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Inhe i ed e inal diseases (IRDs) a e one o he mos he e ogeneous clinical and gene ical diso de s known among
all human medical condi ions, cha ac e ized by he p og essi e loss o pho o ecep o cells, esul ing in se e e
isual impai men 1. IRDs a e classi ied as a e diseases, and hei es ima ed p e alence is abou 1 in 10002–40001.
IRDs can be classi ied acco ding o di e en clinical o gene ic c i e ia, based upon he p ima y e inal cell
a ec ed ( ods, cones, e inal pigmen epi helium (RPE), bipola cells o ganglion cells), he oph halmological
indings and/o he a ec ed gene ound a e he gene ic es ing.
ͷ ǡ × Ǧ × ± Àǡ
Mad id, Spain. ȋȌǡ
III, Mad id, Spain. ǡ × Ǧ
×±ÀǡǡǤͻͿǣǦǡǡ
ǤǡǤ *ƥ
pape . *ǣǤǢƦǤ
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All modes o inhe i ance can be obse ed (au osomal dominan (AD), au osomal ecessi e (AR), X-linked
(XL), including a e non-Mendelian o ms such as mi ochond ial o digenic inhe i ance pa e ns). Age o onse o
i s symp oms ( om ea ly childhood o adul hood), a e o p og ession, associa ion wi h ex a-ocula symp oms
(non-synd omic e sus synd omic o ms) o causa i e gene can also help o subclassi y he di e en pheno ypes3.
The mos p e ailing o m o IRDs is Re ini is Pigmen osa (RP [MIM: 268000]), which is es ima ed o a ec
app oxima ely 1.5 million people wo ldwide4. RP begins wi h he degene a ion o od pho o ecep o s, esul ing
in nigh blindness and cha ac e is ic pigmen a y changes in he pe iphe al e ina. This is also conside ed a od-
cone dys ophy due o he subsequen cone pho o ecep o dea h in la e s ages. O he o ms o IRD, including
cone-domina ed diseases, a e cha ac e ized by pho ophobia, educed isual acui y and impai ed colou ision (i.e.
cone dys ophy (CD), ach oma opsia, blue cone monoch oma ism), as well as gene alized e inal degene a ion
in ol ing simul aneously bo h cones and ods such as in od-cone o cone- od dys ophies (CRD). The mos
se e e o m o non-synd omic IRDs is Lebe congeni al amau osis (LCA) cha ac e ized by congeni al o ea ly
childhood blindness. O he IRD o ms a e cha ac e ized by cen al ision loss a ec ing p ima ily he macula and
a e he e o e acknowledged as Macula Dys ophies (MD), such as S a ga d disease (STGD1 [MIM: 248200])
and Bes Vi elli o m Macula Dys ophy (VMD2 [MIM: 153700])1.
Con e sely, synd omic IRDs a e subclassi ied acco ding o he ype o synd ome. The mos p e alen is Ushe
synd ome (senso ineu al hea ing loss and RP) which can be u he subclassi ied o Ushe ype I (USH1 [MIM:
276900]), ype II (USH2 [MIM: 276901]) and ype III (USH3 [MIM: 276902]), and o he synd omes such as
Ba de -Biedl (BBS [MIM: 209900]) o Als öm (ALMS [MIM: 203800])3.
Since he iden i ica ion o hodopsin (RHO [MIM: 180380]) in 1990 as he i s gene in ol ed in he de elop-
men o AD-RP5, 270 genes ha e been addi ionally desc ibed as causa i e o IRDs (Re Ne , Re inal In o ma ion
Ne wo k; h ps ://sph.u h.edu/ e ne /; accessed on Ma ch 2020). Some o hese genes ha e been epo ed in only
ew amilies wo ldwide; he e o e, hei indi idual con ibu ion o IRD p e alence is ela i ely small. The mos
p e alen IRD-causing genes ac oss all popula ions a e ABCA4 (MIM: 601691), RHO, USH2A (MIM: 608400)
and RPGR (MIM: 312610), which accoun o high pe cen ages o some o he IRD sub ypes, i.e. 70–71% in
STGD1/AR-MD/AR-CRD6, 19–25% o AD-RP, 10% o AR-RP, and 70% o XL-RP, espec i ely1. In addi ion,
al hough he majo i y o he causa i e a ian s a e p i a e, some a e mo e equen , especially in Spanish amilies,
such as USH2A (GenBank: NM_206933.3) c.2299delG (p.Glu767Se sTe 21) and c.2276G > T (p.Cys759Phe)7 o
ABCA4 (GenBank: NM_000350.3) c.3386G > T (p.A g1129Leu) and c.5882G > A (p.Gly1961Glu)6.
The aim o his s udy is o p esen a comp ehensi e o e iew o he la ges coho o IRD pa ien s e e
epo ed wo ldwide and ela ed o IRD in he Spanish popula ion. The p esen ed da a includes he p esumed
inhe i ance pa e n o he di e en pheno ypic sub ypes, mu a ional spec um, p e alence o genes ca ying
likely pa hogenic a ian s and he ecu ence o disease ela ed a ian s.
Ǥ The numbe o cases diagnosed as ha ing IRD in ou hospi al (un il Augus
2019) was 6089, and he las Spanish popula ion egis y accoun ed o 46,722,980 habi an s gi ing us a minimal
p e alence o 1:7673 (con idence in e al (CI):1:7485–1:7871). Regional dis ibu ion o cases and p e alence can
be seen in Fig.1A,B. Conside ing a wo ldwide IRD p e alence o 1:10002–40001, ou coho would ep esen
20–53% o he o al pa ien s wi h IRD in Spain as shown in Supplemen a y TableS1.
Fo non-synd omic IRD, ou coho was g ouped in 2 ca ego ies: 1703 a ec ed indi iduals (1335 amilies), in
cone-domina ed pheno ypes—he ea e “NON-RP”—and 3561 a ec ed cases om 2447 un ela ed amilies wi hin
p ima ily od a ec ion—he ea e “RP”. This esul ed in a minimal p e alence o 1:27,436 (CI:1:26,192–1:28,804)
and 1:13,121 (CI:12,704–13,566) o NON-RP and RP, espec i ely. Addi ionally, synd omic IRD o ms ep e-
sen ed he smalles ac ion accoun ing o 13.6% o he pa ien s wi h 825 a ec ed indi iduals (621 amilies),
esul ing in a minimal p e alence o 1:56,634 (CI:1:53,016–1:60,781).
In ou coho , we ha e a highe p opo ion o cases om Mad id a ea (26.7%; 1625/6089).
Ƥ
Ǥ Non-synd omic NON-RP and RP cases we e ca ego ized by he mode o inhe i ance
(Fig.2A-I and A-II). Synd omic IRD we e ca ego ized by he speci ic ype o suspec ed synd ome (Fig.2A-III),
ins ead o inhe i ance ype, gi en ha mos o hem we e spo adic (53.6%) o had ecessi e inhe i ance (40.2%).
The emaining 6.2% co esponded o dominan (0.5%), X-linked (0.7%), mi ochond ial inhe i ed disease (0.2%)
o non-classi icable cases (4.8%).
Acco ding o his “a p io i” diagnosis based on he clinical and amilial his o y o he pa ien s, he main
inhe i ance pa e n in non-synd omic NON-RP and RP was ecessi e o spo adic, ep esen ing he 68% and
75% o cases, espec i ely. Au osomal dominan and X-linked o ms accoun ed o 21% and 8% o NON-RP
and 15% and 8% o RP, espec i ely (Fig.2A-I). Families wi h no amilia da a we e anno a ed as unclassi ied.
Non-synd omic RP ep esen s he mos common pheno ype, ep esen ing 55.6% o amilies in ou coho .
In he p esen coho , 47% o he synd omic IRD index cases (270/577) su e ed om USH2, ollowed by 17%
USH1 (98/577), as well as o he e y a e synd omes like some a ypical o ms o Ushe synd ome (3%; 16/577)
and ciliopa hies such as BBS o ALMS (16%; 90/577). A miscellanea o non-ciliopa hic synd omes o unclassi ied
symp oms we e p esen ed in 103 index cases.
Ǥ Diagnos ic yield. Gene ic es ing was pe o med in a o al o 3951 index cases wi h
a ailable DNA6–11 (89.7% o he o al coho ), including 1291 NON-RP, 2083 RP, and 577 synd omic IRD pa-
ien s as shown in Supplemen a y Fig.S1. The gene ic analysis p ocedu es e ol ed o e ime since no el ge-
ne ic app oaches we e implemen ed in ou labo a o y. A de ini e gene ic diagnosis was es ablished o 53.2%
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(2100/3951) o cases. Fo NON-RP amilies, 754 ou o 1291 (58.4%) ob ained a gene ic diagnosis; in RP he
molecula cause was iden i ied in 1038 ou o 2083 (49.8%). Simila ly, 53.4% (308/577) o synd omic IRD ami-
lies we e gene ically sol ed (Fig.2B).
ƤǤ The iden i ica ion o he
causa i e gene allowed us o eclassi y he inhe i ance mode in 8.2% (146/1792) o he NON-RP and RP ami-
lies, hus es ablishing he inal inhe i ance ype (Supplemen a y TableS2). A compa ison be ween he “a p io i”
suspec ed inhe i ance based on he pedig ee and he inal inhe i ance sugges ed by he molecula diagnosis was
pe o med in cha ac e ized NON-RP and RP amilies (Fig.2C). As expec ed, mos spo adic NON-RP (n = 378)
and RP cases (n = 379) we e con i med as ha ing AR inhe i ance a e he gene ic es ing. The es o he S cases
we e eclassi ied o AD (n = 43) and XL (n = 36) (Supplemen a y TableS2). Twen y- ou cases (11 NON-RP and
13 RP) wi h an ini ial unknown mode o inhe i ance we e classi ied as: AD (n = 5), AR (n = 16), and XL (n = 3)
a e he molecula es ing.
Gene landscape. In o al, 1549 di e en pa hogenic and likely pa hogenic a ian s we e iden i ied in 142 di e -
en genes. These included SNVs (Single Nucleo ide Va ian s) and CNVs (Copy Numbe Va ian s). As showed in
Figs.3, 4, 5, he e was a wide spec um o genes implica ed in IRD, 121 o hem ep esen ed in 1% o less o he
coho . The 5 mos equen mu a ed genes we e ABCA4, USH2A, RS1 (MIM: 300839), CRB1 (MIM: 604210)
and RHO.
In NON-RP amilies, PRPH2 (MIM: 179605), ABCA4 and RS1 we e he mos commonly mu a ed genes,
explaining 42.2%, 82.7% and 93.3% o he AD, AR and XL o ms, espec i ely as shown in Supplemen a y
TableS3.
Fo non-synd omic RP, 207 AD-RP amilies we e gene ically cha ac e ized wi h he e ozygous a ian s iden i-
ied in one o 23 genes ound. The mos equen mu a ed genes we e RHO (65/207; 31.4%) and PRPF31 (MIM:
606419) (34/207; 16.4%). In AR-RP amilies, he mos common gene mu a ed was USH2A (127/666; 19.1%).
Howe e , he numbe o o he disease genes de ec ed was e y high (N = 70) in he 666 amilies cha ac e ized.
Fo XL-RP amilies, bo h he numbe o cases and a ie y o genes we e low (N = 9) as shown in Supplemen a y
TableS4. Fo hese pa ien s, 41.2% (68/165) o he index cases (all males) ca ied a hemizygous pa hogenic a ian
in RPGR (44 RPGR_ORF15 and 24 in he es o RPGR egions), and 32.7% (54/165) in CHM (MIM: 300390).
A o al o 53 cases ou o he o al 2100 (2.5%) we e clinically econside ed and eclassi ied a e gene ic es -
ing: 12 NON-RP we e eclassi ied as RP (9/754; 1.2%; cha ac e ized wi h RHO, FSCN2, PRPF8, AHI1, CNGB1,
TRPM1 and CHM) o as synd omic IRD (3/754; 0.4%; COL11A1, CDH3 and MYO7A) and 37 RP as NON-
RP (32/1038; 3.1%; C1QTNF5, GUCA1A, CNGB3, CNGA3, ACBD5, GNAT2, PDE6C, ATF6, PDE6H, RS1 and
OPN1LW-OPN1MW), synd omic IRD (1/1038; 0.1%; MYO7A) and o he isual diseases, such as exuda i e
Figu e1. IRD a ec ed cases dis ibu ion and es ima ed p e alence in Spain. (A) Dis ibu ion o RP/NON-RP
a ec ed cases ac oss Spain. To al cases: 6089 (known Spanish o igin: 4668; unknown o igin: 1421). Spanish map
modi ied using image edi o om h ps ://www. eoma pas.com/mapa-mudo-de-las-comun idade s-au on omas-
de-espan a-m103.h ml. (B) IRD es ima ed p e alence in Spanish egions. Da a was ob ained om he numbe o
cases we had in ou Hospi al Se ice and he eco ded popula ion in he di e en egions. *Inconclusi e da a.
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Figu e2. “A p io i” and inal classi ica ion o IRD a ec ed cases. (A) “A p io i” classi ica ion o IRD amilies
wi h da a ob ained om he clinical and amily his o y o he pa ien s, be o e pe o ming molecula es s:
NON-RP (I), RP (II), and synd omic IRD (III), and subclassi ica ion acco ding o he inhe i ance ype in case o
RP and NON-RP, and ype o synd ome in case o synd omic IRD. Da a ob ained wi h he clinical and amilia
his o y o he pa ien s, be o e pe o ming molecula es s. AD Au osomal Dominan , AR au osomal ecessi e,
S spo adic, XL X-linked. (B) P opo ion o gene ically sol ed NON-RP, RP, synd omic IRD and o al IRD.
The diagnos ic a io in he di e en g oup o NON-RP and RP by he ype o “a p io i” inhe i ance and in he
di e en g oup o synd omic IRD by ype o synd ome is indica ed. (C) Gene ically sol ed amilies. Compa ison
o inhe i ance classi ica ion be o e (ligh g ay) and a e (da k g ay) he molecula s udy was pe o med.
Figu e3. Classi ica ion o mu a ed genes in he gene ically sol ed NON-RP a ec ed cases. Below each gene is
gi en he pe cen age ha each gene was mu a ed in he coho . To al cha ac e ized amilies: AD-NON-RP: 121;
AR-NON-RP: 544; XL-NON-RP: 89.
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amilia i eo e inopa hy, op ic a ophy, albinism and gy a e a ophy (4/1038; 0.4%; FZD4, OPA1, GPR143 and
OAT). Mo eo e , 4 synd omic cases we e eclassi ied o RP g oup (4/308; 1.3%; RDH12, PDE6A and RPGRIP1).
Among synd omic IRD amilies, he causa i e gene was iden i ied in 56 amilies wi h a diagnosis o USH1,
145 o USH2, 6 o a ypical Ushe , and 101 o o he synd omes including Ba de -Biedl o Als öm synd ome,
espec i ely. In USH1, biallelic a ian s in MYO7A (MIM: 276903) we e iden i ied in 30 ou o 56 pa ien s
(53.5%). USH2A de ec s we e he main cause o USH2 in 90% (129/145) o he pa ien s. The g oup “o he s” was
a clinically he e ogeneous g oup o non-Ushe cases wi h a o al o 48 in ol ed genes, wi h BBS1 (MIM: 209901)
being he mos equen one (N = 23), as shown in Supplemen a y TableS5.
In addi ion o clinical and gene ic he e ogenei y, he g oup o “o he s” included in synd omic IRD also
p esen ed unusual modes o inhe i ance, such as iallelism. In ou coho , 4 possible iallelic cases ha e been
iden i ied, all o hem diagnosed wi h BBS, 3 pa ien s ca ying biallelic BBS1 a ian s oge he wi h one allele in
MKKS (MIM: 604896)9,12 and one addi ional case ca ying biallelic MKKS a ian s and one allele in BBS5 (MIM:
Figu e4. Classi ica ion o mu a ed genes in he gene ically sol ed RP a ec ed cases. Below each gene is gi en
he pe cen age ha each gene was mu a ed in he coho . To al cha ac e ized amilies: AD-RP: 207; AR-RP: 666;
XL-RP: 165.
Figu e5. Classi ica ion o mu a ed genes in he gene ically sol ed synd omic IRD a ec ed cases. Below each
gene is gi en he pe cen age ha each gene was mu a ed in he coho . To al cha ac e ized amilies: Ushe I: 56;
Ushe II: 145; O he s (including a ypical Ushe ): 107.
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603650). The pheno ypic modi ie e ec o he iallelism could only be s ablished in wo o he 4 amilies. One
has wo a ec ed siblings wi h di e en pheno ypic se e i y ha co ela es wi h he p esence o he hi d allele;
and in he o he bo h a ec ed showed iallelism and ha e he same clinical mani es a ion. Al hough, i could
no be s ablished in he es o he amilies, as hey we e spo adic cases.
Mos equen a ian s. Ou indings e lec he high allelic he e ogenei y in IRD. We iden i ied 458 di e en
disease-causing a ian s in 45 genes in cases “a p io i” classi ied as NON-RP, as well as 836 in 94 genes in he
“a p io i” RP cases and 295 in 55 genes in he “a p io i” synd omic IRD. The mos common pa hogenic a ian
de ec ed in ou NON-RP coho was he p e iously known missense change ABCA4 c.3386G > T (p.A g1129Leu)
(180 mu a ed alleles o 3,618; 5% o he o al pa hogenic alleles), p esen ed in 21.5% (162/754) o he cha ac e -
ized amilies, in homozygous o compound he e ozygous s a e in 18 and 144 amilies, espec i ely (Supplemen-
a y TableS6). Among he RP amilies, he mos p e alen pa hogenic a ian was he missense change USH2A
c.2276G > T (p.Cys759Phe), iden i ied in 106 alleles in 8.4% o he sol ed amilies (87/1038); in 19 cases in
homozygous and in 68 cases in compound he e ozygous s a e. In addi ion, he e we e 15 o he a ian s p esen
in mo e han 10 genes (Supplemen a y TableS6). Some o he mu a ed genes o e lap in NON-RP (Fig.3), RP
(Fig.4) and synd omic IRD (Fig.5).
Disease-causing a ian dis ibu ion in Spain. Analysis o causing a ian s by he di e en Spanish egions
esul ed in a wide a ie y o disease-causing a ian s. Table1 shows a ian s de ec ed in mo e han 5% o cha -
ac e ized amilies, by Spanish egions. All hese a ian s a e depic ed in he Supplemen a y TableS6 o NON-RP
and RP mos equen causing a ian s, excep o he nonsense a ian PRCD (OMIM: 610598) (GenBank:
NM_001077620.3) c.64C > T (p.A g22Te ), ha was ound in homozygosi y in 3 amilies, ep esen ing 7.2%
(6/83) o he iden i ied alleles om Mu cia.
This is he i s and la ges comp ehensi e s udy add essing he p e alence and epidemiology o IRD in he Span-
ish popula ion. The coho he e desc ibed, comp ising 6089 cases om 4403 un ela ed amilies, is no based on
a na ional egis y o IRD pa ien s, bu i is he ou come o a e y wide ec ui ing e o o a single cen e o e
he las 28yea s. An inc easing numbe o cen e s a e cu en ly pe o ming clinical and/o gene ic diagnosis o
IRD in Spain, he e o e ou coho did no e lec all o IRD pa ien s in ou coun y. Hence, o da e, no accu a e
da a abou he IRD p e alence in he Spanish popula ion is a ailable.
In e ms o ep esen a ion o pa ien s om he di e en Spanish egions, ou coho e lec s a biased ec ui -
men , being en iched wi h pa ien s om Mad id and he su ounding egions (i.e. Cas ile and Leon, Cas ile-La
Mancha, and Ex emadu a) p obably due o he ac ha ou hospi al has been hei e e al cen e du ing
mos o he ime o he s udy. O he a eas like Andalusia, Ca alonia, Na a e o he Valencian Communi y had
di e en e e al cen e s and gene ic es ing is pe o med locally. In spi e o hese limi a ions, he la ge sample
size o ou coho and he exhaus i e molecula analysis pe o med o e he yea s, oge he wi h an o e all low
gene ic he e ogenei y in he Spanish popula ion13, ha e allowed a s aigh o wa d ex apola ion o p e alen
genes and/o a ian s in IRD. Conside ing a wo ldwide p e alence o 1:10002–40001 and an es ima ed Spanish
popula ion o 46.7 million, ou coho would ep esen 20–53% o he o al pa ien s wi h IRD in Spain. Despi e
nume ous s udies abou he cha ac e is ics o he di e en IRDs in Spain, such as NON-RP and RP ha e been
pa ially published, s ill no global o e iew o NON-RP and RP diseases using a ep esen a i e coho has been
add essed ye be o e in ou coun y.
Table 1. Va ian s de ec ed in > 5% o NON-RP, RP and synd omic IRD by Spanish egions. Regions wi h
a ian s de ec ed below his pe cen age a e no ep esen ed in he able, and nei he a e egions wi h less han
50 mu a ed alleles in o al o a ian s ha we e p esen ed less han 5 imes. In bold he a ian which no
appea ed as one o he mos equen a ian s in Supplemen a y TableS6.
Spanish egion Gene Nucleo ide change Amino acid change Nº o alleles
Nº o o al alleles o his
gene ound in he egion F equency in he egion
NON-RP
Basque Coun y ABCA4 c.3386G > T p.A g1129Leu 18 93 19.40%
ABCA4 c.5882G > A p.Gly1961Glu 5 5.40%
Cas ile-La Mancha ABCA4 c.3386G > T p.A g1129Leu 8 60 13.30%
Mad id ABCA4 c.3386G > T p.A g1129Leu 47 373 12.60%
Mu cia ABCA4 c.3386G > T p.A g1129Leu 8 71 11.30%
ABCA4 c.5882G > A p.Gly1961Glu 7 9.90%
Non-synd omic RP and
synd omic IRD
Mu cia USH2A c.2276G > T p.Cys759Phe 11 83 13.30%
PRCD c.64C > T p.A g22Te 6 7.20%
Cana y Islands NR2E3 c.932G > A p.A g311Gln 8 83 9.60%
Cas ile-La Mancha USH2A c.2276G > T p.Cys759Phe 18 202 8.90%
Mad id USH2A c.2276G > T p.Cys759Phe 50 714 7.00%
Ex emadu a USH2A c.2276G > T p.Cys759Phe 5 87 5.70%
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Se e al s udies on IRD ha e been pe o med globally (Supplemen a y TableS7) and, in he wo las yea s,
some including big coho s14–16 o me a-analysis17 ha e been published, epo ing mo e han 125 genes explaining
55–62% o he amilies using se e al molecula echniques o achie e ha 14–16 as i could be also seen in his s udy.
O he s udies ocused on s ablishing he p e alence o IRD in ce ain egions has been pe o med in Wes e n
coun ies and in coho s o non-synd omic RP, including Wes e n Aus alia (1:6000)18 o Maine (1:4756)19, as
well as in coho s wi h gene al IRD and an es ima ed p e alence o 1:3454 in Denma k20 o 1:3856 in No way21.
Howe e , his p e alence has been epo ed in a eas and popula ions wi h low a es o consanguini y and could
be highe when consanguini y a e inc eases2, which is no he common scena io in Spain nowadays.
Ou s udy iden i ied AR inhe i ance as he mos common mode o inhe i ance o non-synd omic IRD,
explaining up o 70–75% NON-RP and RP subcoho s (Fig.2A-I and A-II). By con as , only 7% o ou NON-
RP and RP amilies a e explained by X-linked genes. These esul s we e consis en wi h p e ious s udies
published18–22. Besides, some cases could be explained by di e en molecula mechanisms as he pseudodomi-
nance, incomple e pene ance o he p esence o wo a ian s in an AR gene in AD a p io i amilies8, so ex ended
seg ega ion analysis wi hin hese amilies a e needed. Addi ional non-Mendelian ansmission pa e ns we e
only ound in excep ionally a e cases wi h synd omic IRD, including 3 amilies ca ying a ian s a ec ing he
mi ochond ial DNA and 4 cases wi h appa en iallelism in BBS-associa ed genes. Wi hin synd omic IRD g oup,
mos o he cases we e explained by AR biallelic monogenic inhe i ance. Simila o p e ious published s udies
om o he coun ies3,23, Ushe synd ome was he mos p e alen o m o synd omic IRD in ou coho , and
mo e speci ically, USH2, ep esen ing almos hal o he o al synd omic IRD amilies.
The o e all diagnos ic a e o 53.2% ob ained he e is simila o o he s udies p e iously epo ed (50–70%)24–26.
Molecula s udies allowed he iden i ica ion o he genes esponsible o he disease and he eclassi ica ion o
he inhe i ance ype. In ou wo k, 8.2% o he pa ien s we e eclassi ied a e he de ec ion o he disease-causing
a ian s in genes wi h a speci ic inhe i ance pa e n. All o hose we e o ha e been p e iously alida ed. Mo eo-
e , in all he cha ac e ized spo adic cases a mo e accu a e gene ic classi ica ion and counselling could be done8.
Addi ionally, a 2.5% we e clinically eclassi ied a e he gene ic es ing, due o a poo clinical da a acquisi ion
a he o igin cen e . So, iden i ica ion o he gene ic cause o he disease ep esen s a hallma k o he pa ien s,
i s ly, ega ding gene ic counselling and he isk o a ec a ion o o he ela i es; and secondly, gi en he pos-
sibili y o u u e ec ui men s o clinical ials a ge ing speci ic genes and a ian s.
A o al o 142 di e en genes we e iden i ied as he cause o IRD in ou s udy, bu i is impo an o no ice
ha each subg oup o he coho (AD, AR and XL NON-RP and RP) has an en ichmen o cha ac e ized cases
in speci ic genes.
Fo ins ance, PRPH2 was mu a ed in mo e han a hi d o AD-NON-RP amilies, ollowed by BEST1 (MIM:
607854). As expec ed, ABCA4 was he mos p e alen gene in AR-NON-RP amilies. Recen s udies in No way21
and Ko ea22 also iden i ied his gene as one o he mos p e alen mu a ed genes. A s udy published by Bi el
e al.27 in pa ien s wi h MD and cone/cone- od dys ophy showed a simila dis ibu ion o mu a ed genes, wi h
ABCA4, PRPH2 and BEST1 esponsible o 74% o hei sol ed cases. Fo he XL-NON-RP subcoho , RS1 was
he mos equen ly mu a ed gene.
Non-synd omic RP p esen ed a wide spec um o causa i e genes, wi h RHO, USH2A and RPGR (RPGR_
ORF15 and he es o RPGR egions) being he mos p e alen ones in AD-RP, AR-RP and XL-RP subcoho s,
espec i ely. Ou indings a e in line wi h hose published in o he s udies8,28. Fo ins ance, Ha ong e al.3,
showed as well MYO7A, USH2A and BBS1 o be he mos equen ly mu a ed genes in USH1, USH2 and BBS,
espec i ely. O he s udies in di e en popula ions highligh ed di e en genes as he mos ep esen a i e in hei
IRD coho s. Fo example, Eisenbe ge e al.24 desc ibed RP1 (MIM: 603937) (11.3%) and EYS (MIM: 612424)
(9.4%) as he mos equen genes in Ge man pa ien s wi h AR-RP, and Kim e al.22 de ec ed ha EYS (22%)
and PDE6B (MIM: 180072) (17%) a e mos equen ly in ol ed in AR-RP in Ko ean pa ien s. EYS was also he
mos p e alen causa i e gene in he Japanese popula ion s udied by Maeda e al.29, implica ed in 21 ou o 33
AR-RP pa ien s (63.6%), whe eas in ou popula ion EYS was mu a ed in 5.5% o he amilies wi h “a p io i”
AR-RP diagnosis, being he ou h mos equen gene, a e USH2A, CRB1 and ABCA4. Howe e , he o de
o he causa i e genes in AR-RP changes a e e iewing he clinical da a o ABCA4 ela ed IRD pa ien s, since
hey we e mos ly eclassi ied as NON-RP, downg ading EYS as he hi d mos common gene in AR-RP in ou
popula ion. This esul suppo s an eas wa d g adien in he equency o EYS a ian s in pa ien s h oughou
he wo ld and wi hin Eu ope, being mo e equen in Ge many han in Spain.
The mos equen causing a ian s de ec ed in ou s udy appea ed, as expec ed, in ABCA4 and USH2A, he
mos p e alen mu a ed genes in he Spanish popula ion6–8,30. ABCA4 c.3386G > T (p.A g1129Leu) is a a ian
almos exclusi ely ound in Spanish NON-RP pa ien s6,30, being p obably a Spanish ounde mu a ion31,32. How-
e e , USH2A c.2276G > T (p.Cys759Phe) is no exclusi e om he Spanish popula ion and has been epo ed
in o he popula ions33.
Acco ding o he geog aphical dis ibu ion o he a ian s wi hin he coun y, no di e ences be ween egions
we e obse ed. In NON-RP, he wo mos common ABCA4 a ian s we e also he mos ep esen ed in egions
wi h a ian equencies abo e 5%. Meanwhile, in RP we ound a highe ep esen a ion o he mos common
USH2A a ian , which appea ed abo e 5% o he o al alleles in ou egions. Finally, wo a ian s appea ed
o be mo e equen in some egions, i.e. PRCD c.64C > T (p.A g22Te ) in Mu cia and NR2E3 (MIM: 604485)
(GenBank: NM_014249.4) c.932G > A (p.A g311Gln) in he Cana y Islands, whe e a ounde e ec could be
happening.
Ou esul s delinea e he gene ic backg ound o he Spanish IRD pa ien s, indica ing a wide ange o causa i e
genes in ol ed in he disease. Some o he causing a ian s iden i ied a e also equen in Eu ope. Some examples
include he ABCA4 c.5882G > A (p.Gly1961Glu), epo ed wi h high p e alence in he I alian, Ge man and Span-
ish popula ions30,34,35; he c.2276G > T (p.Cys759Phe) as one o he mos equen a ian s in USH2A, especially
in Eu opean coun ies36, USH2A c.2299delG (p.Glu767Se sTe 21), which is possibly an ances al Eu opean
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pa hogenic a ian 37; BBS1 (GenBank: NM_024649.5) c.1169T > G (p.Me 390A g), iden i ied p e iously by
Myky yn e al.38 in 22 No h Ame ican BBS p obands wi h No h Eu opean ances y; and CRB1 (GenBank:
NM_201253.3) c.2843G > A (p.Cys948Ty ), iden i ied epea edly in di e en Eu opean coun ies39,40. Finally,
RHO (GenBank: NM_000539.3) c.1040C > T (p.P o347Leu), desc ibed in he I alian and F ench popula ions41,42
and also in non-Eu opean coho s5,43,44.
Va ian s ound in indi iduals om Eas Medi e anean and Middle-Eas e n coun ies also appea ed in
ou Spanish coho : PRCD c.64C > T (p.A g22Te ) ound by Sha on e al.14 in homozygosis in 15 Is aeli Mus-
lim A ab amilies, and by Behesh ian e al.45 in a Pe sian amily; and FAM161A (MIM: 613596) (GenBank:
NM_001201543.2) c.1355_1356delCA (p.Th 452Se sTe 3), which was iden i ied in Jewish amilies mainly
o igina ing om No h A ican coun ies46. As men ioned abo e, he a ian in PRCD was ound wi h a highe
equency in he egion o Mu cia, and his could be due o he se lemen o Muslim popula ions du ing se e al
cen u ies du ing he Middle Ages13. FAM161A does no ha e a signi ican speci ic geog aphical dis ibu ion in
Spain.
Rema kably, we iden i ied h ee pa hogenic a ian s wi h high equency in Spain: ABCA4 c.3386G > T
(p.A g1129Leu), p e iously men ioned; CERKL (MIM: 608381) (GenBank: NM_201548.5) c.847C > T
(p.Leu283Phe), i s desc ibed by Tuson e al.47, and RP1 c.1625C > G (p.Se 542Te ) p e iously desc ibed o igi-
nally as a Spanish ounde pa hogenic a ian . These h ee a ian s had been sca cely epo ed ou side he Span-
ish popula ion. In he case o RP1 c.1625C > G (p.Se 542Te ) a ian 48, because o i s p esence in 11 ou o 244
un ela ed amilies, we can ex apola e ha i may e y well accoun o app oxima ely 4.5% o all AR-RP cases
in he Spanish popula ion. O he g oups also iden i ied his a ian in Swiss pa ien s26.
In conclusion, his s udy shows he gene al landscape o he gene ic unde pinnings o IRD in Spain and will
help design clinical and p e en i e heal hca e app oaches o his diso de in ou coun y.
Ǥ A e ospec i e analysis was pe o med including all IRD pa ien s om ou Spanish
egis y a he Fundación Jiménez Díaz Uni e si y Hospi al (FJD, Mad id, Spain) om 1991 un il Augus 2019.
This pa ien egis y includes: all pa ien s e e ed o he Gene ic Se ice a he FJD o gene ic diagnos ic es ing
and/o counselling due o a p e ious clinical suspicion o IRD, and pa ien s wi hou gene ic analysis in ou uni
bu iden i ied in he sha ed elec onic clinical his o y o ou same-company hospi als using ICD (In e na ional
Classi ica ion o Diseases) e ms. The comple e coho con ains 6089 IRD a ec ed cases (including index cases
and a ec ed ela i es) belonging o 4403 un ela ed amilies as shown in Supplemen a y Fig.S1.
This s udy was app o ed by he E hics Commi ee o he FJD unde app o al numbe 134/2016_FJD and
ul illed all he ene s o he Decla a ion o Helsinki and i s u he e iews. A w i en in o med consen o m
was ob ained om all he pa ien s o hei legal gua dians.
ƤǤ Du ing his s udy, di e en clinical, demog aphic and amil-
ia da a we e collec ed, including (i) amily pedig ee; (ii) age a onse o isual acui y loss, ex en o isual ield
loss, nigh blindness and/o o he ea ly symp oms o e inal dys ophy; (iii) p esence o any sys emic indings
sugges i e o synd omic o ms o IRD; (i ) geog aphical o igin o he pa ien s.
Clinical diagnosis was based on oph halmic examina ion, including measu emen o bes -co ec ed isual
acui y, isual ield es ing, undus examina ion and, i possible, ull- ield elec o e inog aphy, undus au o luo es-
cence and spec al domain op ical cohe ence omog aphy scan. NON-RP and RP include non-synd omic IRD,
and hei clinical classi ica ion was done acco ding o p e iously desc ibed c i e ia6,8. NON-RP g oup include
mos pa ien s wi h CD, CRD and ach oma opsia, al hough some o hem we e included in he RP g oup due o
incomple e pheno yping a he momen o he diagnosis. Non-synd omic LCA cases we e also included in RP.
Fo NON-RP and RP amilies, an “a p io i” inhe i ance pa e n (AD/AR/XL/spo adic (S)) was es ablished
acco ding o p e iously desc ibed c i e ia1. The subg oup o XL-RP also included cho oide emia cases.
Fo cases no ex ensi ely desc ibed in he i s e e al, a gene ic classi ica ion was made as NON-RP o RP.
C i e ia o synd omic IRD diagnosis we e p e iously desc ibed7,9.
In o ma ion abou he geog aphic o igin o all he IRD cases om Spain was a ailable in 4668. They a e
dis ibu ed h oughou he 17 di e en Spanish communi ies (Fig.1A).
ƤǤ A e molecula diagnosis, inhe i ed pa e ns we e e iewed
and compa ed wi h “a p io i” da a o each amily. S a is ical analysis be ween hese da a se s o assess he global
associa ion in he NON-RP g oup was made using he Fishe ’s exac es wi h a p equal o 0.497. Whe eas o he
RP g oup, Chi-squa e es was used wi h a p below 0.001. Compa isons o each ype o inhe i ance ha e also
been made wi h he Fishe ’s exac es in he NON-RP Unclassi ied subg oup and wi h he Chi-squa e es in
he es . Fishe ’s exac es was used in hose cases in which mo e han 20% o expec ed alues we e below han
5, o a leas one o he expec ed equencies was below 1. Rega ding he signi icance le els chosen, we ha e a
global compa ison o which he signi icance le el is he usual h eshold o 0.05 and p- alue is no co ec ed, and
se e al pos -hoc compa isons o which Bon e oni’s mul iple compa isons adjus men is applied, mul iplying
he p- alues by he numbe o compa isons.
Ǥ In his s udy, we pe o med a e ospec i e analysis o he la ges coho o pa ien s wi h
IRD om Spain, whom we e ec ui ed du ing a pe iod o 28yea s by a single cen e , he FJD. The FJD is a cen e
o e e ence o molecula diagnosis o IRD om all o e he coun y, especially in some speci ic au onomous
communi ies, like Cas ile and Leon, Cas ile-La Mancha, Ex emadu a o Mad id. On he o he hand, as we
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ake in o accoun o he Spanish egions, he e a e o he cen e s o e e ence and he p e alence da a ob ained is
inconclusi e.
P e alence was calcula ed o each clinical ype o he disease by di iding he o al numbe o diagnosed
IRD cases by he o al popula ion in Spain, published by he Spanish S a is ical O ice (INE; h p://www.ine.es)
in Janua y 2019.
Ǥ Genomic DNA samples we e ob ained om he FJD Biobank om a o al o 3951
amilies (89.7%), including 1291 NON-RP, 2083 non-synd omic RP and 577 synd omic IRD amilies. Molecula
s udies we e pe o med using di e en molecula echniques as shown in Supplemen a y TableS8. Acco ding
o he echnology a ailable and he knowledge on he gene ic de e minan s o IRD a he ime o he diagnosis,
a maximum o 291 di e en genes in ol ed in IRD we e p ocessed o he molecula cha ac e iza ion (Supple-
men a y S1 Appendix). In hese s udies, index cases we e ini ially sc eened, analysed ollowing he Ame ican
College o Medical Gene ics and Genomics (ACMG; h ps ://www.acmg.ne /docs/s and a ds_guide lines _ o _
he_in e p e a ion_o _seque nce_ a ia n s.pd ) a ian s classi ica ion guidelines. I po en ially disease-causing
a ian s we e ound, seg ega ion analysis was pe o med when DNA samples om ela i es we e a ailable.
In he gene al desc ip ion o mu a ed genes and equen pa hogenic a ian s, only ully molecula ly cha -
ac e ized index cases we e conside ed. Pa ien s wi h a he e ozygo e allele in a ecessi e gene we e coun ed as
uncha ac e ized.
The equency o ecu en IRD causing a ian s was es ablished conside ing no only he o al Spanish
popula ion, bu also he di e en geog aphical egions o Spain (Fig.1A), in o de o assess he possibili y o
iden i ying any endemic o ounde e ec s. Pa hogenic a ian s wi h a p e alence abo e 5% in a pa icula egion
we e eco ded, and only hose wi h a highe p e alence we e conside ed o u he analysis.
Pa o he NGS da a a e a ailable in public, open access eposi o ies such as he Eu opean Genome-Phenome
A chi e (EGA; h ps ://www.ebi.ac.uk/ega/home; EGAD00001005746 and EGAD00001005498), RD-Connec
(h ps :// d-conne c .eu/) and he Collabo a i e Spanish Va ian Se e (CSVS; h p://cs s.babel omics .o g/) as
agg ega ed da a. The es o he da a a e a ailable upon easonable eques .
Recei ed: 28 Oc obe 2020; Accep ed: 31 Decembe 2020
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