reduces!C3@86s#!as!shown!with!*A$A$!receptor!agonists!R53S.!C3@86s!are! the!)ajor!)otilit+!factor!underl+ing!*@6,!R5FS!and!these!rela>ations!result!in!an! increased!freHuenc+!of!reflu>!episodes!R55S!thereB+!causing!esophageal!)ucosal!da)age. Che!pre-iousl+!puBlished!association!Between!;D33A1!and!*@6,!R17S!represents!a! disease!)echanis)!presu)aBl+!in-ol-ing!tissue!-ulneraBilit+!of!the!esophagus.!Che! A$AC!association!we!present!here!indicates!that!*@6,!in!addition!has!a!neuronal! disease!co)ponent. Dur!results!leading!to!the!identification!of!A$AC!are!in!line!with!the!ongoing!efforts!to! de-elop!antireflu>!drugs!to!treat!*@6,.!Chese!drugs!are!*A$A!analogs!specificall+! directed!against!the!*A$A$!receptor!R53S.!Dur!findings!support!the!in-ol-e)ent!of! *A$A!with!*@6,.!Chis!raises!the!Huestion!of!whether!other!*A$A!signaling! co)ponents!such!as!transporters!and!receptors!are!associated!with!*@6,!as!well. =n!conclusion#!we!show!that!a!pol+)orphic!-ariant!within!the!A$AC!gene!is!associated! with!*@6,!and!that!A$AC!inhiBition!leads!to!a!reduction!of!C3@86s!and!acid!reflu>! e-ents.!Dur!results!raise!the!possiBilit+!of!treating!*@6,!B+!inhiBiting!A$AC!function.

Ac,no0ledgments Top
1e!are!grateful!to!all!the!*@6,!patients!and!their!relati-es!for!participating!in!this!stud+.! 1e!than/!F=68A!?Friends!of!=nfant!6eflu>!8upport!AssociationA!in!8outh!Australia!for! their!support.!1e!also!than/!3ena!$rbndLn!for!e>pert!help!in!running!the!dog!

e>peri)ents.!1e!than/!;hristopher!3elliott!for!reading!the!)anuscript!and!pro-iding! critical!co))ents.

Author Contributions Top

;oncei-ed!and!designed!the!e>peri)ents:!.34F!*,!'(!A3! . ! !$A!.0!A1!3A.! 'erfor)ed!the!e>peri)ents:! !$A! . !A1!.0.!Anal+Ped!the!data:! !$A!.0!A1! . .!;ontriButed!reagents/)aterials/anal+sis!tools:!.34F!A3!*,!'(! . !3A.!1rote!the! paper:! !$A!A1!.0!A3! . !*,!'(!3A.

Re)erences Top
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12. *ilger!.A#!@l48erag!($#!*old!$,#!,ietrich!;3#!Csou!Y#!et!al.!?200GA!'re-alence! of!endoscopic!findings!of!erosi-e!esophagitis!in!children:!A!population4Based! stud+.! !'ediatr!*astroenterol!<utr!F7?2A:!1F1Q1FE.!Find!this!article!online! 13. (u!F5#!'reston!6A#!'ost! ;#!1hite!* #!0i/uchi!31#!et!al.!?2000A!.apping!of!a! gene!for!se-ere!pediatric!gastroesophageal!reflu>!to!chro)oso)e!13H1F.! A.A! 2GF?3A:!325Q33F.!Find!this!article!online! 1F. ;a)eron!A #!3agergren! #!(enri/sson!;#!<+ren!D#!3oc/e!*6#!3rd#!et!al.!?2002A! *astroesophageal!reflu>!disease!in!)onoP+gotic!and!diP+gotic!twins.! *astroenterolog+!122?1A:!55Q59.!Find!this!article!online! 15. .oha))ed!=#!;her/as!3F#!6ile+!8A#!8pector!C,#!Crudgill!< !?2003A!*enetic! influences!in!gastro4oesophageal!reflu>!disease:!A!twin!stud+.!*ut!52?GA:!10G5Q 10G9.!Find!this!article!online! 1E. 3e)Bo!A#!5a)an!.#! ones!.#!Calle+!< !?2007A!=nfluence!of!genetics!on!irritaBle! Bowel!s+ndro)e#!gastro4oesophageal!reflu>!and!d+spepsia:!A!twin!stud+.!Ali)ent! 'har)acol!Cher!25?11A:!13F3Q1350.!Find!this!article!online! 17. Asling!$#! irholt! #!(a))ond!'#!0nutsson!.#!1alentinsson!A#!et!al.!?2009A! ;ollagen!t+pe!===!alpha!=!is!a!gastro4oesophageal!reflu>!disease!susceptiBilit+!gene! and!a!)ale!ris/!factor!for!hiatus!hernia.!*ut!5G?GA:!10E3Q10E9.!Find!this!article! online! 1G. ,ent! #!,odds!1 #!Fried)an!6(#!8e/iguchi!C#!(ogan!1 #!et!al.!?19G0A! .echanis)!of!gastroesophageal!reflu>!in!recu)Bent!as+)pto)atic!hu)an! suBjects.! !;lin!=n-est!E5?2A:!25EQ2E7.!Find!this!article!online! 19. ,odds!1 #!,ent! #!(ogan!1 #!(el)! F#!(auser!6#!et!al.!?19G2A!.echanis)s!of! gastroesophageal!reflu>!in!patients!with!reflu>!esophagitis.!<!@ngl! !.ed! 307?25A:!15F7Q1552.!Find!this!article!online! 20. .ittal!60#!.c;allu)!61!?19GGA!;haracteristics!and!freHuenc+!of!transient! rela>ations!of!the!lower!esophageal!sphincter!in!patients!with!reflu>!esophagitis.! *astroenterolog+!95?3A:!593Q599.!Find!this!article!online! 21. .c.ahon!63#!Ali!A#!;he/an!@*#!;lar+!@.#!*arcia4Dria!. #!et!al.!?2002A!A! canine!)odel!of!gastroesophageal!reflu>!disease!?*@6,A.!8urg!@ndosc!1E?1A:! E7Q7F.!Find!this!article!online! 22. 8taunton!@#!8)id!8,#!,ent! #!$lac/shaw!3A!?2000A!Criggering!of!transient!3@8! rela>ations!in!ferrets:!6ole!of!s+)pathetic!pathwa+s!and!effects!of!Baclofen.!A)! ! 'h+siol!*astrointest!3i-er!'h+siol!279?1A:!*157QE2.!Find!this!article!online! 23. 3eh)ann!A!?200GA!<o-el!treat)ents!of!*@6,:!Focus!on!the!lower!esophageal! sphincter.!@ur!6e-!.ed!'har)acol!8ci!12:!8uppl!1103Q110.!Find!this!article! online! 2F. 'rice!;!?1999A!Australian!population:!@thnic!origins.!'eople!'lace!7?FA:!12Q1E.! Find!this!article!online! 25. <+lander!'D#!$ec/)an!3!?1991A!'opulation!studies!in!northern!sweden.!_Y==.! esti)ates!of!finnish!and!saa)ish!influence.!(u)!(ered!F1?3A:!157Q1E7.!Find!this! article!online! 2E. C+tgat!*!?1990A!@ndoscop+!of!the!esophagus.!=n:!;otton!'#!C+tgat!*#!1illia)s!;#! editors.!Annual!of!*astrointestinal!@ndoscop+.!3ondon:!;urr!8ci!3C,.!pp.!15Q2E.!

27. Yieira!.;#!'isani! ;#!.ulinari!6A!?200FA!,iagnosis!of!reflu>!esophagitis!in! infants:!(istolog+!of!the!distal!esophagus!)ust!co)ple)ent!upper!gastrointestinal! endoscop+.! !'ediatr!?6io! A!G0?3A:!197Q202.!Find!this!article!online! 2G. 0nuff!C@#!$enja)in!8$#!1orsha)!*F#!(ancoc/! @#!;astell!,D!?19GFA! (istologic!e-aluation!of!chronic!gastroesophageal!reflu>.!an!e-aluation!of!Biops+! )ethods!and!diagnostic!criteria.!,ig!,is!8ci!29?3A:!19FQ201.!Find!this!article! online! 29. 3eape!33#!$han!=#!6a)enofs/+!.3!?19G1A!@sophageal!Biops+!in!the!diagnosis!of! reflu>!esophagitis.! !'ediatr!8urg!1E?3A:!379Q3GF.!Find!this!article!online! 30. Aro!'#!6on/ainen! #!8tors/ruBB!C#!$olling48terne-ald!@#!;arlsson!6#!et!al.!?200FA! Yalid!s+)pto)!reporting!at!upper!endoscop+!in!a!rando)!sa)ple!of!the!swedish! adult!general!population:!Che!/ali>anda!stud+.!8cand! !*astroenterol!39?12A:! 12G0Q12GG.!Find!this!article!online! 31. 6on/ainen! #!Aro!'#!8tors/ruBB!C#! ohansson!8@#!3ind!C#!et!al.!?2005A!(igh! pre-alence!of!gastroesophageal!reflu>!s+)pto)s!and!esophagitis!with!or!without! s+)pto)s!in!the!general!adult!swedish!population:!A!/ali>anda!stud+!report.! 8cand! !*astroenterol!F0?3A:!275Q2G5.!Find!this!article!online! 32. 03D''!;C!?1951A!;er-ical!esophagosto)+.! !Chorac!8urg!21?5A:!F90QF91.!Find! this!article!online! 33. $rownstein!. #!;arpten! ,#!8)ith! 6!?199EA!.odulation!of!non4te)plated! nucleotide!addition!B+!taH!,<A!pol+)erase:!'ri)er!)odifications!that!facilitate! genot+ping.!$ioCechniHues!20?EA:!100FQE#!100GQ10.!Find!this!article!online! 3F. 3eh)ann!A#!Antonsson!.#!$re)ner4,anielsen!.#!Flardh!.#!(ansson4$randen! 3#!et!al.!?1999A!Acti-ation!of!the!*A$A?$A!receptor!inhiBits!transient!lower! esophageal!sphincter!rela>ations!in!dogs.!*astroenterolog+!117?5A:!11F7Q115F.! Find!this!article!online! 35. D:;onnell! 6#!1ee/s!,@!?199GA!'ed;hec/:!A!progra)!for!identification!of! genot+pe!inco)patiBilities!in!lin/age!anal+sis.!A)! !(u)!*enet!E3?1A:!259Q2EE.! Find!this!article!online! 3E. 1igginton! @#!ABecasis!*6!?2005A!'@,8CAC8:!,escripti-e!statistics#!graphics! and!Hualit+!assess)ent!for!gene!)apping!data.!$ioinfor)atics!21?1EA:!3FF5Q 3FF7.!Find!this!article!online! 37. ABecasis!*6#!;hern+!88#!;oo/son!1D#!;ardon!36!?2002A!.erlin44rapid! anal+sis!of!dense!genetic!)aps!using!sparse!gene!flow!trees.!<at!*enet!30?1A:!97Q 101.!Find!this!article!online! 3G. 8oBel!@#!3ange!0!?199EA!,escent!graphs!in!pedigree!anal+sis:!Applications!to! haplot+ping#!location!scores#!and!)ar/er4sharing!statistics.!A)! !(u)!*enet! 5G?EA:!1323Q1337.!Find!this!article!online! 39. 8oBel!@#!8engul!(#!1ee/s!,@!?2001A!.ultipoint!esti)ation!of!identit+4B+4 descent!proBaBilities!at!arBitrar+!positions!a)ong!)ar/er!loci!on!general! pedigrees.!(u)!(ered!52?3A:!121Q131.!Find!this!article!online! F0. 8oBel!@#!'app! ;#!3ange!0!?2002A!,etection!and!integration!of!genot+ping!errors! in!statistical!genetics.!A)! !(u)!*enet!70?2A:!F9EQ50G.!Find!this!article!online! F1. $arrett! ;#!Fr+!$#!.aller! #!,al+!. !?2005A!(aplo-iew:!Anal+sis!and! -isualiPation!of!3,!and!haplot+pe!)aps.!$ioinfor)atics!21?2A:!2E3Q2E5.!Find!this! article!online!

F2. 0ong!A#!*udBjartsson!,F#!8ainP! #! onsdottir!*.#!*udjonsson!8A#!et!al.!?2002A! A!high4resolution!reco)Bination!)ap!of!the!hu)an!geno)e.!<at!*enet!31?3A:! 2F1Q2F7.!Find!this!article!online! F3. 0rugl+a/!3#!,al+!. #!6ee-e4,al+!.'#!3ander!@8!?199EA!'ara)etric!and! nonpara)etric!lin/age!anal+sis:!A!unified!)ultipoint!approach.!A)! !(u)!*enet! 5G?EA:!13F7Q13E3.!Find!this!article!online! FF. 0rugl+a/!3#!3ander!@8!?199GA!Faster!)ultipoint!lin/age!anal+sis!using!fourier! transfor)s.! !;o)put!$iol!5?1A:!1Q7.!Find!this!article!online! F5. 3ander!@#!0rugl+a/!3!?1995A!*enetic!dissection!of!co)ple>!traits:!*uidelines!for! interpreting!and!reporting!lin/age!results.!<at!*enet!11?3A:!2F1Q2F7.!Find!this! article!online! FE. Altshuler!,.#!*iBBs!6A#!'eltonen!3#!Altshuler!,.#!et!al.!=nternational!(ap.ap! 3!;onsortiu)!?2010A!=ntegrating!co))on!and!rare!genetic!-ariation!in!di-erse! hu)an!populations.!<ature!FE7?7311A:!52Q5G.!Find!this!article!online! F7. ae/en! #!;asaer!'#!de!;oc/!'#!;orBeel!3#!@ec/els!6#!et!al.!?19GFA!*a))a4 a)inoBut+ric!acid4transa)inase!deficienc+:!A!newl+!recogniPed!inBorn!error!of! neurotrans)itter!)etaBolis).!<europediatrics!15?3A:!1E5Q1E9.!Find!this!article! online! FG. .edina40auwe!30#!CoBin!A #!,e!.eirleir!3#! ae/en! #! a/oBs!;#!et!al.!?1999A!F4 a)inoBut+rate!a)inotransferase!?*A$A4transa)inaseA!deficienc+.! !=nherit! .etaB!,is!22?FA:!F1FQF27.!Find!this!article!online! F9. 0u!;8#!3o+!@d#!'awitan!d#!;hia!08!?2010A!Che!pursuit!of!geno)e4wide! association!studies:!1here!are!we!nowc! !(u)!*enet!55?FA:!195Q20E.!Find!this! article!online! 50. ung!. #!3ippert!$#!.etcalf!$1#!$ohlen!'#!8chechter!' !?1977A!*a))a4-in+l! *A$A!?F4a)ino4he>454enoic!acidA#!a!new!selecti-e!irre-ersiBle!inhiBitor!of! *A$A4C:!@ffects!on!Brain!*A$A!)etaBolis)!in!)ice.! !<euroche)!29?5A:!797Q G02.!Find!this!article!online! 51. ;antu!'#!;ar)agnola!8#!8a-ojardo!,#!Allocca!.#!'enagini!6!?2003A!@ffect!of! non4selecti-e!ga))a4a)inoBut+ric!acid!receptor!sti)ulation!on!)otor!function!of! the!lower!oesophageal!sphincter!and!gastro4oesophageal!reflu>!in!health+!hu)an! suBjects.!Ali)ent!'har)acol!Cher!1G?7A:!E99Q70F.!Find!this!article!online! 52. $eau)ont!(#! onsson46+lander!A;#!;arlsson!0#!'ierrou!8#!Ahlefelt!.#!et!al.! ?200GA!Che!role!of!*A$A?AA!receptors!in!the!control!of!transient!lower! oesophageal!sphincter!rela>ations!in!the!dog.!$r! !'har)acol!153?EA:!1195Q1202.! Find!this!article!online! 53. 3eh)ann!A!?2009A!*A$A$!receptors!as!drug!targets!to!treat!gastroesophageal! reflu>!disease.!'har)acol!Cher!122?3A:!239Q2F5.!Find!this!article!online! 5F. (ollowa+!6(!?2000A!Che!anti4reflu>!Barrier!and!)echanis)s!of!gastro4 oesophageal!reflu>.!$aillieres!$est!'ract!6es!;lin!*astroenterol!1F?5A:!EG1QE99.! Find!this!article!online! 55. ,ent! #!(ollowa+!6(#!Coouli! #!,odds!1 !?19GGA!.echanis)s!of!lower! oesophageal!sphincter!inco)petence!in!patients!with!s+)pto)atic! gastrooesophageal!reflu>.!*ut!29?GA:!1020Q102G.!Find!this!article!online!

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