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.
New Semapho ins Va ian s in HSCR Pa ien s
PLOS ONE | www.plosone.o g 9 Janua y 2013 | Volume 8 | Issue 1 | e54800
View publica ion s a sView publica ion s a s