Advances in the Understanding of Gluten related

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Geneti csofCel i ac Di sease.HLA and NonHLA Genes Let i ci aPl azaI zur i et a,Nor aFer nandezJi menez, Jos eRamonBi l bao Dpt .ofGenet i cs ,Phys i calAnt hr opol ogy and Ani malPhys i ol ogy, Bi oCr uces Heal t h Res ear c hI ns t i t ut e,Uni ver s i t y oft he Bas que Count r yUPV/EHU,Lei oa,Bas queCount r y,Spai n. I mmunogenet i cs . l et @gmai l . com,j os er amon. bi l bao@ehu. es Doi :ht t p: //dx. doi . or g/10. 3926/oms . 249 How to ci te thi schapter Pl az aI z ur i e t a L,Fe r nande z Ji me ne z N,Bi l bao JR.Ge ne t i c s ofCe l i ac Di s e as e .HLA andNonHLA Ge ne s .I n Ar r anzE,Fe r nánde z Bañar e sF, Ros e l lCM,Rodr i goL,Pe ñaAS,e di t or s .Adv anc e si nt heUnde r s t andi ng ofGl ut e n Re l at e d Pat hol o g y and t he Ev ol ut i on ofGl ut e nFr e e Fo ods . Bar c e l ona,Spai n:Omni aSc i e nc e ;2015.p.79104.

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L.Pl az aI z ur i e t a,N.Fe r nande z Ji me ne z ,J. R.Bi l b ao

A bstract Al t hough t hemodeofi nher i t anceofcel i acdi s eas ei ss t i l lunknown, i thasbeen known f ora l ong t i me t hatGenet i cspar t i ci pat esi nt he s us cept i bi l i t yt ot he di s eas e. St udi es on t he pr eval ence of CD i n af f ect ed f ami l i es ,and es peci al l yt hos ecompar i ngt wi n pai r s ,havebeen ver y us ef ult o es t i mat et he pr opor t i on i n whi c h envi r onment aland genet i cf act or scont r i but et ot hedevel opmentoft hi scompl exdi s or der . Accor di ng t ot hes es t udi es ,Genet i csi sa f undament alpl ayerbot hi n t het r i gger i ngandi nt hel at t erdevel opmentofCD. I n gener al ,i ti swel laccept ed t hatt hepr opor t i on ofmonozygot i cor i dent i calt wi nsconc or dantf orCD i sar ound 7586%,whi l ei nt hecas e of di zygot i c t wi ns ,t hi s pr opor t i on i sr educed t o 1620%. Thi s di f f er encebet ween mono-and di zygot i ct wi nshasal l owed s ci ent i s t st o es t i mat et hegenet i ccomponentofCD,whi c hi shi ghert han whathas been cal cul at edf orot heri mmunol ogi calcompl exdi s eas es ,s uc hast ype 1di abet es( T1D)( ar ound 30% concor dancei n monozygot i cand 6% i n 1 di zygot i ct wi ns ) .Mor eover ,concor dance r at esbet ween s i b pai r sand

di zygot i ct wi nsar eal mos tt hes ame,i ndi cat i ngt hatt heenvi r onment al componenthasa mi ni mum cont r i but i on t ot her i s k ofdevel opi ng CD. I ns ummar y,accumul at ed evi dences ugges t st hatCD hasaver ys t r ong genet i ccomponentand i thasbeen cal cul at ed t hatt he her i t abi l i t y of t hi sdi s eas e( pr opor t i onoft her i s kofs uf f er i ngf r om CD at t r i but abl et o genet i cf act or s ,compar ed t o envi r onment aldet er mi nant s )i s ar ound 87%2.Thel ar ges tpor t i onoft hegenet i cr i s kt odevel opCD comesf r om t he pr es ence of cer t ai n Human Leucocyt e Ant i gen ( HLA) al l el es . However ,even i ft he r ol e oft hes e HLA mol ecul esi ses s ent i ali nt he pat hogenes i s of t he di s eas e, t hei r cont r i but i on t ot he her edi t y i s 80

Ge ne t i c sofCe l i acDi s e as e .HLA andNonHLA Ge ne s

modes t ,and t hus ,i thasbeen hypot hes i zed on t heexi s t enceofmany s mal lef f ect ,nonHLA s us cept i bi l i t yl oci .

Keywords Cel i ac di s eas e, aut oi mmune di s eas e, i mmunemedi at ed di s eas e, HLA, l i nkage s t udi es ,genomewi de as s oci at i on s t udi es ( GWAS) ,gene expr es s i on, pat hwayanal ys i s .

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1.HLA Regi on and Cel i ac Di sease 1. 2.HLA Regi on HLA i st he name f or t he Mayor Hi s t ocompat i bi l i t y Compl ex ( MHC) i n humans ;i ti sa s uper l ocus l ocat ed on t he c hr omos omalr egi on 6p21 and cont ai nsal ar genumberofgenesr el at ed t ot hei mmuner es pons e.HLA genes encode ant i gen pr es ent i ng pr ot ei ns t hat ar e expr es s ed i n mos t human cel l s and ar e es s ent i alf ort he abi l i t y oft he or gani s m t o di s t i ngui s h bet ween s el f andf or ei gnmol ecul es . HLA genesar ei nvol ved i n many i nf l ammat or y and aut oi mmunedi s or der s and al s ocont r i but et ot hes us cept i bi l i t yt odevel op i nf ect i ousdi s eas ess uc h as AI DS ormal ar i a.However ,duet ot hehi gh genet i ccompl exi t y oft her egi on, mos toft hepar t i cul argenet i cf act or sand pat hogeni cmec hani s msunder l yi ng t hes us cept i bi l i t yt oeac hoft hes edi s or der sr emai nunknown.I nf act ,t heHLA r egi on pr es ent st he hi ghes t geni c dens i t y oft he ent i r e genome and a ver y s t r onggeneexpr es s i ons eemst obef avor ed3.

1. 2. Contri buti on to the Geneti c Ri sk and Suscepti bi l i ty Genes As pr evi ous l y ment i oned, t he HLA r egi on i s t he mos t i mpor t ant s us cept i bi l i t yl ocusi n CD and expl ai nsar ound 40% oft hegenet i ccomponent oft hedi s eas e.Thef i r s tevi dences uppor t i ngt heas s oci at i onbet weenHLA and 4 CD waspubl i s hed i n 1973 and wasdet ect ed us i ng s er ol ogi calmet hods .Due

t ot he s t r ong l i nkage di s equi l i br i um pr es ent i n t he ar ea, i ni t i al s t udi es i dent i f i ed HLAA1,HLAB8 and HLADR3 ast heet i ol ogi calvar i ant si nt he r egi on, but s ubs equent mol ecul ar s t udi es have r eveal ed t hat t he f act or s di r ect l yi mpl i cat ed ar et heHLA cl as sI Igenesencodi ng bot h HLADQ2 and DQ8 mol ecul es ( Fi gur e 1) .The s t r onges t as s oci at i on has been f ound wi t h HLADQ2, and 90% of cel i ac pat i ent s pr es ent at l eas t one copy of t he HLADQ2. 5 het er odi mer ( f or med by t he combi nat i on of t he pr oduct s of DQA1*05 and DQB1*02 al l el es ,t hat encode t he a and b c hai ns of t he 82


het er odi mer ,r es pect i vel y) . On t he ot her hand, 2030% of t he noncel i ac popul at i on al s o pr es ent st hi s HLADQ2 var i ant ,maki ng i t cl ear t hat ,even t houghi ti sver yi mpor t ant ,i ti snots uf f i ci entt odevel op t hedi s eas e.Mos tof t hepat i ent swhodonotcar r yt heHLADQ2genot ypear eHLADQ8car r i er s and s o haveatl eas tonecopy oft hehapl ot ypecont ai ni ng DQA1*03: 01 and 5 DQB1*03: 02 al l el es .A ver ys mal lpor t i on oft he pat i ent s ar e negat i ve f or

bot h DQ2 and DQ8, but i t has been obs er ved t hat i nt hes ef ew cas es , i ndi vi dual spr es entatl eas toneoft het woal l el esencodi ngt heDQ2mol ecul e 6, 7 ( DQA1*05orDQB1*02) .

Fi g ur e 1.As s o c i at i on oft he HLA l o c us wi t h CD.HLADQ2 mol e c ul ei st he maj or f ac t or c onf e r r i ngr i s kt oCD.Mos tc e l i acp at i e nt se x pr e s st hehe t e r o di me rHLADQ2. 5,e nc o de db yt he al l e l e sHLADQA1*05( c hai n)andHLADQB1*02( c hai n) ,t hatc anb epr e s e nti nc i si nt he DR3DQ2 hapl ot y p e or i n t r ans ,i nt he he t e r oz y g ot e s DR5DQ7 and DR7DQ2. 2. The HLADQ2. 2 di me r ,a v ar i ant ofHLADQ2 e nc o de db yt he al l e l e s HLADQA1*02: 01 and HLADQB1*02: 02,c onf e ra l ow r i s kt o de v e l op t he di s e as e .Mos toft he p at i e nt st hatar e 8 ne g at i v ef orDQ2e x pr e s sHLADQ8,e nc o de db yt heDR4DQ8hapl ot yp e .

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HLADQ2 and DQ8 var i ant sar ei nl i nkagedi s equi l i br i um wi t h DR3 and DR4,r es pect i vel y.Thus ,weof t enr ef ert ot hes er i s kvar i ant sasDR3DQ2and 9 DR4DQ8hapl ot ypes .I ns ever alhapl ot ypes ,asi st hecas eofDR3DQ2,t he

t wo al l el es oft he HLADQ2. 5 het er odi mer ( DQA1*05: 01 and DQB1*02: 01) ar el ocat ed i nt he s ame c hr omos ome and t her ef or e,encoded i n ci s .I nt he het er ozygous i ndi vi dual s car r yi ng DR5DQ7 and DR7DQ2 hapl ot ypes ,t he t wo mol ecul es t aki ng par ti nt he r i s k het er odi mer ar e encoded i n t r ans , becaus et hey ar el ocat ed i n di f f er entc hr omos omes .The di f f er encesbet ween t hes et wot ypesofHLADQ2. 5r el y on a s i ngl eami no aci d oft heDQa c hai n ( DQA1*05: 01 vs .DQA1*05: 05)and anot herr es i dueoft hemembr aner egi on oft he DQb c hai n( DQB1*02: 01 ver s usDQB1*02: 02) ,butt hey s eem nott o haveanyf unct i onalcons equencesand ar eas s oci at ed wi t has i mi l arr i s kef f ect . However ,t he r i s k conf er r ed by anot her HLADQ2 var i ant ,t he HLADQ2. 2 di mer ,i sver yl ow1,10. Ther ei sal s oar el at i ons hi p bet ween t hedegr eeofs us cept i bi l i t yt oCD and t he number of DQ2. 5 het er odi mer s . Homozygous i ndi vi dual s wi t h t wo DR3DQ2 hapl ot ypes as wel l as het er ozygous pat i ent s pr es ent i ng DR3DQ2/DR7DQ2 expr es st he hi ghes tl evel s ofDQ2. 5 het er odi mer s and t hus ,conf ert hemaxi mum genet i cr i s kt odevel op CD11-13.I nt hi ss ens e,i thas t o be ment i oned t hatpat i ent swi t hr ef r act or y CD ( t hos e notr es pondi ng t o GFD)pr es enta hi gherdegr eeofhomozygos i t yf orDR3DQ2 ( 4462%)t han ot hercel i acpat i ent s( 2024%) .A s i mi l ardos edependentef f ecthasal s o been s ugges t edf orDQ8mol ecul es . Apar tf r om t he genes encodi ng DQ mol ecul es ,t he HLA r egi on al s o cont ai nsmany ot hergenest hatpar t i ci pat et ot hei mmuner es pons eand t hat coul d cont r i but et ot hes us cept i bi l i t yt o CD.Sever als t udi eshavepos t ul at ed t hatpol ymor phi s msi n geness uc h asMI CA,MI CB orTNF coul d cont r i but e t ot hegenet i cr i s kt odevel op t hi sdi s or der .Nonet hel es s ,mos toft hes ewor ks have notpai d enough at t ent i on t ot he s t r ong l i nkage di s equi l i br i um among genesandr es ul t sar enotconcl us i ve.Deeps equenci ngandexhaus t i vemappi ng oft her egi on wi l lhel pt o det er mi newhet heri tcont ai nss us cept i bi l i t yf act or s ot her t han HLADQ.Al t hough HLA genes i mpor t ant l y cont r i but et ot he 84


genet i cs us cept i bi l i t y,t heconcor danceoft hedi s eas ei ns i bl i ngsi dent i calf or HLA genot ypeappr oac hesonl y30%,s ot hatwecan concl udet hatHLA genes ar ei mpor t antbutnots uf f i ci entt odevel opCD10.

1. 3.Rol e ofHLA i n the Pathogenesi sofCD Thes t r ongas s oci at i on oft heHLA cl as sI Igeneswi t hCD i sdi r ect l yl i nked t ot hef undament alr ol e ofCD4+ T l ymphocyt esi nt hepat hogenes i soft he di s eas e. I nf act , CD4+ T cel l st hat ar e abl et or ecogni ze gl ut ender i ved pept i desar epr es enti nt hei nt es t i nalmucos aofcel i acpat i ent s ,butnoti nt he cas e of heal t hy, noncel i ac i ndi vi dual s . W hen genet i cal l y s us cept i bl e i ndi vi dual sar eexpos ed t ocer t ai n gl ut ender i ved epi t opes ,t hey ar epr es ent ed by t he HLADQ2/HLADQ8 mol ecul eson t he s ur f ace ofant i gen pr es ent i ng 14 cel l s( APC) ,s t i mul at i ngt hepr ol i f er at i onofgl ut ens peci f i cCD4+ T cel l s .

An i mpor t antl andmar ki nt hemol ecul arbas i sunder l yi ng t heas s oci at i on bet ween HLA and CD wast he di s cover yt hatt he bi ndi ng capaci t y bet ween t he HLADQ2 and/orDQ8 and t he gl i adi n pept i desi ncr eas ess ubs t ant i al l y when t he l at t er have been enzymat i cal l y modi f i ed by t he enzyme t i s s ue t r ans gl ut ami nas et ype 2, or TG2. The enzyme cat al yzes a r eact i on t hat pr ovokes t he i ncr eas e of negat i ve c har ges i nt he gl ut ender i ved pept i des , f avor i ng t hei rbi ndi ng t o cer t ai n HLA mol ecul es( DQ2 and DQ8)and t hus , t r i gger i ngt hepr es ent at i onoft hes egl ut enpept i dest oCD4+ T cel l s . Gi ven t hei mpor t anceofHLA mol ecul esi nt heact i vat i on ofaut or eact i ve gl ut ens peci f i c T cel l s ,i ti s expect ed t hat any modi f i cat i on i nt hei rcodi ng s equencewi l lpr ovokeal t er at i onsi ndi f f er ents t epsoft hi spr oces s .I nt hi sway, pol ymor phi s msi nt hes equenceencodi ngt heant i genbi ndi ngs i t escoul d af f ect af f i ni t y, f avor i ng or hamper i ng t he r ecogni t i on of t he gl ut ender i ved 15 pept i des .On t he ot herhand,s ever alpol ymor phi s msl ocat ed i nr egul at or y

s i t escan r epr es sorenhancet heexpr es s i on oft heHLA mol ecul es ,r educi ngor augment i ngt hei mmuner es pons et ogl ut en.

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2.Search f or Geneti c Suscepti bi l i ty Genesi n CD Gi ven t hef actt hatHLA al onecan onl yexpl ai n ar ound 40% oft hegenet i c component of CD, l ar ge ef f or t s have been done t ol ocal i ze and i dent i f y nonHLA s us cept i bi l i t y genest hatcoul d cl ar i f yt hecompl ex genet i csoft hi s di s or der .Two havebeen t hemaj ors t r at egi esus ed wi t ht hi sai m:on t heone hand,l i nkages t udi esi n af f ect ed f ami l i es ,and on t heot herhand,as s oci at i on s t udi es bas ed on popul at i on s cr eeni ng. Mor er ecent l y, CD has al s o been s t udi ed us i ngGenomeW i deAs s oci at i on St udi es( GWAS) ,i n whi c ht hous ands of Si ngl e Nucl eot i de Pol ymor phi s ms ( SNP) have been genot yped and anal yzed.Thes es t udi eshaveal l owedust oi dent i f ys ever alas s oci at ed l oci ,but f unct i onals t udi eswi l lbeneeded t o conf i r m t hei mpl i cat i on oft hepr opos ed candi dat egenes .

2. 1.Li nkage Studi es Li nkages t udi esi nf ami l i eshaveal l owed t hei dent i f i cat i on ofc hr omos omal r egi ons whi c h ar er epeat edl y and cons i s t ent l y i nher i t ed by t he af f ect ed member s ofa f ami l yt hr ough s ever algener at i ons .Thus ,r egi ons pot ent i al l y r el evant t ot he devel opment oft he di s eas e can be s el ect ed and f enced i n. Genesl ocal i zed i nt hes er egi onsar econs i der ed pos i t i onalcandi dat es ,duet o t he f actt hati ti st hei rpos i t i on i nt he genome t hati sconf er r i ng t hem t he candi dat ei dent i t y. I n t he cas e of CD, apar tf r om t he HLA r egi on ( or CELI AC1)whi c h obvi ous l yi st hemos tcons i s t ent l yr epl i cat ed s i gnaland t he one s howi ng t he s t r onges tl i nkage di s equi l i br i um,t hr ee r egi ons cont ai ni ng pos i t i onalcandi dat es s uc h as a number ofi nt er l euki ns ,t he SPI NK f ami l y, CD28, CTLA4, I COS and MYO9B have been des cr i bed i nt he di f f er ent l i nkage s t udi es ( Fi gur e 2) .However ,even t hough cons i s t ent l yr epl i cat ed i n s ever als t udi es ,t he cer t ai n caus es of as s oci at i on wi t h CD have not been i dent i f i edf ort hes el i nkager egi ons .

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Fi g ur e2.Li nk ag er e g i onsr e pl i c at e di ndi f f e r e ntf ami l i e saf f e c t e db yCD16-18.

2. 2.Functi onalCandi date Genes The candi dat e gene appr oac hf or genet i c as s oci at i on s t udi es f ocus es on as s oci at i ons bet ween genet i c var i at i on wi t hi n pr es peci f i ed genes ofi nt er es t and phenot ypesordi s eas es t at es .Thi si si n cont r as tt o GWA s t udi es ,whi c h s can t he ent i r e genome f or common genet i c var i at i on.Candi dat e genes ar e mos t of t en s el ect ed f or s t udy bas ed on a pri ori knowl edge of t he gene' s bi ol ogi calf unct i onali mpacton t het r ai tordi s eas ei n ques t i on.Thi sappr oac h hasbeencommonl yus edi ncompl exdi s eas es t udi es ,andal s oi nCD. Mos t of t he candi dat e gene s t udi es t o dat e has f ocus ed on i mmune r es pons e,s i ncei ti sgener al l y accept ed t hatCD i sa T cel lmedi at ed di s eas e, i n whi c h gl i adi nder i ved pept i des ,ei t her i n nat i ve f or m or deami dat ed by t r ans gl ut ami nas e,act i vat el ami na pr opri ai nf i l t r at i ngT l ymphocyt es ,l eadi ng t o bot h Th1 and Th17 i nf l ammat or yr es pons es of t he adapt i ve i mmune s ys t em19. Thus , bot ht he mos t Th1 r es pons ec har act er i s t i c cyt oki ne I NFg 20 ( encodedbyI NFG) and I L23R t her ecept oroft hebes tknown i nt er l euki ni n

t he Th17 cas cade21-23 have been s t udi ed among many ot her s ,wi t h notmany s t r ongas s oci at i onevi dencesasconcl us i on. Dur i ng t he l as t decade,however ,a gr owi ng i nt er es t has f ocus ed on t he pos s i bl ei mpl i cat i on oft he i nnat ei mmune r es pons e,bas ed on t he f actt hat gl i adi n pept i desar eal s oabl et ot r i ggeranonTcel l dependentr es pons et hat coul d es t abl i s ht he pr oi nf l ammat or y envi r onment neces s ar yf or s ubs equent 87


Tcel lact i vat i on and t i s s uedes t r uct i on24.Di f f er enti nnat ei mmunegenesand genef ami l i eshavebeen pr opos ed asput at i ves us cept i bi l i t y candi dat est oCD s uc h as t he i nf l ammat or y medi at or sI L1A,I L1B,I L1RN,I L18,RANTES and MCP125, t he Ki l l er I mmunogl obul i nl i ke r ecept or ( KI R) f ami l y26, t he Tol l l i ke r ecept or ( TLR) f ami l y27,28 and t he s t r es s mol ecul es MI CA and MI CB29 butal t houghagener alact i vat i onoft hei nnat ei mmunes ys t em i swel l known t ooccuri n CD,noneoft hepr opos ed candi dat eshaves hown as t r ong as s oci at i onwi t ht hedi s eas e. Fi nal l y, f unct i onal pl ayer si nvol ved i nt he r emodel i ng of t he i nt es t i nal epi t hel i a and i nt he mai nt enance oft he ext r acel l ul armat r i x have al s o been pr opos ed asput at i ves us cept i bi l i t y genes ,butagai n,no as s oci at i on hasbeen conf i r medf oranyoft hem.

2. 3.Genom ewi de associ ati on and f ol l owup studi esi n CD Mi l l i ons of SNPs have been i dent i f i ed t hanks t ot he Human Genome s equenci ngpr oj ect s .Someoft hos eSNPs ,cal l ed t agSNPs ,havebeen us ed as genet i c mar ker si n GWAS and al l ow t he i dent i f i cat i on of t hous ands of s us cept i bi l i t y var i ant sf ormany compl ex di s eas es .Thet wo GWAS per f or med i nCD,t oget herwi t hs ever alf ol l owups t udi es ,r eveal edat ot alof26nonHLA 3032 as s oci at ed r egi ons . The mos tr ecent l ar ges cal e pr oj ect per f or med t o

i dent i f y var i ant s as s oci at ed wi t h CD and ot her aut oi mmune di s eas es i st he I mmunoc hi p Pr oj ect ,i n whi c h a dens er genot ypi ng of 186 GWAS l oci as s oci at ed wi t h 12 i mmuner el at ed di s eas es i dent i f i ed 13 addi t i onalr egi ons as s oci at edwi t hCD33. Hence, t her ei sa t ot al of 39 nonHLA r egi ons as s oci at ed wi t h CD, cont ai ni ng 57 i ndependent as s oci at i on s i gnal s . Ni net een of t hos er egi ons pi npoi ntt o as i ngl ecandi dat egene,butonl y 3as s oci at ed SNPsar el i nked t o pr ot ei nal t er i ng var i ant sl ocat ed i n exoni cr egi ons ,al t hough s omepot ent i al l y caus at i vegeneshavebeen pr opos ed duet ot heexi s t enceofs i gnal sneart he5’ or3’r egul at or yr egi ons ( Fi gur e3) . Even t hough mos t SNPs l ocal i ze t o nonpr ot ei n codi ng i nt er geni c and i nt r oni cr egi ons ,CD as s oci at ed var i ant ss eem t o be l ocat ed i n expr es s i on 88


quant i t at i vet r ai tl ocioreQTLs ,genomi cl ocit hatr egul at eexpr es s i on l evel s ofmRNAsorpr ot ei ns .W hen eQTLsmap t o a genomi cl ocat i on cl os et ot he r egul at ed genet heyar er ef er r ed t oasci s eQTLs ;i n cont r as t ,when t heeQTL mapsf arf r om t he gene ( even on di f f er entc hr omos ome) ,i ti sr ef er r ed t o as t r ans eQTL.Af t era met aanal ys i sofa genomewi de eQTL dat as etof1, 469 human whol e bl ood s ampl es ,s uppos ed t or ef l ect pr i mar yl eukocyt e gene expr es s i on,38genomewi deCD as s oci at ed nonHLA l ociwer eas s es s ed f orci s expr es s i ongenot ype cor r el at i on32.Twent ys i gni f i cant eQTLs wer ei dent i f i ed, mor et han expect ed by c hance,i ndi cat i ng t hat CD as s oci at ed r egi ons ar e gr eat l y enr i c hed f oreQTLs .Thes edat a may i ndi cat et hats omer i s k var i ant s coul d have an i nf l uence i n CD s us cept i bi l i t y by al t er i ng gene expr es s i on, however ,t her e ar e many evi dencesi ndi cat i ng t hatci s eQTLsdi f f erbet ween di f f er entt i s s uesandcanevenhavecompl et el yoppos i t eef f ect s . Ther eby,i ti si mpor t ant t o per f or m f unct i onalanal ys i s oft he pr opos ed candi dat e genesi nt he di s eas et i s s ue.The ei ghtas s oci at i on peaksf r om t he f i r s tCD GWAS wer er epl i cat ed i n a Spani s h popul at i on i n 2011,i dent i f yi ng f our genes ( I L12A, LPP, SCHI P1 and SH2B3) whos e expr es s i on i nt he i nt es t i nalmucos a var i ed accor di ng t o di s eas es t at usand t hegenot ypeoft he 34 as s oci at ed var i ant . Thes e r es ul t s s ugges t t hat t hes e genes may be

cons t i t ut i vel y al t er ed i n cel i ac pat i ent s , pr obabl y bef or e t he ons et of obs er vabl es ympt omsoft hedi s eas e,and t her ef or ecoul d havea pr i mar yr ol e i ni t spat hogenes i s .

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Fi g ur e3.Ce l i acdi s e as eas s o c i at e dr e g i onsandpr op os e dc andi dat eg e ne s .Ge ne shi g hl i g ht e di n r e ds howe ddi f f e r e nt i ale x pr e s s i oni nf unc t i onalanal y s i s .

As econd wor kt ook as t ep f or war d and i dent i f i ed t wogenes( PTPRK and THEMI S) ,l ocat ed i nt he s ame as s oci at ed r egi on,whi c h wer e coexpr es s ed bot hi n act i ve di s eas e and i nr es pons et oi n vi t r os t i mul at i on by gl i adi n of i nt es t i nalbi ops i esofcel i acpat i ent swi t hi nact i vedi s eas ewhohaveadher ed t o t hegl ut enf r eedi etf oratl eas tt woyear s35.Ther ef or e,i ts eemst hatas s oci at ed var i ant si n t hi sr egi on af f ect t he expr es s i on of di f f er ent genes , but not cons t i t ut i vel yf r om t het i meofbi r t h oft hef ut ur ecel i acpat i ent ,butonl yi n t he pr es ence of a t oxi cs t i mul us t hat t r i gger s an i mmune r es pons e. The i mpl i cat i onsoft hi sf i ndi ngar eofgr eati mpor t ancebecaus et heyhi ghl i ghtt he exi s t ence ofcommon r egul at or y mec hani s msf ordi f f er entgenesi nt he DNA

90


s equence t hat onl y have an ef f ect i nt he pr es ence of a di s eas epr ovoki ng i mmunogeni cs t i mul us . I n or der t o el uci dat et he s ubs t ant i al f r act i on of her i t abi l i t y t hat r emai ns unexpl ai ned i n mos t compl ex di s eas es ,a novelhypot hes i s has r ecent l y been pos t ul at ed. I t has been cal l ed t he “r ar evar i ant s ynt het i c genomewi deas s oci at i on hypot hes i s ” and i ti sbas ed on t heas s umpt i on t hat unobs er ved r ar e caus alvar i ant sl ead t o as s oci at i on det ect ed atcommon t ag var i ant s .However ,a r ecent wor ki n whi c hs equenci ng and genot ypi ng f or codi ng exonsof25 GWAS r i s k geneswer eper f or med i n 41, 911 UK r es i dent s of whi t e Eur opean or i gi n ( 24, 892 s ubj ect s wi t h s i x aut oi mmune di s eas e phenot ypesand 17, 019cont r ol s )hasr eveal ed t hatr ar ecodi ngr egi on var i ant s at known l oci have a negl i gi bl e r ol e i n common aut oi mmune di s eas e s us cept i bi l i t y,i ncl udi ngCD36. A di f f er entappr oac hwast akent of i nemapt heLPP l ocusi nt hes ear c hf or pos s i bl ef unct i onal var i ant s . Thi ss t r at egy r eveal ed 6 SNPs t hat over l ap r egul at or y s i t es , wi t h r s 4686484 havi ng a pos s i bl e ef f ect on LPP gene 37 expr es s i oni npat i ent s .

Al mos t al l as s oci at ed r egi ons cont ai n genes wi t h an i mmunol ogi cal f unct i on, many of whi c h act i n t he s ame bi ol ogi cal pat hways . Tcel l devel opment i n t he t hymus , a pat hway pr evi ous l y not expl or ed i n CD pat hogenes i s ,i soneoft hos epat hways .A s t udy car r i ed outby Amunds en et al .ai med t o expl or et he r egul at or y pot ent i aloft he CDas s oci at ed SNPsby eQTL anal ys i si n t hymi ct i s s ue38. They f ound 43 nomi nal l y s i gni f i cant ( p4) wer e under r epr es ent ed among pat i ent s ,s ugges t i ng t hat i ncr eas ed copy number s coul d pr ot ect f r om CD, pos s i bl y by i mpedi ng bact er i al i nf i l t r at i on mor e ef f i ci ent l yandpr es er vi nggutepi t hel i ali nt egr i t y. Ont heot herhand,genomewi deexpr es s i on anal ys i shavecons i s t ent l ybeen us ed t o dr aw maps oft he mos t common f unct i onalal t er at i ons i n di f f er ent compl exdi s eas es .Somet i mes ,t hes eappr oac heshaveal s obeenus edt oi dent i f y as s oci at ed var i ant st hatcoul d expl ai n di f f er entpat hol ogi cals i t uat i ons .I nt he cas e of CD, Cas t el l anos Rubi o et al . des i gned i n 2008 a s t r at egy t hat combi ned gene expr es s i on pr of i l i ng of i nt es t i nalbi ops ys peci mens ,l i nkage r egi on i nf or mat i on, and di f f er ent bi oi nf or mat i cs t ool sf or t he s el ect i on of 42 pot ent i al l yr egul at or ys i ngl enucl eot i depol ymor phi s ms .Amongot herr es ul t s ,

t hey f ound evi dence of as s oci at i on wi t h s ever al SNPs and i dent i f i ed SERPI NE2i n 2q33,and PBX3orPPP6C i n9q34aspot ent i alr ol epl ayer si n t hedevel opmentoft hedi s eas e. Fol l owi ng t her es ul t sf r om t heENCODE pr oj ect ,i ti snow known t hata s ubs t ant i al f r act i on of genet i c var i ant s cont r i but i ng t o compl ex t r ai t si n humansar ei nvol ved i n gener egul at i on43.Mos tphenot ypeas s oci at ed var i ant s di s cover edi nGWA s t udi esar ef arawayf r om pr ot ei ncodi ngr egi ons ,andeven 44 appeari n genedes er t s .Thi sdi s t r i but i on i ss i mi l art ot hats hown bymos tof

t he ci sr egul at or y modul es s uc h as pr omot er s and enhancer s , and i ti s expect ed t hatmany var i ant sas s oci at ed wi t h compl ex t r ai t smay af f ectgene 93


expr es s i on. Fur t her mor e, vi r t ual l y any noncodi ng s equence i nt he human genome coul d pot ent i al l y be a r egul at or y el ement45 and even act f ar away f r om i t s genomi cl ocat i on and gl obal l y al t er whol e pat hways and s i gnal i ng r out es . Thus , over s i mpl i s t i c and ar bi t r ar ys el ect i on of near by and s i ngl e candi dat egeness houl d beavoi ded and Sys t emsBi ol ogyappr oac hess houl d be i mpl ement ed t o f i nd t he r el at i ons and common r egul at or y mec hani s ms conj ugat i ngt hegenest hati nt er actt ogener at et hecel i acphenot ype. I nt hi ss ens e,i n a genomewi de expr es s i on mi cr oar r ay car r i ed outs ever al year sago,s omes i gnal i ng r out eswer ef ound t obeal t er ed i n CD,s uc h ast he 46 JakSt at ,NFkB,MAPK orTGFB pat hways .Someoft hegenespar t i ci pat i ng

i n t hes er out es have been s t udi ed t o det er mi ne whet her t hey cont ai n CDas s oci at ed var i ant s .One oft hes e genes i s STAT1,whos e expr es s i on i s al t er ed i n t he di s eas e. However , no as s oci at ed SNPs have been f ound47. NFkB1 hasal s o been s t udi ed but ,al t hough i ti scons t i t ut i vel y act i vei nt he i nt es t i nalmucos a ofCD pat i ent s ,i t does not s eem t o cont ai n any genet i c al t er at i on t hatcoul d expl ai ni t sover expr es s i on.I thasbeen s ugges t ed t hat t he pat hogeni c ef f ect s as s i gned t ot hi st r ans cr i pt i on f act or ( TF) coul d be caus ed by a r egul at or y def ect and t hat var i ant s or al t er at i ons i n genes ups t r eam NFkB coul dt r i ggert he enhanced t r ans cr i pt i onalact i vi t y obs er ved i n CD.I thasbeen s pecul at ed t hatt wo oft hegenesi dent i f i ed i n af ol l owup s t udy af t ert heGWAS ( REL and TNFAI P3)coul d under l i et heder egul at i on oft hi sbi ol ogi calr out e48.A r egul at or y SNP i nt heUBD genet hati si nvol ved i nt heact i vat i on ofNFkB hasbeen as s oci at ed t o CD i n Spani s h popul at i on. Thi sgenei sover expr es s ed i n act i vedi s eas eand t heal l el i cdi s t r i but i on oft he as s oci at ed pol ymor phi s m pr es ent sa s i gni f i cant cor r el at i on wi t h expr es s i on 49 l evel s .

I n t hi s cont ext ,a r ecent s t udy t hat t r i ed t o nor mal i ze t he al t er ed expr es s i on of t he NFkB pat hway i n vi t r o us i ng a MALT1 par acas pas e i nhi bi t ordi s cover edas t r ongcoexpr es s i onamonggenesoft her out ei nheal t hy gut mucos a,whi l ei nt es t i nalbi ops i es f r om act i ve CD pat i ent s pr es ent ed a 50 compl et el y de r egul at ed pat hway ( Fi gur e4) .Thi sdi s r upt i on ofcoexpr es s i on

per s i s t ed i n t r eat ed, i nact i ve pat i ent s , es peci al l y af t er acut e gl i adi n 94


s t i mul at i on i n vi t r o,and coul d ber ever t ed t oar egul at ed pat t er ns i mi l art o t he one s een i n cont r ol st hr ough MALT1 i nhi bi t i on.Thes er es ul t ss t r ongl y s ugges tt hatunknown r egul at or ymec hani s msbehi nd t het i ghtcoexpr es s i on of t he NFkB pat hway obs er ved i n noni nf l amed gutmucos a coul d be t he ones af f ect ed by put at i vegenet i corepi genet i cal t er at i onsr at hert han s i ngl egenes t aki ngpar ti nt heact i vat i ngcas cade.

Fi g ur e4.Ge nep ai rc o e x pr e s s i on mat r i x e sf ort hedi f f e r e ntdi s e as es t at us e s .Eac hs mal ls q uar e r e pr e s e nt st hepv al uef ort hec or r e l at i on oft hee x pr e s s i on l e v e li n as p e c i f i cg e nep ai r .Whi t e , l i g htg r ay ,dar kg r ayandb l ac ki ndi c at ePe ar s on' sc or r e l at i on pv al ue s>0. 05,