ARTICLE
S uc u al Va ian s C ea e New Topological-Associa ed
Domains and Ec opic Re inal Enhance -Gene Con ac
in Dominan Re ini is Pigmen osa
Suzanne E. de B uijn,
1,2,26
Alessia Fio en ino,
3,4,5,26
Daniele O a iani,
3
S ephanie Fanucchi,
6
Ui a
´S. Melo,
7,8
Julio C. Co al-Se ano,
3
Timo Mulde s,
2,9
Michalis Geo giou,
3,10
Ca lo Ri ol a,
11,12,13
Nikolas Pon ikos,
3,4,5
Ga in A no,
3,4,5,10
Lisa Robe s,
14
Jacquie G eenbe g,
14
Sil ia Albe ,
1
Ch is ian Gilissen,
1
Ma co Aben,
1
Geo ge Rebello,
14
Simon Mead,
15
F. Lucy Raymond,
16,17
Jo di Co ominas,
1
Clai e E.L. Smi h,
18
Hannie K eme ,
1,2,19
Susan Downes,
4,5,20
G aeme C. Black,
4,5,21
And ew R. Webs e ,
3,4,5,10
Ch is F. Inglehea n,
4,5,18
L. Ingebo gh an den Bo n,
22
Robe K. Koenekoop,
23
Michel Michaelides,
3,4,5,10
Raj S. Ramesa ,
14
Ca el B. Hoyng,
2,9
S e an Mundlos,
7,8
Musa M. Mhlanga,
1,6,24,25
F ans P.M. C eme s,
1,2
Michael E. Chee ham,
3,4,5
Susanne Roosing,
1,2,27,
*and Alison J. Ha dcas le
3,4,5,27
Summa y
The cause o au osomal-dominan e ini is pigmen osa (adRP), which leads o loss o ision and blindness, was in es iga ed in amilies
lacking a molecula diagnosis. A e ined locus o adRP on Ch 17q22 (RP17) was delinea ed h ough geno yping and genome
sequencing, leading o he iden i ica ion o s uc u al a ian s (SVs) ha seg ega e wi h disease. Eigh di e en complex SVs we e cha -
ac e ized in 22 adRP-a ec ed amilies wi h >300 a ec ed indi iduals. All RP17 SVs had b eakpoin s wi hin a genomic egion spanning
YPEL2 o LINC01476. To in es iga e he mechanism o disease, we ep og ammed ib oblas s om a ec ed indi iduals and con ols in o
induced plu ipo en s em cells (iPSCs) and di e en ia ed hem in o pho o ecep o p ecu so cells (PPCs) o e inal o ganoids (ROs). Hi-C
was pe o med on ROs, and di e en ial exp ession o egional genes and a e inal enhance RNA a his locus was assessed by qPCR. The
epigene ic landscape o he egion, and Hi-C RO da a, showed ha YPEL2 si s wi hin i s own opologically associa ing domain (TAD),
ich in enhance s wi h binding si es o e inal ansc ip ion ac o s. The Hi-C map o RP17 ROs e ealed c ea ion o a neo-TAD wi h
ec opic con ac s be ween GDPD1 and e inal enhance s, and modeling o all RP17 SVs was consis en wi h neo-TADs leading o ec opic
e inal-speci ic enhance -GDPD1 accessibili y. qPCR con i med inc eased exp ession o GDPD1 and inc eased exp ession o he e inal
enhance ha en e s he neo-TAD. Al e ed TAD s uc u e esul ing in inc eased e inal exp ession o GDPD1 is he likely con e gen
mechanism o disease, consis en wi h a dominan gain o unc ion. Ou s udy highligh s he impo ance o SVs as a genomic mecha-
nism in unsol ed Mendelian diseases.
In oduc ion
Despi e ecen ad ances in nex -gene a ion sequencing,
app oxima ely 30%–40% o indi iduals wi h inhe i ed
e inal diseases (IRDs) lack a molecula diagnosis. This is
p obably due o a combina ion o a e no el disease genes,
which equi e la ge coho s o alida ion, and p e iously
in ac able mu a ion classes, such as in onic a ian s,
s uc u al a ian s (SVs), and a ian s in egula o y e-
gions.
1,2
1
Depa men o Human Gene ics, Radboud Uni e si y Medical Cen e , Nijmegen, 6500 HB, he Ne he lands;
2
Donde s Ins i u e o B ain Cogni ion and
Beha iou , Radboud Uni e si y Medical Cen e , Nijmegen, 6500 HB, he Ne he lands;
3
UCL Ins i u e o Oph halmology, London, EC1V 9EL, UK;
4
UK In-
he i ed Re inal Disease Conso ium;
5
Genomics England Clinical In e p e a ion Pa ne ship;
6
Gene Exp ession and Biophysics G oup, Di ision o Chem-
ical, Sys ems and Syn he ic Biology, Depa men o In eg a i e Biomedical Science, Ins i u e o In ec ious Disease & Molecula Medicine, Facul y o Heal h
Sciences, Uni e si y o Cape Town, Cape Town, 7935, Sou h A ica;
7
Max Planck Ins i u e o Molecula Gene ics, RG De elopmen & Disease, Be lin,
14195, Ge many;
8
Ins i u e o Medical and Human Gene ics, Cha i e
´– Uni e si a
¨ smedizin, Be lin, 10117, Ge many;
9
Depa men o Oph halmology,
Radboud Uni e si y Medical Cen e , Nijmegen, 6500 HB, he Ne he lands;
10
Moo ields Eye Hospi al, London, EC1V 2PD, UK;
11
Depa men o Gene ics
and Genome Biology, Uni e si y o Leices e , Leices e , LE1 7RH, UK;
12
Clinical Resea ch Cen e , Ins i u e o Molecula and Clinical Oph halmology Basel
(IOB), Basel, 4031, Swi ze land;
13
Depa men o Oph halmology, Uni e si y Hospi al Basel, Basel, 4001, Swi ze land;
14
Uni e si y o Cape Town/MRC
Genomic and P ecision Medicine Resea ch Uni , Di ision o Human Gene ics, Depa men o Pa hology, Ins i u e o In ec ious Disease and Molecula Med-
icine, Facul y o Heal h Sciences, Uni e si y o Cape Town, Cape Town, 7935, Sou h A ica;
15
MRC P ion Uni a UCL, UCL Ins i u e o P ion Disease, Lon-
don, W1W 7FF, UK;
16
NIHR BioResou ce, Camb idge Uni e si y Hospi als, Camb idge, CB2 0QQ, UK;
17
Depa men o Medical Gene ics, Camb idge Ins i-
u e o Medical Resea ch, Uni e si y o Camb idge, Camb idge, CB2 OXY, UK;
18
Di ision o Molecula Medicine, Leeds Ins i u e o Medical Resea ch,
Uni e si y o Leeds, Leeds, LS2 9JT, UK;
19
Depa men o O o hinola yngology, Radboud Uni e si y Medical Cen e , Nijmegen, 6500 HB, he Ne he lands;
20
Ox o d Eye Hospi al, Ox o d Uni e si y Hospi als NHS T us and Nu ield Labo a o y o Oph halmology, Uni e si y o Ox o d, Ox o d, OX3 9DU, UK;
21
Manches e Cen e o Genomic Medicine, S . Ma y’s Hospi al, Manches e , M13 9WL, UK;
22
The Ro e dam Eye Hospi al, Ro e dam, 3011 BH, he
Ne he lands;
23
Depa men o Paedia ic Su ge y, Human Gene ics and Oph halmology, McGill Uni e si y, Mon e
´al, QC H4A 3J1, Canada;
24
Gene Exp es-
sion and Biophysics Uni , Ins i u o de Medicina Molecula , Faculdade de Medicina Uni e sidade de Lisboa, Lisbon, 1649-028, Po ugal;
25
Epigenomics &
Single Cell Biophysics G oup, Radboud Ins i u e o Molecula Li e Sciences (RIMLS), Radboud Uni e si y, Nijmegen, 6525 GA, he Ne he lands
26
These au ho s con ibu ed equally
27
These au ho s con ibu ed equally
*Co espondence: susanne. oosi[email p o ec ed]
h ps://doi.o g/10.1016/j.ajhg.2020.09.002.
802 The Ame ican Jou nal o Human Gene ics 107, 802–814, No embe 5, 2020
Ó2020 The Au ho s. This is an open access a icle unde he CC BY license (h p://c ea i ecommons.o g/licenses/by/4.0/).
The mos common o m o IRD is e ini is pigmen osa
(RP [MIM: 268000]), which is gene ically he e ogeneous,
wi h a p e alence o 1 in 4,000.
3
RP is de ined as a
e inal degene a ion ha p ima ily a ec s od pho o e-
cep o s, esul ing in nigh blindness and p og essi e
loss o pe iphe al ision, o en p og essing in o he
cen al e ina and a ec ing cone pho o ecep o s,
leading o se e e isual impai men o blindness (see
‘‘Nonsynd omic Re ini is Pigmen osa O e iew’’ in Web
Resou ces). Au osomal-dominan RP (adRP) accoun s
o 25%–40% o cases, depending on he popula ion
s udied, and has been associa ed wi h mu a ions
in 30 genes, including CA4 (MIM: 114760) on
Ch 17q23.1 (RP17 [MIM: 600852])(see ‘‘Re Ne ’’ in Web
Resou ces).
4,5
Following ini ial publica ions de ining
his locus
6,7
a a ian inCA4 wasimplica edas hecause
o adRP in amilies o Sou h A ican o igin, howe e
pa hogenici y o he epo ed a ian has been ques-
ioned because i has a popula ion equency o 4% in
heal hy con ols in no he n Sweden.
8–10
Subsequen ly
epo ed CA4 a ian s in indi iduals wi h RP we e iden-
i ied by a ge ed Sange sequencing and do no ully
exclude a ian s in o he genes as a cause o disease
(Table S1).
We in es iga ed he cause o adRP in unsol ed amilies,
including he i s pedig ee (GC1, e e ed o as UK1)
d awn up a Moo ields Eye Hospi al o e 35 yea s ago
and he o iginal Du ch amily (W97-079, e e ed o as
NL1) ha showed linkage o he RP17 locus bu lacked a
mu a ion in CA4.
7
He e, we epo iden i ica ion and cha ac e iza ion o
complex SVs on Ch 17q22, h ough whole-genome
sequencing (WGS), as he genomic cause o adRP a
he RP17 locus in a la ge numbe o amilies, including
he amilies o Sou h A ican o igin. To explo e a
con e gen mechanism o disease, we in es iga ed
he e ec o RP17 SVs on h ee-dimensional (3D)
ch oma in o ganiza ion ha esul s in he compa -
men aliza ion o he genome in o opologically associ-
a ing domains (TADs) and he epigene ic landscape o
he egion. TADs a e ch oma in domains wi hin he
genome ha acili a e enhance p omo e con ac s
wi hin he nuclea 3D space.
11
Dis up ion o TAD
s uc u es can lead o loss o ch omosomal con ac
be ween egula o y egions and hei a ge genes o
he o ma ion o no el ac i e domains wi h ec opic
con ac s occu ing be ween egula o y egions and a
new a ge gene, esul ing in pa hogenic al e a ions
in gene exp ession.
12–15
We demons a e ha al e ed
TAD s uc u e a he RP17 locus leads o ec opic
e inal enhance -gene in e ac ions, consis en wi h a
dominan gain o unc ion. Ou s udy highligh s he
pa hogenici y o SVs ha al e 3D ch oma in o ganiza-
ion and gene exp ession by ea anging TAD s uc u es
and he need o e isi a e Mendelian diseases o which
genes and a ian s ha e no been subs an ia ed in o he
coho s.
Ma e ial and Me hods
S udy Coho
The s udy was app o ed by he medical e hics commi ee o he
E asmusMC Ro e dam, Radboudumc Nijmegen, and Moo ields
Eye Hospi al and was pe o med in acco dance wi h he p inciples
o he Wo ld Medical Associa ion Decla a ion o Helsinki.
In o med consen was ob ained om all pa icipan s o hei legal
ep esen a i es.
Gene ic Analyses
We pe o med SNP geno yping o index amilies NL1 and UK1
o de ine and e ine he RP17 locus. Genomic DNA om a ec ed
indi iduals and hei amily membe s was analyzed by whole-
exome sequencing (WES) and WGS. Sequence da a was aligned
o he Human Re e ence Genome build hg19. Va ian s we e
p io i ized on he basis o a mino allele equency (MAF) %
0.0001 in gnomAD. SVs we e called wi h ExomeDep h, Man a
S uc u al Va ian Calle , Can as Copy Numbe Va ian Calle ,
and Con ol-FREEC. De ails o geno yping, sequencing, and
analysis pipelines a e p o ided in he Supplemen al Ma e ial
and Me hods.
Cha ac e iza ion and Valida ion o S uc u al Va ian s
SV b eakpoin junc ions we e PCR ampli ied and alida ed wi h
Sange sequencing. P ime sequences and coo dina es a e lis ed
in Table S2. SV b eakpoin egions we e assessed o he p esence
o mic ohomology and epe i i e elemen s. To alida e a ipli-
ca ed egion o UK-SV6, we pe o med quan i a i e eal- ime
PCR (qPCR) on genomic DNA om a ec ed indi iduals om am-
ily UK13 and una ec ed con ols (Supplemen al Ma e ial and
Me hods).
Clinical Analysis
A ailable clinical no es o cases o he pedig ees iden i ied a Rad-
boudumc, Moo ields Eye Hospi al, Uni e si y o Cape Town, and
McGill Uni e si y Heal h Cen e we e e iewed, as well as de ailed
e inal imaging, undus au o luo escence, and op ical cohe ence
omog aphy. Age o onse is de ined as he age a which symp oms
we e i s expe ienced.
In e oga ion o he Genomic Region
We in e oga ed ch oma in and genome egula ion da ase s o
explo e he epigenomic landscape o he egion. A ailable da ase s
we e ob ained and analyzed ia he UCSC genome b owse (de ails
o da ase s used a e p o ided in Supplemen al Ma e ial and
Me hods).
Rep og amming Fib oblas s in o iPSCs and
Di e en ia ion in o Pho o ecep o P ogeni o Cells and
3D Re inal O ganoids
Fib oblas s we e cul u ed om skin biopsies o wo indi iduals
wi h NL-SV1, one indi idual wi h UK-SV2, and i e anonymous
con ol indi iduals. Cell lines we e ep og ammed in o induced
plu ipo en s em cells (iPSCs) and di e en ia ed in o pho o e-
cep o p ogeni o cells (PPCs) ollowing he p e iously desc ibed
60 day p o ocol (Supplemen al Ma e ial and Me hods).
16,17
3D
e inal o ganoids (ROs) we e di e en ia ed o UK-SV2 and
con ols, as p e iously desc ibed (Supplemen al Ma e ial and
Me hods).
18
The Ame ican Jou nal o Human Gene ics 107, 802–814, No embe 5, 2020 803
P epa a ion o Low Inpu Hi-C Lib a ies (Low-C)
Hi-C was pe o med on UK-SV2 and con ol 3D ROs ia a low
inpu p o ocol (Low-C) wi h ew modi ica ions (Supplemen al
Ma e ial and Me hods).
19
Two lib a ies pe sample we e sequenced
o 200 million agmen s in a 100 bp pai ed-end un on a No a-
Seq 6000 (Illumina). Pai ed-end sequencing da a was p ocessed
ia Juice
20
and he Hi-C maps we e c ea ed wi h a bin size wi h
10 kb esolu ion. Fu he in o ma ion abou he bioin o ma ics
pipeline is de ailed in Melo e al., 2020.
21
Exp ession Analysis o Genes and Enhance RNA wi hin
he RP17 Locus
To assess exp ession o genes, we pe o med qPCR o di e en hu-
man issues, including e ina (Table S3 and Supplemen al Ma e ial
and Me hods). Single-cell RNA sequencing da a o human
22
and
p ima e
23
e inal cell ypes we e ob ained and isualized ia he
B oad Ins i u e Single Cell Po al (Supplemen al Ma e ial and
Me hods).
cDNA was syn hesized om o al RNA ex ac ed om PPCs,
ROs, and ib oblas s. Di e en ial exp ession o genes implica ed
in he SVs, and con ol housekeeping and e inal p ogeni o
genes, was assessed by qPCR (Table S3 and Supplemen al Ma e ial
and Me hods). We designed p ime s o he enhance egion con-
aining mul iple e inal ansc ip ion ac o binding si es impli-
ca ed in all SVs o analyze a ge ed enhance RNA exp ession by
qPCR (Table S3 and Supplemen al Ma e ial and Me hods).
Resul s
Re inemen o he RP17 Locus in Two Un ela ed adRP-
A ec ed Families
The a ec ed haplo ype o a Du ch adRP-a ec ed amily
(NL1) (Figu e 1A) was p e iously mapped o a 7.18 Mb
egion spanning he RP17 locus on ch omosome 17.
7
The RP17 locus was e ined o a 5.16 Mb in e al by SNP
haplo yping in an ex ended pedig ee (Figu e 1D and
Supplemen al In o ma ion). No a e coding o splice si e
he e ozygous a ian s (MAF %0.0001) sha ed be ween
a ec ed indi iduals we e ound h ough WES. Subse-
quen ly, WGS was pe o med, and simila ly, no a e
candida e coding, splice si e, in onic, o in e genic he e o-
zygous single-nucleo ide a ian s we e iden i ied (Table S4
and Supplemen al In o ma ion).
In pa allel, WES and WGS we e pe o med o a ec ed
indi iduals om a gene ically unexplained UK adRP-
a ec ed amily (UK1) (Figu e 1B). This also ailed o iden-
i y a a e causa i e a ian ; howe e , a disease-associa ed
haplo ype on ch omosome 17 was iden i ied (Figu e 1E,
Table S5, and Supplemen al In o ma ion). In e oga ion
o unsol ed IRD sequence da a gene a ed h ough he UK
IRDC, UCL-Ex, NIHR-Bio esou ce, and Genomics England
iden i ied o he adRP p obands ha sha ed he same
haplo ype o Ch 17 SNVs and es ablished his as a ounde
haplo ype in ele en addi ional UK adRP-a ec ed amilies
(Figu e 1C). The adRP locus was e ined o a 4.4 Mb in e al
on Ch 17q22 (Figu e 1E). This genomic in e al o e laps
he p e iously desc ibed RP17 locus in amilies o Du ch
and Sou h A ican o igin (Figu e 1F).
A missense a ian in CA4 [c.40C>T (p.A g14T p);
GenBank: NM_000717.4] was p e iously desc ibed as
he cause o adRP a he RP17 locus in amilies o Sou h
A ican o igin.
8
No a e coding, in onic, o ups eam
a ian s in CA4 we e iden i ied in he Du ch and UK
amilies.
Iden i ica ion o S uc u al Va ian s wi hin he RP17
Locus
Nex , we analyzed genome and exome da a o copy num-
be a ian s and SVs (Supplemen al In o ma ion). In amily
NL1, WGS e ealed a 226 kb duplica ion wi hin he RP17
locus: ch 17: 57,291,905_57,518,137dup (NL-SV1). This
SV in ol es wo duplica ed genes (GDPD1 [MIM: 616317]
and YPEL2 [MIM: 609723]), an in agenic mic oRNA
(MIR4729), and pa ial duplica ion o SMG8 (MIM:
613175) and he long non-coding RNA LINC01476.
The duplica ion c ea es a b eakpoin junc ion (ch 17:
g.57,518,137–57,291,905) speci ic o he mu a ed allele in
NL1 (Figu es 2A, 2B, and S1), which was used o con i m
seg ega ion o he SV wi h he adRP pheno ype in his am-
ily. No o e lapping SVs in he RP17 locus we e obse ed
in WES o ~7,500 indi iduals wi hou e inal disease gene -
a ed in-house a he Depa men o Human Gene ics,
Radboudumc.
Fo he wel e UK RP17 ounde haplo ype am-
ilies, WGS e ealed a duplica ed in e sion: ch 17:
57,456,098–57,468,960delins57,275,839_57,559,114in
(UK-SV2) (Figu e 2B). The SV was cha ac e ized, and
b eakpoin junc ions we e alida ed (Figu e S1 and Sup-
plemen al In o ma ion). This SV in ol ed ou coding
genes (PRR11 [MIM: 615920], SMG8,GDPD1,and
YPEL2) and wo non-coding RNA genes (MIR4729 and
LINC01476)(Figu e 2B). UK-SV2 seg ega ed wi h adRP
in all amilies o which DNA was a ailable o analysis.
UK-SV2 was absen in WGS con ol genome da a gene -
a ed o 58,000 UK indi iduals (Genomics England).
Di e en S uc u al Va ian s wi hin he RP17 Locus in
Mul iple adRP-A ec ed Families
These da a p omp ed us o in es iga e whe he SVs we e
p esen in he wo o iginal Sou h A ican amilies (SA1
and SA2) ha we e linked o he RP17 locus
(Figu e S2A).
6,8
In addi ion, a Canadian adRP-a ec ed am-
ily (CA1) was also mapped o he RP17 locus (unpublished
da a, Figu e S2B). WGS was pe o med o a ec ed indi id-
uals om hese amilies, and in e sion duplica ion e en s
we e iden i ied in all samples analyzed (Figu e 2C). In SA1
and SA2, an iden ical SV, SA-SV3, was e ealed, sugges ing
his is a ounde a ian in his popula ion. SA-SV3 was
also ound by b eakpoin PCR in wo addi ional amilies
o Sou h A ican o igin (SA3 and SA4), con i ming he
ounde e ec (Figu e S2A). In he Canadian amily, a
di e en in e sion duplica ion e en was iden i ied, CA-
SV4. SA-SV3 and CA-SV4 b eakpoin s we e cha ac e ized
and alida ed (Figu e S1), and seg ega ion o he SVs wi h
he adRP pheno ype was con i med.
804 The Ame ican Jou nal o Human Gene ics 107, 802–814, No embe 5, 2020
Ou da a sugges ed ha SVs a he RP17 locus a e an
impo an cause o adRP. The e o e, WGS and WES da a
o gene ically unexplained adRP-a ec ed amilies we e
analyzed o SVs wi hin his locus. In ou un ela ed am-
ilies o Du ch o UK o igin, ou addi ional unique com-
plex SVs we e disco e ed (Figu es 2D and S2C). Fo
Figu e 1. Mapping o he RP17 Locus in Two Un ela ed Families
(A) Pedig ee o Du ch NL1 amily.
(B) Pedig ee o UK1 amily.
(C) Pedig ees o addi ional UK amilies wi h he ounde haplo ype on Ch 17q. WGS o WES was pe o med in indi iduals highligh ed in
blue o ed, espec i ely.
(D) SNP haplo yping esul s o NL1. The e ined RP17 locus ( s8078110– s9910672) is sha ed by all a ec ed indi iduals (n ¼35) and no
p esen in una ec ed indi iduals (n ¼28, only indi iduals wi h ecombina ion close o o e ining he c i ical egion a e depic ed) wi h a
maximum LOD sco e o 15.0. The ho izon al numbe s ep esen he numbe o indi iduals wi h his haplo ype.
(E) UK ounde haplo ype e ining he RP17 locus in UK amilies. Rep esen a i e haplo ypes om se e al un ela ed amilies a e shown
wi h a ec ed (a ) indi iduals compa ed o an una ec ed (una ) indi idual. Black lines and a ows indica e ecombina ion e en s.
Sha ed haplo ype in indi iduals is shaded ed.
(F) O e lap o e ined RP17 loci in UK, NL, and p e iously desc ibed SA amilies.
8
The Ame ican Jou nal o Human Gene ics 107, 802–814, No embe 5, 2020 805
PRR11 SMG8 GDPD1 YPEL2
LINC01476
NL-SV1
AB
SMG8
GDPD1 YPEL2 LINC01476
BC
PRR11 SMG8 GDPD1 YPEL2
UK-SV2
DE
SMG8GDPD1YPEL2LINC01476 PRR11
GF E
YPEL2 LINC01476
GH
PRR11 SMG8 GDPD1 YPEL2 LINC01476
SA-SV3
IJ KL
PRR11 SMG8 GDPD1
JM
LINC01476 LINC01476
MNL
CA-SV4
PRR11
OP
LINC01476 YPEL2 GDPD1 SMG8 PRR11
RQP
LINC01476
RS
PRR11 SMG8 GDPD1 YPEL2 LINC01476
NL-SV5
TU
PRR11 SMG8 GDPD1 YPEL2
U
LINC01476
V
PRR11 SMG8 GDPD1 YPEL2 LINC01476
UK-SV6
WX
GDPD1 YPEL2 LINC01476
X
GDPD1 YPEL2
X
LINC01476
Y
UK-SV7
UK-SV8
GDPD1 YPEL2
Z AA
GDPD1YPEL2LINC01476DHX40
CLTC
CLTC
AC AB AA AC AD
DHX40
LINC01476
YPEL2
B
C
D
MIR4729 MIR4729
MIR4729 MIR4729
MIR4729
MIR4729
MIR4729 MIR4729
MIR4729MIR4729
MIR4729
MIR4729
APRR11 SMG8 GDPD1 YPEL2 LINC01476
WT
MIR4729
CLTCDHX40
PRR11 SMG8 PRR11SMG8
MIR4729
M
PRR11 SMG8 GDPD1
AE AF
SMG8GDPD1YPEL2LINC01476
AH AG AF
PRR11 YPEL2 LINC01476
AH AIMIR4729 MIR4729
E
PRR11 SMG8 GDPD1 YPEL2 LINC01476
MIR4729
NL-SV1
UK-SV2
SA-SV3
CA-SV4
NL-SV5
DHX40 CLTC
UK-SV6
UK-SV7
UK-SV8
Figu e 2. O e iew o S uc u al Va ian s wi hin he RP17 Locus in adRP-A ec ed Families
B eakpoin s a e indica ed wi h dashed lines. Blue segmen s ep esen duplica ed o iplica ed egions, whe eas in e sions a e high-
ligh ed in pu ple.
(A) Wild- ype (WT) ch omosomal o ganiza ion.
(B) S uc u al a ian s iden i ied in NL1 (NL-SV1) and UK ounde haplo ype amilies (UK-SV2).
(C) S uc u al a ian s iden i ied in adRP-a ec ed amilies ha we e p e iously linked o he RP17 locus; SA-SV3
8
and CA-SV4 (unpub-
lished da a).
(legend con inued on nex page)
806 The Ame ican Jou nal o Human Gene ics 107, 802–814, No embe 5, 2020

indi iduals ha had only unde gone WES, we pe o med
WGS o de e mine he b eakpoin junc ions and iden i y
po en ial in e sions o o he SVs. In all amilies, b eak-
poin s we e alida ed, and seg ega ion analysis was pe -
o med whe e possible. T iplica ion o UK-SV6 was
con i med by qPCR in amily UK13 (Figu e S3 and Supple-
men al In o ma ion).
De ails o all SVs iden i ied in his s udy a e shown in
Table S6,Figu e 2, and Figu e S4, and an o e iew o SV-
speci ic b eakpoin junc ions is shown in Figu e S1. All
RP17 SVs sha e a common duplica ed (o iplica ed) e-
gion o 11.5 kb and ha bo unique b eakpoin s dis up ing
he genomic egion spanning YPEL2 o LINC01476 (ch 17:
57,499,214–57,510,765) (Figu e S4). We analyzed all b eak-
poin junc ion sequences o in es iga e he po en ial
mechanism(s) ha c ea ed RP17 SVs. No single mecha-
nism could accoun o he RP17 SVs because a combina-
ion o (mic o)homology-media ed epai and non-homol-
ogous end joining e en s we e iden i ied (Table S7 and S8,
Figu e S5, and Supplemen al In o ma ion).
Consis en Au osomal-Dominan Re ini is Pigmen osa
Pheno ype o RP17-A ec ed Families
The SVs iden i ied we e ully pene an in all amilies.
A ailable clinical da a a e p esen ed in Table S9. Twen y-
ou a ec ed indi iduals om se en een pedig ees we e
e alua ed. The e is signi ican co ela ion o pheno ype
ac oss all geno ypes wi h ela i ely mild disease, dec eased
isual acui y, isual ield cons ic ion, nyc alopia, and slow
p og ession consis en wi h adRP. Many a ec ed indi id-
uals ha e p ese ed cen al isual unc ion and acui y un il
he 6
h
–7
h
decade. Fo eal spa ing and cys oid macula
edema we e a common inding in indi iduals wi h UK-
SV2. On he basis o a small numbe o a ec ed indi iduals
(n ¼2), UK-SV6 (wi h a iplica ed SV) may be associa ed
wi h an ea lie age o onse and mo e se e e pheno ype
(Figu e S6).
Topologically Associa ing Domain S uc u e and
Epigene ic Landscape o he RP17 Genomic Region
Allo heRP17SVslead odis up iono hegenomic e-
gion spanning YPEL2 o LINC01476 (Figu e 2E). SVs
ha in e e e wi h genome s uc u e can ha e dis inc e -
ec s on gene egula ion depending on he ype and
ex en o he SV and landscape o he genomic egion.
15
TADs a e sepa a ed by bounda ies, egions o low ch o-
ma in in e ac ion ha insula e he egula o y ac i i ies
o neighbo ing TADs. The ansc ip ion ac o CTCF
(CCTC-binding ac o ) ypically binds in hese egions
whe e i plays a pi o al ole in he main enance o bound-
a ies. SVs can cause loss o unc ion by disconnec ing en-
hance s om hei a ge genes; howe e , dis up ion o
TAD s uc u es and bounda ies can also exe a gain-o -
unc ion e ec . Dele ions, o example, can lead o he
using o wo p e iously sepa a ed TADs (TAD- usion), in-
e sions can esul in he exchange o egula o y ma e ial
be ween TADs (TAD-shu ling), whe eas duplica ions can
gi e ise o he gene a ion o no el domains, so-called
neo-TADs.
12,13
In each case, SVs esul in he gene a ion
o ec opic con ac s o enhance s wi h he p omo e s o
no el a ge genes esul ing in abe an gene ac i a ion.
The human limb mal o ma ions caused by SVs ha al e
he CTCF-associa ed bounda y o he WNT6/IHH/
EPHA4/PAX3 locus a e a p ominen example. The SVs
esul in ec opic in e ac ions be ween EPHA4 (MIM:
602188) limb enhance s and he neighbo ing de elop-
men al genes ha a e no mally insula ed, d i ing ec opic
exp ession in he limb.
14
Simila ly, he dele ion o a CTCF
si e loca ed be ween he Xis (MIM: 314670) and Tsix
(MIM: 300181) TADs on he X ch omosome esul ed in
a no el domain by usion o he adjacen TADs ( used-
TAD).
24
As a consequence, p e iously insula ed en-
hance s ac i a ed genes in he adjacen TAD, leading o
he dys egula ion o hese genes.
Hi-C da a we e no a ailable o human e ina, and
he e o e, we gene a ed Hi-C maps o con ol human 3D
ROs o ob ain maps o he ch oma in o ganiza ion o
ou egion o in e es . Hi-C e ealed a s uc u ed domain
con aining YPEL2 (YPEL2 TAD) lanked by less s uc u ed
neighbo ing domains (Figu e 3A). CTCF binding is p e-
sen on bo h bounda ies (Figu e 3B) suppo ing he TAD
s uc u e a his locus. CTCF ChIA-PET da a highligh ed
in e ac ions be ween he CTCF binding si es a he 50
and he 30bounda y o he YPEL2 TAD (Figu e S7B).
Assay o ansposase-accessible ch oma in using seqenc-
ing (ATAC-seq) da a om human e ina show ha he
ch oma in in he YPEL2 TAD is accessible, and H3K27Ac
ChIP-seq da a e ealed ha he e a e se e al ac i e en-
hance s loca ed wi hin he YPEL2 TAD ha a e expec ed
o d i e YPEL2 exp ession in he e ina (Figu e 3B).
25
Impo an ly, he YPEL2 TAD ha bo s wo egions o ac i e
enhance s wi h binding si es o ansc ip ion ac o (TFs)
known o be equi ed o pho o ecep o unc ion,
including NRL, CRX, and OTX2 (Figu e 3B). NRL is a TF
ha is p e e en ially exp essed in od pho o ecep o s.
These TF binding si es co ela ed wi h H3K27Ac and
ATAC-seq peaks in e ina. The published GeneHance da a-
se shows ha hese egula o y elemen s ha e in e ac ions
wi h he YPEL2 p omo e (Figu e S7C).
26
Collec i ely, hese
analyses e ealed ha YPEL2 is loca ed wi hin an ac i e
compa men ha con ains e inal-speci ic enhance s
(Figu e 3C).
(D) S uc u al a ian s ound in a coho o unsol ed adRP-a ec ed amilies; NL-SV5, UK-SV6, UK-SV7, and UK-SV8. Le e s A–AI depic
he genomic in e als o each SV used o analyze and anno a e SV b eakpoin s.
(E) O e iew o all SV b eakpoin s iden i ied in he RP17 locus. An o e lapping genomic egion ha is duplica ed o iplica ed in all SVs
was iden i ied (ch 17: 57,499,214–57,510,765) and is highligh ed by a ligh blue e ical ba . The size o DHX40 is educed, and CLTC is
pa ially shown o he pu pose o his igu e.
The Ame ican Jou nal o Human Gene ics 107, 802–814, No embe 5, 2020 807
Exp ession o YPEL2 and GDPD1
Exp ession o YPEL2 and GDPD1 was assessed by qPCR
in mul iple heal hy human issues, including e ina
(Figu e S8). YPEL2 is ubiqui ously exp essed in he is-
sues s udied, including e ina, wi h highes ela i e
exp ession in b ain. Single-cell e ina RNA-seq da ase s
e ealed YPEL2 is exp essed a highe le els in od pho o-
ecep o cells, which is he p ima y cell ype a ec ed in
e ini is pigmen osa, compa ed o cone pho o ecep o s
(Figu e S8).
23
GDPD1 is de ec ed a low exp ession in
all issue ypes bu has highe exp ession in es is and
he b ain. These da a suppo he hypo hesis ha
YPEL2 exp ession is egula ed by e inal enhance s
wi hin he YPEL2 TAD.
RP17 SVs C ea e New Topologically Associa ing
Domains and Ec opic Enhance -Gene In e ac ions
Using he wild- ype e inal o ganoid Hi-C map, we
modeled he TAD bounda ies, CTCF si e o ien a ion, and
e inal TF binding si e posi ions o each unique RP17 SV
LINC01476
55 _
0
_
200 _
0
_
0 _
-5
_
55 _
0
_
55 _
0
_
55 _
0
_
55 _
0
_
55 _
0
_
10.0
20.0
0.0
CTCF
Re ina-speci ic exp ession
H3K27ac
ATAC-seq
NRL
CRX
OTX2
Re inal o ganoid Hi-C mapA
BHuman e ina ChIP-seq and RNA-seq da a
C
LINC01476
B
B
YPEL2
TAD
B
Re inal enhance
Bounda y
SMG8 GDPD1
Scale
ch 17:
200 kb hg19
57
,
300
,
000 57
,
400
,
000 57
,
500
,
000 57
,
600
,
000 57
,
700
,
000
SKA2
PRR11
S
MG8
GDPD1 YPEL2 MIR4729 DHX40 CLTC PTRH2
VMP1
Figu e 3. YPEL2 is Loca ed wi hin a S uc u ed Ac i e Compa men ha Con ains Re inal-Speci ic Enhance s
(A) The TAD landscape o he genomic egion dis up ed by he RP17 SVs. Hi-C map o con ol e inal o ganoids e ealed a s uc u ed
domain con aining YPEL2.
(B) YPEL2 TAD bounda ies co espond wi h CTCF si es iden i ied in human e ina. Analysis o RNA-seq and assay o ansposase-
accessible ch oma in using seqencing (ATAC-seq) da a ac oss he YPEL2 egion shows YPEL2 e inal exp ession and an accessible ch o-
ma in con igu a ion. Analysis o H3K27Ac ChIP-seq da a in he same egion e ealed se e al ac i e enhance s loca ed wi hin he YPEL2
TAD, which a e en iched o e inal ansc ip ion ac o binding si es, including NRL, CRX, and OTX2.
25
These enhance s we e loca ed 50
o he CTCF bounda y si e wi hin LINC01476.
(C) Schema ic ep esen a ion o he YPEL2 TAD s uc u e.
808 The Ame ican Jou nal o Human Gene ics 107, 802–814, No embe 5, 2020
Figu e 4. RP17 SVs C ea e No el Domains (neo-TADs) and Hype -ac i a ion o Re inal Enhance s
(A) Schema ic modeling o he genome a chi ec u e spanning he RP17 egion using Hi-C maps. The wild- ype Hi-C map de i ed om
neu onal issue shows a TAD wi h CTCF bounda ies con aining YPEL2 and e inal enhance s, lanked by uns uc u ed domains. TAD
models o NL-SV1 and UK-SV2 (do ed e ical lines ep esen SV b eakpoin s) p edic he o ma ion o neo-TADs and ec opic in e ac-
ions o he e inal enhance wi h GDPD1.
(B) Hi-C pe o med on e inal o ganoids (ROs) de i ed om con ol ( op) and RP17 UK-SV2 indi iduals (bo om) (10 kb esolu ion; aw
coun map). The ch oma in o ganiza ion in con ol ROs shows he YPEL2 TAD (indica ed by dashed lines). Two no el domains (neo-
TAD 1 and 2) a e isible in he UK-SV2 ROs, and neo-TAD 2 allows ec opic e inal enhance con ac s o GDPD1 and SMG8. The dashed
ci cle indica es he s ong ch oma in con ac be ween e inal enhance s and he GDPD1 p omo e .
(C) qPCR e ealed signi ican ly up egula ed e inal enhance RNA exp ession in UK-SV2 ROs compa ed o con ols (n ¼3, mean 5s an-
da d e o o he mean, **p %0.01).
The Ame ican Jou nal o Human Gene ics 107, 802–814, No embe 5, 2020 809
Wild ype
GDPD1SMG8 YPEL2 LN 01476
><
NL-SV1
GDPD1SMG8 YPEL2 01476
GDPD1
SMG8 LN 01476
GDPD1 YPEL2 LN
YPEL2 SMG8GDPD1
Bounda y
CTCF o ien a ion
B
Enhance in e ac ion
>
UK-SV2
Re inal enhance
YPEL2
YPEL2
GDPD1
>
<
>
><
<
A
B
D
GDPD1
CTL
NL-SV1
0.0
0.5
1.0
1.5
2.0
Fold changes
SMG8
CTL
NL-SV1
0.0
0.5
1.0
1.5
2.0
Fold changes
YPEL2
CTL
NL-SV1
0.0
0.5
1.0
1.5
2.0
Fold changes
GDPD1
SMG8
GDPD1
CTL
UK-SV2
0.0
1.0
2.0
3.0
Fold changes
SMG8
CTL
UK-SV2
0.0
1.0
2.0
3.0
Fold changes
YPEL2
CTL
UK-SV2
0.0
1.0
2.0
3.0
Fold changes
C
E
**
**
**
****
Re inal o ganoid exp ession
Pho o ecep o p ecu so s exp ession
Neo-TAD
Neo-TAD
No el TAD domain
Gene ansc ip ion
BB
BB
B
B
B
B
B
B
B B
B
B
B
BBBB
BBBB
B
B
B
B
YPEL2 TAD
(legend on nex page)
810 The Ame ican Jou nal o Human Gene ics 107, 802–814, No embe 5, 2020