ORIGINAL ARTICLE
Deciphe ing in a amilial pheno ypic a iabili y by exome
sequencing in a Ba de –Biedl amily
Ma 
ıa Gonz
alez-del Pozo
1,2
, C is ina M
endez-Vidal
1,2
, Ja ie San oyo-Lopez
3
, Alicia Vela-Boza
3
,
Ne eida B a o-Gil
1
, An onio Rueda
3
, Luz Ga c
ıa-Alonso
4
, Ca men V
azquez-Ma ouschek
5
,
Joaqu
ın Dopazo
3,4,6
, Salud Bo ego
1,2
& Guille mo An i~
nolo
1,2,3
1
Depa 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 Rocio/Consejo
Supe io de In es igaciones Cien 
ı icas/Uni e si y o Se ille, Se ille, Spain
2
Cen e o Biomedical Ne wo k Resea ch on Ra e Diseases (CIBERER), Se ille, Spain
3
Medical Genome P ojec , Genomics and Bioin o ma ics Pla o m o Andalusia (GBPA), Se ille, Spain
4
Depa men o Compu a ional Genomics, Cen o de In es igaci
on P 
ıncipe Felipe (CIPF), Valencia, Spain
5
Depa men o Oph halmology, Uni e si y Hospi al Vi gen del Roc
ıo, Se ille, Spain
6
Func ional Genomics Node (INB), CIPF, Valencia, Spain
Keywo ds
Ba de –Biedl Synd ome, in a amilial
a iabili y, MKKS, NGS, NPHP4.
Co espondence
Guille mo An i~
nolo, Depa men o Gene ics,
Rep oduc ion and Fe al Medicine, Uni e si y
Hospi al Vi gen del Rocio, A enida Manuel
Siu o s/n, 41013, Se ille. Spain.
Tel: + 34-955-012-772; Fax: +34-955-013-
473; E-mail: guille mo.an inolo.sspa@
jun adeandalucia.es
Funding In o ma ion
This wo k was suppo ed by he Ins i u o de
Salud Ca los III (ISCIII), Spanish Minis y o
Economy and Compe i i eness, Spain
(PI1102923), Regional Minis y o Economy,
Inno a ion, Science and Employmen o he
Au onomous Go e nmen o Andalusia
(CTS-03687), Regional Minis y o Heal h o
he Au onomous Go e nmen o Andalusia
(PI100154), (PCT-30000-2009-12),
INNPLANTA (PCT-300000-2010-007), Cibe
de En e medades a as (CIBERER), he
Founda ion Ramon A eces (CIVP16A1856),
he Spanish Minis y o Science and
Inno a ion (BIO2011-27069), he Conselle ia
de Educacio o he Valencia Communi y
(PROMETEO/2010/001), and F. Ho man-
LaRoche.
Recei ed: 10 Sep embe 2013; Re ised: 9
Oc obe 2013; Accep ed: 16 Oc obe 2013
doi: 10.1002/mgg3.50
Abs ac
Ba de –Biedl synd ome (BBS) is a model ciliopa hy cha ac e ized by a wide
ange o clinical a iabili y. The he e ogenei y o his condi ion is e lec ed in
he numbe o unde lying gene de ec s and he epis a ic in e ac ions be ween
he p o eins encoded. BBS is gene ally inhe i ed in an au osomal ecessi e ai .
Howe e , in some amilies, mu a ions ac oss di e en loci in e ac o modula e
he exp essi i y o he pheno ype. In o de o in es iga e he magni ude o epis-
asis in one BBS amily wi h ema kable in a amilial pheno ypic a iabili y, we
designed an exome sequencing–based app oach using SOLID 5500xl pla o m.
This s a egy allowed he eliable de ec ion o he p ima y causal mu a ions in
ou amily consis ing o wo no el compound he e ozygous mu a ions in
McKusick–Kau man synd ome (MKKS) gene (p.D90G and p.V396F). Addi ion-
ally, exome sequencing enabled he de ec ion o one no el he e ozygous NPHP4
a ian which is p edic ed o ac i a e a c yp ic accep o splice si e and is only
p esen in he mos se e ely a ec ed pa ien . He e, we p o ide an exome
sequencing analysis o a BBS amily and show he po en ial u ili y o his ool,
in combina ion wi h ne wo k analysis, o de ec disease-causing mu a ions and
second-si e modi ie s. Ou da a demons a e how nex -gene a ion sequencing
(NGS) can acili a e he dissec ion o epis a ic phenomena, and shed ligh on
he gene ic basis o pheno ypic a iabili y.
ª2013 The Au ho s. Molecula Gene ics & Genomic Medicine published by Wiley Pe iodicals, Inc.
This is an open access a icle unde he e ms o he C ea i e Commons A ibu ion License, which pe mi s use,
dis ibu ion and ep oduc ion in any medium, p o ided he o iginal wo k is p ope ly ci ed.
1
In oduc ion
Ciliopa hies a e an eme ging g oup o clinical diso de s
cha ac e ized by la ge gene ic he e ogenei y and clinical
o e lap. Due o he ubiqui ous na u e o he p ima y cil-
ium, ciliopa hies can a ec many o gan sys ems (Fliegau
e al. 2007). Ba de –Biedl synd ome (BBS; MIM 209900)
is conside ed a model o ciliopa hy (Badano e al. 2006a),
and i s p e alence a ies om 1:160,000 in Eu ope (Klein
and Ammann 1969; Beales e al. 1997) o 1:13,500 and
1:17,500 in Kuwai and New oundland, espec i ely (Fa ag
and Teebi 1989; G een e al. 1989). BBS is a pleio opic
diso de ha has a iable exp essi i y and a wide ange o
clinical a iabili y obse ed bo h wi hin and be ween am-
ilies (Beales e al. 1999). P ima y ea u es include e ini is
pigmen osa (RP), polydac yly, obesi y, geni al de ec s,
enal anomalies, and lea ning disabili ies. Seconda y ea-
u es include speech delay, de elopmen al delay, diabe es
melli us, den al anomalies, congeni al hea disease,
b achydac yly/syndac yly, a axia/poo coo dina ion, and
anosmia/hiposmia (Fo sy he and Beales 2013).
The clinical diagnosis o BBS equi es he p esence o
a leas ou p ima y o h ee p ima y and wo seconda y
ea u es (Beales e al. 1999; Roo yck and Lacombe 2008;
Pu oux e al. 2012). The spec um o clinical ea u es o
BBS sha es common cha ac e is ics wi h o he ciliopa hies
such as Joube synd ome (JBTS; MIM 213300), Lebe
congeni al amau osis (LCA; MIM 204000), McKusick–
Kau man synd ome (MKKS; MIM 236700), Meckel–
G ube synd ome (MKS; MIM 249000), neph onoph hisis
(NPH; MIM 256100), o o aciodigi al synd ome ype 1
(OFD1; MIM 311200), and Senio –Løken synd ome (SLS;
MIM 266900). Al hough hese a e conside ed as dis inc
clinical en i ies, in many cases i is e y di icul o assign
a speci ic clinical diagnosis due o pheno ypic o e lap.
Acco dingly, o he pheno ypic o e lap, gene ic o e lap
also exis s. Thus, mu a ions in MKKS (MIM 604896) also
cause MKKS –cha ac e ized by geni ou ina y mal o ma-
ions (hyd ome ocolpos), polydac yly, and mo e a ely
gas oin es inal abno mali ies (McKusick e al. 1964;
S one e al. 2000).
To da e, mu a ions in 18 genes ha e been associa ed
wi h BBS (Re inal In o ma ion Ne wo k websi e. A ail-
able: h ps://sph.u h.edu/ e ne /) accoun ing o ~70% o
a ec ed indi iduals (Mulle e al. 2010). BBS is ypically
inhe i ed in an au osomal ecessi e manne . Howe e , he
in ol emen o mu a ions in mo e han one locus has
been subs an ially g owing. Digenic iallelic inhe i ance
has been epo ed in some pa ien s wi h h ee mu a ions
ac oss wo BBS loci ha in e ac o cause disease (Ka san-
is e al. 2001; Badano e al. 2003; Beales e al. 2003). In
addi ion, he p esence o second-si e modi ie s ha may
modula e he exp ession o he clinical pheno ype has
been p oposed o explain, in pa , he signi ican in e -
and in a amilial clinical a iabili y in BBS (Ka sanis
2004; Badano e al. 2006b; Khanna e al. 2009).
In he las ew yea s, he in oduc ion o he nex -gene -
a ion sequencing (NGS) has e olu ionized clinical gene -
ics, making whole-exome sequencing (WES) a apid way
o elucida e he gene ic basis o clinically and gene ically
he e ogeneous Mendelian diso de s (Bamshad e al. 2011;
Ioni a-Laza e al. 2011; Rabbani e al. 2012). A p esen , i
is expec ed ha NGS, in combina ion wi h ne wo k analy-
sis (Minguez e al. 2009) and o he ad anced bioin o ma -
ics ools ha allows p io i izing candida e genes because
o hei unc ional ela ionships (Ideke and Sha an 2008),
will play an inc easingly impo an ole in he diagnosis o
complex and oligogenic diso de s. Mo eo e , WES p o-
ides a unique possibili y o in es iga ing he p esence o
addi ional mu a ions ha may modi y he exp essi i y o
BBS pheno ype. O he au ho s ha e been in e es ed in
de e mining he con ibu ion o epis asis in BBS (Ka sanis
e al. 2002; Hich i e al. 2005; Smaoui e al. 2006; Abu-Sa-
ieh e al. 2012), bu hei app oaches ha e been based on
Sange sequencing o known BBS loci (Ka sanis e al.
2002; Hich i e al. 2005; Smaoui e al. 2006; Abu-Sa ieh
e al. 2012) o on a ge ed exon cap u e sequencing o a
numbe o genes (Redin e al. 2012).
In his s udy, we conduc ed a WES app oach in a Span-
ish amily wi h signi ican in a amilial a iabili y in he
BBS pheno ype, om ull blown o ela i ely mild o ms
o BBS. Using his echnology, compound he e ozygous
mu a ions ha e been ound in MKKS and he con ibu ion
o addi ional changes has been dissec ed.
Ma e ial and Me hods
Subjec s and clinical assessmen
Ou s udy in ol ed one Spanish amily (RP42) com-
p ising h ee a ec ed and ou una ec ed indi iduals
(Fig. 1), all de i ed om he Oph halmology Depa -
men o ou Gene ic Depa men . The s udy was ca ied
ou in acco dance wi h he ene s o he Decla a ion o
Helsinki and he e hical guidelines o ou ins i u ions. A
g oup o 200 ma ching con ol indi iduals was also
ec ui ed. W i en in o med consen was ob ained om
all pa icipan s.
Clinical diagnosis o e inal degene a ion was based on
isual acui y, undus pho og aphy, compu e ized es ing
o cen al and pe iphe al isual ields, and elec o e inog-
aphy (ERG) indings. Typical ocula ea u es include ini-
ial nigh blindness, es ic ion o isual ield, bone
spicule pigmen a ion, a enua ion o e inal essels, waxy
disk pallo , and abno mal ERG indings in a od-cone
pa e n when eco dable.
2ª2013 The Au ho s. Molecula Gene ics & Genomic Medicine published by Wiley Pe iodicals, Inc.
No el Mu a ions in MKKS in a Ba de –Biedl Family M. Gonz
alez-del Pozo e al.
All subjec s unde wen a pe iphe al blood ex ac ion
o genomic DNA isola ion om leukocy es using s an-
da d p o ocols. DNA samples om indi iduals I:1, I:2,
II:3, and II:4 we e p ocessed o NGS.
A e he iden i ica ion o he MKKS gene de ec s,
a ec ed indi iduals unde wen clinical ee alua ion
ocused on he iden i ica ion o ex aocula ea u es asso-
cia ed wi h BBS.
Desc ip ion o DNA lib a y p epa a ion
and sequencing
Lib a y p epa a ion and exome cap u e we e pe o med
acco ding o a p o ocol based on he Baylo College o
Medicine p o ocol e sion 2.1 wi h se e al modi ica ions.
B ie ly, 5 lg o inpu genomic DNA was shea ed, end
epai ed, and liga ed wi h speci ic adap o s. A agmen
size dis ibu ion anging om 160 bp o 180 bp a e
shea ing and 200–250 bp a e adap o liga ion was
e i ied by Bioanalyze (Agilen Technologies, San a Cla a,
CA). The lib a y was ampli ied by p ecap u e linke -med-
ia ed polyme ase chain eac ion (LM-PCR) using Fas -
S a High Fideli y PCR Sys em (Roche, Indianapolis,
IN). A e pu i ica ion, 2 lg o LM-PCR p oduc was
hyb idized o NimbleGen SeqCap EZ Exome lib a ies V3.
A e washing, ampli ica ion was pe o med by pos cap u e
LM-PCR using Fas S a High Fideli y PCR Sys em
(Roche). Cap u e en ichmen was measu ed by qPCR
acco ding o NimbleGen p o ocol. The success ully cap-
u ed DNA was measu ed by Quan -iT
TM
PicoG een

dsDNA eagen (In i ogen, Ca lsbad, CA) and subjec ed
o s anda d sample p epa a ion p ocedu es o sequenc-
ing wi h SOLiD 5500xl pla o m as ecommended by he
manu ac u e . Sho ly, emulsion PCR was pe o med on
E80 scale (abou 1 billion empla e beads) using a con-
cen a ion o 0.616 PM o en iched cap u ed DNA. A e
b eaking and en ichmen , abou 276 million en iched
empla e beads we e sequenced pe lane on a six-lane
SOLiD 5500xl slide.
Analysis o da a om deep sequencing
SOLiD 5500xl eads we e aligned agains he human gen-
ome e e ence (hg19) using he p og am BFAST (Bla -like
Fas Accu a e Sea ch Tool) allowing eads o map only o a
unique posi ion in he e e ence genome. Imp ope ly
mapped eads we e il e ed ou wi h he SAM ools package,
which was also used o so SAM iles and o gene a e and
index BAM iles. Va ian calling was pe o med wi h he
so wa e GATK (Genome Analysis Toolki ) aking in o
accoun a ian s om NCBI da abase o Single Nucleo ide
Polymo phisms (dbSNP) o ecalib a ion and ealignmen .
Seconda y analysis was pe o med by a cus om shell sc ip
ha que ies VARIANT da abase (Medina e al. 2012),
which includes SIFT (Kuma e al. 2009) and Polyphen-2
(Adzhubei e al. 2010) sco es, o anno a e all single-nucleo-
ide a ia ions (SNVs) and small inse ions and dele ions
(INDELS). Va ian s wi h an allele equency highe han
5% in he 1000 Genomes P ojec da abase we e disca ded.
Only, exonic a ian s which p oduce a synonymous change
in he open eading ame we e disca ded, whe eas o he
ype o a ian s in he exomes and a ian s in splice si es
we e kep o u he analysis. Then, a ian s ound in
a ec ed indi iduals we e compa ed wi h a ian s p esen in
no a ec ed ela i es. A las s ep was pe o med o compa e
he emaining a ian s wi h a ian s ob ained om a g oup
o heal hy con ols om he same local popula ion as he
amily in s udy ob ained om The Medical Genome P ojec
(www.medicalgenomep ojec .com). Finally, genes wi h
a ian s in bo h alleles p esen in a ec ed bu no in heal hy
indi iduals o he amily no in he con ol local popula ion
and wi h a a ian only in one allele in he ca ie we e
anked based on he analysis o he in e ac ome, using Ne -
wo kMine (Ga cia-Alonso e al. 2012), o gene a e a lis o
candida e genes.
Ve i ica ion and assessmen o he
pa hogenici y o a ian s
Each p edic ed disease-causing a ian was con i med by
Sange sequencing, and coseg ega ion analysis was pe -
o med in he es o he amily membe s DNA samples.
As men ioned abo e, we used Polyphen-2 and SIFT
sco es o e alua e he po en ial impac o no el missense
subs i u ions on he unc ion o he encoded p o ein.
E olu iona y conse a ion ac oss species was assessed
I:1
[M2];[=]
[M3];[=]
I:2
[M1];[=]
II:1
[M2];[=]
II:2
[M1];[=]
[M2];[=]
II:3
[M1];[=]
II:4
[M1];[=]
[M2];[=]
[M3];[=]
II:5
[M1];[=]
[M2];[=]
M1: MKKS: c.269A>G p.D90G
M2: MKKS: c.1186G>T p.V396F
M3: NPHP4: c.992+71G>T (splice si e)
RP 42
Figu e 1. Family coseg ega ion analysis. RP42 amily ee showing
he seg ega ion o he sequence a ian s iden i ied du ing he
molecula analysis o MKKS and NPHP4. [M];[M]: homozygous; [M];
[=]: he e ozygous.
ª2013 The Au ho s. Molecula Gene ics & Genomic Medicine published by Wiley Pe iodicals, Inc. 3
M. Gonz
alez-del Pozo e al.No el Mu a ions in MKKS in a Ba de –Biedl Family
h ough he alignmen o o hologous MKKS p o ein
sequences (Pan oglody es,Mus musculus,Canis lupus a-
milia is,Gallus gallus,Xenopus opicalis, and Danio e io)
wi h he human MKKS p o ein sequence, using Clus al
Omega Tool (Sie e s e al. 2011). Fu he mo e, splice si e
ool P edic ion by Neu al Ne wo k (Reese 1997; Reese
e al. 1997), exonic splicing enhance p edic ion p og ams
ESE Finde (Ca egni e al. 2003; Smi h e al. 2006), and
Ne Gene2 se e (Hebsgaa d e al. 1996) we e applied o
es ima e he pa hogenic na u e o in onic sequence a i-
an s ha could a ec he splicing p ocess. The co ec
nomencla u e o mu a ion was checked applying Mu a-
lyze (Wildeman e al. 2008) using he co esponding
Genbank e e ence sequences (MKKS; NG_009109.1 and
NPHP4; NG_011724.2). No el a ian s included in his
a icle we e submi ed o he espec i e Locus Speci ic
Da abase (LSDB) (h p://g enada.lumc.nl/LOVD2/eye/home.
php?selec _db=MKKS).
Ne wo k analysis
Ne wo k en ichmen analysis has been pe o med using
he p og am SNOW (Minguez e al. 2009) included in
he Babelomics (Medina e al. 2010) package (h p://
www.babelomics.o g). Genes a e mapped on o he in e -
ac ome (ob ained om he STRING [F anceschini e al.
2013] da abase), and he subne wo k connec ing hem is
ob ained. Se e al ele an pa ame e s a e calcula ed o
his subne wo k, such as he connec i i y o he numbe
o componen s. An empi ical dis ibu ion o he andom
expec a ion o hese pa ame e s is ob ained by epea edly
sampling andom se s o he same numbe o genes om
he comple e genome and calcula ing he a e age connec-
i i y o hei co esponding subne wo ks. Thus, eal al-
ues o he pa ame e s ob ained o he genes analyzed can
be con as ed wi h espec o hei andom expec a ions
(Minguez e al. 2009; Minguez and Dopazo 2010).
Resul s
Clinical assessmen
The clinical indings o he a ec ed indi iduals (II:2, II:4,
and II:5) a e epo ed in Table 1. Ocula mani es a ions
o he disease in he h ee siblings we e ai ly ypical o
an ea ly onse and se e e o m o RP. Nigh blindness
was epo ed om he i s decade. The ea e , he disease
apidly p og essed, and by he age o 20, II:2, II:4, and
II:5 we e se e ely isually disabled. The undus examina-
ion showed he ypical signs o RP, including pale waxy
disks, a enua ion o he e inal essels, and bone spicule
pigmen a ion in he midpe iphe y. Pho opic and sco opic
ERGs we e ex inguished (II:2) o diminished (II:4) on
examina ion a ages 21 and 16, espec i ely. Pa ien II:4
showed ex aocula ea u es commonly associa ed wi h
BBS such as pos axial polydac yly, o e weigh , polycys ic
kidney, lea ning disabili ies, and mild psychomo o delay.
Pa ien s II:2 and II:5 exhibi ed a less se e e pheno ype
consis ing o RP, pos axial polydac yly, and mild lea ning
disabili ies. Subjec II:4 unde wen enal ansplan a ion
due o polycys ic kidney disease, whe eas he o he wo
did no mani es any enal abno mali ies. The e o e,
pa ien II:4 ul illed he c i e ia o he diagnosis o classic
BBS, whe eas II:2 and II:5 we e diagnosed o mild BBS.
Iden i ica ion o causal mu a ions
Upon exome sequencing, 43,570 sequence a ian s we e
iden i ied in pa ien II:4 (Table 2). The wo mos p oba-
bly pa hogenic mu a ions loca ed on exons 3 and 5 o
Table 1. Summa y o pheno ype ea u es documen ed in he
a ec ed membe s o he amily RP 42.
Fea u e II:2 II:4 II:5
Fi s symp om Nigh
blindness
Nigh
blindness
Nigh
blindness
Onse age 3 10 8
P ima y ea u es
Re ini is pigmen osa +++
Pos axial polydac yly Bo h ee Bo h ee One oo
Weigh gain anomaly No mal
weigh
Obese O e weigh
Geni al de ec s,
hyd ome ocolpos

Lea ning disabili ies +++
Renal anomalies
Kidney cys s +
Kidney ansplan +
Seconda y ea u es
Poo coo dina ion +
De elopmen al delay 
Speech delay 
B achydac yly/Syndac yly 
Den al anomalies
Tee h c owding 
Congeni al hea disease 
Table 2. Va ian s iden i ied by exome sequencing in he RP 42
amily.
I:1 I:2 II:3 II:4
To al SNVs 45,068 42,243 43,269 43,570
Nonsynonymous SNVs 6114 5855 5892 5942
Fil e ed dbSNP 5979 5735 5756 5821
Fil e ed dbSNP and 1000 g 5913 5692 5710 5760
Fil e ed dbSNP and 1000 g
and p edic ed dele e ious
357 373 355 367
SNVs, single-nucleo ide a ia ions.
4ª2013 The Au ho s. Molecula Gene ics & Genomic Medicine published by Wiley Pe iodicals, Inc.
No el Mu a ions in MKKS in a Ba de –Biedl Family M. Gonz
alez-del Pozo e al.
MKKS gene (Fig. 2) ha e ne e been epo ed in public
a ian da abases such as dbSNP, Exome Va ian Se e
(h p://e s.gs.washing on.edu/EVS/), o 1000 genomes
(h p://www.1000 genomes.o g/). Sange sequencing o
hese exons con i med he no el missense a ian s
(c.269A>G; p.D90G and c.1186G>T; p.V396F) in a com-
pound he e ozygous s a e in he a ec ed siblings. The
a ian s coseg ega ed wi h he disease in he en i e amily
(Fig. 1) and we e absen in 400 con ol ch omosomes.
Mu a ion D90G lay wi hin he p edic ed equa o ial
domain and mu a ion V396F lay wi hin he p edic ed
in e media e domain which connec s he equa o ial and
c.269A>G
Sample II:4
c.1186G>T
Re e ence
29.04 Kbs andRe e se
123456
MKKS
Ch 20 p12.2
A
B
C
Figu e 2. De ec ion o no el mu a ions in he MKKS gene. (A) Ch omosome o e iew o he ch omosome 20, MKKS is mapped on egion
20p12.2 (black ba ). (B) MKKS spans app oxima ely 29 Kb and is composed o six exons. Filled boxes e lec coding exons (3–6) and un illed
boxes e lec UTR. (C) Elec ophe og am depic ion o he index pa ien (II:4) con i ming he he e ozygous mu a ions in exons 3 and 5 o MKKS
gene. IUPAC SNP codes used o designa e he e ozygous subs i u ions (“k” om Ke o o G/T and “ ” om puRine o A/G). MKKS Genbank
accession numbe : NG_009109.1.
Figu e 3. MKKS p o ein depic ion. (A) Schema ic ep esen a ion o he iden i ied a ian s wi hin he MKKS domains, including he ypical
chape onin g oup II domains (equa o ial, in e media e, and apical). The domain o ganiza ion was modi ied om (S oe zel e al. 2007). (B)
Alignmen o he o hologs om di e en species showing conse a ion o he mu a ed esidues. An *(as e isk) indica es posi ions which ha e a
single, ully conse ed esidue. A: (colon) indica es conse a ion be ween g oups o s ongly simila p ope ies. A. (pe iod) indica es conse a ion
be ween g oups o weakly simila p ope ies.
ª2013 The Au ho s. Molecula Gene ics & Genomic Medicine published by Wiley Pe iodicals, Inc. 5
M. Gonz
alez-del Pozo e al.No el Mu a ions in MKKS in a Ba de –Biedl Family

he apical domains ia lexible hinges (S one e al. 2000)
(Fig. 3A). Alignmen o MKKS amino acid sequence o
a ious o hologs showed ha he subs i u ed esidues
(aspa ic acid a posi ion 90 and aline a posi ion 396)
a e highly conse ed ac oss species om di e en
e olu iona y b anches (Fig. 3B). In silico ools p edic ed
ha bo h p.D90G and p.V396F a e p obably damaging by
Polyphen-2 (sco e =1 o p.D90G and sco e =0.987 o
p.V396F) and SIFT (sco e =0.01 o p.D90G and
sco e =0 o p.V396F).
Epis asis e alua ion
Whole-exome da a we e subjec ed o exhaus i e e alua-
ion paying special a en ion in he iden i ica ion o addi-
ional a ian s ha may be ac ing as second-si e modi ie s
making pa ien ’s II:4 BBS pheno ype mo e se e e.
Al hough se e al SNPs we e iden i ied in he emaining
16 known BBS genes: BBS1 (MIM 209901), BBS2 (MIM
606151), ARL6 (BBS3; MIM 608845), BBS4 (MIM
600374), BBS5 (MIM 603650), MKKS (BBS6), BBS7
(MIM 607590), TTC8 (BBS8; MIM 608132), BBS9 (MIM
607968), BBS10 (MIM 610148), TRIM32 (BBS11;
MIM 602290), BBS12 (MIM 610683), MKS1 (BBS13;
MIM 609883), CEP290 (BBS14; MIM 610142), SDCCAG8
(BBS16; MIM 613524), LZTFL1 (BBS17; MIM 606568),
and INPP5E (MIM 613037) (Table S1); none o hem was
p edic ed o be pa hogenic.
To explo e he possibili y ha he polycys ic kidney
disease (PKD), a ec ing only pa ien II:4, may be
caused by mu a ions in o he loci such as PKD genes:
PKD1 (MIM 601313), PKD2 (MIM 173910), and
PKHD1 (MIM 606702); o neph onoph hisis genes:
NPHP1 (MIM 607100), INVS (NPHP2; MIM 243305),
NPHP3 (MIM 608002), NPHP4 (MIM 607215), IQCB1
(NPHP5; MIM 609237), GLIS2 (NPHP7; MIM 608539),
RPGRIP1L (NPHP8; MIM 610937), NEK8 (NPHP9;
MIM 609799), NEK1 (MIM 604588), MKS1 (MIM
609883), and TMEM67 (NPHP11; MIM 609884) (Table
S1), we e alua ed he exome da a and ound one no el
a ian in in on 8-9 o he NPHP4 gene
(c.992 +71G>T). This a ian has ne e been epo ed
in da abases such as dbSNP and EVS. As co e age
in o ma ion o his posi ion was no clea in he EVS
da a se , we checked he absence o c.992 +71G>Tin
addi ional genomic da abases which also include in on-
ic a ian s (1000 genomes and 5000 genomes). Family
seg ega ion showed he p esence o he he e ozygous
NPHP4 a ian only in pa ien II:4 and in his una -
ec ed pa en (I:1) (Fig. 1). In silico ools p edic ed ha
he subs i u ion o a G >T in his posi ion ac i a es a
c yp ic splice accep o si e in he in on 8-9 o NPHP4
(Table 3). The comple e coding sequence was scanned
o elimina e he possibili y o he p esence o o he
mu a ion in NPHP4. The absence in 400 con ol
ch omosomes and he esul s o he in silico p edic ion
suppo ed he pa hogenic ole o he NPHP4 c.992 +
71G>T a ian .
Ne wo k analysis
In o de o explo e he possible physical in e ac ions
among he BBS p o eins and NPHP4, ne wo k en ich-
men analysis, using he p og am SNOW (Minguez e al.
2009), has been pe o med. Figu e 4 shows he p o eins
signi ican ly connec ed (signi ican ly smalles numbe o
componen s, P- alue =0.01) allowing he in oduc ion o
one ex a connec ing node among he p o eins s udied.
The ne wo k documen s he dense ne wo k o physical
p o ein–p o ein in e ac ions ha connec BBS p o eins
and also documen s how NPHP4 connec s o se e al BBS
p o eins h ough di e en in e media es. O special in e -
es is RPGR, which connec s NPHP4 o CEP290.RPGR,
he RP GTPase egula o , is also in ol ed in RP X-Linked,
cone- od dys ophy X-linked, and macula dys ophy
X-linked. Ano he in e es ing gene is INVS, In e sin,
which encodes a p o ein ha p o ein may unc ion in
enal ubula de elopmen and unc ion. Such connec-
ions ein o ce he possible ole o NPHP4 as modula o
o he pene ance o he disease.
Discussion
In his epo , a Spanish BBS amily wi h h ee a ec ed sib-
lings is desc ibed. The mode o inhe i ance and he main
clinical ea u es co espond o au osomal ecessi e BBS.
Table 3. Splice accep o si e p edic ion sco es o NPHP4
c.992 +71G>T mu a ion e sus wild ype.
Allele
Wild ype
agg ca g g
ca g cagg g
c.992 +71G>T
in onEXON
agg ca g ca g cagGTGT
NNSPLICE 0 0.71
ESE inde
3SS_U2_human
( h eshold: 6.632)
0 7.735500
3SS_U2_mouse
( h eshold: 6.724)
0 7.26420
B anchSi e
( h eshold: 0)
0 2.07090 ( g ca )
Ne Gene2 0 0.28
Sequence a ian s a e highligh ed in bold; po en ial splice accep o
si e a e unde lined; p edic ed exonic sequence is in capi al le e s.
3SS_U2_Human: 3’s splice si es (accep o ) o human (U2 ype).
3SS_U2_mouse: 3’s splice si es (accep o ) o mouse (U2 ype). B anch
si e: mammalian b anch si e (U2 ype). NPHP4 Genbank accession
numbe : NG_011724.2.
6ª2013 The Au ho s. Molecula Gene ics & Genomic Medicine published by Wiley Pe iodicals, Inc.
No el Mu a ions in MKKS in a Ba de –Biedl Family M. Gonz
alez-del Pozo e al.
Exome analysis o ou indi iduals (I:1, I:2, II:3, and
II:4) led o he iden i ica ion o wo no el compound he -
e ozygous mu a ions in he MKKS gene (c.269A>G;
p.D90G and c.1186G>T; p.V396F) o a ec ed indi iduals.
These a ian s we e absen in 200 con ol indi iduals and
showed seg ega ion wi h disease in he en i e amily.
Mu a ion D90G lay wi hin he p edic ed equa o ial
domain, he mos conse ed egion among g oup I and II
chape onins. In he g oup II chape onin amily, his
domain is esponsible o ATP hyd olysis and he subs i-
u ion migh al e i s s uc u e. Mu a ion V396F lay
wi hin he p edic ed in e media e domain which connec s
he equa o ial and he apical domains ia lexible hinges
(S one e al. 2000). Bioin o ma ic analysis p edic ed pa h-
ogenic consequences o bo h missense mu a ions.
The molecula diagnosis is gene ally help ul o con i m
a clinical diagnosis. Al hough many s udies ha e dissec ed
he clinical o e laps due o MKKS mu a ions, geno ype–
pheno ype co ela ions a e s ill no well unde s ood. The
clinical e alua ion o he a ec ed membe s o he amily
did no e eal hyd ome ocolpos, sugges ing ha hese
wo missense mu a ions in he MKKS gene did no cause
geni al mal o ma ions ypical o MKKS pheno ype. In his
amily, mu a ions in MKKS esul ed in a spec um o BBS
pheno ypes, anging om BBS wi h se e e enal in ol e-
men in pa ien II:4 o milde o ms o BBS (pa ien s II:2
and II:5). The exp ession o he pheno ype and he dis-
ease p og ession can a y g ea ly om pa ien o pa ien ,
e en among membe s o he same amily. Whe eas e inal
dys ophy, digi anomaly, and lea ning disabili ies we e
highly pene an ai s o all pa ien s, kidney abno mali-
ies and obesi y showed incomple e pene ance in ou
amily.
The index pa ien in es iga ed he e had p e iously
unde gone selec ed geno yping (APEX analysis, Aspe
h p://www.aspe bio.com/aspe -oph halmics), bu his
mu a ional sc eening app oach ailed o iden i y he
unde lying gene de ec in his amily. The implemen a ion
o a eliable diagnos ic sys em able o de ec no el dis-
ease-causing mu a ions, e en in genes no p e iously
associa ed wi h an assumed clinical diagnosis, is necessa y.
Exome sequencing has p o en o be an impo an diag-
nos ic ool o diso de s ha a e cha ac e ized by signi i-
can gene ic he e ogenei y. In he case o BBS, besides he
high numbe o genes in ol ed, oligogenic inhe i ance is
also well documen ed (Ka sanis 2004) adding a laye o
complexi y o he gene ic cha ac e iza ion o such
pa ien s.
One ad an age o analyzing he whole exome simul a-
neously is he unique possibili y o in es iga e he
in ol emen o second-si e mu a ions which may be mod-
ula ing he exp ession o he BBS pheno ype (Ka sanis
e al. 2001; Badano e al. 2003; Beales e al. 2003; Ka sanis
2004; Hjo shoj e al. 2010). Howe e , he use o unbi-
ased me hodologies o en p oduces many candida es ha
mus be il e ed ou wi h in silico p io i iza ion ech-
niques. Thus, ne wo k analysis cap u es he ela ionships
o mu a ed genes wi h al eady known disease genes allow-
ing a a ional p io i iza ion o candida e genes. The com-
bina ion o NGS wi h ne wo k analysis app oach
Figu e 4. Signi ican p o ein–p o ein in e ac ion ne wo k: Ne wo k analysis allows ela ing NPHP4 o BBS p o eins, suppo ing in his way i s’
possible ole as modi ie gene.
ª2013 The Au ho s. Molecula Gene ics & Genomic Medicine published by Wiley Pe iodicals, Inc. 7
M. Gonz
alez-del Pozo e al.No el Mu a ions in MKKS in a Ba de –Biedl Family
conduc ed he e allowed us o iden i y a no el a ian –
loca ed on in on 8-9 o NPHP4 –which was p edic ed
o enhance he use o a c yp ic splice accep o si e. This
would p obably cause he in oduc ion o a p ema u e
e mina ion codon and he educ ion in NPHP4 mRNA
le els. Only he mos se e ely a ec ed pa ien (II:4) ca -
ies he a ian (c.992 +71G>T) in he e ozygous s a e. In
addi ion, he absence o he a ian in con ol indi iduals
seems o indica e ha i is a pa hogenic allele.
NPHP4 is a cilia y p o ein ha is known o belong o
a mul i unc ional complex and colocalizes wi h RPGRIP1,
RPGR, and he se ologically de ined colon cance an igen-
8 (SDCCAG8), a p o ein hough o pa ake in he
RPGRIP1 in e ac ome and implica ed also in e inal– enal
ciliopa hies (Roepman e al. 2005; Schae e e al. 2011;
Won e al. 2011; Pa il e al. 2012). Mu a ions in NPHP4
ha e been associa ed wi h ela ed ciliopa hies such as
NPH (Molle e al. 2002; Hoe ele e al. 2004) and SLS
(O o e al. 2002; Schue mann e al. 2002).
Few splice si e a ian s ha e been ound o modula e
he pa hogenesis o BBS. One example is he allele C430T
o MGC1203 which enhances he use o a c yp ic splice
accep o si e, causing he in oduc ion o a p ema u e e -
mina ion codon and he educ ion in he s eady-s a e
MGC1203 mRNA le els (Badano e al. 2006b). Al hough
he MGC1203 mu a ions a e p obably insu icien o
cause BBS, his gene may be in ol ed in he pa hogenesis
o BBS by con ibu ing hypomo phic mu a ions o an
al eady sensi ized gene ic backg ound. The au ho s con-
clude ha supp ession o MGC1203 exe s an epis a ic
e ec on he de elopmen al BBS pheno ype. I is emp -
ing o specula e ha NPHP4 allele epo ed he ein may
also show a simila e ec . We hypo hesize ha he
NPHP4 a ian may be ac ing as a second-si e modi ie
a ec ing he likelihood o enal degene a ion in he con-
ex o he o he MKKS mu a ions. This hypo hesis is also
suppo ed by he known physical ela ionships e ealed
by ne wo k analysis. The MKKS p o ein o ms a signi i-
can clus e o p o eins wi h se e al BBS p o eins, as
shown in Figu e 4. The se e e BBS pheno ype obse ed in
pa ien II:4 may be due o an epis a ic e ec o MKKS
and NPHP4 mu a ions. This would p o ide an explana-
ion o one o he dis inc clinical mani es a ions
obse ed in his amily. Mo eo e , i is possible ha o he
in a amilial pheno ypic a ia ions may be due o al e a-
ions in addi ional loci as well as nons ic ly gene ic ac-
o s no e alua ed in his s udy. The e o e, ou indings
wa an u he s udies o e alua e he ole o pu a i e
disease-modi ying a ian s.
Iden i ying he p esence o epis a ic a ian s can be chal-
lenging, gi en ha a la ge numbe o common and a e
alleles –loca ed in known and in no el genes –has been
shown o in luence he exp essi i y o he BBS pheno ype.
Ou da a demons a e ha NGS is a powe ul app oach o
iden i y a e and high pene an disease a ian s and o
s udy he gene ic con ibu ion o pheno ypic a iabili y.
This gene ic diagnos ic ool can now be applied o la ge
coho s o Mendelian and oligogenic diso de s and should
apidly p o ide he molecula diagnosis and he p e a-
lence o associa ed genes.
In summa y, we show how WES has clea ly imp o ed
he molecula diagnosis o he e ogeneous diso de s such
as BBS. In addi ion, ou s udy highligh s he use ulness o
NGS app oaches o he dissec ion o epis a ic phenom-
ena and p o ides p omising indings o deciphe he
gene ic basis o pheno ypic a iabili y.
Acknowledgmen s
The au ho s a e g a e ul o he amily desc ibed in his
s udy. This wo k was suppo ed by he Ins i u o de Salud
Ca los III (ISCIII), Spanish Minis y o Economy and
Compe i i eness, Spain (PI1102923), Regional Minis y o
Economy, Inno a ion, Science and Employmen o he
Au onomous Go e nmen o Andalusia (CTS-03687),
Regional Minis y o Heal h o he Au onomous Go e n-
men o Andalusia (PI100154), he Founda ion Ramon
A eces (CIVP16A1856), he Spanish Minis y o Science
and Inno a ion (BIO2011-27069), he Conselle ia de Edu-
cacio o he Valencia Communi y (PROMETEO/2010/
001), and F. Ho man-LaRoche. The CIBERER is an ini-
ia i e o he ISCIII, Spanish Minis y o Economy and
Compe i i eness. 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 .
Con lic o In e es
None decla ed.
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