Mutational Spectrum of Semaphorin 3A and Semaphorin 3D Genes in Spanish Hirschsprung patients

Mu a ional Spec um o Semapho in 3A and Semapho in 3D Genes in Spanish Hi schsp ung pa ien s Be a Luzo ´n-To o 1,2 , Raquel M Fe na ´ndez 1,2 , Ana To oglosa 1,2 , Juan Ca los de Agus ı ´n 3 , C is ina Me ´ndez-Vidal 1,2 , Dolo es Isabel Segu a 4 , Guille mo An in ˜olo 1,2 , Salud Bo ego 1,2 * 1Depa men o Gene ics, Rep oduc ion and Fe al Medicine, Ins i u e o Biomedicine o Se ille, Uni e si y Hospi al Vi gen del Rocı ´o/Consejo Supe io de In es igaciones Cien ı ´ icas/Uni e si y o Se ille, Se ille, Spain, 2Cen e o Biomedical Ne wo k Resea ch on Ra e Diseases, Se ille, Spain, 3Depa men o Pedia ic Su ge y, Uni e si y Hospi al Vi gen del Rocı ´o, Se ille, Spain, 4Depa men o Pa hology, Uni e si y Hospi al Vi gen del Rocı ´o, Se ille, Spain Abs ac Hi schsp ung disease (HSCR, OMIM 142623) is a de elopmen al diso de cha ac e ized by he absence o ganglion cells along a iable leng hs o he dis al gas oin es inal ac , which esul s in onic con ac ion o he aganglionic colon segmen and unc ional in es inal obs uc ion. The RET p o o-oncogene is he majo gene associa ed o HSCR wi h di e en ial con ibu ions o i s a e and common, coding and noncoding mu a ions o he mul i ac o ial na u e o his pa hology. In addi ion, many o he genes ha e been desc ibed o be associa ed wi h his pa hology, including he semapho ins class III genes SEMA3A (7p12.1) and SEMA3D (7q21.11) h ough SNP a ay analyses and by nex -gene a ion sequencing echnologies. Semapho ins a e guidance cues o de eloping neu ons implica ed in he axonal p ojec ions and in he de e mina ion o he mig a o y pa hway o neu al-c es de i ed neu al p ecu so s du ing en e ic ne ous sys em de elopmen . In addi ion, i has been desc ibed ha inc eased SEMA3A exp ession may be a isk ac o o HSCR h ough he up egula ion o he gene in he aganglionic smoo h muscle laye o he colon in HSCR pa ien s. He e we p esen he esul s o a comp ehensi e analysis o SEMA3A and SEMA3D in a se ies o 200 Spanish HSCR pa ien s by he mu a ional sc eening o i s coding sequence, which has led o ind a numbe o po en ially dele e ious a ian s. RET mu a ions ha e been also de ec ed in some o hose pa ien s ca ying SEMAs a ian s. We ha e e alua ed he A131T-SEMA3A, S598G- SEMA3A and E198K-SEMA3D mu a ions using colon issue sec ions o hese pa ien s by immunohis ochemis y. All mu an s p esen ed inc eased p o ein exp ession in smoo h muscle laye o ganglionic segmen s. Mo eo e , A131T-SEMA3A also main ained highe p o ein le els in he aganglionic muscle laye s. These indings s ongly sugges ha hese mu an s ha e a pa hogenic e ec on he disease. Fu he mo e, because o hei coexis ence wi h RET mu a ions, ou da a subs an ia e he addi i e gene ic model p oposed o his a e diso de and u he suppo he associa ion o SEMAs genes wi h HSCR. Ci a ion: Luzo ´n-To o B, Fe na ´ndez RM, To oglosa A, de Agus ı ´n JC, Me ´ndez-Vidal C, e al. (2013) Mu a ional Spec um o Semapho in 3A and Semapho in 3D Genes in Spanish Hi schsp ung pa ien s. PLoS ONE 8(1): e54800. doi:10.1371/jou nal.pone.0054800 Edi o : S acey Che ny, Uni e si y o Hong Kong, Hong Kong Recei ed Ap il 1, 2012; Accep ed Decembe 17, 2012; Published Janua y 23, 2013 Copy igh : ß2013 Luzo ´n-To o e al. This is an open-access a icle dis ibu ed unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s un es ic ed use, dis ibu ion, and ep oduc ion in any medium, p o ided he o iginal au ho and sou ce a e c edi ed. Funding: This s udy was unded by he Ins i u o de Salud Ca los III (ISCIII), Spain (PI1001290) and Conseje ı ´a de Economı ´a, Inno acio ´n y Ciencia de la Jun a de Andalucı ´a (CTS-7447). The CIBER de En e medades Ra as is an ini ia i e o he ISCIII, Minis e io de Economı ´a y Compe i i idad. The unde s had no ole in s udy design, da a collec ion and analysis, decision o publish, o p epa a ion o he manusc ip . Compe ing In e es s: The au ho s ha e decla ed ha no compe ing in e es s exis . * E-mail: salud.bo ego.sspa@jun adeandalucia.es In oduc ion Hi schsp ung disease (HSCR, OMIM 142623) is a de el- opmen al diso de occu ing in 1 o 5,000 li e bi hs. HSCR mos commonly p esen s as isola ed cases and displays a complex pa e n o inhe i ance wi h low, sex dependen pene ance and a iable exp ession. I is cha ac e ized by he absence o ganglion cells along a iable leng hs o he dis al gas oin es inal ac , which esul s in onic con ac ion o he aganglionic colon segmen and unc ional in es inal obs uc- ion. Such aganglionosis is a ibu ed o a ailu e o neu al c es cells (NCC) o mig a e, p oli e a e, and/o di e en ia e du ing en e ic ne ous sys em (ENS) de elopmen in he emb yonic s age [1,2]. The RET p o o-oncogene (OMIM 164761) is he majo gene associa ed o HSCR. RET has been ex ensi ely s udied in HSCR pa ien s and o e 100 mu a ions ha e been iden i ied along he gene (see Human Gene Mu a ion Da abase). Howe e , mu a ions in he RET coding sequence accoun o only up o 50% o 7–20% o amilial and spo adic cases, espec i ely [2]. The in ol emen o RET in he pa hogenesis o HSCR is u he suppo ed by he exis ence o a speci ic haplo ype, cons i u ed by common RET polymo phisms, which seems o be esponsible o he majo i y o spo adic o ms [3,4,5]. This HSCR-associa ed RET haplo ype is cha ac e ized by a common allele (c.73+9277T, s2435357) wi hin a conse ed enhance -like sequence in in on 1 (MCS+9.7) [5,6], making a 20- old g ea e con ibu ion o isk han coding mu a ions [5]. I has been demons a ed a di e ence in abili y o SOX10 o bind o MCS+9.7 and ansac i a e RET depending on he bea ing allele a such speci ic locus [6]. On he o he hand, nume ous molecula gene ic s udies ha e iden i ied a e coding mu a ions in many o he genes (GDNF, NRTN, PSPN, EDNRB, EDN3, ECE1, NTF3, NTRK3, SOX10, PHOX2B, L1CAM, ZFHX1B, KIAA1279, TCF4, PROK1, PROKR1, PROKR2, GFRA1, NRG1 and SEMAs) ela ed o HSCR [2,7–13]. Howe e , he con en ional mu a ions ela ed o HSCR epo ed so a only explain a ound 5% o cases, being he as majo i y o PLOS ONE | www.plosone.o g 1 Janua y 2013 | Volume 8 | Issue 1 | e54800 . hem long segmen HSCR and/o o al colonic aganglionosis and synd omic o ms o he disease [2]. HSCR is ega ded as a complex and mul i ac o ial gene ic diso de , in which he con ibu ion o se e al di e en loci ac ing in an addi i e o mul iplica i e manne is usually equi ed o cause he disease [3]. Based on his e idence, se e al HSCR-associa ed egions (9q31 [14], 19q12 [15], 3p21 [15,16], 16q23 [17], 21q21 [18], o 4q31.3-q32.3 [19]) ha e been iden i ied by genome wide linkage and genome wide associa ion s udies (GWAS), al hough he genes unde lying such associa ions ha e no been iden i ied ye in mos o he cases. Th ough a GWAS pe o med by he In e na ional HSCR Conso ium, in which ou g oup akes pa , a signi ican clus e o SNPs was iden i ied in a egion on ch omosome 7, con aining s ong associa ion wi h HSCR wi h allelic e ec s independen o RET, ha ell downs eam om he p o ein SEMA3D (7q21.11; OMIM 609907) and ups eam om SEMA3A (7p12.1; OMIM 603961) (S. A nold e al., 2 nd In e na ional Symposium: De elopmen o he Table 1. SEMA3A sequence a ian s de ec ed in he cu en s udy. Nucleo ide Change Aminoacid Change No el/Desc ibed Allelic equency (%) in con ol popula ion c.112+52del No el 0 c.112+52 C.G No el 0 c.112+63 T.C s13231702 0 c.113-110 A.C s12671857 52.2 c.113-42 G.C s17241389 0 c.201T.C S67S No el 1 c.267A.G Q89Q A nold e al., 2009 0 c.270+96A.G No el 0 c.333+84T.A No el 0 c.333+92G.T s6955597 0.3 c.334-24delTT No el 0 c.391G . A A131T s143007146 c.453+24A.G s1990044 47.9 c.547+167G.A s2527039 15.8 c.548-45C.T No el 0 c.548-67T.C No el 1 c.548-79G.C No el 0 c.548-92 T.C No el 0 c.668-199_201del No el 2.5 c.668-138A.T No el 0 c.668-20 C.T s2272221 10.5 c.668-14 T.A s2272222 2.2 c.705T.C S235S s34541339 6.8 c.732C.T Y244Y No el 0 c.811-139C.T No el 0 c.945C.T N315N No el 0 c.1140+46C.G No el 0 c.1302T.C I434I No el 15 c.1303G.A V435I A nold e al., 2009 3 c.1361-52T.A s10250165 23.8 c.1361-14A.G s3735513 14.7 c.1453-9delA No el 0 c.1495+132A.C s17246251 61.5 c.1563G.C G521G s10487865 0 c.1652-85insTA No el 0 c.1653-6C.T s701320 84 c.1792A . G S598G No el 0 c.1860+30A.G s7809708 26.5 c.2151A.G T717T s797821 32.5 Compila ion o all he SEMA3A sequences a ian s ound by dHPLC. No e ha he missense a ian s a e shaded. doi:10.1371/jou nal.pone.0054800. 001 New Semapho ins Va ian s in HSCR Pa ien s PLOS ONE | www.plosone.o g 2 Janua y 2013 | Volume 8 | Issue 1 | e54800 ENS: Cells, signals and genes. Feb 2009). In such s udy, sho -HSCR ios we e analyzed wi h he 500K SNP a ay pla o m (A yme- ix) and he SEMA SNPs clus e was iden i ied and subsequen ly e ined. The wo SEMA amily III membe s demons a ed e y simila empo o-spa ial pa e ns o exp ession h oughou he colon. No ably, hey we e co-exp essed wi h RET in his issue, suppo ing he possibili y ha any o hem migh modi y RET unc ion in he de eloping ENS. In addi ion, based in hese indings, nex -gene a ion sequenc- ing echnologies and unc ional analyses ha e allowed he iden i ica ion o semapho ins class III genes (SEMAs) 3A and 3D mu a ions po en ially in ol ed in he pa hogenesis o HSCR [13]. SEMAs ep esen he la ges amily o axonal guidance cues iden i ied so a , ha p o ides di ec ional in o ma ion o g owing axons. The ole o SEMAs pa e ing senso y p ojec ions in he pe iphe al [20] and cen al ne ous sys em [21] is well known. P e ious s udies ha e also sugges ed a ole o membe s o he SEMA amily in NCC de elopmen , p oli e a ion, mig a ion, and/o di e en ia ion, which a e p ocesses ela ed wi h HSCR e iology [22–25]. Mo e speci ically, SEMA3A is exp essed in he mesenchyme o dis al la ge in es ine and i also ac s as a epulsi e signal o neu i es o Remak ganglia [26]. Al hough ENS p ecu so s de i ed om sac al NCCs exp ess he ecep o Neu opilin1 (NP1), i emains o be demons a ed i SEMA3A ac s on mig a ion by a di ec e ec on he mig a ing ENS p ecu so s, o by an indi ec ac ion o e ex insic axons ha accompany hose cells when colonizing he bowel, and a e epelled i SEMA3A is p esen in he ou e segmen s o colon mesenchyme [22]. Mo e ecen ly, di e en in i o and in i o app oaches ein o ce he impo an ole o SEMAs and hei ecep o s as key playe s in he immunological and he neu olog- ical sys ems [27–29]. Fu he mo e, i has been p oposed ha inc eased SEMA3A exp ession may be a isk ac o o HSCR pa hology in a subse o HSCR pa ien s, based on he up egula ion o his gene in he aganglionic smoo h muscle laye o he colon [30]. In addi ion, he associa ion be ween wo SEMA3A polymo phisms ( s7804122 and s797821) and he isk o HSCR in he No heas e n Chinese popula ion has been alida ed, as i was p e iously demons a ed in Caucasian popula ion [31]. Based on he e idences o he implica ion o SEMA class III genes in HSCR, we ha e pe o med a sc eening o he coding egion o SEMA3A and SEMA3D genes in a se ies o 200 isola ed Spanish HSCR cases, o de e mine hei mu a ional spec um in ou popula ion. Th ee mu a ions, A131T- SEMA3A, S598G-SEMA3A and E198K-SEMA3D we e u he s udied using an immunohis ochemical app oach o e alua e hei exp ession le els in he HSCR samples. All mu an s p esen ed an inc eased amoun o he p o eins in colon issue sec ions in compa ison wi h he no mal p o eins, indica ing he pa hogenic ole o hese mu a ions. Ma e ials and Me hods Pa ien s and Con ols Subjec s In his s udy we ha e included a o al o 200 Spanish HSCR pa ien s (23% emale, 77% male), and hei pa en s when a ailable. 180 we e spo adic cases, while 20 we e amilial cases belonging o 13 di e en amilies. In addi ion, we ha e also analyzed a g oup o 200 no mal con ols comp ising unselec ed, un ela ed, ace, age, and sex-ma ched indi iduals. E hics S a emen A w i en in o med consen was ob ained om all he pa icipan s o clinical and molecula gene ic s udies. The s udy con o med o he ene s o he decla a ion o Helsinki as Table 2. SEMA3D sequence a ian s de ec ed in he cu en s udy. Nucleo ide Change Aminoacid Change No el/Desc ibed Allelic equency (%) in con ol popula ion c-151-158_154del No el 1 c.376-32G.A No el 0 c.496-12T.C No el 0 c.589+37G.A s17159594 0 c.590-33T.A No el 3.5 c.718+26T.C No el 0 c.636C.T D212D No el 0 c.592G . A E198K No el 0 c.861+67_71del s56131427 4.8 c.1545+91G.A s7780132 28.3 c.1546-9 G.A No el 0 c.1578C.T L526L s17559084 43.8 c.1703+28G.C s6468008 27 c.1843C.A P615T No el 0.5 c.1901G . A R634Q TMP_ESP_7:84636125* 0 c.1906+147T.C No el 0 c.2103G.T K701Q s7800072 35.8 Compila ion o all he SEMA3D sequences a ian s ound by dHPLC. No e ha he missense a ian s a e shaded. *Sou ce: Ensemble. doi:10.1371/jou nal.pone.0054800. 002 New Semapho ins Va ian s in HSCR Pa ien s PLOS ONE | www.plosone.o g 3 Janua y 2013 | Volume 8 | Issue 1 | e54800 well as he equi emen s es ablished by ou Ins i u ional Re iew Boa d. Mu a ional Analysis Genomic DNA was ex ac ed om pe iphe al blood leukocy es om all he indi iduals included in he s udy, using s anda d p o ocols. The mu a ional sc eening o he comple e coding sequence o SEMA3A and SEMA3D was ca ied ou by dena u ing high-pe o mance liquid ch oma og aphy in a WAVE DNA F agmen Analysis sys em (T ansgenomic). Those agmen s wi h abe an p o iles we e subjec ed o sequence analysis using an ABI P ismH3730 Gene ic Analyze and he SeqScapeH 2.5 so wa e (Applied Biosys ems). When a change was de ec ed, he app op i- a e DNA agmen was also sc eened in a g oup o 200 no mal con ols o de e mine i s allelic equency in ou popula ion. P ime s and PCR-dHPLC condi ions used a e a aliable unde eques . Immunohis ochemis y (IHQ) Fo malin- ixed pa a in embedded (FFPE) colon issue blocks we e collec ed om pa ien s wi h he ollowing mu a ions: A131T-SEMA3A (bo h ganglionic and aganglionic sec ions), S598G-SEMA3A and E198K-SEMA3D (only ganglionic issues). Un o una ely, no ma e ial om pa ien s wi h R634Q-SEMA3D mu a ions was a ailable. Fou mm hick pa a in sec ions we e dewaxed in xylene and ehyd a ed in a se ies o g aded alcohols. Endogenous pe oxidase ac i i y was blocked wi h wa e con ain- ing 3% H 2 O 2 o 30 minu es. An igen e ie al was done by mic owa ing using ci a e phospha e bu e (pH 6.0). Sec ions we e incuba ed a 4uC o e nigh wi h he p ima y an ibodies an i-SEMA3A (1:50 dilu ion, an i- abbi , An ibodyBcn, Ba ce- lona, Spain) and an i-SEMA3D (1:5 dilu ion, an i- abbi , No us Biologicals, Camb idge, UK). A e se e al washes in T is bu e , pe oxidase-labelled seconda y an ibodies and 3,39-diaminobenzi- dine we e applied o de elop immuno eac i i y, acco ding o manu ac u e ’s p o ocol (EnVision; Dako, Glos up, Denma k). The slides we e hen coun e s ained wi h hema oxylin and moun ed in DPX (BDH Labo a o ies, Poole, UK). Colon issue sec ion in which p ima y an ibody was omi ed was used as nega i e con ol. Resul s and Discussion Membe s o SEMA class III p o ein sub amily a e inhibi o y axon guidance molecules, which could help o de e mine he axonal p ojec ion pa e ns o se e al neu ons and ganglia h oughou he cen al and pe iphe al ne ous sys em [32]. Mo eo e , SEMA3A has been ela ed o HSCR h ough di e en app oaches [13,30,31]. We ha e de ec ed a o al o 56 sequence a ian s in he mu a ional sc eening o SEMA3A and SEMA3D genes (Tables 1 and 2). The mos in e es ing inding among hese esul s was he de ec ion o ou missense a ian s (SEMA3A: A131T, S598G; SEMA3D: E198K, R634Q) in he e ozygosis. Two o hem we e al eady desc ibed (A131T-3A wi h a MAF = 0,01 and R634Q-3D wi h a MAF = 0,0093) in public da abases (1000 Genomes, EVS and NCBI) bu he o he wo (S598G-3A and E198K-3D) we e no el. All ou a ian s a e loca ed in he coding egion o bo h SEMA p o eins and ound exclusi ely in HSCR se ies bu absen in he con ol popula ion (Table 3). They we e de ec ed wi h a mu a ional equency ange o 0.005%-0.01%, which was compa able wi h he pe cen ages o SEMA mu a ions p e iously desc ibed in Caucasian popula ion (0.004%–0.011%) [13]. Table 3. SEMA3A and SEMA3D missense a ian s de ec ed in isola ed HSCR pa ien s. Mu a ion in o ma ion Pa ien s in o ma ion Gene Nucleo ide Change Aminoacid Change P o ein domain Gende Leng h o aganglionosis Inhe i ance O he mu a ional e en s in he pa ien SEMA3A c.391G.A A131T SEMA Male No a ailable Pa e nal RET enhance mu a ion in homozygosis SEMA3A c.391G.A A131T SEMA Male No a ailable Ma e nal RET R313W mu a ion inhe i ed om he a he SEMA3A c.1792A.G S598G Ig-like Female Sigmoid Da a no a ailable* 1 RET W543R mu a ion* 1 SEMA3D c.592G.A E198K SEMA Male Rec osigmoid De no o o pa e nal* 2 RET enhance mu a ion inhe i ed om he mo he SEMA3D c.1901G.A R634Q Ig-like Male Hepa ic Flexu e Ma e nal EDNRB K15X mu a ion inhe i ed om he a he SEMA3D c.1901G.A R634Q Ig-like Male Sigmoid Ma e nal RET enhance mu a ion in homozygosis De ailed in o ma ion ega ding he ou SEMA3A and SEMA3D missense a ian s de ec ed in isola ed HSCR pa ien s. * 1 Bo h RET and SEMA3A mu a ions could no be e i ied o be inhe i ed o de no o e en s, since DNA samples om he pa en s o his pa ien we e no a ailable. * 2 The E198K-SEMA3D mu a ion could no be e i ied o be pa e nally inhe i ed o a de no o e en , since pa e nal DNA was no a ailable. doi:10.1371/jou nal.pone.0054800. 003 New Semapho ins Va ian s in HSCR Pa ien s PLOS ONE | www.plosone.o g 4 Janua y 2013 | Volume 8 | Issue 1 | e54800 The e a e se e al e idences ha led us o p opose he ou missense a ian s in wo SEMAs genes (SEMA3A: A131T, S598G; SEMA3D: E198K, R634Q) as mu a ions associa ed o HSCR. Fi s , he mu a ions we e de ec ed in wo main p o ein domains (Figu e 1): he SEMA domain, which is impo an o p o ein- p o ein in e ac ion (A131T-SEMA3A and E198K-SEMA3D) and he domain implica ed in ecep o binding (S598G-SEMA3A and R634Q-SEMA3D). Second, in silico p edic ions (SIFT, Polyphen) had shown he damaging e ec o hose ou mu an p o eins. He e, we ha e also pa ially cha ac e ized he mu a ions A131T- SEMA3A and S598G-SEMA3A and E198K-SEMA3D by immu- nohis ochemis y. In his ological analyses, bo h SEMA3A (Figu e 2) and SEMA3D (Figu e 3) we e exp essed in he ganglion cells o he myen e ic and submucosal plexuses as well as in he smoo h muscle laye s o he ganglionic colon o ou pa ien s and in he con ol samples. Rega ding he A131T-SEMA3A mu a ion, we ob ained bo h ganglionic and aganglionic segmen s om he pa ien . A highly exp essed mu an SEMA3A p o ein in he smoo h muscle laye s in he aganglionic segmen was de ec ed. This inc eased amoun o p o ein seemed o be highe han he inc ease ound in bo h he co esponding ganglionic segmen and he con ol sample (Figu e 4). This is in acco dance wi h p e iously published esul s o he wild ype SEMA3A p o ein, ha i is up egula ed in he aganglionic colon issue [30]. In he case o S598G-SEMA3A and E198K-SEMA3D mu an s, a mo e in ense immunos aining in all ganglionic issue laye s in compa ison wi h he con ol sample was de ec ed (Figu e 4). Based on he inc eased amoun de ec ed o bo h SEMA3A and SEMA3D p o eins in colon issue o HSCR pa ien s, we p opose ha hose a ian s esul s in he accumula ion o SEMAs p o eins leading o impai ed en e ic axonal p ojec ion du ing NCCs mig a ion and/o di e en ia ion, wi h unc ional consequences in ENS o ma ion. Gi en he ole o SEMAs as c ucial modula o s du ing emb yonic de elopmen [32], abno mal SEMAs exp ession o unc ion may ha e a pleio opic e ec , as i has been obse ed in mouse null mu an s o genes encoding SEMA3A and i s ecep o NP1 [33]. This hypo hesis i s wi h he lack o null mu a ions in ou mu a ional sc eening, being mo e plausible ha he missense mu a ions de ec ed on SEMA3A and SEMA3D genes cause a mo e sub le e ec on p o ein unc ion han null mu a ions do. Thus, we p opose ha he gene ic backg ound o he indi idual in combina ion wi h he p esence o hose hypomo phic mu a ions in he SEMA genes would gene a e he speci ic pheno ype obse ed in ou pa ien s, in acco dance wi h he addi i e model p e iously p oposed o HSCR [34]. We ailed o de ec any o he coding mu a ion in he p e iously associa ed HSCR genes in he pa ien s wi h SEMA mu a ions (da a no shown), excep o RET and EDNRB genes (Table 3), al hough we canno disca d he con ibu ion o addi ional mu a ional e en s in s ill uniden i ied HSCR genes. I is wo h o men ion he high incidence o co-occu ence o coding RET mu a ions and SEMA3A mu a ions in ou se ies o pa ien s, as 50% o he pa ien s wi h mu a ions on his locus p esen coding mu a ions in RET. All pa ien s ca ying mu a ions ei he in SEMA3A o SEMA3D genes ha e inhe i ed Figu e 1. Schema ic domain loca ion o he mu a ions analyzed in human Sema3A and Sema3D p o eins. An N- e minal se en-bladed b-p opelle Sema domain ollowed by a cys eine- ich PSI (plexin, semapho in, in eg in) domain is a signa u e ea u e in he ec odomains o semapho in and plexin amily membe s. The composi ion o he emainde o he semapho in ec odomain a ies acco ding o class: he ec odomains o he sec e ed class 3 semapho ins con ain an Ig (immunoglobulin-like) domain and a basic C- e minal ail. doi:10.1371/jou nal.pone.0054800.g001 New Semapho ins Va ian s in HSCR Pa ien s PLOS ONE | www.plosone.o g 5 Janua y 2013 | Volume 8 | Issue 1 | e54800 Figu e 2. Immunos aining o Semapho in 3A in colon om con ols and HSCR pa ien s. The SEMA3A s aining illus a ed ha he exp ession was p esen a smoo h muscle (D, E, F) and submucous (G, H, I) laye s, as well as in myen e ic (J, K) and submucous plexuses (M, N) ei he in no mal colon (A, D, G, J, M) and pa ien s wi h A131T-3A (B, E, H, K, N) and S598G-3A mu a ions (C, F, I). The FFPE issue block om pa ien wi h S598G had no all issue laye s. Scale ba s: A–C = 200 mm and he es o pic u es = 10 mm. doi:10.1371/jou nal.pone.0054800.g002 New Semapho ins Va ian s in HSCR Pa ien s PLOS ONE | www.plosone.o g 6 Janua y 2013 | Volume 8 | Issue 1 | e54800 ei he RET enhance a ian o a coding mu a ion associa ed o HSCR. I is well es ablished ha ac i a ion o RET is essen ial o p oli e a ion, mig a ion and di e en ia ion o en e ic neu al p ecu so s [35]. The as majo i y o en e ic neu ons and glial cells a ise om agal NCCs, al hough sac al NCCs also gi e ise o some neu ons and glia, p incipally in he dis al hindgu , a e he caecum [36,37]. Mig a ion o en e ic p ecu so s de i ed om agal NCCs, wi hin he colon has been shown o depend mainly on he p oli e a ion a he mig a ion wa e on , a he han on he p esence o ac o s ha p omo e mig a ion [38]. Fo ha eason, mu a ions in RET cause a ailu e o hose cells o colonise he dis al colon in an app op ia e numbe o o m he en e ic ganglia, leading o HSCR [2]. The exp ession o GDNF in he hindgu is lowe han in he caecum [39] and he e o e many au ho s ha e p oposed ha mig a ion and p oli e a ion o sac al NCCs is less dependen on GDNF. In addi ion, sac al NCCs a e able o mig a e h ough p e-caecum colon in a slowe manne compa ed o agal de i ed cells [22,39–42]. In such scena io, we p opose ha he absence o en e ic ganglia in he dis al pa o he bowel, due o a ail o agal NCCs, could be escued by he mig a ion and di e en ia ion o sac al NCCs. I has been demons a ed ha SEMA3A egula es he en y o ex insic axons in o he hindgu and also sac al NCCs, as hey accompany hose axons when mig a ing o he colon. In ac , de ec s on SEMA3A exp ession impai he en y o sac al en e ic p ecu so s in he hindgu [22]. Fo ha eason, we sugges ha a ail on sac al NCCs o colonize he hindgu in he app op ia e momen , due o he p esence o mu a ions in SEMA3A, would esul s in a mo e d ama ic pheno ype in pa ien s also ha bou ing mu a ions in RET,asSEMA3A has no e ec s on agal NCCs [22]. In ag eemen wi h his hypo hesis, we did no obse e an en e ic pheno ype in amily membe s o ou HSCR pa ien s wi h isola ed mu a ions in RET o SEMA3A. Seg ega ion analysis o hese mu a ions in bo h genes oge he wi h he a ailable da a o mu a ions in o he genes associa ed o HSCR, led us o specula e wi h he po en ial syne gis ic e ec o mu a ions in bo h RET and SEMA genes. We belie e ha he majo con ibu ing mu a ion could be ei he on RET o SEMA, o in bo h genes a he same ime alone o oge he wi h some o he mu a ions in unknown genes, due o he complexi y o he gene ic basis o HSCR and he addi i e model p oposed o he disease. Fu u e analyses will be needed o cla i y he ole o bo h genes. In summa y, in his s udy we ha e analyzed h ee aminoacidic subs i u ions, A131T-SEMA3A, S598G-SEMA3A and E198K- SEMA3D. These a ian s esul in an inc ease o SEMA p o eins le els in he HSCR colon issue. Ou indings u he suppo he unc ional implica ion o SEMAs as signalling molecules in he de elopmen o ENS and indica e a syne gis ic e ec o mu a ions in bo h SEMA and RET genes, o in luence he pheno ype o ou HSCR pa ien s. Acknowledgmen s We would like o hank he pa ien s and amilies ha ha e pa icipa ed in his s udy. We hank D . Julia Ma ı ´nez Lapen˜a om he Hospi al Regional Uni e si a io Ca los Haya o Ma´laga (Spain), o sha e hei FFPE issue blocks wi h us. Au ho Con ibu ions Concei ed and designed he expe imen s: BLT RF AT SB. Pe o med he expe imen s: BLT RF AT. Analyzed he da a: BLT RF AT DIS SB. Con ibu ed eagen s/ma e ials/analysis ools: BLT AT JCdA. W o e he pape : BLT RF AT CMV GA SB. Figu e 3. Immunos aining o Semapho in 3D in colon om con ols and HSCR pa ien s. The SEMA3D s aining illus a ed was p esen a smoo h muscle (C, D) and submucous (E, F) laye s, as well as in myen e ic (G, H) and submucous plexuses (I, J) ei he in no mal colon (A, C, E, G, I) and pa ien s wi h E198K-3D mu a ion (B,D, F, H, J). Scale ba s: A, B = 200 mm and he es o pic u es = 10 mm. doi:10.1371/jou nal.pone.0054800.g003 New Semapho ins Va ian s in HSCR Pa ien s PLOS ONE | www.plosone.o g 7 Janua y 2013 | Volume 8 | Issue 1 | e54800 Figu e 4. Immunohis ochemical de ec ion o A131T- SEMA3A, S598G- SEMA3A and E198K- SEMA3 D mu an p o eins in FFPE colon samples. Immunohis ochemis y analysis o A131T-3A, S598G-3A and E198K-3D mu an p o eins in smoo h muscle laye o con ol samples (3A: A, D; 3D: H, K) and colon issue om pa ien s wi h A131T-3A (ganglionic: B, E; aganglionic: C, F), S598G-3A (G, J) and E198K-3D mu a ions (I, L) was pe o med. Scale ba s: A, B, C, G, H, I = 200 mm and he es o he pic u es = 10 mm. doi:10.1371/jou nal.pone.0054800.g004 New Semapho ins Va ian s in HSCR Pa ien s PLOS ONE | www.plosone.o g 8 Janua y 2013 | Volume 8 | Issue 1 | e54800 Re e ences 1. Chak a a i A, Lyonne S (2002) Hi schsp ung disease. The Me abolic and Molecula Bases o Inhe i ed disease. Eds McG aw-Hill Companies. Chap e 251. 2. Amiel J, Sp oa -Emison E, Ga cia-Ba celo M, Lan ie i F, Bu zynski G, e al (2008) Hi schsp ung disease, associa ed synd omes and gene ics: a e iew. J Med Gene 45: 1–14. 3. Bo ego S, W igh FA, Fe na´ndez RM, Williams N, Lo´pez-Alonso M, e al (2003) A ounding locus wi hin he RET p o o-oncogene may accoun o a la ge p opo ion o appa en ly spo adic Hi schsp ung disease and a subse o cases o spo adic medulla y hy oid ca cinoma. Am J Hum Gene 72(1): 88–100. 4. Fe nandez RM, Bo u G, Pecin˜a A, Jones K, Lo´pez-Alonso M, e al (2005) Ances al RET haplo ype associa ed wi h Hi schsp ung’s disease shows linkage disequilib ium b eakpoin a -1249. J Med Gene 42(4): 322–7. 5. Emison ES, McCallion AS, Kashuk CS, Bush RT, G ice E, e al (2005) A common sex-dependen mu a ion in a RET enhance unde lies Hi schsp ung disease isk. Na u e 14;434(7035): 857–863. 6. Emison ES, Ga cia-Ba celo M, G ice EA, Lan ie i F, Amiel J, e al (2010) Di e en ial con ibu ions o a e and common, coding and noncoding Re mu a ions o mul i ac o ial Hi schsp ung disease liabili y. Am J Hum Gene 87(1): 60–74. 7. Bo ego S, Fe na´ndez RM, Dziema H, Niess A, Lo´pez-Alonso M, e al (2003) In es iga ion o ge mline GFRA4 mu a ions and e alua ion o he in ol emen o GFRA1, GFRA2, GFRA3, and GFRA4 sequence a ian s in Hi schsp ung disease. J Med Gene 40(3): e18. 8. Ruiz-Fe e M, Fe nandez RM, An in˜olo G, Lopez-Alonso M, Bo ego S (2008) NTF-3, a gene in ol ed in he en e ic ne ous sys em de elopmen , as a candida e gene o Hi schsp ung disease. J Pedia Su g 43: 1308–1311. 9. Fe na´ndez RM, Sa´nchez-Mejı ´as A, Mena MD, Ruiz-Fe e M, Lo´pez-Alonso M, e al (2009) A no el poin a ian in NTRK3, R645C, sugges s a ole o his gene in he pa hogenesis o Hi schsp ung disease. Ann Hum Gene 73: 19–25. 10. Ruiz-Fe e M, To oglosa A, Luzo´n-To o B, Fe na´ndez RM, An in˜olo G, e al (2011) No el mu a ions a RET ligand genes p e en ing ecep o ac i a ion a e associa ed o Hi schsp ung’s disease. J Mol Med (Be l) 89: 471–480. 11. Ruiz-Fe e M, To oglosa A, Nu´n˜ez-To es R, de Agus ı ´n JC, An in˜olo G, e al (2011). Exp ession o PROKR1 and PROKR2 in human en e ic neu al p ecu so cells and iden i ica ion o sequence a ian s sugges a ole in HSCR. PLoS One 6(8): e23475. 12. Tang CS, Ngan ES, Tang WK, So MT, Cheng G, e al (2012) Mu a ions in he NRG1 gene a e associa ed wi h Hi schsp ung disease. Hum Gene 131(1): 67– 76. 13. Jiang Q, Tu ne T, Sosa MX, Rakha A, A nold S, e al (2012) Rapid and e icien human mu a ion de ec ion using a bench- op nex -gene a ion DNA sequence . Hum Mu a 33(1): 281–9. 14. Bolk S, Pele A, Ho s a RM, Ang is M, Salomon R, e al (2000) A human model o mul igenic inhe i ance: pheno ypic exp ession in Hi schsp ung disease equi es bo h he RET gene and a new 9q31 locus. P oc Na l Acad Sci USA 97: 268–273. 15. Gab iel SB, Salomon R, Pele A, Ang is M, Amiel J, e al (2002) Seg ega ion a h ee loci explains amilial and popula ion isk in Hi schsp ung disease. Na Gene 31: 89–93. 16. Ga cia-Ba celo´ MM, Fong PY, Tang CS, Miao XP, So MT, e al (2008) Mapping o a Hi schsp ung’s disease locus in 3p21. Eu J Hum Gene 16: 833– 840. 17. Ca asquillo MM, McCallion AS, Pu enbe ge EG, Kashuk CS, Nou i N, e al (2002) Genome-wide associa ion s udy and mouse model iden i y in e ac ion be ween RET and EDNRB pa hways in Hi schsp ung disease. Na Gene 32: 237–244. 18. Lin S, Chak a a i A, Cu le DJ (2004) Exhaus i e allelic ansmission disequilib ium es s as a new app oach o genome-wide associa ion s udies. Na Gene 36: 1181–1188. 19. B ooks AS, Leegwa e PA, Bu zynski GM, Willems PJ, de G aa B, e al (2006) A no el suscep ibili y locus o Hi schsp ung’s disease maps o 4q31.3-q32.3. J Med Gene 43(7)e35. 20. Taniguchi M, Yuasa S, Fujisawa H, Na use I, Saga S, e al (1997) Dis up ion o semapho in III/D gene causes se e e abno mali y in pe iphe al ne e p ojec ion. Neu on 19(3): 519–30. 21. Shephe d IT, Luo Y, Le co F, Reicha d LF, Rape JA (1997) A senso y axon epellen sec e ed om en al spinal co d explan s is neu alized by an ibodies aised agains collapsin-1. De elopmen 124(7): 1377–85. 22. Ande son RB, Be gne AJ, Taniguchi M, Fujisawa H, Fo ai A, e al (2007) E ec s o di e en egions o he de eloping gu on he mig a ion o en e ic neu al c es -de i ed cells: A ole o Sema3A, bu no Sema3F. De Biol 305: 287–299. 23. Yu HH, Moens CB (2005) Semapho in signaling guides c anial neu al c es cell mig a ion in zeb a ish. De Biol 15: 280(2): 373–85. 24. Be nd JD, Hallo an MC (2006) Semapho in 3d p omo es cell p oli e a ion and neu al c es cell de elopmen downs eam o TCF in he zeb a ish hindb ain. De elopmen 133(20): 3983–92. 25. Lwigale PY, B onne -F ase M (2009) Semapho in3A/neu opilin-1 signaling ac s as a molecula swi ch egula ing neu al c es mig a ion du ing co nea de elopmen . De Biol 336(2): 257–65. 26. Shephe d IT, Rape JA (1999) Collapsin-1/semapho in D is a epellen o chick ganglion o Remak axons. De Biol 212(1): 42–53. 27. Maden CH, Gomes J, Schwa z Q, Da idson K, Tinke A, e al (2012) NRP1 and NRP2 coope a e o egula e gangliogenesis, axon guidance and a ge inne a ion in he sympa he ic ne ous sys em. De Biol 15;369(2): 277–85. doi: 10.1016/j.ydbio.2012.06.026. 28. Goshima Y, Sasaki Y, Yamashi a N, Nakamu a F (2012) Class 3 semapho ins as a he apeu ic a ge . Expe Opin The Ta ge s 16(9): 933–44. doi: 10.1517/ 14728222.2012.710201. 29. Cha oy C, Nawabi H, Reynaud F, De ing on E, Bozon M, e al (2012) Gdn Ac i a es Midline Repulsion by Semapho in3B ia NCAM du ing Commissu al Axon Guidance. Neu on 75(6): 1051–66. doi: 10.1016/j.neu on.2012.08.021. 30. Wang LL, Fan Y, Zhou FH, Li H, Zhang Y, e al (2011) Semapho in 3A Exp ession in he Colon o Hi schsp ung Disease. Bi h De ec s Res A Clin Mol Te a ol 91(9): 842–847. 31. Wang LL, Zhang Y, Fan Y, Li H, Zhou FH, e al (2012) SEMA3A s7804122 polymo phism is associa ed wi h Hi schsp ung disease in he No heas e n egion o China. Bi h De ec s Res A Clin Mol Te a ol 94(2): 91–95. 32. Ro h L, Koncina E, Sa kauskas S, C e´mel G, Aunis D, e al (2009) The many aces o SEMAs: om de elopmen o pa hology. Cell Mol Li e Sci 66: 649–666. 33. Su o F, I o K, Uemu a M, Shimizu M, Shinkawa Y, e al (2005) Plexin-a4 media es axon- epulsi e ac i i ies o bo h sec e ed and ansmemb ane semapho ins and plays oles in ne e ibe guidance. J Neu osci 25(14): 3628– 3637. 34. Ho s a RM, Osinga J, Buys CH (1997) Mu a ions in Hi schsp ung disease: When does a mu a ion con ibu e o pheno ype. Eu J Hum Gene 5: 180–185. 35. Ta a i as S, Ma cos-Gu ie ez CV, Du bec P, Jani H, G igo iou M, e al (1999) Signalling by he RET ecep o y osine kinase and i s ole in he de elopmen o he mammalian en e ic ne ous sys em. De elopmen 126: 2785–2797. 36. Ge shon MD, Ra cli e EM (2006) De elopmen o he En e ic Ne ous Sys em. Physiology o he Gas oin es inal T ac (Fou h Edi ion) 499–521. 37. Ande son RB, S ewa AL, Young HM (2006) Pheno ypes o neu al-c es de i ed cells in agal and sac al pa hways. Cell Tissue Res 323: 11–25. 38. Simpson MJ, Zhang DC, Ma iani M, Landman KA, Newg een DF (2007) Cell p oli e a ion d i es neu al c es cell in asion o he in es ine. De Biol 302: 553– 568. 39. D uckenb od NR, Eps ein ML (2005) The pa e n o neu al c es ad ance in he cecum and colon. De Biol 287: 125–133. 40. Na a ajan D, Ma cos-Gu ie ez C, Pachnis V, de G aa E (2002) Requi emen o signalling by ecep o y osine kinase RET o he di ec ed mig a ion o en e ic ne ous sys em p ogeni o cells du ing mammalian emb yogenesis. De elopmen 129: 5151–5160. 41. D uckenb od NR, Eps ein ML (2007) Beha io o en e ic neu al c es -de i ed cells a ies wi h espec o he mig a o y wa e on . De Dyn 236: 84–92. 42. Young HM, Be gne AJ, Ande son RB, Enomo o H, Milb and J, e al (2004) Dynamics o neu al c es -de i ed cell mig a ion in he emb yonic mouse gu . De Biol 270: 455–473. 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